In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by ...In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.展开更多
Glutamate decarboxylase(GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate(GABA), which is in great demand in some foods and pharmaceuticals. In our previous study,gad, the gene ...Glutamate decarboxylase(GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate(GABA), which is in great demand in some foods and pharmaceuticals. In our previous study,gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51 H with high activity [55.4 mmol·L-1·min-1·(mg protein)-1, 120% higher than that of the wild type at p H 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out,and three specific mutants, N-terminal truncated GAD, Q51 P, and Q51 L, were identified. The kinetic parameters of the three mutants and Q51 H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.展开更多
Hirudin gene was mutated by error\|prone PCR and DNA shuffling.The shuffled genes were inserted into phagemid pCANTAB\|5G8.After the introduction of the recombinant DNA into E.coli TG1,the hirudin mutants were display...Hirudin gene was mutated by error\|prone PCR and DNA shuffling.The shuffled genes were inserted into phagemid pCANTAB\|5G8.After the introduction of the recombinant DNA into E.coli TG1,the hirudin mutants were displayed on the surface of bacteriophage M13.A mutant with increased specific activity of antithrombin was selected by two rounds of panning with decreased amounts of thrombin,and was identified as N47K.Activity analysis of HV2 and its mutants N47K,N26S,D5A,P46L/S50G suggested that K47,P46 were important to the interaction of hirudin and thrombin.Hirudin mutants with improved properties could be hopefully aquired through directed evolution by DNA shuffling.展开更多
DNA家族改组(DNA family shuffling)是一项高效的体外进化技术,该技术利用自然界中具有同源序列的基因家族——如来自不同种属的相同基因以及同一种属的相关基因等进行改组,为蛋白质定向进化和结构——功能关系分析等研究提供了有效获...DNA家族改组(DNA family shuffling)是一项高效的体外进化技术,该技术利用自然界中具有同源序列的基因家族——如来自不同种属的相同基因以及同一种属的相关基因等进行改组,为蛋白质定向进化和结构——功能关系分析等研究提供了有效获得突变文库的方法。近年来,DNA家族改组技术不断取得新的发展,应用领域日益扩展,在生物合成途径的改造、植物抗病能力的加强、环境污染的治理、医药工业的发展、异源表达体系的建立等方面都发挥了重要作用。文章综述了这项技术在近年来的新发展和新应用,并展望了DNA家族改组技术的发展前景。展开更多
基金Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of ZhejiangProvince (No. Z304076), China
文摘In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.
基金Supported by the National Natural Science Foundation of China(30970638,21176220,and 31240054)the National Natural Science Foundation of Zhejiang Province(LZ13B060002)
文摘Glutamate decarboxylase(GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate(GABA), which is in great demand in some foods and pharmaceuticals. In our previous study,gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51 H with high activity [55.4 mmol·L-1·min-1·(mg protein)-1, 120% higher than that of the wild type at p H 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out,and three specific mutants, N-terminal truncated GAD, Q51 P, and Q51 L, were identified. The kinetic parameters of the three mutants and Q51 H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.
文摘Hirudin gene was mutated by error\|prone PCR and DNA shuffling.The shuffled genes were inserted into phagemid pCANTAB\|5G8.After the introduction of the recombinant DNA into E.coli TG1,the hirudin mutants were displayed on the surface of bacteriophage M13.A mutant with increased specific activity of antithrombin was selected by two rounds of panning with decreased amounts of thrombin,and was identified as N47K.Activity analysis of HV2 and its mutants N47K,N26S,D5A,P46L/S50G suggested that K47,P46 were important to the interaction of hirudin and thrombin.Hirudin mutants with improved properties could be hopefully aquired through directed evolution by DNA shuffling.
文摘DNA家族改组(DNA family shuffling)是一项高效的体外进化技术,该技术利用自然界中具有同源序列的基因家族——如来自不同种属的相同基因以及同一种属的相关基因等进行改组,为蛋白质定向进化和结构——功能关系分析等研究提供了有效获得突变文库的方法。近年来,DNA家族改组技术不断取得新的发展,应用领域日益扩展,在生物合成途径的改造、植物抗病能力的加强、环境污染的治理、医药工业的发展、异源表达体系的建立等方面都发挥了重要作用。文章综述了这项技术在近年来的新发展和新应用,并展望了DNA家族改组技术的发展前景。
