Abstract:酶工程是酶学与工程科学融合的综合性科学技术，新酶的发掘、对酶的结构与功能的认知及酶的改造是合成生物学、生物制造技术的重要科学与技术基础。在合成生物学的发展方兴未艾的今天，酶工程更是不可或缺的研究领域。

Abstract:[Background] Cas9 nuclease is a site-specific RNA-guided endonuclease that can form a stable ribonucleoprotein complex with single guide RNA (sgRNA), which recognizes and cleaves target DNA molecules. Due to its high flexibility and efficiency, Cas9 is the most widely used gene-editing tool in both basic research and clinical treatment. [Objective] To provide a theoretical basis for the rational development and utilization of Cas9 nuclease. [Methods] The wild type Cas9 nuclease from Streptococcus pyogenes was expressed in the Escherichia coli system. The expressed enzyme was then purified by ammonium sulfate precipitation and Ni2+-affinity chromatography. Finally, the purified Cas9 nuclease was characterized for its thermal stability, pH stability and the influence of metal ions. [Results] The results showed that the wet weight of the bacteria was 191.0 g/L after high-density fermentation. The specific activity was 641.29 U/mg and the bacteria was purified up to 16.02 times with a recovery of 46.40% after purification. Cas9 nuclease retained over 65% of its initial activity after incubation between 25 and 42 °C for 2 h, but it was completely deactivated after treatment at 45 °C for 15 min. The enzyme was stable between pH 6.0 and 10.0 with residual enzyme activity more than 68%, and especially the highest stability at pH 9.0. Mg2+ activated the enzyme between 0.5 and 20.0 mmol/L, and 10.0 mmol/L Mg2+ increased the enzyme activity by 23%. Besides, this enzyme was inhibited by some metal ions such as Ba2+, Co2+, Ca2+, Mn2+, Cu2+, Fe2+ and Zn2+, wherein Cu2+ and Fe2+ completly inhibited Cas9 nuclease at a concentration of 0.5 mmol/L. [Conclusion] Cas9 nuclease from Streptococcus pyogenes was heterologously expressed and purified with high purity and high activity. The purified Cas9 nuclease were characterized, serving as reference for further promotion and application of this enzyme in CRISPR/Cas9 technology.

Abstract:[Background] Zearalenone (ZEN) and its derivatives are a series of mycotoxins with estrogenic activity which are widespread in mold contaminated cereals. Mycotoxin contamination has caused huge loss to food and feed industries. To develop an efficient solution is an urgent task. [Objective] In order to promote the research and application of zearalenone hydrolase, we constructed the recombinant plasmid that highly expressed zearalenone hydrolase in Pichia pastoris. [Methods] The Rmzhd gene from Rhinocladiella mackenziei CBS 650.93 was transformed into Pichia pastoris GS115 for heterologous expression. The high expression strain was screened and the characterization of recombinant RmZHD was analyzed by high performance liquid chromatography (HPLC). [Results] The ZEN and α-ZOL degradation activities of the recombinant RmZHD from the supernatant were 16.67 U/mL and 9.85 U/mL, respectively. The molecular mass of RmZHD was about 30.7 kD, and the purity of protein was very high, as shown by SDS-PAGE. The optimal pH and temperature of recombinant RmZHD were determined as pH 9.6 and 45 °C, and RmZHD has a good thermostability. [Conclusion] The results can provide guidance for the heterologous expression and potential industrial application of zearalenone hydrolase.

Abstract:[Background] N-heterocyclic aromatic compounds (NHACs) are one of the important environmental pollutants. Long-term accumulation in the human body can lead to diseases. The degradation of N-heterocyclic aromatic compounds by microorganism is an effective approach. [Objective] The 6-hydroxynicotinic acid 3-monooxygenase gene nicC was cloned and expressed in Escherichia coli, the NicC protein was purified and the crystallization conditions were studied. [Methods] The nicC gene was amplified from the genomic DNA of Pseudomonas putida KT2440 and a recombinant expression vector pET28a-nicC was constructed and expressed in E. coli BL21(DE3). Affinity and gel filtration chromatography were used to purify NicC. The preliminary screening and optimization of crystals were done by using hanging drop diffusion method. [Results] The pET28a-nicC was constructed successfully and the purified NicC protein was obtained. Through preliminary screening of crystallization conditions and orthogonal optimization experiments, the optimal crystallization conditions were 0.2 mol/L NH4Cl, 0.01 mol/L CaCl2·2H2O, 31% PEG4000, 0.05 mol/L Tris-HCl ph 8.0, 4 °C for NicC, and 0.2 mol/L NH4Cl, 0.01 mol/L CaCl2·2H2O, 0.05 mol/L Tris-HCl ph 7.9, 31% PEG3350, 4 °C for SeMet-NicC and cocrystals of NicC and 6-HNA. [Conclusion] The construction of NicC protein purification system and study of crystallization conditions provided favorable conditions for the final analysis of the three-dimensional structure of NicC protein. The results laid a foundation for revealing the molecular mechanism of the pyridine ring to β-position monooxygenase to recognize the pyridine ring-containing substrate and catalyze the hydroxylation of β-position on the pyridine ring of the substrate.

Abstract:[Background] β-glucan is a non-starch polysaccharide, widely existed in nature, especially in cell wall of cereal plants. β-glucanase can catalyze β-glucan into β-glucooligosaccharides, it plays an important role in food, feed and papermaking industry. [Objective] gene of β-1,3(4)-glucanase from marine bacteria Microbulbifer arenaceous was cloned and expressed in Escherichia coli, and its enzymatic characteristics and hydrolysis properties were studied. [Methods] The β-1,3(4)-glucanase gene (MaGlu16A) was cloned from the genomic DNA of Microbulbifer arenaceous and the recombinant plasmid (pET-28a-MaGlu16A) was constructed. The recombination strain was successfully expressed in E. coli BL21(DE3), and purified by Ni-NTA affinity chromatography, then enzymatic characteristics was studied. [Results] The optimal pH of MaGlu16A was 6.0 and optimal temperature was 40 °C. It was stable between pH 5.0 and 10.5, and below 35 °C. The enzyme showed obvious resistance to EDTA, and it could maintain 99.3% and 82.5% activity at the concentration of 1 mmol/L and 10 mmol/L. In addition, MaGlu16A showed broad substrate specificity. It could hydrolyze not only curdlan and laminarin but also barley β-glucan, lichenan, oat β-glucan, and yeast β-glucan, whose hydrolytic products were mainly glucose, disaccharide, trisaccharide and tetragasaccharide. [Conclusion] Cloning, expression and characterization of β-1,3(4)-glucanase from marine bacteria Microbulbifer arenaceous could provide a basis for the exploration of β-glucanase and preparation of β-glucooligosaccharides.

Abstract:[Background] γ-lactam is an important pharmaceutical intermediate and its chiral property makes against drug synthesis. The preparation of optically pure γ-lactam can be realized by γ-lactamases’ selective resolution. [Objective] To explore the gene of γ-lactamase from Bacillus thuringiensis and the technology of optically pure γ-lactam preparation. [Methods] The genome sequence of Bacillus thuringiensis was searched by the key words “acetamidase/formamidase”, two sequences were selected and heterologously expressed. The degradation of γ-lactam by recombinant bacteria was monitored by HPLC method to determine whether the cloned gene was a γ-lactamase gene. The application of 5 L fermentation, substrate separation and product recovery were also performed using the constructed recombinant bacteria. [Results] The skA2 recombinant strain had activity of (?)γ-lactamase and showed good performance in small scale production. [Conclusion] The A gene is an unreported new (?)γ-lactamase gene, which enriches producers’ enzyme toolbox. The skA2 strain can meet the need of industrial production of (+)γ-lactam and lay a solid foundation for the preparation of optically pure drugs.

Abstract:[Background] β-glucosidase (EC 3.2.1.21) is an important component of cellulase system. At present, most β-glucosidases used in industry come from plants and fungi, but few come from bacteria, and there are some problems such as low enzyme activity, poor thermal stability, narrow reaction conditions, products inhibition, which increase the economic cost. Thermophilic microorganisms have special genetic information resources, so it is possible to excavate novel β-glucosidases with good enzymatic characterization from the genome to solve the industrial problems. [Objective] A novel β-glucosidase gene was extracted from the genome of Bacillus thermoamylovorans, and purified protein was obtained by gene recombination, heterologous expression and protein purification. The enzymatic characterization was studied systematically. It can lay the foundation for the application of β-glucosidase in the fields of cellulose hydrolysis. [Methods] The recombinant plasmid pET22b-bgl52 was constructed and transformed into Escherichia coli BL21(DE3) by electric pulse method. The recombinant protein was expressed in soluble form and purified by Ni-NTA affinity chromatography. [Results] The recombinant plasmid pET22b-bgl52 was expressed in E. coli BL21(DE3) and purified β-glucosidase Bgl52 protein was obtained. The molecular weight of the Bgl52 was 52 kD and it showed the best activity at 70 °C and pH 6.5. When p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate, the specific enzyme activity was 223.7±5.3 U/mg, Km was 9.3±1.2 mmol/L, Vmax was 270.3±4.3 μmol/(min·mg). Bgl52 preferred substrate for hydrolysis of β-1,4 glycosidic bond. Fe2+ and Mg2+ activated the enzyme activity obviously, Co2+, Cu2+ and SDS inhibited the activity of enzyme. Bgl52 is one of the few glucose and xylose-activated glucosidases. A maximal 2.84-fold stimulation by glucose was observed at 0.2 mol/L, and a maximal 3.24-fold stimulation by xylose was found at 0.4 mol/L. At the same time, under physiological conditions, Bgl52 was not substantially inhibited by the feedback of the product glucose. [Conclusion] Using the genetic information resources contained in the genomes of thermophilic microorganisms, and through modern biotechnological methods such as gene synthesis, we can excavate the β-glucosidase with excellent enzymatic characterization, it lays a foundation for its application in cellulose degradation and other industrial fields.

Abstract:[Background] 4-vinylphenols produced by phenolic acid decarboxylase catalytic degradation of phenolic acid can be used in food additives, flavor and fragrance industries. The expression level of phenolic acid decarboxylase is relatively low, so high levels of phenolic acid decarboxylase are a prerequisite for the industrial scale production of 4-vinylphenols. [Objective] The gene of phenolic acid decarboxylase from Bacillus amyloliquefaciens was cloned to achieve efficient heterologous expression in E. coli. The substrate specificity of phenolic acid decarboxylase was analyzed and its expression conditions were optimized. [Methods] The gene of phenolic acid decarboxylase was constructed using PCR to construct a recombinant genetically engineered bacterium. The sequencing results were compared with the phenolic acid decarboxylase family, and IPTG was used to induce protein expression. It was reacted with four different substrates, and response surface experiments were designed to optimize the inducing conditions. [Results] The specific enzyme activity ratios of phenolic acid decarboxylase to p-coumaric acid, ferulic acid, caffeic acid, and sinapic acid were: 100, 23.33, 15.39, 10.51. Combined with the comparison results with other phenolic acid decarboxylase, it was found that the amino acid sequence of the C-terminal region had the highest rate of variation, which was related to the substrate specificity and catalytic mechanism of the phenolic acid decarboxylase. The optimal conditions for expression of phenolic acid decarboxylase by designing response surface experiments were: 2×YT medium, inducting temperature 30 °C, inoculation amount 1.78%, inducing point 3.8 h, IPTG 1.25 mmol/L , inducing time 18 h, the predicted value and actual enzyme activity were 47.61 IU/mL and 47.55 IU/mL, respectively. [Conclusion] It is feasible to optimize the induced expression of phenolic acid decarboxylase by response surface methodology. This study provides an important theoretical basis for the production of stable and high-yield phenolic acid decarboxylase and understanding of its catalytic mechanism.

Abstract:[Background] Microbial protease has become the main source of industrial enzyme preparation. Screening microorganisms with special environmental adaptability has become a research hotspot. [Objective] To isolate and screen the strain with high protease activity from the alpine meadow soil on the Tibetan Plateau, and optimize the enzyme production conditions and explore enzymatic properties. [Methods] Strains were identified by morphology and molecular biological identification. The fermentation condition and enzymatic properties were studied by single factor and/or orthogonal test. [Results] Strain XC2 with high protease activity was identified as Bacillus subtilis. The optimum fermentation conditions were: soluble starch 4.0%, beef extract 1.0%, K+ 0.6%, 34 °C, pH 7.0, inoculum amount 2.0%, rotation speed 200 r/min after 13 h. The highest protease activity of XC2 reached 638.5 U/mL. The optimum reaction temperature and pH of the protease was 60 °C and 9.0 respectively. The enzyme activity is relative stable between 40 and 50 °C or pH 8.0?10.0. The protease activity of XC2 was significantly enhanced by Mn2+, but significantly reduced by Zn2+, Cu2+, Fe2+ and Fe3+. [Conclusion] Bacillus subtilis XC2 can efficiently produce alkaline protease, with good application prospect.

Abstract:[Background] The protein-protein interaction between catalytic domains and the cognate acyl carrier protein (ACP) of modular polyketide synthases (PKSs) are essential for PKSs to function normally, but difficult to trapping due to the transient nature. [Objective] In this research, we tried to obtain stable protein complex of ketoreductase and ACP domains of modular PKSs. [Methods] Bifunctional maleimide agents, BMH, were used to cross-link KR and ACP domains while a tobacco etch virus (TEV) protease was inserted into the linker between KR and ACP to help the confirmation of the cross-linked complex. Reaction conditions were optimized to increase the cross-linker efficiency. Stable pure KR-ACP complex was obtained by affinity and size exclusion chromatography according to the tags of proteins and their molecular weight. [Results] The cross-linking of isolated KR and ACP was unsuccessful. However, the fused di-domain of KR and ACP could be cross-linked efficiently and generated stable pure complex following affinity and size exclusion chromatography. This strategy is useful for KR and ACP domains from different modular PKSs. [Conclusion] A method for capturing and purifying stable KR and ACP complexes was established.

Abstract:[Background] As a kind of broad-spectrum insecticides, organophosphorus compounds have caused serious environmental pollution due to their high dosage, strong toxicity and non-degradability. [Objective] To degrade the organophosphorus compounds, organophosphohydrolase (OpdA) was immobilized on NiCo2O4 and the properties of the catalyst in degrading organophosphorus compounds were investigated. [Methods] Specifically, histidine tag (His-tag) was introduced into OpdA to construct His-tagged OpdA, and the His-tagged OpdA was efficiently expressed in Escherichia coli Rosetta(DE3) using pET-28a(+) as vector under the induction of 1.0 mmol/L isopropyl-β-D-thiogalactopyranoside. The immobilized OpdA (OpdA@NiCo2O4) was prepared by one-step purification and immobilization method. [Results] NiCo2O4 was prepared by hydrothermal treatment and calcination. Based on the coordination between the transition metal ions on the surface of NiCo2O4 and the imidazole group of histidine on the surface of enzyme, one-step purification and immobilization of His-tagged OpdA were achieved. OpdA@NiCo2O4 with high stability was obtained under optimized conditions, and then it was applied for organophosphorus compounds degradation. The high degradation efficiency of organophosphorus compounds was realized by cascade reaction in the presence of NaBH4 under the optimal degradation conditions. [Conclusion] In conclusion, one-step purification and immobilization of recombinant enzyme were achieved, and it’s also proved that this study provides a safe, efficient and environmental new way for the degradation of organophosphorus compounds.

Abstract:[Background] Candida antarctica lipase B (CALB) exhibits excellent ester synthesis activity and is used widely in biochemical synthesis. [Objective] This study aimed to improve the thermostability of CALB based on retaining its excellent catalytic performance. [Methods] The potential thermostability mutation sites of CALB were calculated by the prediction software PoPMuSiC and FoldX, and further selected according to the spatial position of amino acid residues. Ten single point mutations were introduced into gene calb via the overlap extension PCR and expressed in Pichia pastoris GS115. [Results] The point mutations A146G, A151P and L278M could effectively improve the thermostability of CALB. Based on single point mutations, the thermostability of the combined mutants A146G-L278M and A146G-L278M-A151P was further improved. Compared with the wild-type, the optimum reaction temperature for A146G-L278M and A146G-L278M-A151P were increased by 5 °C, and the Tm value were increased by 3.3 °C and 4.2 °C, respectively. Besides, the kinetic study of the enzymatic reaction of synthesis ethyl caproate shows that mutants A146G-L278M and A146G-L278M-A151P had higher affinity to hexanoic acid and ethanol than the wild-type, and the catalytic efficiency kcatA/KmA to hexanoic acid was 4.1-fold over that of the wild-type. The mechanism of the thermostability of the mutants A146G-L278M and A146G-L278M-A151P were elucidated at molecular level through molecular dynamics simulation. [Conclusion] The rational design strategy adopted in this study is effective for improving the thermostability of CALB. This strategy can also be used as a reference for other industrial enzymes to improve their thermostability.

Abstract:[Background] Efficient biosynthesis of D-amino acids is highly desired. Meso-diaminopimelate dehydrogenase (DAPDH) synthesizes D-amino acids from 2-keto acids and ammonia. [Objective] To increase the catalytic activity against the alkyl substituted 2-keto acids. [Methods] Based on structural analysis and mutation results from previously selected sites, the saturation mutagenesis was carried out at the amino acid residue H227 of DAPDH from Symbiobacterium thermophilum (StDAPDH). The resulting mutant library was subjected to screening using D-alanine, D-2-aminobutyric acid, D-norvaline, and D-glutamic acid as substrates. [Results] The mutants H227Q and H227N were obtained. Mutant H227Q was found to have 10.9-, 11.5-, 8.6- and 7.6-folds improved enzyme activity toward pyruvic acid, 2-oxobutyric acid, 2-oxovaleric acid and 2-ketoglutaric acid, respectively, compared to that of wild-type enzyme. The kinetic parameters indicated that mutant H227Q increased the turnover number of the enzyme and the affinity of the enzyme for the substrate simultaneously, so that the catalytic efficiency (kcat/Km) of pyruvic acid was 9.4 folds higher than that of wild-type enzyme. Molecular modeling analysis of interaction between mutant H227Q and product amino acid, indicating that glutamine at position H227 forms a hydrogen bond with the carboxylic acid of the amino acid, so that the distance between the α-hydrogen atom of product amino acid and C4 of coenzyme nicotinamide ring was shortened. [Conclusion] Directed evolution technology has been successfully used to improve the catalytic activity of DADPH for alkyl-substituted 2-keto acids, which is helpful for the development of new high-efficiency biocatalysts. These efforts also provide guidance for our future engineering of this enzyme about more challenging D-amino acids.

Abstract:[Background] Xylan is the second most abundant polysaccharide in nature, and second only to cellulose. The structure of xylan is complex, and its complete degradation needs many xylanases. Endo-β-1,4-xylanase is the key enzyme in the hydrolysis process of xylan main chain. It has been widely used in feed, papermaking, energy, food and medicine industries. However, in practical application, due to the poor thermal stability of fungal xylanase, its application in industry is limited. [Objective] The purpose of this study is to improve the thermal stability of endo-β-1,4-xylanase (xynB) from Aspergillus niger. [Methods] Firstly, an N-glycosylation site was introduced into xynB by amino acid virtual mutation technology to obtain the mutants. Then the mutants and wild-type enzymes were expressed in Pichia pastoris SMD1168. Finally, the purified wild-type and mutant enzymes were characterized by enzymatic properties. [Results] Five candidate mutants were obtained by virtual mutation and screening. Four mutants were successfully expressed in Pichia pastoris SMD1168, and three of which were glycosylated. The mutants and wild-type enzyme showed wide pH tolerance, and the stability of xynBA92N/D94T at pH 4.0–11.0 was significantly better than that of the wild-type; xynBA92N/D94T, xynBG66N/A68T and xynBG66F/D67N/G69T, which were glycosylated, showed significantly higher thermal stability at 60–80 °C than that of the wild-type, and the residual enzyme activity of xynBG66N/A68T at 80 °C after incubation for 30 min was about 30% higher than that of the wild-type. [Conclusion] The method of this study can provide reference for the thermal stability molecular modification of other xylanases and other industrial enzymes.

Abstract:[Background] Lipase is widely used in textile, food, medicine, leather and other industrial fields. The study on the heterologous expression of lipase in microorganisms further promotes the production and application of lipase products. [Objective] To perform the efficient heterologous expression of lipase from Aspergillus fischeri in Pichia pastoris. Screening suitable strategies for expression and fermentation of the engineering strain to increase yield and reduce production cost of lipase. [Methods] After codon optimization of the lipase coding gene of Aspergillus fischeri, plasmid pPIC9k was used to integrate the codon optimized gene (lip605) into the genome of Pichia pastoris GS115 to construct an engineering strain for high yield of the recombinant lipase. The lipase expression was further increased by optimizing the fermentation conditions, screening the optimal chaperone protein and performing high-density fermentation, successively. [Results] The optimal fermentation conditions for the engineering strain with high lipase production in shake flask were determined as 3.103% (v/v) methanol, 0.4 mg/L biotin, 11.5 g/L yeast powder, and 13.4 g/L YNB; the initial pH was 6.4, the filling volume was 50 mL/250 mL, the speed was 220 r/min, the temperature was 24 °C, and the culture time was 40 h. After optimization, the activity of extracellular lipase reached 72.34 U/mL, which was 5.8 times higher than that of initial fermentation. Furthermore, 12 chaperones were selected for co-expression with Lip605, among which the co-expression of chaperone Rpl10 (pPICZA-RPL10) had the best effect, which further increased the expression of Lip605 by 46.8%. On this basis, after 142 h high-density fermentation with batch feeding in a 10 L fermenter, the maximum extracellular lipase activity reached 680 U/mL, with a protein concentration of 15.89 g/L. [Conclusion] In this paper, compound strategies were used to effectively improve the fermentation yield of lipase Lip605 in Pichia pastoris, which laid a good foundation for its further industrial production.

Abstract:[Background] Enzymatic transformation for production of D-mannose has attracted considerable attention. [Objective] The production conditions of D-mannose were investigated using the co-expressed E. coli cells harboring D-glucose isomerase (D-GIase) and D-lyxose isomerase (D-LIase). [Methods] The synthesized D-GIase and D-LIase gene fragments were digested and ligated into the vector pCDFDuet-1. Then, the resultant recombinant plasmid pCDFDuet-Acce-DGI/Peba-DLI was transformated into E. coli BL21(DE3) strain to co-express the two enzymes. After collecting the cells co-expressing D-GIase and D-LIase by shake flask culture, the reaction conditions of the co-expression cells were determined. [Results] The optimal temperature and pH of enzymes in the co-expressing system were 70 °C and 6.0 in the presence of Co2+ (1 mmol/L), respectively. After the reaction reached equilibrium, 13.8, 38.1 and 62.6 g/L of D-mannose were obtained from 100, 300 and 500 g/L of D-glucose, which corresponded to a conversion of 13.8%, 12.7% and 12.5%, respectively, and the equilibrium ratio of D-glucose, D-fructose and D-mannose was about 50?37.5?12.5. [Conclusion] A co-expression system consisted of D-GIase and D-LIase in E. coli cells can be used for one-pot production of D-mannose from D-glucose.

Abstract:In recent years, the nanotechnology has provided a variety of nanoscale materials for enzyme immobilization. The immobilized enzyme of nanomaterials not only has high enzyme load, but also has excellent enzyme stability. Based on the immobilized enzyme of nanomaterials, this paper summarized the types of nanomaterials, and the effects of nanomaterials on the performance of immobilized enzymes were analyzed. Immobilized methods and application of nanobiocatalysis in the fields of biological transformation, biosensors and biofuel cells were also introduced.

Abstract:Enzyme is a green and natural biocatalyst, with the advantages of high activity under moderate conditions and high specificity. However, the high molecular complexity and intrinsic fragility are the challenges for applying the enzymes in the tough reaction conditions for the industry process. The advanced enzyme immobilization technology has great chance to improve the enzymatic activity and stability, bringing new opportunities for the engineering application of enzymes. Porous nanomaterials with large surface area, high porosity, stable mechanical, chemical resistance and superior cost-effectiveness, are ideal carriers of immobilizing enzyme. In this paper, the recent research progress and applications of nanomaterials such as metal organic framework, covalent organic framework and porous microspheres for enzymatic immobilization are reviewed. We mainly introduced the method of carrier for immobilization of enzymes, summarized the characteristics of each carrier. The challenges and future development of the immobilization technology for enzymes by porous nanomaterials have been finally discussed and concluded.

Abstract:The research on the application of enzyme drugs has become a hot spot in the field of biopharmaceuticals. Increasing number of enzymatic drugs have contained in pharmacopoeias in various countries, and most of them have become effective treatment drugs for various major diseases. Enzymes have great affinity and specificity for substrates and can catalyze the transformation of a variety of target molecules into desired products that make enzymes as special and effective drugs and perform therapeutic biochemical reactions, which can’t be performed by small molecules. Utilizing biotechnology to promote the development of new enzymatic drugs, and chemical modification to reduce the immune prototype of therapeutic enzymes and improve their relative stability are the current research direction of exploring enzymatic drugs. This review systematically summarizes the latest research progress and prospects of enzymes as therapeutic drugs at home and abroad.

Abstract:Asymmetric synthesis of unnatural chiral D-amino acids is a research hotspot, using meso-diaminopimelate dehydrogenase, in the field of biocatalysis. Meso-diaminopimelate dehydrogenase has excellent stereoselectivity, and it was used for enzymatic asymmetric synthesis of optically pure chiral D-amino acids, which is widely applied in medicine, food, cosmetics, fine chemicals field. To efficiently synthesize chiral D-amino acids, the methods of biocatalysis should be further developed. In this review, the research status was introduced about the synthesis of D-amino acids catalyzed by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum, Symbiobacterium thermophilum, Ureibacillus thermosphaericus. It focuses on research progress of meso-diaminopimelate dehydrogenase in mining of new enzyme, catalytic performance, crystal structure, molecular modification, function and catalytic mechanism, new way of synthesizing D-amino acid, etc. Finally, this paper prospected the future research directions and strategies for meso-diaminopimelate dehydrogenase. Therefore, this review has deepened our understanding of meso-diaminopimelate dehydrogenase, and it also provides guidance and information for challenging biosynthetic tasks.

Abstract:3-ketosteroid-Δ1-dehydrogenase is one of the key enzymes in microbial steroid catabolism, which catalyzes Δ1-dehydrogenation of A ring of 3-ketosteroid substrates. The enzyme not only plays a critical role in the early step of the degradation of the steroid nucleus, but also introduces the double bond at the C1,2 position of the A ring to significantly improve the physiological activity of the steroidal compound. In this paper, the species distribution in microorganisms and sequence characteristics, biological properties, physiological role, catalytic mechanism and molecular modification of 3-ketosteroid-Δ1-dehydrogenase are reviewed. It provides reference for further study of the 3-ketosteroid- Δ1-dehydrogenase application in the steroid biotransformation field.

Abstract:Glutamate decarboxylase, a widely distributed in plants, animals and microorganisms in nature, is a pyridoxal-5′-phosphate-dependent enzyme. It undergoes structural changes in an acidic environment and can irreversibly catalyze the α-decarboxylation of l-glutamic acid or glutamate to produce γ-aminobutyric acid. γ-aminobutyric acid, as an inhibitory neurotransmitter in human body, has important physiological functions and can be widely used in the food and pharmaceutical industries. In this paper, the research progress of the structure and catalytic mechanism of glutamate decarboxylase was summarized.

Abstract:Cytochrome P450 (CYP), membrane-bound hemoglobin enzymes, are widely present in microorganisms, animals, plants, and human. CYPs have a variety of biocatalytic activities such as oxidation, epoxidation hydroxylation, demethylation et al. Therefore, they play a key role in detoxification of biotin, cell metabolism and maintaining homeostasis. More importantly, the catalytic activity of CYPs is a hot issue in the fields of drug interaction and endocrine function. Furthermore, they are also widely utilized in the metabolism of drugs, carcinogens, steroids, fat-soluble vitamins and many other types of chemicals. This review provides a comprehensive overview of the structure, function, clinical application, and development prospects of CYPs. Besides, we aim to discuss the latest research and development prospects of CYPs.

Abstract:Carboxylic acid reductases (CARs) are capable of reducing carboxylic acids to the respective aldehydes under mild conditions, and have a broad substrate scope with no side products. Herein, we summarize recent advances in the research of phylogeny divergence, protein structure, catalytic mechanism and protein engineering of CARs, revealing its application prospect as an important tool enzyme in biological transformation and synthetic biology.

Abstract:The bacterial phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) is widely found in bacteria, fungi and some archaea, but not in plants and animals. PTS is composed of phosphotransferases such as enzyme I (EI), histidine phosphate carrier protein (HPr or NPr), and enzyme II complex. It has both a catalytic transport function and a very extensive regulatory function. PTS mainly phosphorylates various sugars and their derivatives through a phosphate cascade and then transports them into the cell. It not only participates in the metabolism of carbon and nitrogen sources, regulates the homeostasis of iron and potassium, regulates the virulence of certain pathogens, but it also mediates stress responses. During these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which changes according to the availability of the PTS substrate and the metabolic state of the cell. This article reviews the composition and regulatory network of phosphotransferase systems in bacteria, with a goal to provide a knowledge base for the study of the overall regulatory mechanism of the PTS and its effect on the overall metabolism of bacteria.

Abstract:Xylan is the most abundant non-cellulose polysaccharide in plant cell walls, accounting for about 20%?35% of terrestrial biomass resources. Xylan shows structural heterogeneity due to different substitution, which poses a major challenge for the conversion of biomass resources to biofuels and other value-added products efficiently. Therefore, it is necessary to develop optimal mixtures composed of different types of enzymes for efficient degrading xylan substrates. However, it is difficult to customize efficient degradation enzyme systems for specific types of substrates. The types of substrates, the composition and physical properties of substrates, the degree of polymerization of polysaccharides as well as the performance of different degrading enzyme components should be considered. This paper shows the recalcitrant barrier of plant xylan from structural heterogeneity and synthesis complexity. Moreover, the diversity of xylan degrading enzymes and their synergistic degradation are analyzed, as well as the mixed enzymes produced by microbial flora in nature habitats, the efficient enzyme system produced by degrading dominant microorganism, and the simplified efficient enzyme system modified and customized based on specific xylan substrates. With the deeper study on the fine structure of different types of xylan and the substrate specificity of the xylan-degrading enzymes, the green and efficient xylanase system was customized for specific substrate to accelerate the degradation of xylan substrate, so as to realize the friendly and high-value utilization of lignocellulosic resources.

Abstract:β-xylosidases are the members of xylanases. Their functions have been recognized to degrade xylan that is the most common hemicellulosic polysaccharide. In recent years, some microbial β-xylosidases show the function of bioactive substances transformation, including transxylosylation and dexylosylation of xylosylated substances such as notoginsenosides R1 and R2, astragaloside IV, 7-xylosyl-10-deacetylpaclitaxel and anthocyanins to produce bioactive substances. These β-xylosidases have huge potential applications in food, pharmaceutical and many other industries. Furthermore, some mechanisms involved in bioactive substances transformation by β-xylosidases have been revealed. This review mainly introduces the bioactive substances transformation functions, sources, family classification, transformation mechanisms and applications of β-xylosidases. The aim of the review is to provide useful information for the further development and utilization of β-xylosidases.

Abstract:XIP-type xylanase inhibitor proteins have inhibitory effects on most fungal xylanases of the GH10 and GH11 families, but they cannot inhibit xylanase produced by plants and bacteria. The inhibitory effect of XIP-type xylanase inhibitor protein on xylanase is mainly through simulating substrate contacting the active site of the enzyme, and quickly blocking the channel of the substrate into the active site region. However, in the crystal structures of GH10 and GH11 xylanase that are resistant to XIP-type xylanase inhibitor proteins, the loop conformations linking secondary structures obviously hinder the inhibition of XIP-type xylanase inhibitor proteins. Compared with XIP-sensitive xylanase, the insertion mutation of amino acid residues results in a prominent conformation of the loop of resistant xylanase. In XIP-resistant GH11 xylanase, the substitution of some amino acids in the thumb structure prevents the XIP-type xylanase inhibitor protein from forming a stable hydrogen bond and hydrophobic structure with the thumb structure, thereby weakening the inhibitory effect of XIP.

Abstract:The lytic polysaccharide monooxygenase (LPMO) of AA9 is widely distributed in fungi and plays an important role in the bioconversion of biomass because it can degrade crystalline polysaccharide of lignocellulose. The detailed information of the structural features, catalytic mechanism, structure and function and microbial expression and regulation of AA9 LPMO is summarized. Finally, the applications of AA9 LPMO in the conversion of lignocellulose are also discussed.