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An acid polysaccharide, named HP, was produced by endophytic Penicillium javanicum MSC-R1 isolated from southern medicine Millettia speciosa Champ. The molecular weight of HP was 37.8 kDa and consisted of Ara f, Galр, Glcр, Manр, and GlcрA with a molar ratio of 1.09: 3.47: 68.48: 16.59: 8.85. The glycosidic linkage of HP was proven to be →3, 4)-α-D-Glcр-(1→6)-α-D-Manр-(1→, →3, 4)-α-D-Glcр-(1→4)-α-D-Glcр-(1→, →3), →6)-α-D-Manр-(1→4)-α-D-Glcр-(1→, →3), β-D-Galр-(1→3)-α-D-Glcр-(1→, →4), →5)-α-L-Ara f -(1→3)-α-D-Glcр-(1→, →4), →6)-α-D-Manр-(1→4)-α-D-GlcAр-(1→ and →4)-α-D-GlcAр-(1→4)-α-D-Glcр-(1→, →3). Additionally, 250 μg/mL of HP possessed nontoxicity to RAW 264.7 cells and exhibited anti-inflammation activity. HP could significantly restrain the amount of tumor necrosis factor-α, interleukin-6 and NO release in RAW264.7, which property is possibly associated with its abundant glucosidic linkage. These results indicated that HP could be regarded as a ponderable ingredient for the health-beneficial functional foods.

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.

Nanozyme with intrinsic enzyme-like activity has emerged as favorite artificial catalyst during recent years. However, current nanozymes are mainly limited to inorganic-derived nanomaterials, while biomolecule-sourced nanozyme (bionanozyme) are rarely reported. Herein, inspired by the basic structure of natural hydrolase family, we constructed 3 oligopeptide-based bionanozymes with intrinsic hydrolase-like activity by implementing zinc induced self-assembly of histidine-rich heptapeptides. Under mild condition, divalent zinc (Zn

CuAsp with a fiber-like structure exhibited both NADH peroxidase- and glycerol dehydrogenase (GlyDH)-mimetic activities.

Lignocellulose, the most abundant biomass resource, was a promising alternative to oil resources. The acid catalyst can hydrolyze lignocellulose to reduce sugars, the raw material for lipid production via fermentation. However, the by-product yield during hydrolysis would inhibit yeast activity when directly applying lignocellulose hydrolysate from dilute acid-catalyzed to microbial lipid production. In this paper, a solid acid catalyst highly selective for lignocellulose (rice straw) hydrolysis was prepared to minimize the effects of by-products. Also, a combination of solid acid catalysts to generate glucose with subsequent microbial fermentation was studied to obtain the higher efficiency of lipid production. A carbon-based solid acid catalyst with an enhanced hydrolysis capacity of cellulose and lignocellulose was efficiently prepared. It resulted in 71% yield of glucose with 97% of selectivity with optimal catalyst in hydrolyzing milled cellulose. The optimal preparation conditions of the catalyst were: carbonization temperature 642 K, sulfonation temperature 412 K, initial sulfonation time 12.5 h, and liquid-to-solid ratio 11.5. To expand the catalyst’ application, the rice straw hydrolysate catalyzed by the prepared catalyst was then applied to the microbial lipid production for the first time. Both dilute sulfuric acid and Amberlyst-15 resin as hydrolysis catalysts caused a decrease in yeast biomass, lipid content, and lipid yield. However, when the hydrolysate was obtained from co-heating rice straw with sucralose-derived solid acid catalyst, the yeast biomass, lipid content, and lipid yield in the presence of 5-HMF (0.39 g/L) were 12.6 g/L, 48.1%, and 7.6 g/L, respectively, and were almost similar to the control group. Our results showed that a combination of solid acid catalysts with subsequent microbial fermentation to generate lipid was confirmed to be environmentally friendly and low cost.

In this study, three kinds of Maillard reaction products (MRPs) have been, for the first time, successfully prepared by conjugating soy protein isolate (SPI) with isomaltooligosaccharide, xylooligosaccharide, or galactooligosaccharide at 80 °C for 30 or 60 min and applied for the construction of Lactobacillus casei (L. casei) microcapsules. The results showed that MRPs exhibited enhanced antioxidative activities compared with their physically mixed counterparts. The digested MRPs displayed excellent resistance to pathogenic bacteria and promoted the growth of L. casei. Moreover, MRP-encapsulated L. casei showed a higher survival rate than free L. casei under tested adverse conditions including heat treatment, storage, and mechanical forces. Under simulated digestion conditions, the viability of L. casei decreased from 8.8 log cfu/mL to 1.6 log cfu/mL, while that of MRP-encapsulated L. casei was maintained at 7.4 log cfu/mL. Thus, MRP-based SPI-oligosaccharide conjugates exhibited great potential for microencapsulation of probiotics.

In this study, five strains of lactic acid bacteria (LAB) with excellent cholesterol-lowering ability were screened from fermented foods. The gastrointestinal stress resistance, intestinal adhesion, and bacteriostasis abilities were evaluated to obtain the best LAB. And then, high-cholesterol HepG2 cell model was further prepared to explore the cholesterol-lowering mechanism of the LAB. Finally, pH-sensitive hydrogel prepared by Millettia speciosa Champ. carboxymethyl cellulose and Millettia speciosa Champ. cellulose was first applied to the microencapsulation of LAB. As a result, Lactobacillus paracasei BY2 (LP-BY2) exhibited higher cholesterol-lowering activity, intestinal adhesion, and bacteriostasis abilities compared with other LAB. Furthermore, it was found that LP-BY2 could reduce the cholesterol level by regulating the expression of key genes that involved in cholesterol synthesis (HMGCR and SREBP-2), uptake (LDLR), and outflow (LXR-α, ABCA1, ABCG5, ABCG8, and CYP7A1) in liver. At the same time, microencapsulation significantly enhanced the survival rate and cholesterol-lowering ability of LP-BY2 after gastrointestinal digestion. This study will provide an available reference for the application of Lactobacillus in prevention and treatment of hypercholesterolemia.

Natural plant-derived protein with excellent bioactivities has attracted much attention so a functional protein with molecular weight of 15.2 kDa was extracted from Millettia speciosa Champ. leaf for the first time. Under the pH of 12.0, solid-liquid ratio of 1:40 (w/v), extraction time of 2.0 h, and extraction temperature of 50 °C, the highest extracting efficiency (79.25 ± 0.78%) of the Millettia speciosa Champ. leaf protein (MLP) was achieved. The main structure of MLP contained β-fold and β-corner by Fourier transform infrared spectroscopy (FTIR) and Circular dichroism (CD) spectra analysis. Additionally, MLP was predominant with glutamic acid, aspartic acid, and leucine, which could be considered as a high quality natural protein. MLP showed great water holding capacity (WHC), oil absorption capacity (OAC), as well as emulsifying and foaming properties. Simultaneously, MLP exhibited considerable antioxidant activity. These results suggested that MLP could be utilised as a promising ingredient of functional foods.

A copper analogue of Prussian blue incorporated multienzyme system was successfully established via a biomimetic mineralization process, whereas CPB with peroxidase-like activity provided protecting effect for encapsulated glucose oxidase from attack in hostile conditions. The hybrid composite was developed as a colorimetric biosensor for ultrasensitive detection of glucose, with a low detection limit (0.48 μM).

A sucralose-derived magnetically separable carbon-based solid acid catalyst with cellulose-binding and catalytic sites has been successfully prepared by incompletely carbonizing the sulfonated sucralose with Fe3+ at high temperature to load ɤ-Fe2O3 nanoparticles onto the surface of the catalyst. The resultant magnetic catalyst was fully characterized and found to be suitable in the hydrolysis of cellulose. Microcrystalline cellulose pretreated by ball milling achieved the highest glucose yield of 32% using this neoteric catalyst at 363 K. Catalysts retained 90% of their activity after five runs recycle. The introduction of magnetic properties into the catalyst system will combine the high catalytic capacity for hydrolyzing cellulose and magnetic materials’ separation convenience.

In the recent past, numerous new types of nanostructured carriers, as support matrices, have been engineered to advance the traditional enzyme immobilization strategies. The current research aimed to develop a robust enzyme-based biocatalytic platform and its effective deployment in the industrial biotechnology sectors at large and catalysis area, in particular, as low-cost biocatalytic systems. Suitable coordination between the target enzyme molecules and surface pendent multifunctional entities of nanostructured carriers has led an effective and significant contribution in myriad novel industrial, biotechnological, and biomedical applications. As compared to the immobilization on planar two-dimensional (2-D) surface, the unique physicochemical, structural and functional attributes of nano-engineered matrices, such as high surface-to-volume ratio, surface area, robust chemical and mechanical stability, surface pendant functional groups, outstanding optical, thermal, and electrical characteristics, resulted in the concentration of the immobilized entity being substantially higher, which is highly requisite from applied bio-catalysis perspective. Besides inherited features, nanostructured materials-based enzyme immobilization aided additional features, such as (1) ease in the preparation or green synthesis route, (2) no or minimal use of surfactants and harsh reagents, (3) homogeneous and well-defined core-shell nanostructures with thick enzyme shell, and (4) nano-size can be conveniently tailored within utility limits, as compared to the conventional enzyme immobilization. Moreover, the growing catalytic needs can be fulfilled by multi-enzymes co-immobilization on these nanostructured materials-based support matrices. This review spotlights the unique structural and functional attributes of several nanostructured materials, including carbon nanotubes, graphene, and its derivate constructs, nanoparticles, nanoflowers, and metal-organic frameworks as robust matrices for laccase immobilization. The later half of the review focuses on the applied perspective of immobilized laccases for the degradation of emergent contaminants, biosensing cues, and lignin deconstruction and high-value products.

Artificial multienzyme biocatalysts have played a crucial role in biosynthesis because they allow for conducting complex reactions. Here, we reorted a facile approach to fabricate multienzyme nanodevices with high catalytic activity and stability based on protein assembly and chemical crosslinking. The self-assembled partner SpyCatcher and SpyTag were genetically fused with 2,3-butanediol hydrogenase and formate hydrogenase to produce KgBDH-SC (SpyCatcher-fused 2,3-butanediol hydrogenase) and FDH-ST (SpyTag-fused formate hydrogenase), respectively. After assembling the two fusion proteins, the complexes were then immobilized on the functionalized silicon dioxide nanoparticles by glutaraldehyde, forming KgBDH-SC-ST-FDH-SiO2 with the capability of reducing 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol with cofactor regeneration. Under the optimal conditions, the created co-immobilized enzymes performed 49% activity recovery compared with the mixture of free enzymes as well as showed 2.9-fold higher catalytic activity than the traditional random co-immobilized enzymes. Moreover, KgBDH-SC-ST-FDH-SiO2 showed better pH stability and organic solvents stability than the free enzymes, and remained 52.5% overall catalytic activity after 8 cycles. Finally, the co-immobilized enzymes can reduce 60 mM HAP for fabrication of (R)-PED with cofactor regeneration in the phosphate buffer reaction system, affording 83.9% yield and above 99% optical purity.

In this study, the purification and characterization of a novel polysaccharide-based bioflocculant BM2 produced by a bacterium Bacillus megaterium strain PL8 with self-flocculating property were investigated. The results showed that BM2 was an acidic polysaccharide composed of Gal, GalUA, Glc, GlcUA and Man at a molar ratio of 45.1: 33.8:9.3:9.2:2.4, respectively. The molecular weight of BM2 was 4.55 × 106 Da. BM2 had high flocculation efficiencies across a wide pH ranged from 4 to 11 and a wide temperature ranged from 20 to 100 °C towards kaolin clay. BM2 was a cation-independent bioflocculant which could achieve high flocculation activity without the addition of other cations. Adsorption bridging was the main mechanism in the flocculation process of BM2 towards kaolin clay. The BM2 also displayed a high removal efficiency in terms of Congo red (88.14%) and Pb2+ ions (82.64%). These results suggested that BM2 had a great potential as an efficient bioflocculant candidate in wastewater treatment.

Nanostructured materials constitute an interesting and novel class of support matrices for the immobilization of peroxidase enzymes. Owing to the high surface area, robust mechanical stability, outstanding optical, thermal, and electrical properties, nanomaterials have been rightly perceived as immobilization matrices for enzyme immobilization with applications in diverse areas such as nano-biocatalysis, biosensing, drug delivery, antimicrobial activities, solar cells, and environmental protection. Many nano-scale materials have been employed as support matrices for the immobilization of different classes of enzymes. Nanobiocatalysts, enzymes immobilized on nano-size materials, are more stable, catalytically robust, and could be reused and recycled in multiple reaction cycles. In this review, we illustrate the unique structural/functional features and potentialities of nanomaterials-immobilized peroxidase enzymes in different biotechnological applications. After a comprehensive introduction to the immobilized enzymes and nanocarriers, the first section reviewed carbonaceous nanomaterials (carbon nanotube, graphene, and its derivatives) as a host matrix to constitute robust peroxidases-based nanobiocatalytic systems. The second half covers metallic nanomaterials (metals, and metal oxides) and some other novel materials as host carriers for peroxidases immobilization. The next section vetted the potential biotechnological applications of the resulted nanomaterials-immobilized robust peroxidases-based nanobiocatalytic systems. Concluding remarks, trends, and future recommendations for nanomaterial immobilized enzymes are also given.

The epoxide hydrolase gene (SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity (E-value) of the recombinant SpEH was 7.7 and the yield of the remaining (R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether (rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues (V195, V196, F218, N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEHV196A/N226A/M332A had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg−1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and pH for purified SpEHV196A/N226A/M332A to catalyze the hydrolysis of rac-PGE were 25 °C and 7.0 with 200 U·mg−1. The enantioselectivity and yield of the remaining (R)-PGE of E. coli_SpEHV196A/N226A/M332A increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEHV196A/N226A/M332A has a greater affinity toward (S)-PGE than (R) - PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of (R)-PGE, resulting in its improved enantioselectivity.

Human milk fat substitutes (HMFSs) are the structured lipids intended for infant formula. It provides energy and essential fatty acid for infant. HMFSs are mainly prepared by enzymatic method. In this study, we aim to explore the potential for producing HMFSs by fermentation using R. opacus. The results indicated that different compounds with chain length from 12 to 18, used as carbon source, could be incorporated into triacylglycerols directly. Polyunsaturated fatty acids in term of ARA, EPA, DHA could enter the kennedy pathway directly and involved in the biosynthesis of triacylglycerols. GC, UPLC-MS and 13C-NMR analysis demonstrated that typical structured lipids β-OPL (40.09%) was synthesized in R. opacus. Transcriptome analysis revealed that β-oxidation, fatty acid elongation and kennedy pathways existed in R. opacus. It was concluded that fatty acid supplied as carbon source could enter the kennedy pathways directly or via the de novo fatty acid biosynthesis pathway depending on the chain length, thus, affect the triacylglycerol species formed in the Rhodococcus opacus.

Microbial contamination, especially in large-scale processes, is partly a life-or-death issue for industrial fermentation. Therefore, the aim of this research was to create an anti-microbial contam...

Human milk fat substitute (HMFS) is a class of structured lipids widely used in infant formulas. Herein, HMFS was prepared by Rhodococcus opacus fermentation. The substrate oils suitable for HMFS production were coconut oil (66.1-57.5%), soybean oil (17.5-26.5%), high oleic acid sunflower oil (5.4-4.5%), Antarctic krill oil (9-9.5%), and fungal oil (2%). Six HMFSs were prepared, among which HMFS V and VI were similar to human milk fat from Chinese in terms of fatty acid composition and triacylglycerol species. The sn-2 position of HMFS was occupied by palmitic acid (49.31 and 43.48% in HMFS V and VI, respectively). The major triacylglycerols were OPL, OPO, and LPL, accounting for 15.90, 9.49, and 6.84 and 17.52, 8.44, and 8.55% in HMFS V and VI, respectively. This study is the first to prepare structured lipids intended for infant formula through fermentation, providing a novel strategy for the edible oil industry.

Papain (PA) immobilized onto magnetic nanocrystalline cellulose (PA@MNCC) was successfully fabricated and adopted as an efficient biocatalyst for the synthesis of N-(benzyloxycarbonyl)-alanyl-histidine (Z-Ala-His) dipeptide. Introducing deep eutectic solvents (DESs) as reaction media promoted the synthesis of the Z-Ala-His dipeptide. The effects of reaction conditions on the yield of papain catalytic Z-Ala-His were systematically investigated with the highest yield of 68.4%, which was higher than free papain (63.3%). Besides, this novel PA@MNCC composite can be easily recycled from the reaction system by magnetic forces. In a word, the PA@MNCC composite exhibited great potential for efficient biosynthesis of dipeptide in DESs.

In the study, monodispersed silver nanoparticles (AgNPs) with an average diameter of 9.57 nm were efficiently and controllably biosynthesized by a reductase from Fusarium solani DO7 only in the presence of β-NADPH and polyvinyl pyrrolidone (PVP). The reductase responsible for AgNP formation in F. solani DO7 was further confirmed as 1,4-α-glucosidase. Meanwhile, based on the debate on the antibacterial mechanism of AgNPs, this study elucidated in further depth that antibacterial action of AgNPs was achieved by absorbing to the cell membrane and destabilizing the membrane, leading to cell death. Moreover, AgNPs could accelerate the catalytic reaction of 4−nitroaniline, and 86.9% of 4-nitroaniline was converted to p-phenylene diamine in only 20 min by AgNPs of controllable size and morphology. Our study highlights a simple, green, and cost-effective process for biosynthesizing AgNPs with uniform sizes and excellent antibacterial activity and catalytic reduction of 4-nitroaniline.

In this study, three kinds of Maillard reaction products (MRPs) have been, for the first time, successfully prepared by conjugating soy protein isolate (SPI) with isomaltooligosaccharide, xylooligosaccharide, or galactooligosaccharide at 80 °C for 30 or 60 min and applied for the construction of

In this study, a natural antioxidant emulsifier, Millettia speciosa Champ polysaccharide conjugates (MSC-PC), was used for fabricating oil-in-water emulsion, and the influences of MSC-PC on β-carotene stability and bioaccessibility were studied. Results suggested that MSC-PC stabilized emulsion exhibited excellent resistance to a wide range of salt levels (0-500 mM of Na

Enzymatic asymmetric amination addition is seen as a promising approach for synthesizing amine derivatives, especially unnatural amino acids, which are valuable precursors to fine chemicals and drugs. Despite the broad substrate spectrum of methylaspartate lyase (MAL), some bulky substrates, such as caffeic acid, cannot be effectively accepted. Herein, we report a group of variants structurally derived from Escherichia coli O157:H7 MAL (EcMAL). A combined mutagenesis strategy was used to simultaneously redesign the key residues of the entrance tunnel and binding pocket to explore the possibility of accepting bulky substrates with potential application to chiral drug synthesis. Libraries of residues capable of lining the active center of EcMAL were then constructed and screened by an effective activity solid-phase color screening method using tyrosinase as a cascade catalyst system. Activity assays and molecular dynamics studies of the resultant variants showed that the substrate specificity of EcMAL was modified by adjusting the polarity of the binding pocket and the degree of flexibility of the entrance tunnel. Compared to M3, the optimal variant M8 was obtained with a 15-fold increase in catalytic activity. This structure-based protein engineering of EcMAL can be used to open new application directions or to develop practical multi-enzymatic processes for the production of various useful compounds.

Microbial contamination is a serious challenge that needs to be overcome for the successful biosynthesis of 2,3-butanediol (2,3-BD). However, traditional strategies such as antibiotic administration or sterilization are costly, have high energy demands, and may increase the risk of antibiotic resistance. Here, we intend to develop a robust strategy to achieve non-sterilized fermentation of 2,3-BD. Briefly, the robust strain can metabolize unconventional chemicals as essential growth nutrients, and therefore, outcompete contaminant microbes that cannot use unconventional chemicals. To this end, Klebsiella pneumoniae OU7, a robust strain, was confirmed to rapidly exploit urea and phosphite (unconventional chemicals) as the primary sources of nitrogen (N) and phosphorus (P), and withstand deliberate contamination in the possibly contaminated systems. Secondly, metabolic engineering, pathogenicity elimination and adaptive laboratory evolution were successively performed, endowing the best strain with an excellent fermentation performance for safe 2,3-BD production. Finally, 84.53 g L−1 of 2,3-BD was synthesized with a productivity of 1.17 g L−1 h−1 and a yield of 0.38 g g−1 under the non-sterilized system. In summary, our technique reduces labor and energy costs and simplifies the fermentation process because sterilization does not need to be performed. Thus, our work will be beneficial for the sustainable synthesis of 2,3-BD.

The asymmetric addition of ammonia to unsaturated acids using engineered methylaspartate ammonia lyase (MAL) is a particularly attractive and atom-economic method for the synthesis of unnatural ami...