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The 1,4-α-glucan branching enzymes (GBEs, EC 2.4.1.18), is reported to catalyze the formation of α-1,6 branching points in carbohydrates, which has ability of changing the structure of starch by adjusting the points and frequency of branch points in starch. In practical uses, GBEs are expected to be used at relatively high temperature because of high temperature requirement of starch gelatinization process. However, the present GBE does not meet the requirements, which limits the application of GBEs in starch modification industry. In order to enhance the thermostability of GBE, we combined the values of B-factor and accessible surface area, as well as the opportunity of building potential salt bridges in this enzyme. As a result, amino acid sites 73 and 137 in GBE from Geobacillus thermoglucosidans STB02 were selected in our research. The results show that substitution of Lys with Glu at amino acid 137 site resulted in an approximately 36.6 % enhancement in half-time; replacement of Gln with Asp at this site resulted in a 26 % improvement in thermostability. In addition to thermostability, catalytic ability is another factor to be concerned in our study. We constructed combined mutants with high catalytic efficiency and high thermostability. Compared with wild type, the resulting mutants have advantages in maltodextrin modification, of which product exhibited stronger stability than unmodified maltodextrin. Our study provides a new approach to increase thermostability of present GBE and create mutants with high thermostability and catalytic ability.

Biocatalysis has become a powerful alternative for green chemistry. Expanding the range of amino acids used in protein biosynthesis can improve industrially appealing properties such as enantioselectivity, activity and stability. This review will specifically delve into the thermal stability improvements that non-canonical amino acids (ncAAs) can confer to enzymes. Methods to achieve this end, such as the use of halogenated ncAAs, selective immobilization and rational design, will be discussed. Additionally, specific enzyme design considerations using ncAAs are discussed along with the benefits and limitations of the various approaches available to enhance the thermal stability of enzymes.

We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH

The β-galactosidase was extracted and purified from 100 g of C. arvensis seeds using a variety of protein purification procedures such as ammonium sulphate fractionation, gel filtration, and finally chromatography on a cationic ion exchanger. The effects of metal ions, kinetics parameters, and glycoprotein nature were determined, as well as the optimal pH and temperature of the purified enzyme. With a high specific activity (72 units/mg), β-galactosidase was isolated to a 24-fold apparent electrophoretic homogeneity. The molecular mass of β-galactosidase was determined as monomeric, which was further confirmed by SDS-PAGE and MALDI-TOF/MS analysis, with a 45 kDa molecular weight. The enzyme has a K

A key aspect of adaptation to cold environments is the production of cold-active enzymes by psychrophilic organisms. These enzymes not only have high activity at low temperatures, but also exhibit remarkable structural flexibility and thermolability. In this context, the role of metal ions has been little explored, and the few available studies seem to suggest that metal binding counteracts structural flexibility. This article reports an investigation into the role of the binding of manganese ion (Mn2+) in the thermal adaptation of an esterase (M-Est) of the GDSx family, identified in the genome of the Antarctic bacterium Marinomonas sp. ef1. M-Est is specific for esters containing acetate groups and turned out to be a highly thermolabile cold-active enzyme, with a catalysis optimum temperature of 5 °C and a melting temperature of 31.7 °C. A combination of biochemical and computational analyses, including molecular dynamics simulations, revealed that M-Est binds Mn2+ ions via a single binding site located on the surface of the enzyme, close to the active site. Although the interaction between M-Est and Mn2+ induces only local conformational changes involving the active site, quite surprisingly they trigger an improvement in both thermal stability and catalytic efficiency under mild temperature conditions. These results, together with the conservation of the Mn2+ binding site among psychrophilic and psychrotolerant homologues, suggest that Mn2+ binding may be a useful, albeit atypical, strategy to mitigate the detrimental effects of temperature on true cold-active enzymes.

Ammonia lyases have great application potential in food and pharmaceuticals owing to their unique ammonia addition reaction and atom economy. A novel methylaspartate ammonia-lyase, EcMAL, from E. coli O157:H7 showed high catalytic activity. To further strengthen its thermostability and activity, disulfide bond and backbone cyclization (cyclase) variants were constructed by rational design, respectively. Among them, variant M3, with a disulfide bond introduced, exhibited a 2.3-fold increase in half-life at 50 °C, while cyclase variant M8 showed better performance, with 25.9-fold increases. The synergistic promotion effect of this combinational strategy on activity and stability was also investigated, and the combined mutant M9 exhibited a 1.1-fold improvement in catalytic efficiency while maintaining good thermostability. Circular dichroism analysis and molecular dynamics simulation confirmed that the main sources of improved thermostability were reduced atomic fluctuation and a more stable secondary structure. To our knowledge, this is the first example of combining the introduction of disulfide bonds with cyclase construction to improve enzyme stability, which was characterized by modification away from the enzyme active center, and provided a new method for adjusting enzyme thermostability.

A preferable pullulanase with high thermostability and catalytic activity at pH 4.5-5 is desired to match with glucoamylase in the starch-saccharification process. However, most of them exhibit low activity under such low pH conditions. Here, the optimal pH of the hyperthermostable pullulanase from Pyrococcus yayanosii (Pul

In this study, magnetic nanoparticles (MNPs) were combined with graphene oxide (GO) to effectively improve the immobilization effect of lipase, and magnetic graphene oxide (MGON) was modified to immobilize Candida rugosa lipase (CRL) with functionalized MGON as a carrier. The optimal magnetic carrier was screened. Characterization and analysis revealed that the specific surface area of MGON was 263.75 m

As a new generation of green solvents, deep eutectic solvents (DESs) are considered a promising alternative to current harsh organic solvents and find application in many chemical processing methods such as extraction and synthesis. DESs, normally formed by two or more components via various hydrogen bond interactions, offer high potential as medium for biocatalysis reactions where they can improve efficiency by enhancing substrate solubility and the activity and stability of the enzymes. In the current study, the stabilization of Humicola insolens cutinase (HiC) in natural deep eutectic solvents (NADESs) was assessed. The best hydrogen bond donor among sorbitol, xylitol, erythritol, glycerol and ethylene glycol, and the best acceptor among betaine, choline chloride, choline acetate, choline dihydrogen citrate and tetramethylammonium chloride, were selected, evaluating binding energies and molecular orientations through molecular docking simulations, and finally used to prepare NADES aqueous solutions. The effects of component ratio and NADES concentration on HiC thermostability at 90 °C were also investigated. The choline dihydrogen citrate:xylitol, in a 1:1 ratio with a 20 wt% concentration, was selected as the best combination in stabilizing HiC, increasing its half-life three-fold.

Type of cross-linking agents influence the stability and active cross-linked enzyme aggregates (CLEA) immobilization. The information of molecular interaction between enzyme-cross linker is not well explored thus screening wide numbers of cross-linker is crucial in CLEA development. This study combined the molecular modeling and experimental optimization to investigate the influences of different cross-linking agents in developing CLEA of cyclodextrin glucanotranferase G1 (CGTase G1) for cyclodextrins (CDs) synthesis. Seven types of cross-linkers were tested and CGTase G1 cross-linked with chitosan (CS-CGTG1-CLEA) displayed the highest activity recovery (84.6 ± 0.26%), aligning with its highest binding affinity, radius of gyration and flexibility through in-silico analysis towards CGTase G1. CS-CGTG1-CLEA was characterized and showed a longer half-life (30.06 ± 1.51 min) and retained a greater thermal stability (52.73 ± 0.93%) after 30 min incubation at optimal conditions compared to free enzyme (10.30 ± 1.34 min and 5.51 ± 2.10% respectively). CS-CGTG1-CLEA improved CDs production by 33% and yielded cumulative of 52.62 g/L CDs after five cycles for 2 h of reaction. This study reveals that abundant of hydroxyl group on chitosan interacted with CGTase G1 surface amino acid residues to form strong and stable CLEA thus can be a promising biocatalyst in CDs production.

In this research, a magnetic reusable nickel nanoparticle (NiNPs) supporting materials were prepared for cellulase enzyme immobilization. The immobilized cellulase showed high activity recovery, largefast immobilization capacity and improved pHtemperature tolerance. The excellent stability and reusability enabled the immobilized cellulase to retain 84% of its initial activity after ten cycles. At 2 mg/mL enzyme concentration, highest 93% immobilization efficiency was achieved within two hours of immobilization. When the treatment temperature reached 40 °C and pH 5, the immobilized cellulase exhibited highest residual activity. The immobilized cellulase could be separated from the solution by a magnetic force. This study introduced a novel supporting material for cellulase immobilization, and the immobilized cellulase poses a great potential in the hydrolysis of lignocellulosic biomass which can used as an easily applicable and sustainable pre-treatment step for advanced biofuel production.

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R

Lipase B from Candida antarctica (CALB) and lipases from Candida rugosa (CRL) and Rhizomucor miehei (RML) have been coimmobilized on octyl and octyl-Asp agarose beads. CALB was much more stable than CRL, that was significantly more stable than RML. This forces the user to discard immobilized CALB and CRL when only RML has been inactivated, or immobilized CALB when CRL have been inactivated. To solve this problem, a new strategy has been proposed using three different immobilization protocols. CALB was covalently immobilized on octyl-vinyl sulfone agarose and blocked with Asp. Then, CRL was immobilized via interfacial activation. After coating both immobilized enzymes with polyethylenimine, RML could be immobilized via ion exchange. That way, by incubating in ammonium sulfate solutions, inactivated RML could be released enabling the reuse of coimmobilized CRL and CALB to build a new combi-lipase. Incubating in triton and ammonium sulfate solutions, it was possible to release inactivated CRL and RML, enabling the reuse of immobilized CALB when CRL was inactivated. These cycles could be repeated for 3 full cycles, maintaining the activity of the active and immobilized enzymes.

Trypsin (Try, EC. 3.4.21.4) was effectively immobilized on the surface of glutaraldehyde(GA)-activated ZnO/Chitosan nanocomposite through covalent attachment via Schiff-base linkages. Size, structure, surface morphology,percentage elemental composition of the prepared ZnO nanoparticles and chitosan-coated ZnO nanocomposite were studied by UV-Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-Ray diffraction analysis (XRD), transmission electron microscopy (TEM), Scanning electron microscopy (SEM), and Energy-Dispersive X-Ray Microanalysis (EDAX) techniques. Optimal immobilization conditions (incubation time (16 h), enzyme concentration (1.8 mg/ml), and pH (7.8)) were investigated to obtain the maximum expressed activity of the immobilized trypsin. Immobilizedsolubilized trypsin exhibited the optimum catalytic activity at pH 8.5, 60 °C, and pH 7.8, 45 °C respectively. Kinetic parameters (K

Feruloyl esterases are indispensable biocatalysts catalyzing the cleavage of ester bonds between polysaccharides and their hydroxycinnamoyl cross-links. GthFAE from Geobacillus thermoglucosidasius was identified as a thermophilic alkaline feruloyl esterase with potential applications in paper manufacturing. To improve the enzymatic properties rationally and efficiently, the structure of GthFAE was solved at 1.9 Å, revealing a core domain of classical α/β hydrolase fold and an inserted α/β cap domain. In silico analysis based on it helped us to investigate whether the residues at the active center have positive effects on the stability, and how. Several site-directed mutations were conducted, of which substitutions at residues T41 and T150 apparently improved the thermostability. The combination mutant T41N/T150R exhibited an optimal temperature of 65 °C, a 6.4 °C higher T

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.

Among the matrices for enzyme immobilization, activated carbon has been standing out in immobilization processes due to its properties and to its characteristics that provide superficial modification by inserting new functional groups capable of binding the enzymes forming covalent bonds. In this study the effect of different modification methods of activated carbon (functionalization with genipin, metallization, metallization in the presence of chelating agent, and functionalization with glutaraldehyde) on efficiency of pepsin immobilization was evaluated. The effect of immobilization pH and the reaction medium on hydrolysis activity of bovine casein was also evaluated. The functionalization of activated carbon using iron ions allowed an immobilization capacity of 98.93 mg·g

This study aimed to develop single-step purification and immobilization processes on cellulosic supports of β-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/g

SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis.

The hydrophobic nature of wool induced by its surface lipid barrier hinders its wettability during processing. Scouring of wool is conducted to remove this lipid barrier and facilitate any wet processes. Scouring of wool is conducted using soda ash followed by rinsing with huge amount of water to ensure complete removal of alkali. This work aimed at utilization of thermophilic lipase enzyme for removal of wool surface lipid barrier without deterioration on the fibre interior. A thermally stable lipase enzyme was produced from thermophilic microorganism; namely Bacillus aryabhattai B8W22, and was utilized in bio-scouring of wool. The produced enzyme was immobilized on sericin-based discs to enhance its stability and to make it reusable. The activity of both free and immobilized lipase enzymes at different conditions was assessed. The effects of bio-scouring of wool on its dyeability with acid, basic, and reactive dyes, as well as on some of its inherent properties, were monitored. Results showed that the bio-scoured wool exhibits enhanced dyeability with the said classes of dyes more than that of conventionally scoured samples. One-bath scouring and dyeing of wool fibres in two successive steps was conducted to reduce consumption of water and energy during wet processing of wool.

The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (kcat /Km ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities.

The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL−1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg−1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7–9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the diaminopimelate pathway of bacteria, yielding amino acids required for cell wall and protein biosyntheses. The essentiality of the enzyme to bacteria, coupled with its absence in humans, validates DHDPS as an antibacterial drug target. Conventional drug design efforts have thus far been unsuccessful in identifying potent DHDPS inhibitors. Here, we make use of contemporary molecular dynamics simulation and Markov state models to explore the interactions between DHDPS from the human pathogen Staphylococcus aureus and its cognate substrate, pyruvate. Our simulations recover the crystallographic DHDPS-pyruvate complex without a priori knowledge of the final bound structure. The highly conserved residue Arg140 was found to have a pivotal role in coordinating the entry of pyruvate into the active site from bulk solvent, consistent with previous kinetic reports, indicating an indirect role for the residue in DHDPS catalysis. A metastable binding intermediate characterized by multiple points of intermolecular interaction between pyruvate and key DHDPS residue Arg140 was found to be a highly conserved feature of the binding trajectory when comparing alternative binding pathways. By means of umbrella sampling we show that these binding intermediates are thermodynamically metastable, consistent with both the available experimental data and the substrate binding model presented in this study. Our results provide insight into an important enzyme-substrate interaction in atomistic detail that offers the potential to be exploited for the discovery of more effective DHDPS inhibitors and, in a broader sense, dynamic protein-drug interactions., Author Summary Interactions between proteins and ligands underpin many important biological processes, such as binding of substrates to their cognate enzymes in the process of catalysis. These interactions are complex, often requiring several intermediate steps to fully transition into the bound state. Here, we have used computational simulation to study binding of pyruvate to Dihydrodipicolinate synthase (DHDPS), an enzyme in the bacterial diaminopimelate pathway. In bacteria, such as the human pathogen S. aureus, DHDPS functions to make building blocks necessary for protein and bacterial cell wall biosyntheses. As the enzyme is absent in humans, yet essential for bacterial growth, DHDPS is a valid target for broad-range antibiotics. However, known DHDPS inhibitors show poor potency. One avenue that has not yet been taken into consideration for inhibitor design is the dynamics of DHDPS’s interaction with its reaction substrates (e.g. pyruvate). Using molecular dynamics simulation, we find that pyruvate binding to DHDPS must pass through a transition intermediate ‘hotspot’ in which the substrate is held in place by a dense network of noncovalent bonds. Given that many of the protein residues involved in this interaction are also shared by DHDPS from many pathogenic bacteria, this binding intermediate ‘hotspot’ may help in development of better broad-range DHDPS inhibitors.

The polyextremophilic β-galactosidase enzyme of the haloarchaeon Halorubrum lacusprofundi functions in extremely cold and hypersaline conditions. To better understand the basis of polyextremophilic activity, the enzyme was studied using steady-state kinetics and molecular dynamics at temperatures ranging from 10 °C to 50 °C and salt concentrations from 1 M to 4 M KCl. Kinetic analysis showed that while catalytic efficiency (kcat/Km) improves with increasing temperature and salinity, Km is reduced with decreasing temperatures and increasing salinity, consistent with improved substrate binding at low temperatures. In contrast, kcat was similar from 2–4 M KCl across the temperature range, with the calculated enthalpic and entropic components indicating a threshold of 2 M KCl to lower the activation barrier for catalysis. With molecular dynamics simulations, the increase in per-residue root-mean-square fluctuation (RMSF) was observed with higher temperature and salinity, with trends like those seen with the catalytic efficiency, consistent with the enzyme’s function being related to its flexibility. Domain A had the smallest change in flexibility across the conditions tested, suggesting the adaptation to extreme conditions occurs via regions distant to the active site and surface accessible residues. Increased flexibility was most apparent in the distal active sites, indicating their importance in conferring salinity and temperature-dependent effects.

Enzymatic conversion of pharmaceutically active ingredients (API), using immobilized enzymes should be considered as a promising industrial tool due to improved reusability and stability of the biocatalysts at harsh process conditions. Therefore, in this study horseradish peroxidase was immobilized into sodium alginate capsules and then trapped into poly(vinyl chloride) electrospun fibers to provide additional enzyme stabilization and protection against the negative effect of harsh process conditions. Due to encapsulation immobilization, 100% of immobilization yield was achieved leading to loading of 25 μg of enzyme in 1 mg of the support. Immobilized in such a way, enzyme showed over 80% activity retention. Further, only slight changes in kinetic parameters of free (Km = 1.54 mM) and immobilized horseradish peroxidase (Km = 1.83 mM) were noticed, indicating retention of high catalytic properties and high substrate affinity by encapsulated biocatalyst. Encapsulated horseradish peroxidase was tested in biodegradation of two frequently occurring in wastewater API, sulfamethoxazole (antibiotic) and carbamazepine (anticonvulsant). Over 80% of both pharmaceutics was removed by immobilized enzyme after 24 h of the process from the solution at a concentration of 1 mg/L, under optimal conditions, which were found to be pH 7, temperature 25 °C and 2 mM of H2O2. However, even from 10 mg/L solutions, it was possible to remove over 40% of both pharmaceuticals. Finally, the reusability and storage stability study of immobilized horseradish peroxidase showed retention of over 60% of initial activity after 20 days of storage at 4 °C and after 10 repeated catalytic cycles, indicating great practical application potential. By contrast, the free enzyme showed less than 20% of its initial activity after 20 days of storage and exhibited no recycling potential.

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL−1 of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL−1 PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL−1. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.

The group of hydrolytic enzymes synonymously known as proteases is predominantly most favored for the class of industrial enzymes. The present work focuses on the thermostable nature of these proteolytic enzymes that occur naturally among mesophilic and thermophilic microbes. The broad thermo-active feature (40-80 °C), ease of cultivation, maintenance, and bulk production are the key features associated with these enzymes. Detailing of contemporary production technologies, and controllable operational parameters including the purification strategies, are the key features that justify their industrial dominance as biocatalysts. In addition, the rigorous research inputs by protein engineering and enzyme immobilization studies add up to the thermo-catalytic features and application capabilities of these enzymes. The work summarizes key features of microbial proteases that make them numero-uno for laundry, biomaterials, waste management, food and feed, tannery, and medical as well as pharmaceutical industries. The quest for novel and/or designed and engineered thermostable protease from unexplored sources is highly stimulating and will address the ever-increasing industrial demands.

Microbial urate oxidase has emerged as a potential source of therapeutic properties for hyperuricemia in arthritic gout and renal disease. The thermostability and long-term thermal tolerance of the enzyme need to be established to prolong its therapeutic effects. Here, we present the biochemical and structural aspects of a hyperthermostable urate oxidase (TbUox) from the thermophilic microorganism Thermobispora bispora. Enzymatic characterization of TbUox revealed that it was active over a wide range of temperatures, from 30 to 70 °C, with optimal activity at 65 °C and pH 8.0, which suggests its applicability under physiological conditions. Moreover, TbUox exhibits high thermostability from 10 to 65 °C, with Tm of 70.3 °C and near-neutral pH stability from pH 7.0 to 8.0 and high thermal tolerance. The crystal structures of TbUox revealed a distinct feature of the C-terminal loop extensions that may help with protein stability via inter-subunit interactions. In addition, the high thermal tolerance of TbUox may be contributed by the extensive inter-subunit contacts via salt bridges, hydrogen bonds, and hydrophobic interactions. The findings in this study provide a molecular basis for the thermophilic TbUox urate oxidase for application in hyperuricemia and gout therapy.

Leifsonia alcohol dehydrogenase (LnADH) is a promising biocatalyst for the synthesis of chiral alcohols. However, limitations of wild-type LnADH observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve its thermostability and catalytic efficiency by altering the subunit interfaces. Residues of T100 and S148 were identified to be significant for thermostability and activity, and the melting temperature (ΔTm) and catalytic efficiency of the mutant T100R/S148I toward ketone substrates were improved by 18.7 °C and 1.8-5.5-fold, respectively. Solving the crystal structures of the wild-type enzyme and T100R/S148L reveals the beneficial effects of mutations on stability and catalytic activity. The most robust mutant T100R/S148I is promising for industrial applications and can produce 200 g liter -1 day -1 chiral alcohols at 50 °C by only a 1:500 ratio of enzyme to substrate.

Marine macroalgae are potential renewable feedstocks for valuable biomaterials. Among them, alginate is a primary component in brown algae that can be nonenzymatically converted and enzymatically degraded by alginate lyases to 4-deoxy- l -erythro-5-hexoseulose uronic acid (DEH). Here, we constructed alginolytic enzyme complexes comprising two different alginate lyases for synergistic alginate degradation. The complexes showed good thermostability with 60% of the residual activity at high temperature (60 °C). Furthermore, they produced 0.85 and 0.18 mg/mL DEH from alginate and natural brown algae as substrates, respectively. The enzyme complex successfully decomposed brown algal biomass, resulting in a 3.15-fold improvement in DEH when compared to free enzymes. The Ralstonia eutropha strain with alginolytic enzyme complexes on the cell surface showed higher Polyhydroxybutyrate (PHB) production and produced 2.58 g/L PHB from alginate. After the use of alginate, remaining biomass such as fucoidan and laminaran can also be used in the future for high value ingredients in nutritional, medical device, skincare and dermatological products. These results demonstrate that it is possible to create more efficient strategies for producing biodegradable PHB and functional polysaccharides from brown algal substrates.

Two morphologies of laccase-mineral hybrid complexes, i.e., laccase-mineral hybrid nanoflowers (La-HNF) and nanopetals (La-HNP), were synthesized via biomineralization using Cu3 (PO4)2·3H2O as the mineral for Evans Blue (EB) dye biodegradation. XRD patterns and FT-IR spectra results revealed the successful immobilization of laccase via in-situ formed Cu3(PO4)2·3H2O crystals. Compared with free laccase, laccase-mineral hybrid complexes showed higher enzymatic activity due to the activation effect induced by copper ions of Cu3(PO4)2·3H2O, further, the improved kinetic parameters of laccase-mineral hybrid complexes could be ascribed to nanoscale-dispersed laccase molecules within hybrid complexes. For EB dye biodegradation, the reason why the biodegradation efficiency (94.9%) of La-HNF was higher than that (86.8%) of La-HNP could be synergistic effect of immobilized laccase within 3D hierarchical structure of La-HNF. In addition, the optimized biodegradation conditions (pH 4.6 and 40 °C) of La-HNF were obtained, moreover, 93.2% and 48.1% of EB dye were biodegraded by La-HNF after stored for 30 days and reused for 10 cycles, respectively, demonstrating La-HNF have good practicability.

The EccC enzyme of ESX-1 system contains (i) a membrane bound Rv3870 with single ATPase domain and (ii) a cytoplasmic Rv3871 with two ATPase domains and involved in secretion of ESAT6/CFP10 factor out of the cell. In current study, we have structurally and biochemically characterized the ATPase domain (442–747 residues) of Rv3870 enzyme. The ΔRv3870 eluted as oligomer (~813 kDa) from Superdex 200 (16/60) column, as identified based on molecular mass standard and dynamics light scattering. The SAXS analysis yielded a tetrameric ring envelope of ΔRv3870, quite consistent to dynamic light scattering data. The ΔRv3870 exhibited ATPase activity having kinetic parameters, Km ~ 100 ± 40 μM, kcat ~ 1.81 ± 0.27 min−1 and Vmax ~ 54.41 μM/min/mg. ATPase activity using nine ΔRv3870 mutants showed 70–91% decrease in catalytic efficiency of the enzyme. ΔRv3870 binds Rv3871 with KD ~ 484.0 ± 10.3 nM and its catalytic efficiency is enhanced ~6.7-fold in presence of Rv3871. CD data revealed the high TM ~ 82.2 ± 0.5 °C for ΔRv3870 and enhanced in presence of ATP + Mg2+, as observed in dynamics simulation on ΔRv3870 hexameric models. Overall, our structural and biochemical studies on ΔRv3870 have explained the mechanism, which will contribute in development of antivirulence inhibitors against M. tuberculosis.

Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable.

Hen egg white lysozyme (HEWL) is a structural homolog of human lysozyme and is widely used as a model protein to investigate protein aggregation. The effect of N-benzylisatin on stress-induced aggregation of HEWL has been investigated in the present study. Interaction of the isatin derivative with HEWL induced changes in protein secondary and tertiary structural conformation as evident from different biophysical and spectroscopic studies. In addition, modification with N-benzylisatin was found to increase the conformational stability of HEWL and afford considerable resistance to the protein to stress-induced aggregation as indicated from subsequent experimental studies, including thioflavin T fluorescence, microscopic imaging and dynamic light scattering analysis. Protein modification was analysed and confirmed by MALDI-TOF and ESI-MS studies. The results highlight possible clinical implications of isatin derivative in the treatment of protein misfolding and conformational disorders.

The development of new biocatalytic systems to replace the chemical catalysts, with suitable characteristics in terms of efficiency, stability under high temperature reactions and in the presence of organic solvents, reusability, and eco-friendliness is considered a very important step to move towards the green processes. From this basis, the use of lipase as a catalyst is highly desired for many industrial applications because it offers the reactions in which could be used, stability in harsh conditions, reusability and a greener process. Therefore, the introduction of temperature-resistant and solvent-tolerant lipases have become essential and ideal for industrial applications. Temperature-resistant and solvent-tolerant lipases have been involved in many large-scale applications including biodiesel, detergent, food, pharmaceutical, organic synthesis, biosensing, pulp and paper, textile, animal feed, cosmetics, and leather industry. So, the present review provides a comprehensive overview of the industrial use of lipase. Moreover, special interest in biotechnological and biochemical techniques for enhancing temperature-resistance and solvent-tolerance of lipases to be suitable for the industrial uses.

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol−1, −76.8 kJ.mol−1.°K−1, and 84.1 kJ.mol−1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0–10.0) and temperatures (35–75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

The glucose-regulated protein GRP94 is a molecular chaperone that is located in the endoplasmic reticulum (ER). Here, we demonstrate in pull down experiments an interaction between GRP94 and sucrase-isomaltase (SI), the most prominent disaccharidase of the small intestine. GRP94 binds to SI exclusively via its mannose-rich form compatible with an interaction occurring in the ER. We have also examined the interaction GRP94 to a panel of SI mutants that are associated with congenital sucrase-isomaltase deficiency (CSID). These mutants exhibited more efficient binding to GRP94 than wild type SI underlining a specific role of this chaperone in the quality control in the ER. In view of the hypoxic milieu of the intestine, we probed the interaction of GRP94 to SI and its mutants in cell culture under hypoxic conditions and observed a substantial increase in the binding of GRP94 to the SI mutants. The interaction of GRP94 to the major carbohydrate digesting enzyme and regulating its folding as well as retaining SI mutants in the ER points to a potential role of GRP94 in maintenance of intestinal homeostasis by chaperoning and stabilizing SI.

Because of the essential roles of SARS-CoV-2 papain-like protease (PLpro) in the viral polyprotein processing and suppression of host immune responses, it is a crucial target for drug discovery against COVID-19. To develop robust biochemical methodologies for inhibitor screening against PLpro, extensive characterization of recombinant protein is important. Here we report cloning, expression, and purification of the recombinant SARS-CoV-2 PLpro, and explore various parameters affecting its stability and the catalytic activity. We also report the optimum conditions which should be used for high-throughput inhibitor screening using a fluorogenic tetrapeptide substrate. Supplementary Information The online version contains supplementary material available at 10.1007/s12033-021-00383-y.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using β-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant β-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized β-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant β-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Cellulose microcrystalline (MCC) was widely used in pharmaceutical and chemical industries because of its low degree of polymerization and large specific surface area. As its modified form, dialdehyde cellulose (DAC) was used for cross-linking and immobilizing Rhizopus lipase together with magnetic nanoparticles (MNPs) due to its active aldehyde groups. In this study, in order to maintain the original enzyme activity as much as possible and improve the stability of lipase, the Rhizopus lipase was successfully immobilized on the magnetic dialdehyde cellulose nanoparticles (MDC). Specifically, the immobilization conditions including dosage of DAC, concentration of enzyme, immobilization time and temperature together with pH value of the reaction medium were optimized. Maximum immobilization yield (60.03 ± 0.49%) and recovery activity (88.88 ± 0.61%) can be obtained under the optimal process conditions. The changes in secondary structures of immobilized enzyme revealed the increment in conformational rigidity, which can be reflected in temperature and pH stability as well as tolerance of organic reagents. Additionally, the recovery activity of immobilized enzyme still reached 50.60 ± 0.59% after 30 d of storage and 52.10 ± 0.57% retained after 6 cycles. These results indicated the ideal application prospect of MDC in immobilized enzymes.

Herein, our suggestion is to immobilize enzymes in-situ on absorbable shape-memory stents instead of injecting therapeutic enzymes into the blood. Chitosan (CHI)-based stents were tailored as novel support and the enzyme-immobilizing ability was elucidated using L-asparaginase (L-ASNase). For developing shape-memory stents, CHI-glycerol (GLY) solution was prepared and further blended with different ratios of polyethylene glycol (PEG), and polyvinyl alcohol (PVA). Afterward, the blends were modified by ionic crosslinking with sodium tripolyphosphate to obtain a shape-memory character. L-ASNase was included in the blends by using in-situ method before ionic crosslinking. The prepared stents, with or without L-ASNase, were comprehensively characterized by using several techniques. Collectively, immobilized L-ASNase exhibited much better performance in immobilization parameters than free one, thanks to its improved stability and reusability. For instance, CHI/GLY/PEG-3@L-ASNase retained about 70% of the initial activity after storage at 30 °C for 2 weeks, whereas the free form lost half of its initial activity. Besides, it retained 73.4% residual activity after 15 consecutive cycles. Most importantly, stent formulations exhibited ~60% activity in the bioreactor system after 4 weeks of incubation. Given the above results, shape-memory stents can be a promising candidate as a new platform for immobilization, especially in the blood circulation system.

15 pags., 7 figs., 1 tab. -- This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs, One of the major drawbacks of the industrial implementation of enzymatic processes is the low operational stability of the enzymes under tough industrial conditions. In this respect, the use of thermostable enzymes in the industry is gaining ground during the last decades. Herein, we report a structure-guided approach for the development of novel and thermostable 2'-deoxyribosyltransferases (NDTs) based on the computational design of disulfide bonds on hot spot positions. To this end, a small library of NDT variants from Lactobacillus delbrueckii (LdNDT) with introduced cysteine pairs was created. Among them, LdNDTS104C (100% retained activity) was chosen as the most thermostable variant, displaying a six- and two-fold enhanced long-term stability when stored at 55 °C (t1/255 °C ≈ 24 h) and 60 °C (t1/260 °C ≈ 4 h), respectively. Moreover, the biochemical characterization revealed that LdNDTS104C showed >60% relative activity across a broad range of temperature (30-90 °C) and pH (5-7). Finally, to study the potential application of LdNDTS104C as an industrial catalyst, the enzymatic synthesis of nelarabine was successfully carried out under different substrate conditions (1:1 and 3:1) at different reaction times. Under these experimental conditions, the production of nelarabine was increased up to 2.8-fold (72% conversion) compared with wild-type LdNDT., The authors gratefully acknowledge the financial support provided by the Spanish Ministry of Science and Innovation (grant PID2020-117025RB-I00) and Santander Foundation (grant XSAN192006).