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Lignocellulolytic clostridia employ multiple pairs of alternative σ/anti‐σ (SigI/RsgI) factors to regulate cellulosomal components for substrate‐specific degradation of cellulosic biomass. The current model has proposed that RsgIs use a sensor domain to bind specific extracellular lignocellulosic components and activate cognate SigIs to initiate expression of corresponding cellulosomal enzyme genes, while expression of scaffoldins can be initiated by several different SigIs. Pseudobacteroides cellulosolvens contains the most complex known cellulosome system and the highest number of SigI–RsgI regulons yet discovered. However, the function of many RsgI sensor domains and their relationship with the various enzyme types are not fully understood. Here, we report that RsgI4 from P. cellulosolvens employs a C‐terminal module that bears distant similarity to the fibronectin type III (Fn3) domain and serves as the sensor domain. Substrate‐binding analysis revealed that the Fn3‐like domain of RsgI4 represents a novel carbohydrate‐binding module (CBM) that binds to a wide range of polysaccharide types. Structure determination further revealed that the Fn3‐like domain belongs to the type B group of CBMs with a predicted concave face for substrate binding. Promoter sequence analysis of cellulosomal genes revealed that SigI4 is responsible for cellulosomal regulation of major scaffoldins rather than enzymes, consistent with the broad substrate specificity of the RsgI4 sensor domain. Notably, scaffoldins are invariably required as cellulosome components regardless of the substrate type. These findings suggest that the intricate cellulosome system of P. cellulosolvens comprises a more elaborate regulation mechanism than other bacteria and thus expands the paradigm of cellulosome regulation. [ABSTRACT FROM AUTHOR]

This study aimed to obtain a lentinan-specific fluorescent probe for the in situ visualization of lentinan. A carbohydrate-binding module with potential lentinan-binding capacity was discovered in a β-1,3-D-glucanase by using the bioinformatics techniques, and further recombinantly expressed. The binding specificity of the expressed protein, which was named as LBM was determined by the microtiter plate assays. The results indicated that LBM displayed a desired specificity for lentinan. The protein exhibited positive binding signals to lentinan, while could not bind to several examined polysaccharides including barley β-glucan, curdlan, or konjac glucomannan. Furthermore, the first lentinan-specific fluorescent probe was successfully constructed by fusing LBM with a green fluorescent protein EmGFP, based on which the in situ visualization of lentinan in Lentinula edodes was realized. It was shown that lentinan was not only presented in the mycelial cell wall, but also amorphously distributed in the interstitial space and inside the cell. The construction of the EmGFP-LBM provided a promising tool for the in situ visualization of lentinan. [ABSTRACT FROM AUTHOR]

In tailed phages, the baseplate is the macromolecular structure located at the tail distal part, which is directly implicated in host recognition and cell wall penetration. In myophages (i.e., with contractile tails), the baseplate is complex and comprises a central puncturing device and baseplate wedges connecting the hub to the receptor-binding proteins (RBPs). In this work, we investigated the structures and functions of adsorption-associated tail proteins of Deep-Blue and Vp4, two Herelleviridae phages infecting members of the Bacillus cereus group. Their interest resides in their different host spectrum despite a high degree of similarity. Analysis of their tail module revealed that the gene order is similar to that of the Listeria phage A511. Among their tail proteins, Gp185 (Deep-Blue) and Gp112 (Vp4) had no structural homolog, but the C-terminal variable parts of these proteins were able to bind B. cereus strains, confirming their implication in the phage adsorption. Interestingly, Vp4 and Deep-Blue adsorption to their hosts was also shown to require polysaccharides, which are likely to be bound by the arsenal of carbohydrate-binding modules (CBMs) of these phages’ baseplates, suggesting that the adsorption does not rely solely on the RBPs. In particular, the BW Gp119 (Vp4), harboring a CBM fold, was shown to effectively bind to bacterial cells. Finally, we also showed that the putative baseplate hub proteins (i.e., Deep-Blue Gp189 and Vp4 Gp110) have a bacteriolytic activity against B. cereus strains, which supports their role as ectolysins locally degrading the peptidoglycan to facilitate genome injection. IMPORTANCE The Bacillus cereus group comprises closely related species, including some with pathogenic potential (e.g., Bacillus anthracis and Bacillus cytotoxicus). Their toxins represent the most frequently reported cause of food poisoning outbreaks at the European level. Bacteriophage research is undergoing a remarkable renaissance for its potential in the biocontrol and detection of such pathogens. As the primary site of phage-bacteria interactions and a prerequisite for successful phage infection, adsorption is a crucial process that needs further investigation. The current knowledge about B. cereus phage adsorption is currently limited to siphoviruses and tectiviruses. Here, we present the first insights into the adsorption process of Herelleviridae Vp4 and Deep-Blue myophages preying on B. cereus hosts, highlighting the importance of polysaccharide moieties in this process and confirming the binding to the host surface of Deep-Blue Gp185 and Vp4 Gp112 receptor-binding proteins and Gp119 baseplate wedge. [ABSTRACT FROM AUTHOR]

This study aimed to obtain a lentinan-specific fluorescent probe for the in situ visualization of lentinan. A carbohydrate-binding module with potential lentinan-binding capacity was discovered in a β-1,3-D-glucanase by using the bioinformatics techniques, and further recombinantly expressed. The binding specificity of the expressed protein, which was named as LBM was determined by the microtiter plate assays. The results indicated that LBM displayed a desired specificity for lentinan. The protein exhibited positive binding signals to lentinan, while could not bind to several examined polysaccharides including barley β-glucan, curdlan, or konjac glucomannan. Furthermore, the first lentinan-specific fluorescent probe was successfully constructed by fusing LBM with a green fluorescent protein EmGFP, based on which the in situ visualization of lentinan in Lentinula edodes was realized. It was shown that lentinan was not only presented in the mycelial cell wall, but also amorphously distributed in the interstitial space and inside the cell. The construction of the EmGFP-LBM provided a promising tool for the in situ visualization of lentinan.

The biocatalytic degradation of poly(ethylene terephthalate) (PET) through enzymatic methods has garnered considerable attention due to its environmentally friendly and non-polluting nature, as well as its high specificity. While previous efforts in enhancing IsPETase performance have focused on amino acid substitutions in protein engineering, we introduced an amino acid insertion strategy in this work. By inserting a negatively charged acidic amino acid, Glu, at the right-angle bend of IsPETase, the binding capability between the enzyme's active pocket and PET was improved. The resulted mutant IsPETase9394insE exhibited enhanced hydrolytic activity towards PET at various temperatures ranging from 30 to 45 ℃ compared with the wild-type IsPETase. Notably, a 10.04-fold increase was observed at 45 ℃. To further enhance PET hydrolysis, different carbohydrate-binding modules (CBMs) were incorporated at the C-terminus of IsPETase9394insE. Among these, the fusion of CBM from Verrucosispora sioxanthis exhibited the highest enhancement, resulting in a 1.82-fold increase in PET hydrolytic activity at 37 ℃ compared with the IsPETase9394insE. Finally, the engineered variant was successfully employed for the degradation of polyester filter cloth, demonstrating its promising hydrolytic capacity. In conclusion, this research presents an alternative enzyme engineering strategy for modifying PETases and enriches the pool of potential candidates for industrial PET degradation. [ABSTRACT FROM AUTHOR]

Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce nonproductive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca) fused with endocellulase Cel5A (from T. fusca) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning -14 to +6. These enzymes were recombinantly expressed in E. coli, purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (~3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes. [ABSTRACT FROM AUTHOR]

Abstract Background Degradation via enzymatic processes for the production of valuable β-1,3-glucooligosaccharides (GOS) from curdlan has attracted considerable interest. CBM6E functions as a curdlan-specific β-1,3-endoglucanase, composed of a glycoside hydrolase family 128 (GH128) module and a carbohydrate-binding module (CBM) derived from family CBM6. Results Crystallographic analyses were conducted to comprehend the substrate specificity mechanism of CBM6E. This unveiled structures of both apo CBM6E and its GOS-complexed form. The GH128 and CBM6 modules constitute a cohesive unit, binding nine glucoside moieties within the catalytic groove in a singular helical conformation. By extending the substrate-binding groove, we engineered CBM6E variants with heightened hydrolytic activities, generating diverse GOS profiles from curdlan. Molecular docking, followed by mutation validation, unveiled the cooperative recognition of triple-helical β-1,3-glucan by the GH128 and CBM6 modules, along with the identification of a novel sugar-binding residue situated within the CBM6 module. Interestingly, supplementing the CBM6 module into curdlan gel disrupted the gel’s network structure, enhancing the hydrolysis of curdlan by specific β-1,3-glucanases. Conclusions This study offers new insights into the recognition mechanism of glycoside hydrolases toward triple-helical β-1,3-glucans, presenting an effective method to enhance endoglucanase activity and manipulate its product profile. Furthermore, it discovered a CBM module capable of disrupting the quaternary structures of curdlan, thereby boosting the hydrolytic activity of curdlan gel when co-incubated with β-1,3-glucanases. These findings hold relevance for developing future enzyme and CBM cocktails useful in GOS production from curdlan degradation.

Laminaripentaose (L5)‐producing β‐1,3‐glucanases can preferentially cleave the triple‐helix curdlan into β‐1,3‐glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, β‐1,3‐glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a β‐1,3‐glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 °C, and exhibited strict substrate specificity toward curdlan (83.1 U·mg−1). It efficiently hydrolyzed curdlan to produce L5 as the end product. The overall structure of SpGlu64A consisted of a barrel domain and a mixed (α/β) domain, which formed an unusually wide groove with a crescent‐like structure. In the two complex structures (SpGlu64A–L3 and SpGlu64A–L4), two oligosaccharide chains were captured and the triple‐helical structure was relatively compatible with the wide groove, which suggested the possibility of binding to the triple‐helical β‐1,3‐glucan. A catalytic framework (β6–β9–β10) and the steric hindrance formed by the side chains of residues Y161, N163, and H393 in the catalytic groove were predicted to complete the exotype‐like cleavage manner. On the basis of the structure, a fusion protein with the CBM56 domain (SpGlu64A–CBM) and a mutant (Y161F; by site‐directed mutation) were obtained, with 1.2‐ and 1.7‐fold increases in specific activity, respectively. Moreover, the combined expression of SpGlu64A–CBM and ‐Y161F improved the enzyme activity by 2.63‐fold. The study will not only be helpful in understanding the reaction mechanism of β‐1,3‐glucanases but will also provide a basis for further enzyme engineering. [ABSTRACT FROM AUTHOR]

BACKGROUND: Agarose, mainly composed of 3,6-anhydro-α-L-galactopyranose (LA) and β-D-galactopyranose (G) units, is an important polysaccharide with wide applications in food, biomedical and bioengineering industries. Carbohydrate-binding modules (CBMs) are favorable tools for the investigations of polysaccharides. Few agarose-binding CBMs have been hitherto reported, and their binding specificity is unclear. RESULTS: An unknown domain with a predicted α-sandwich fold was discovered from a β-agarase of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. The expressed protein WfCBM101 could bind to agarose and exhibited relatively weak affinity for porphyran, with no affinity for the other seven examined polysaccharides. The protein binds to the tetrasaccharide (LA-G)2, but not to the major tetrasaccharide contained in porphyran. The sequence novelty and well-defined binding function of WfCBM101 shed light on a novel CBM family (CBM101). Furthermore, the feasibility of WfCBM101 for visualizing agarose in situ was confirmed. CONCLUSION: A novel CBM, WfCBM101, with a desired specificity for agarose was discovered and characterized, which represents a new CBM family. The CBM could be utilized as a promising tool for studies of agarose. [ABSTRACT FROM AUTHOR]

Background: Degradation via enzymatic processes for the production of valuable β-1,3-glucooligosaccharides (GOS) from curdlan has attracted considerable interest. CBM6E functions as a curdlan-specific β-1,3-endoglucanase, composed of a glycoside hydrolase family 128 (GH128) module and a carbohydrate-binding module (CBM) derived from family CBM6. Results: Crystallographic analyses were conducted to comprehend the substrate specificity mechanism of CBM6E. This unveiled structures of both apo CBM6E and its GOS-complexed form. The GH128 and CBM6 modules constitute a cohesive unit, binding nine glucoside moieties within the catalytic groove in a singular helical conformation. By extending the substrate-binding groove, we engineered CBM6E variants with heightened hydrolytic activities, generating diverse GOS profiles from curdlan. Molecular docking, followed by mutation validation, unveiled the cooperative recognition of triple-helical β-1,3-glucan by the GH128 and CBM6 modules, along with the identification of a novel sugar-binding residue situated within the CBM6 module. Interestingly, supplementing the CBM6 module into curdlan gel disrupted the gel's network structure, enhancing the hydrolysis of curdlan by specific β-1,3-glucanases. Conclusions: This study offers new insights into the recognition mechanism of glycoside hydrolases toward triple-helical β-1,3-glucans, presenting an effective method to enhance endoglucanase activity and manipulate its product profile. Furthermore, it discovered a CBM module capable of disrupting the quaternary structures of curdlan, thereby boosting the hydrolytic activity of curdlan gel when co-incubated with β-1,3-glucanases. These findings hold relevance for developing future enzyme and CBM cocktails useful in GOS production from curdlan degradation. [ABSTRACT FROM AUTHOR]

Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca) fused with endocellulase Cel5A (from T. fusca) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning −14 to +6. These enzymes were recombinantly expressed in E. coli, purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (∼3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 μM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 μM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

ntroduction: The depolymerization of lignocellulose biomass by white-rot fungi has been an important research topic. However, few simulated in-situ analyses have been conducted to uncover the decay. Methods: In this study, the white-rot Lentinula edodes was used to colonize the wood and non-wood substrates, and then hyphal transcriptional response and substrate degradation were analyzed during the spatial-temporal colonization on different type substrates to better understand the depolymerization of lignocellulose. Results and discussion: Faster growth and thicker mat of hyphae on corn stalk were observed in comparison to oak wafer. Coincide with the higher levels of gene transcripts related to protein synthesis on corn stalk. The higher lignin oxidase activity of hyphae was detected on oak wafer, and the higher cellulase activity was observed on corn stalk containing a much higher content of soluble sugars. A large number of carbohydrate-binding module (CBM1 and CBM20)-containing enzyme genes, including lytic polysaccharide monooxygenase (AA9), cellobiohydrolase (GH6 and GH7), glucanase (GH5), xylanase (GH10 and GH11), glucoamylase (GH15), and alpha-amylase (GH13), were significantly upregulated in the back-distal hyphae colonized on corn stalk. The hyphae tended to colonize and degrade the secondary cell wall, and the deposited oxalate crystal suggested that oxalate may play an important role during lignocellulose degradation. In addition, lignin was degraded in priority in oak wafer. Of note, three lignin monomers were degraded simultaneously in oak wafer but sequentially in corn stalk. This growth Our results indicated that the white-rot degradation pattern of lignocellulose is determined by the chemical composition and structure of the colonized biomass. [ABSTRACT FROM AUTHOR]

In order to clarify the effect of the non-catalytic carbohydrate-binding module (CBM) and F5/8 type C domains on the enzymatic properties of AlgL7, an alginate lyase from Microbulbifer sp. ALW1, the full-length enzyme AlgL7 and two truncated enzymes: CD1 (catalytic domain) and CD2 (containing F5/8 type C domain and catalytic domain) were constructed and characterized. The results showed that the truncated enzyme CD2 exhibited higher specific activity, thermostability, Michaelis constant (Km), and maximum reaction velocity (Vmax) compared to the full-length AlgL7, indicating that the CBM domain played an important role in maintaining the substrate affinity of the enzyme, but reduced the catalytic activity, thermostability, and Vmax value the enzyme. Compared to the truncated enzyme CD1, CD2 exhibited higher specific activity, optimal reaction temperature, thermostability, Vmax, and Km, indicating that the F5/8 type C domain contributed to improve the enzymatic activity, optimum reaction temperature, thermostability, and Vmax, but reduced the substrate affinity of the enzyme. Using sodium alginate as the substrate, the specific activity of CD2 was 183.9 U/mg. The optimal reaction temperature and pH were 40 ℃ and 7.0, respectively. The Km and Vmax were 39.80 mg/mL and 2 000 U/mg, respectively. The major enzymatic hydrolysates were disaccharides and trisaccharides. This study promotes the understanding of the structure-activity relationship between the non-catalytic domains and the properties of alginate lyase, and lays a theoretical basis for using the non-catalytic domains to improve the catalytic properties of alginate lysate.

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Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98–CBM48–GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut. [ABSTRACT FROM AUTHOR]

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM‐polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM‐based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type‐A CBMs from families 3a and 64 with crystalline cellulose‐I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose‐I using equilibrium binding assays. To compute the adsorption (nkon) and desorption (koff) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide‐field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts. [ABSTRACT FROM AUTHOR]

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes. [ABSTRACT FROM AUTHOR]

The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β‐1,3‐glucan and β‐1,6‐glucan. While several fungal β‐1,6‐glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C. pinensis shows strong growth using β‐1,6‐glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C. pinensis secretome to hydrolyse the β‐1,6‐glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β‐1,6‐glucanase activity and which are classified into different GH families. Our data show that C. pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β‐1,6‐glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates. [ABSTRACT FROM AUTHOR]

Lam16A is a novel GH16 β-1,3–1,4-lichenase isolated from the genus Caldicellulosiruptor which can utilize untreated carbohydrate components of plant cell walls. Its catalytic module has been characterized that the six carbohydrate-binding modules (CBMs) were queued in the C-terminus, but their roles were still unclear. Here, full-length and CBM-truncated mutants of Lam16A were purified and characterized through heterologous expression in Escherichia coli. The profiles of these proteins, including the enzyme activity, degrading efficiency, substrate-binding affinity, and thermostability, were explored. Full-length Lam16A with six CBMs showed excellent thermostability and the highest activity against barley β-glucan and laminarin with optimum pH of 6.5. The CBMs stimulated degrading ability of the catalytic module, especially against β-1,3(4)-glucan-based polysaccharides. The released products from β-1,3–1,4-glucan by Lam16A or its truncated mutants revealed an endo-type glycoside hydrolase. Lam16As exhibited strong binding affinities to the insoluble polysaccharides, especially Lam16A-1CBM. The degradation of yeast cell walls by Lam16A enzyme solution relative to the control reduced the absorbance values at OD800 by ~ 85% ± 1.2, enabling the release of up to ~ 0.057 ± 0.0039 µg/mL of the cytoplasmic protein into the supernatant, lowering the viability of the cells by ~ 70.3% ± 6.9, thus causing significant damage in the cell wall structure. Taken together, CBMs could influence the substrate specificity, thermal stability, and binding affinity of β-1,3–1,4-glucanase. These results demonstrate the great potential of these enzymes to promote the bioavailability of β-1,3-glucan oligosaccharides for health benefits. Key points: • Carbohydrate-binding modules strongly influenced the enzyme activity and binding affinity, and further impacted glycoside hydrolase activity. • Lam16A enzymes have sufficient ability to hydrolyze β-1,3–1,4-glucan-based polysaccharides. • Lam16As provide a powerful tool to promote the bioavailability of β-1,3-glucan oligosaccharides. [ABSTRACT FROM AUTHOR]

Although more than 12,000 bacteriophages infecting mycobacteria (mycobacteriophages) have been isolated so far, there is a knowledge gap on their structure-function relationships. Here, we have explored the architecture of host-binding machineries from seven representative mycobacteriophages of the Siphoviridae family infecting Mycobacterium smegmatis, Mycobacterium abscessus, and Mycobacterium tuberculosis, using AlphaFold2 (AF2). AF2 enables confident structural analyses of large and flexible biological assemblies resistant to experimental methods, thereby opening new avenues to shed light on phage structure and function. Our results highlight the modularity and structural diversity of siphophage host-binding machineries that recognize host-specific receptors at the onset of viral infection. Interestingly, the studied mycobacteriophages' host-binding machineries present unique features compared with those of phages infecting other Gram-positive actinobacteria. Although they all assemble the classical Dit (distal tail), Tal (tail-associated lysin), and receptor-binding proteins, five of them contain two potential additional adhesion proteins. Moreover, we have identified brush-like domains formed of multiple polyglycine helices which expose hydrophobic residues as potential receptor-binding domains. These polyglycine-rich domains, which have been observed in only five native proteins, may be a hallmark of mycobacteriophages' host-binding machineries, and they may be more common in nature than expected. Altogether, the unique composition of mycobacteriophages' host-binding machineries indicate they might have evolved to bind to the peculiar mycobacterial cell envelope, which is rich in polysaccharides and mycolic acids. This work provides a rational framework to efficiently produce recombinant proteins or protein domains and test their host-binding function and, hence, to shed light on molecular mechanisms used by mycobacteriophages to infect their host. IMPORTANCE Mycobacteria include both saprophytes, such as the model system Mycobacterium smegmatis, and pathogens, such as Mycobacterium tuberculosis and Mycobacterium abscessus, that are poorly responsive to antibiotic treatments and pose a global public health problem. Mycobacteriophages have been collected at a very large scale over the last decade, and they have proven to be valuable tools for mycobacteria genetic manipulation, rapid diagnostics, and infection treatment. Yet, molecular mechanisms used by mycobacteriophages to infect their host remain poorly understood. Therefore, exploring the structural diversity of mycobacteriophages' host-binding machineries is important not only to better understand viral diversity and bacteriophage-host interactions, but also to rationally develop biotechnological tools. With the powerful protein structure prediction software AlphaFold2, which was publicly released a year ago, it is now possible to gain structural and functional insights on such challenging assemblies. [ABSTRACT FROM AUTHOR]

ABSTRACT In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one β-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a β-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL’s outer membrane sensor protein, we suggest that the FCWUL is activated by β-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade. IMPORTANCE We report that the genome of the soil bacterium Chitinophaga pinensis encodes three multi-modular carbohydrate-active enzymes that work together to hydrolyze the major polysaccharide components found in fungal cell walls (FCWs). The enzymes are all encoded by one polysaccharide utilization locus and are co-expressed, potentially induced in the presence of β-1,3-glucans. We present biochemical characterization of each enzyme, including the appended carbohydrate-binding modules that likely tether the enzymes to a FCW substrate. Finally, we propose a model for how this so-called fungal cell wall utilization locus might enable C. pinensis to metabolize both chitin and β-1,3-glucans found in complex biomass in the soil.

Abstract Background The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags. Results In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g− 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg− 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade. Conclusion CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9.

Poly(ethylene terephthalate) (PET) is a manufactured plastic broadly available, whereas improper disposal of PET waste has become a serious burden on the environment. Leaf-branch compost cutinase (LCC) is one of the most powerful and promising PET hydrolases, and its mutant LCCICCG shows high catalytic activity and excellent thermal stability. However, low binding affinity with PET has been found to dramatically limit its further industrial application. Herein, TrCBM and CfCBM were rationally selected from the CAZy database to construct fusion proteins with LCCICCG, and mechanistic studies revealed that these two domains could bind with PET favorably via polar amino acids. The optimal temperatures of LCCICCG-TrCBM and CfCBM-LCCICCG were measured to be 70 and 80 °C, respectively. Moreover, these two fusion proteins exhibited favorable thermal stability, maintaining 53.1% and 48.8% of initial activity after the incubation at 90 °C for 300 min. Compared with LCCICCG, the binding affinity of LCCICCG-TrCBM and CfCBM-LCCICCG for PET has been improved by 1.4- and 1.3-fold, respectively, and meanwhile their degradation efficiency on PET films was enhanced by 3.7% and 24.2%. Overall, this study demonstrated that the strategy of constructing fusion proteins is practical and prospective to facilitate the enzymatic PET degradation ability. [ABSTRACT FROM AUTHOR]

Corn xylan is resistant to enzymatic hydrolysis due to its complex structure. We characterized PsXyn5A, an enzyme highly active for corn xylan, isolated from Paenibacillus sp. H2C. PsXyn5A is a modular xylanase with a catalytic domain belonging to the glycoside hydrolase family 5 subfamily 35 (GH5_35) and a carbohydrate-binding module family 13 (CBM13) domain. The substrate recognition mechanism of GH5_35 xylanase has not been reported. Analysis of the hydrolysate from rye arabinoxylan (RAX) has shown that the GH5_35 catalytic domain of PsXyn5A recognizes an arabinofuranosyl (Ara f) side residue and cleaves the reducing terminal side of Ara f -linked xylopyranose. This cleavage specificity is the same as reported for the GH5_34 xylanase from Hungateiclostridium thermocellum (HtXyl5A). Unlike HtXyl5A, PsXyn5A produced Ara f -xylopyranose from RAX and did not hydrolyze 3 3 - a-l -Ara f -xylotetraose. Deletion of the CBM13 domain significantly decreased the activity toward insoluble corn xylan, indicating that CBM13 plays an essential role in hydrolyzing corn xylan. [ABSTRACT FROM AUTHOR]

Hyaluronic acid is a commercially important polysaccharide with wide applications. Along with the rapid development of hyaluronic acid-based products, their authenticity has aroused considerable attention from consumers. In the present study, a carbohydrate-binding module (CBM) SrCBM70 was cloned and expressed. The protein could specifically bind to hyaluronic acid with a strong affinity. A novel method for the identification of hyaluronic acid was subsequently established by integrating SrCBM70 into the lateral flow immunoassay (LFIA). Its detection limit for hyaluronic acid was approximately 0.1 μg/mL, and the assay could be completed in 5 min. The feasibility of this method in the authenticity identification of commercialized products containing hyaluronic acid was confirmed. The establishment of the SrCBM70-based LFIA method provided a solution for the on-site authenticity identification and would facilitate the market supervision of hyaluronic acid-based products. • A hyaluronic acid-specific CBM SrCBM70 was discovered and characterized. • A novel method was established by integrating SrCBM70 into LFIA. • This method was competent in the on-site identification of hyaluronic acid. • Its detection limit was about 0.1 μg/mL, and detection could be completed in 5 min. • The CBM-based LFIA strategy was proposed for identifying polysaccharides. [ABSTRACT FROM AUTHOR]

N -glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N -acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N -glycans in vertebrates. This enzyme attaches N -acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N -glycan core structure via a β-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind β- N -acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with β-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a β-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with β-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of β- N -acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability. [ABSTRACT FROM AUTHOR]

Abstract Background Glucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost. Results ReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4–5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch. Conclusions Our results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

The important role of Carbohydrate-binding module (CBM) in the cellulases catalytic activity has been widely studied. CBM3 showed highest affinity for cellulose substrate with 84.69 % adsorption rate among CBM1, CBM2, CBM3, and CBM4 in this study. How CBM affect the catalytic properties of GH5 endoglucanase III from Trichoderma viride (Tv EG3) was systematically explored from two perspectives: the deletion of its own CBM(Tv EG3dc) and the replacement of high substrate affinity CBM3 (Tv EG3dcCBM3). Compared with Tv EG3, Tv EG3dc lost its binding ability on Avicel and filter paper, but its catalytic activity did not change significantly. The binding ability and catalytic activity of Tv EG3dcCBM3 to Avicel increased 348.3 % and 372.51 % than that of Tv EG3, respectively. The binding ability and catalytic activity of Tv EG3dcCBM3 to filter paper decreased 51.7 % and 33.33 % than that of Tv EG3, respectively. Further structural analysis of Tv EG3, Tv EG3dc, and Tv EG3dcCBM3 revealed no changes in the positions and secondary structures of the key amino acids. These results demonstrated that its own CBM1 of Tv EG3 did not affect its catalytic activity center, so it had no effect on its catalytic activity. But CBM3 changed the adsorption affinity for different substrates, which resulted in a change in the catalytic activity of the substrate. [ABSTRACT FROM AUTHOR]

Alginate is a commercially important polysaccharide widely distributed in brown algae. Carbohydrate-binding modules (CBMs), a class of commonly used polysaccharide-binding proteins, have greatly facilitated the investigations of polysaccharides. Few alginate-binding CBMs have been hitherto reported and structurally characterized. Herein, an unknown domain from a potential PL6 family alginate lyase in the marine bacterium Vibrio breoganii was discovered and recombinantly expressed. The obtained protein, designated VbCBM106, displayed the favorable specificity to alginate. The unique sequence and well-defined function of VbCBM106 reveal a new CBM family (CBM106). Moreover, the structure of VbCBM106 was determined at a 1.5 Å resolution by the X-ray crystallography, which shows a typical β-sandwich fold comprised of two antiparallel β-sheets. Site-directed mutagenesis assays confirmed that positively charged polar residues are crucial for the ligand binding of VbCBM106. The discovery of VbCBM106 enriches the toolbox of alginate-binding proteins, and the elucidation of critical residues would guide the future practical applications of VbCBM106. • A novel CBM domain was discovered, expressed, and characterized. • The CBM exhibits the desired specificity to alginate. • The sequence novelty and defined function of the CBM reveal a new CBM family. • Its structure was resolved at a 1.5 Å resolution by X-ray crystallography. • Its critical binding residues were confirmed by site-directed mutagenesis assays. [ABSTRACT FROM AUTHOR]

Enzyme-driven recycling of PET has now become a fully developed industrial process. With the right pre-treatment, PET can be completely depolymerized within workable timeframes. This has been realized due to extensive research conducted over the past decade, resulting in a large set of engineered PET hydrolases. Among various engineering strategies to enhance PET hydrolases, fusion with binding domains has been used to tune affinity and boost activity of the enzymes. While fusion enzymes have demonstrated higher activity in many cases, these results are primarily observed under conditions that would not be economically viable at scale. Furthermore, the wide variation in PET substrates, conditions, and combinations of PET hydrolases and binding domains complicates direct comparisons. Here, we present a self-consistent and thorough analysis of two leading PET hydrolases, LCCICCG and PHL7. Both enzymes were evaluated both without and with a substrate-binding domain across a range of industrially relevant PET substrates. We demonstrate that the presence of a substrate-binding module does not significantly affect the affinity of LCCICCG and PHL7 for PET. However, significant differences exist in how the fusion enzymes act on different PET substrates and solid substrate loading, ranging from a 3-fold increase in activity to a 6-fold decrease. These findings could inform the tailoring of enzyme choice to different industrial scenarios. [Display omitted] • Detailed study of PET hydrolase fusion enzymes on different PET substrates • Binding modules do not increase the affinity of PET hydrolases for PET • Binding modules are detrimental to PET hydrolase activity on powdered PET • NMR experiments are used to show fusion enzymes do not differ in endo/exo activity [ABSTRACT FROM AUTHOR]

Abstract We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline–threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540–588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

Abstract Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein– carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM–substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose–CBM bond rupture forces exceeding 15 pN. [ABSTRACT FROM AUTHOR]

Background. Cellulolytic, hemicellulolytic, and amylolytic (CHA) enzyme-producing halophiles are understudied. The recently defined taxon Iocasia fonsfrigidae consists of one well-described anaerobic bacterial strain: NS-1T. Prior to characterization of strain NS-1T, an isolate designated Halocella sp. SP3-1 was isolated and its genome was published. Based on physiological and genetic comparisons, it was suggested that Halocella sp. SP3-1 may be another isolate of I. fronsfrigidae. Despite being geographic variants of the same species, data indicate that strain SP3-1 exhibits genetic, genomic, and physiological characteristics that distinguish it from strain NS-1T. In this study, we examine the halophilic and alkaliphilic nature of strain SP3-1 and the genetic substrates underlying phenotypic differences between strains SP3-1 and NS-1T with focus on sugar metabolism and CHA enzyme expression. Methods. Standard methods in anaerobic cell culture were used to grow strains SP3-1 as well as other comparator species. Morphological characterization was done via electron microscopy and Schaeffer-Fulton staining. Data for sequence comparisons (e.g., 16S rRNA) were retrieved via BLAST and EzBioCloud. Alignments and phylogenetic trees were generated via CLUTAL_X and neighbor joining functions in MEGA (version 11). Genomes were assembled/annotated via the Prokka annotation pipeline. Clusters of Orthologous Groups (COGs) were defined by eegNOG 4.5. DNA-DNA hybridization calculations were performed by the ANI Calculator web service. Results. Cells of strain SP3-1 are rods. SP3-1 cells grow at NaCl concentrations of 5-30% (w/v). Optimal growth occurs at 37 ℃, pH 8.0, and 20% NaCl (w/v). Although phylogenetic analysis based on 16S rRNA gene indicates that strain SP3-1 belongs to the genus Iocasia with 99.58% average nucleotide sequence identity to Iocasia fonsfrigida NS-1T, strain SP3-1 is uniquely an extreme haloalkaliphile. Moreover, strain SP3-1 ferments D-glucose to acetate, butyrate, carbon dioxide, hydrogen, ethanol, and butanol and will grow on L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-raffinose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, xylan and phosphoric acid swollen cellulose (PASC). D-rhamnose, alginate, and lignin do not serve as suitable culture substrates for strain SP3-1. Thus, the carbon utilization profile of strain SP3-1 differs from that of I. fronsfrigidae strain NS-1T. Differences between these two strains are also noted in their lipid composition. Genomic data reveal key differences between the genetic profiles of strain SP3-1 and NS-1T that likely account for differences in morphology, sugar metabolism, and CHA-enzyme potential. Important to this study, I. fonsfrigidae SP3-1 produces and extracellularly secretes CHA enzymes at different levels and composition than type strain NS-1T. The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery. [ABSTRACT FROM AUTHOR]

Quantification is essential in the research and development of polysaccharides. However, achieving simplicity and specificity in polysaccharide quantification remains a challenging task. Enzyme-linked immunosorbent assay (ELISA) based on antibodies provides a straightforward and specific quantification strategy. Nevertheless, acquiring antibodies for polysaccharides is complicated. Carbohydrate-binding modules (CBMs), which can be efficiently obtained, exhibit the capability to specifically recognize and bind carbohydrates. In this study, we verified the feasibility of a CBM-based ELISA for the quantification of hyaluronic acid (HA) by replacing an antibody with a CBM. The CBM-based ELISA, which employed a HA-specific CBM, exhibited a linear detection range spanning from 10 to 100 μg/mL. Both intra-assay and inter-assay coefficients of variation remained below 15 % and recoveries ranged from 96.26 % to 98.22 %, indicating favorable precision and accuracy. The method exhibited specificity exclusively to HA, suggesting its reliance on CBM binding specificity. The effectiveness of the method in analyzing commercial products was confirmed. Additionally, a comparison with the carbazole assay revealed a highly significant correlation (r = 0.994). By integrating CBMs with ELISA, the study presented a novel and easily implementable solution for simple and specific quantification of HA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Enzymatic degradation offers a promising eco-friendly solution to plastic pollution, especially for polyethylene terephthalate (PET). Current efforts have focused on screening PET-degrading enzymes from microbial and metagenomic sources and obtaining superior candidates with a limited set of templates. More efficient PET hydrolases are required for PET-waste biorefinery. Here, using a structure-guided bioinformatic workflow, we identified a novel PET hydrolase, LSPET4, from Micromonospora sp. HM5-17, by screening the AlphaFold protein structure database. LSPET4 features a unique carbohydrate-binding module (CBM) and a distinctive linear substrate binding conformation. The intrinsic CBM in LSPET4 exhibited superior binding ability on PET surfaces and enhanced PET hydrolysis performance compared to the previously reported most effective CBM3. Through rational protein engineering focused on stabilizing and modifying the linear substrate binding conformation, we developed LSPET4 M6 (D130P, N127F, Y96F, Q209E, A238K, D241S), a variant that achieved a 38.79-fold improvement in activity compared to the wild type, and was comparable to the reported most effective PET hydrolase derived from IsPETase, FAST-PETase at 45 ℃. This variant also demonstrated effectiveness in degrading various commercial PET materials, including PET food sealing films, PET strawberry boxes, and PET tomato boxes used in the food industry. This study not only provides a new template for protein engineering endeavors to create efficient biocatalysts for PET recycling but also offers an effective enzyme discovery approach to uncover enzymes of interest from the AlphaFold protein structure database., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lignocellulolytic clostridia employ multiple pairs of alternative σ/anti-σ (SigI/RsgI) factors to regulate cellulosomal components for substrate-specific degradation of cellulosic biomass. The current model has proposed that RsgIs use a sensor domain to bind specific extracellular lignocellulosic components and activate cognate SigIs to initiate expression of corresponding cellulosomal enzyme genes, while expression of scaffoldins can be initiated by several different SigIs. Pseudobacteroides cellulosolvens contains the most complex known cellulosome system and the highest number of SigI-RsgI regulons yet discovered. However, the function of many RsgI sensor domains and their relationship with the various enzyme types are not fully understood. Here, we report that RsgI4 from P. cellulosolvens employs a C-terminal module that bears distant similarity to the fibronectin type III (Fn3) domain and serves as the sensor domain. Substrate-binding analysis revealed that the Fn3-like domain of RsgI4 represents a novel carbohydrate-binding module (CBM) that binds to a wide range of polysaccharide types. Structure determination further revealed that the Fn3-like domain belongs to the type B group of CBMs with a predicted concave face for substrate binding. Promoter sequence analysis of cellulosomal genes revealed that SigI4 is responsible for cellulosomal regulation of major scaffoldins rather than enzymes, consistent with the broad substrate specificity of the RsgI4 sensor domain. Notably, scaffoldins are invariably required as cellulosome components regardless of the substrate type. These findings suggest that the intricate cellulosome system of P. cellulosolvens comprises a more elaborate regulation mechanism than other bacteria and thus expands the paradigm of cellulosome regulation., (© 2024 The Protein Society.)

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation has been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

Cellulomonas flavigena is a saprotrophic bacterium that encodes, within its genome, four predicted lytic polysaccharide monooxygenases (LPMOs) from Auxiliary Activity family 10 (AA10). We showed previously that three of these cleave the plant polysaccharide cellulose by oxidation at carbon-1 (J. Li, L. Solhi, E.D. Goddard-Borger, Y. Mattieu et al., Biotechnol Biofuels 14:29, 2021, https://doi.org/10.1186/s13068-020-01860-3). Here, we present the biochemical characterization of the fourth C. flavigena AA10 member (CflaLPMO10D) as a chitin-active LPMO. Both the full-length CflaLPMO10D-Carbohydrate-Binding Module family 2 (CBM2) and catalytic module-only proteins were produced in Escherichia coli using the native general secretory (Sec) signal peptide. To quantify chitinolytic activity, we developed a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAECPAD) method as an alternative to the established hydrophilic interaction liquid ion chromatography coupled with UV detection (HILIC-UV) method for separation and detection of released oxidized chito-oligosaccharides. Using this method, we demonstrated that CflaLPMO10D is strictly active on the b-allomorph of chitin, with optimal activity at pH 5 to 6 and a preference for ascorbic acid as the reducing agent. We also demonstrated the importance of the CBM2 member for both mediating enzyme localization to substrates and prolonging LPMO activity. Together with previous work, the present study defines the distinct substrate specificities of the suite of C. flavigena AA10 members. Notably, a cross-genome survey of AA10 members indicated that chitinolytic LPMOs are, in fact, rare among Cellulomonas bacteria. [ABSTRACT FROM AUTHOR]

Carbohydrate-binding modules (CBMs) are important substrate-binding domains that are mainly contained within carbohydrate-active enzymes. To elucidate the mechanism of enzyme-carbohydrate recognition and to promote the process of enzymatic engineering, it is important to explore more potential CBMs. However, the information and analytic tools of CBMs provided by current databases are limited. Here, a simple, user-friendly, and comprehensive CBM database (CBMDB) that integrates multidimensional information and analysis tools was constructed. Based on a data query function and analysis tools provided by the CBMDB, including sequence similarity searches, pairwise alignment, multiple sequence alignment, structure similarity searches, and phylogenetic visualization, information retrieval and analysis of known CBMs could be easily performed. Notably, unknown proteins with potential CBM functions could also be examined based on existing CBM data. [ABSTRACT FROM AUTHOR]

Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a ß-xylosidase, was cloned and sequenced. Sequence analysis revealed that this ß-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-ß-D-xylopyranoside (pNPX) and p-nitrophenyl-a-L-arabinofuranoside at specific activities of 250 and 310 mU/mg, respectively. The optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54 °C, respectively. At pH 7.0 and 54 °C, the Km and kcat for pNPX were 1.2 mM and 2.8 ± 0.15 s-1, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UM's binding constant value Ka for oat spelt xylan was 2.0 × 10-5 M-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBM91, specific for xylan-containing polysaccharides. [ABSTRACT FROM AUTHOR]

Background: The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags. Results: In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g− 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg− 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade. Conclusion: CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9. [ABSTRACT FROM AUTHOR]

Successful bacteriophage infection starts with specific recognition and adhesion to the host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limits their structural analyses by experimental approaches such as X-ray crystallography and electron microscopy. However, the protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of whole adhesion devices of five representative siphophages infecting Streptococcus thermophilus, one of the main lactic acid bacteria used in dairy fermentations. The predictions highlight the mosaic nature of these devices that share functional domains for which active sites and residues could be unambiguously identified. Such AlphaFold2 analyses of phage-encoded host adhesion devices should become a standard method to characterize phage-host interaction machineries and to reliably annotate phage genomes.

Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that TfXyl10A, a xylanase from Thermobifida fusca, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that TfXyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of TfXyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme’s temperature tolerance was also weakened. While truncating only the CBM domain (TfXyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase TfXyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that TfXyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of TfXyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme TfXyl10A-CD (the catalytic domain of TfXyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.

Abstract Extensive decoration of backbones is a major factor resulting in resistance of enzymatic conversion in hemicellulose and other branched polysaccharides. Employing debranching enzymes is the main strategy to overcome this kind of recalcitrance at present. A carbohydrate-binding module (CBM) is a contiguous amino acid sequence that can promote the binding of enzymes to various carbohydrates, thereby facilitating enzymatic hydrolysis. According to previous studies, CBMs can be classified into four types based on their preference in ligand type, where Type III and IV CBMs prefer to branched polysaccharides than the linear and thus are able to specifically enhance the hydrolysis of substrates containing side chains. With a role in dominating the hydrolysis of branched substrates, Type III and IV CBMs could represent a non-catalytic approach in overcoming side-chain recalcitrance.

Broad-acting antiviral strategies to prevent respiratory tract infections are urgently required. Emerging or re-emerging viral diseases caused by new or genetic variants of viruses such as influenza viruses (IFVs), respiratory syncytial viruses (RSVs), human rhinoviruses (HRVs), parainfluenza viruses (PIVs) or coronaviruses (CoVs), pose a severe threat to human health, particularly in the very young or old, or in those with pre-existing respiratory conditions such as asthma or chronic obstructive pulmonary disease (COPD). Although vaccines remain a key component in controlling and preventing viral infections, they are unable to provide broad-spectrum protection against recurring seasonal infections or newly emerging threats. HEX17 (aka Neumifil), is a first-in-class protein-based antiviral prophylactic for respiratory viral infections. HEX17 consists of a hexavalent carbohydrate-binding module (CBM) with high affinity to sialic acids, which are typically present on terminating branches of glycans on viral cellular receptors. This allows HEX17 to block virus engagement of host receptors and inhibit infection of a wide range of viral pathogens and their variants with reduced risk of antiviral resistance. As described herein, HEX17 has demonstrated broad-spectrum efficacy against respiratory viral pathogens including IFV, RSV, CoV and HRV in multiple in vivo and in vitro studies. In addition, HEX17 can be easily administered via an intranasal spray and is currently undergoing clinical trials. • Exacerbation of chronic respiratory diseases, such as COPD, can be driven by a range of different viruses. • HEX17(Neumifil) is an engineered, multivalent carbohydrate-binding protein with high affinity to sialic acid. • Sialic acid is a ubiquitous cell-surface glycan and is also present on some viruses. • By targeting sialic acid, HEX17(Neumifil) provides broad-acting protection against respiratory viruses. • Data show efficacy against influenza, respiratory syncytial virus, rhinoviruses, parainfluenza viruses and coronaviruses. [ABSTRACT FROM AUTHOR]