Effects of site-directed mutagenesis of A79Y and T81Q at Loop structure on thermal stability of xylanase XynASP / 中国生物制品学杂志

@#ObjectiveTo identify the sites of thermal stability at Loop structure of xylanase Xyn ASP in Aspergillus saccharolyticus JOP 1030-1 and improve the thermal stability.MethodsThe amino acid sites related to thermal stability of xylanase were predicted,and beneficial mutation sites at Loop structure were screened by Fireprot online server.Singlepoint mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and double-point mutants Xyn(T81Q)and double-point mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(T81Q)retained 37.7%,Xyn(T81Q)retained 37.7%,Xyn(A79Y)and Xyn(A79Y)and Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)at pH 3.0～8.0 showed no significant change compared with the wild type.ConclusionSite-directed mutagenesis(A79Y and T81Q)was carried out at Loop structure,and xylanase mutants with obviously improved thermal stability were obtained,which laid a foundation of the later research on the structure,function and relationship of the enzyme and its industrial application.

Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum / 生物工程学报

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Identification and verification of key salt-tolerant amino acid sites of banana MaNHX5 / 生物工程学报

In order to improve the salt tolerance of banana NHX genes, we cloned a MaNHX5 gene from Musa acuminata L. AAA group and predicted the key salt-tolerant amino acid sites and mutant protein structure changes of MaNHX5 by using bioinformatics tools. The 276-position serine (S) of MaNHX5 protein was successfully mutated to aspartic acid (D) by site-directed mutagenesis, and the AXT3 salt-sensitive mutant yeast was used for a functional complementation test. The results showed that after the mutated MaNHX5 gene was transferred to AXT3 salt-sensitive mutant yeast, the salt tolerance of the mutant yeast was significantly improved under 200 mmol/L NaCl treatment. It is hypothesized that Ser276 of MaNHX5 protein plays an important role in the transport of Na+ across the tonoplast.

Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations / 生物工程学报

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene / 生物工程学报

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Optimized inverse PCR strategy for constructing multilocus mutants efficiently / 生物工程学报

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.

Site-directed mutagenesis of long gene by partial amplification combining with double fragments ligation / 生物工程学报

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

Identifying protein epitopes recognized by monoclonal antibodies / 生物工程学报

To establish a method for identifying protein epitopes recognized by therapeutic monoclonal antibodies, the programmed death receptor-1 (PD-1) was selected as the target protein. Based on the alanine scanning strategy, a rapid expression method of antigen mutants combining site-directed mutagenesis with mammalian cell expression system was established, the conditions for eukaryotic expression element amplification and cell transfection expression were established. 150 PD-1 protein mutants were co-expressed, and the binding ability of these mutants to anti-PD-1 antibody Pembrolizumab was identified. The epitopes of Pembrolizumab were determined based on the binding ability of protein mutants to antibodies and combined with protein structure analysis, which was highly consistent with the reported crystal structure-based epitopes, indicating that this method is simple and accurate and can be used for epitope mapping of therapeutic monoclonal antibodies.

Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli / 生物工程学报

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.

Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase β-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.

Screening of 14-3-3τ protein inhibitors from natural products based on fluorescence spectroscopy, surface plasmon resonance and molecular docking technique / 国际药学研究杂志

Objective:To study and establish a method for the efficient screening of 14-3-3τ protein inhibitors from natural products, and analyze the sites of their interactions with the 14-3-3τ protein. Methods: The binding activity of natural compounds with the 14-3-3τ protein was tested by the liquid chromatography-fluorescence spectroscopy, and the binding activity of the potential compounds was further verified by the surface plasmon resonance(SPR)technique. The binding sites of the active compounds were predicted by the molecular docking technique. Furthermore, the key binding sites were selected and then validated using amino acid site-directed mutants. Results: A total of 17 different type compounds with potential 14-3-3τ binding activity were screened out from 82 natural products. The binding activi- ties of 10 compounds were verified by the SPR experiments. Then the binding sites of interactions between the 14-3-3τ protein and the 10 compounds were predicted by the molecular docking technology to be mainly at the Arg56, Arg127 and Y128A, dem- onstrate that the binding of five of the 10 compounds with the target protein was associated with the three sites Arg56, Arg127 and Tyr128. Conclusion: The established method is accurate and efficient, which could be used for rapid screening of small molecule 14-3-3τ inhibitors from natural products. The present study provides a reference and a new approach for the rapid screening of 14-3-3τ inhibitors for the new breast cancer therapeutic drugs.

Advances in structure-function relation of plant type Ⅲ polyketide synthases by site-directed mutagenesis / 生物工程学报

Plant type Ⅲ polyketide synthases (PKSs), the pivotal enzymes in the biosynthesis of polyketides, produce backbones of many structurally diverse and functionally different polyketides. So far, a variety of functionally diverse plant type Ⅲ PKSs have been cloned and identified from plant origin. Site-directed mutagenesis is a useful technique to study the complex relationship between protein structure and function. This review summarized advances in the structure-function relation of plant type Ⅲ polyketide synthases by site-directed mutagenesis in recent years, including the modification of the amino acid residues influencing enzyme architectures (such as controlling the specificity of starter substrates, the number of condensation reactions, and the cyclization reactions of the intermediate product). This review provides information to study the structure-function relation of plant type Ⅲ polyketide synthases.

Molecular modification of β-glucosidase from the midgut of Macrotermes barneyi / 生物工程学报

Cellulose hydrolysis to glucose requires a series of cellulase enzymes, of which β-glucosidases play a crucial role. β-glucosidase (MbmgBG1) derived from the midgut of Macrotermes barneyi has higher glucose tolerance (maintaining more than 60% enzyme activity at 1.5 mol/L glucose). However, low enzyme activity and poor thermal stability limit the applications of β-glucosidase in food industries. Point mutants (F167L, T176C, E347I, R354K, N393G and V425M) were obtained by site-directed mutagenesis of non-conserved amino acids near conserved amino acids. Among them, the specific activities against to substrate pNPG of two mutants (F167L and R354K) were about 2-fold and 4-fold higher than that of MbmgBG1. Kcat/Km values were also higher than that of the wild-type, reflecting stronger affinity to the substrate and higher catalytic ability of mutants than MbmgBG1. When the glucose concentration was 1.5 mol/L, the enzyme activity of MbmgBG1 was about 60% of the original activity. F167L and R354K kept 60% enzymatic activity when the glucose concentrations of was 2.0 mol/L and 3.0 mol/L, respectively. These results lay a foundation for further studies on the catalytic efficiency of β-glucosidase.

Improvement of catalytic activity of Aspergillus terreus lipase by site-directed mutagenesis / 生物工程学报

The catalytic activity of Aspergillus terreus lipase (ATL) was improved by rational design. According to the sequence analysis and homologous modeling, several amino acids involved in the lid domain and substrate binding pocket domains of the acidic lipase ATL were mutated by site-directed mutagenesis, and eight mutants were constructed. These mutants and the wild type lipase ATL were expressed in Pichia pastoris GS115 and the enzymatic properties were characterized. The mutants ATLLid and ATLV218W exhibited higher hydrolytic activity than ATL towards p-nitrophenyl laurate. The kcat values of ATLLid and ATLV218W towards p-nitrophenyl laurate were 39.37- and 50.79-fold higher, and the kcat/Km values were 2.85- and 8.48-fold higher than the wild type, respectively. Although thermostability of these mutants decreased slightly, ATLLid and ATLV218W still exhibited the maximum activity at pH 5.0 and high stability in a broad range of pH (4.0-8.0), which were similar to the wild type. Using homologous modeling and molecular docking technology the mechanism for the improvement of catalytic activity was analyzed. These findings not only shed light on the relationship between the lid domain/substrate binding pocket domain and catalytic activity but also provided comprehensive scheme for further engineering to gain more efficient lipases.

Improving the thermostability of α-amylase from Rhizopus oryzae by rational design / 生物工程学报

Fungal α-amylases are widely used in the production of maltose syrup, while additional production costs may be required in the syrup production process due to the loss of enzyme activity, because of the poor thermostability exhibited in this type of enzyme. After deeply studying the importance of thermostability of fungal α-amylases applied in industrial production, with attempt to improve the thermostability of Rhizopus oryzae α-amylase (ROAmy), single-point mutations and combined mutations that based on analysis of B-factor values and molecular dynamics simulations were carried out for amino acid residues G128, K269 and G393 of ROAmy by overlapping PCR. The results showed that all the 7 mutants obtained presented better thermostability than the wild-type enzyme, and the best mutant was G128L/K269L/G393P which showed a 5.63-fold increase in half-life at 55 ℃ compared with the wild-type enzyme. Meanwhile, its optimum temperature increased from 50 ℃ to 65 ℃, the maximum reaction rate (Vmax) and catalytic efficiency (kcat/Km) increased by 65.38% and 99.86%. By comparing and analyzing the protein structure and function between the mutants and the wild-type enzyme, it was found that the increase of the number of hydrogen bonds or the introduction of proline in special position may be the main reasons for the improved thermostability that found in the mutants.

Construction of EGFR gene G719S and T790M mutation vector and preliminary clinical application / 临床检验杂志

Objective To construct mutant recombinant vector of epidermal growth factor receptor ( EGFR) gene G719S and T790M sites associated with cervical cancer, lay the foundation for the detection of EGFR gene mutation in cervical cancer. And using it to es-tablish a molecular switch platform to detect cervical cancer EGFR gene mutations. Methods Using the wild-type recombinant plasmid as template, the mutant fusion target fragment were amplified by overlap PCR, then connect this target fragment into the vector pMD19-T. The constructed mutant recombinant plasmid was finally transformed into competent cells E.coli DH5αfurther identified by PCR with bacterial solution and genome sequencing. Establishing the molecular switch for the detection of clinical cervical cancer samples. Re-sults The G719S and T790M mutations were successfully certified by genome sequencing, and the site-directed mutant vector was successfully constructed. In addition, a molecular switch detection platform was also successfully established for the detection of cervical cancer tissue DNA. Conclusion We successfully constructed an EGFR gene mutant recombinant vector by overlap PCR technique, which providing a new technical means for gene site-directed mutagenesis. And the molecular switch detection platform was successfully established based on it, which furnishing a new method for clinical detection of EGFR gene mutations.

Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris

Background: Laccases are copper-containing enzymes which have been used as green biocatalysts for many industrial processes. Although bacterial laccases have high stabilities which facilitate their application under harsh conditions, their activities and production yields are usually very low. In this work, we attempt to use a combinatorial strategy, including site-directed mutagenesis, codon and cultivation optimization, for improving the productivity of a thermo-alkali stable bacterial laccase in Pichia pastoris. Results: A D500G mutant of Bacillus licheniformis LS04 laccase, which was constructed by site-directed mutagenesis, demonstrated 2.1-fold higher activity when expressed in P. pastoris. The D500G variant retained similar catalytic characteristics to the wild-type laccase, and could efficiently decolorize synthetic dyes at alkaline conditions. Various cultivation factors such as medium components, pH and temperature were investigated for their effects on laccase expression. After cultivation optimization, a laccase activity of 347 ± 7 U/L was finally achieved for D500G after 3 d of induction, which was about 9.3 times higher than that of wild-type enzyme. The protein yield under the optimized conditions was about 59 mg/L for D500G. Conclusions: The productivity of the thermo-alkali stable laccase from B. licheniformis expressed in P. pastoris was significantly improved through the combination of site-directed mutagenesis and optimization of the cultivation process. The mutant enzyme retains good stability under high temperature and alkaline conditions, and is a good candidate for industrial application in dye decolorization.

Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.

Studies on site-directed mutagenesis of BmK AngM1 from scorpion venom and its anti-inflammatory activity / 药学学报

Scorpion toxin BmK AngM1 has been reported to have a strong analgesic effect. However, its anti-inflammatory activity was unknown. In this study, the recombinant BmK AngM1 (rBmK AngM1) was expressed in Escherichia coli BL21 trxB (DE3). The purified rBmK AngM1 was obtained efficiently through the IMPACTTM-TWIN system. The anti-inflammatory activity of the recombinant protein was investigated. In order to improve the anti-inflammatory activity of rBmK AngM1, the potential active sites (Y5, Y42, R58) were substituted with different amino acids. The results showed that rBmK AngM1 and its mutants all have significant anti-inflammatory activity. The activities were significantly increased in the single mutant R58N and mutants Y5F/R58N, Y42F/R58N over the wild type protein. The data suggest that position 58 in BmK AngM1 plays a functional role in the anti-inflammatory activity. This study lays a foundation for the protein engineering design of BmK AngM1 to improve its pharmacological activity.

The Clone, Expression and Site-directed Mutagenesis of 3-Ketosteroid-Delta (1)-Dehydrogenase from Arthrobacter Simplex / 现代生物医学进展

Objective:In this study,a prokaryotic expression of the 3-ketosteroid-Delta (1)-dehydrogenase (KSDD) which came from Arthrobacter simplex was built.Moreover,in order to investigate the catalytic mechanism of KSDD and improve its stability,the structure of KSDD was predicted by computer and the critical sites were confirmed by site-directed mutations.Methods:The recombinant plasmid was constructed by eukaryotic expression vector pET-22b and the recombinant strain was constructed and expressed in Escherichia coli BL21 (DE3).High-performance liquid chromatography was used to determine the transformation rate of 4-AD to ADD.The KSDD structure and key sites were predicted by SWISS-MODEL.Site-directed mutations for the amino acid residues of key sites were constructed and activities of the mutations were detected.Results:The recombinant strain E.coli pET-22-ksdd was successfully constructed.It was induced to express the dehydrogenase by IPTG and the conversion rate of 4-AD to ADD was 27％ at 21 ℃.The structure of 3-ketosteroid-Delta (1)-dehydrogenase and the four key sites was analyzed by SWISS-MODEL.Four mutants,Y120R,Y320L,Y488F and G492Y were constructed.Mutants Y120R and Y488F were inactivated,so they were proved to be the key active sites of KSDD.The conversion rate of mutant Y320L was consistent with that of wild type,but the stability at 37 ℃ was improved.The conversion rate of mutant G492Y was 1.2 times of the wild type and the stability has been improved at 37 ℃.Conclusions:At present,there are few studies about the structure and catalytic mechanism of dehydrogenase.The active sites of the enzyme were verified by this study,which laid the foundation for the further study of the properties of the enzyme KSDD.