外源表达γ-内酰胺酶菌株的构建及发酵条件优化
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摘要
我们构建了重组γ-内酰胺菌株E. coli BL21/pCDFDuet-γ-lactam,研究了发酵产γ-内酰胺酶的条件并进行了优化。研究结果表明,将pCDFDuet-1表达载体转化到含有γ-内酰胺酶基因的感受态E. coli中,得到重组γ-内酰胺菌株E. coli BL21/pCDFDuet-γ-lactam,通过IPTG诱导表达和HPLC检测发现,重组酶可以水解(+)γ-内酰胺,其e.e.值比E. coli BL21/γ-lactam表达提高了约30%。重组菌中(+)γ-内酰胺酶蛋白表观分子量为50 kD。对重组菌株发酵条件进行了初步研究,结果进一步表明,当选择碳源葡萄糖5 g/L、温度30℃、接种量4%、装液量100 mL/500 mL,转速180 r/min条件下,(+)γ-内酰胺酶含量最高,e.e.值约98%。本研究为探索出一条适合规模化生产的酶催化反应工艺提供参考,该工艺的意义不仅可以节约经济,而且对环境友好,可以替代化学工艺。
We constructed the recombinant γ-lactam strains of E. coli BL21/pCDFDuet-γ-lactam, and we studied and optimized the fermentation condition for producing gamma lactamase. The results showed that the pCDFDuet-1 expression vector was transformed into competent cell of E. coli containing a gamma-lactamase gene, generating the recombinant strain of the γ-lactam strains E. coli BL21/pCDFDuet-γ-lactam. By using IPTG induction expression and HPLC detection, the recombinase can hydrolyze(+)γ-lactam, and its e.e. value can reasch about 30% higher than that of E. coli BL21/γ-lactam. The molecular weight of γ-lactamase protein is 50 kD.Furthermore, we studied the fermentation conditions of recombinant strains preliminarily. The results indicated that under the conditions of 5 g/L glucose as carbon source, 30℃ temperature, 4% inoculation amount, 100 m L/500 m L loading liquid volume, 180 r/min rotation speed,(+)γ-lactamase content is able to be the highest, and the e.e. value is upto 98% as well. This study might provide a reference for exploring an enzyme-catalyzed reaction process suitable for large-scale production. The significance of this process is not only saving money, but also being friendly environment as well as it might be alternative to the chemical process.
We constructed the recombinant γ-lactam strains of E. coli BL21/pCDFDuet-γ-lactam, and we studied and optimized the fermentation condition for producing gamma lactamase. The results showed that the pCDFDuet-1 expression vector was transformed into competent cell of E. coli containing a gamma-lactamase gene, generating the recombinant strain of the γ-lactam strains E. coli BL21/pCDFDuet-γ-lactam. By using IPTG induction expression and HPLC detection, the recombinase can hydrolyze(+)γ-lactam, and its e.e. value can reasch about 30% higher than that of E. coli BL21/γ-lactam. The molecular weight of γ-lactamase protein is 50 kD.Furthermore, we studied the fermentation conditions of recombinant strains preliminarily. The results indicated that under the conditions of 5 g/L glucose as carbon source, 30℃ temperature, 4% inoculation amount, 100 m L/500 m L loading liquid volume, 180 r/min rotation speed,(+)γ-lactamase content is able to be the highest, and the e.e. value is upto 98% as well. This study might provide a reference for exploring an enzyme-catalyzed reaction process suitable for large-scale production. The significance of this process is not only saving money, but also being friendly environment as well as it might be alternative to the chemical process.
引文
Abusco A.S.,Ammendola S.,Scandurra R.,and Politi L.,2001,Molecular and biochemical characterization of the recombinant amidase from hyperthermophilic archaeon,Sulfolobus solfataricus,Extremophiles,5(3):183-192
Brabban A.D.,Littlechild J.,and Wisdom R.,1996,Stereospecificγ-lactamase activity in a Pseudomonas fluorescens species,Journal of Industrial Microbiology,16(1):8-14
Chand P.,Kotian P.L.,Dehghani A.,El-Kattan Y.,Lin T.H.,Hutchison T.L.,Babu Y.S.,Bantia S.,Elliott A.J.,and Montgomery J.A.,2001,Systematic structure based design and stereoselective synthesis of novel multisubstitu tedcyclo-pentane derivatives with potent antiinfluenzaactivity,Med.Chem.,44(25):4379-4392
Abusco A.S.,Casadio R.,Tasco G.,Tasco G.,Giangiacomo L.,Giartosio A.,Calamia V.,and Marco S.,2014,Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature,Archaea-an International Microbiological Journal,1(6):411-423
Lagoja I.M.,and Clercq E.D.,2008,Anti-influenzavirus agents:synthesis and mode of action,Med.Res.Rev.,28(1):1-38
Simon H.,Bader J.,and Gunther H.,1985,Chiral compounds synthesized by biocatalyic reductions,Angewandte Chemie International Edition,24(7):539-553
Taylor S.J.C.,McCague R.,Wisdom R.,Lee C.,Dickson K.,Ruecroft G.,Littlechild J.,Bevan J.,Roberts S.,and Evans C.T.,19 93,Development of the biocatalytic resolution of2-azabicyclo[2.2.1]hept-5-en-3-one as an entry to singleeantiomer carbocyclic nucleosides,Tetrahedron Asymmetry,4(6):1117-1128
Toogood H.S.,Brown R.,Line K.,and Littlechild J.,2004,The use of a thermostable signature amidase in the resolution of the bicyclic synthon(rac)γ-lactam,Tetrahedron,60(3):711-716
Wang J.J.,Zhu J.G.,Min C.,and Wu S.,2014,CBD binding domain fusedγ-lactamase from Sulfolobus solfataricus is an efficient catalyst for(-)γ-lactam production,Bmc Biotechnology,14(1):1-8
Whitesides G.M.,and Wong C.H.,2010,Enzymes as catalysts in synthetic organic chemistry,New Synthetic Methods,24(8):617-638
Xiao L.,Wang Y.J.,and Cao Z.,Liu Z.Q.,and Zheng Y.G.,2013,Development of biocatalytic process for t-butyl 6-cyano-(3R,5R)-dihydroxylhexanoate,Shenwu Jiagong Guochen(Chinese Journal of Bioprocess Engineering),11(1):29-34(肖黎,王亚军,曹政,柳志强,郑裕国,2013,生物催化法合成6-氰基-(3R,5R)-二羟基己酸叔丁酯,生物加工过程,11(1):29-34)
Xie X.L.,Cheng J.S.,Wang X.M.,Mei X.H.,Liu L.,and Li J.,2014,One-Step preparation of biotinylated protein using prokaryotic expression system,Zhongguo Shengwu Gongcheng Zazhi(Journal of Chinese Biotechnology),34(1):36-41(谢小丽,程剑松,王晓敏,梅香寒,刘琳,李静,2014,利用原核表达系统一步法制备生物素标记蛋白质,中国生物工程杂志,34(1):36-41)
Zhang X.J.,Chi N.Y.,Wang Q.,Wang X.H.,Jin L.D.,and Zhang Q.F.,2016,Isolation and identification of a(+)γ-lactamases producing strain and preliminary study on its enzymatic properties,Zhongguo Ninagzao(China Brewing),35(5):52-55(张旭姣,迟乃玉,王强,王晓辉,金连豆,张庆芳,2016,(+)γ-内酰胺酶菌株的筛选鉴定及其部分酶学特性研究,中国酿造,35(5):52-55)
Brabban A.D.,Littlechild J.,and Wisdom R.,1996,Stereospecificγ-lactamase activity in a Pseudomonas fluorescens species,Journal of Industrial Microbiology,16(1):8-14
Chand P.,Kotian P.L.,Dehghani A.,El-Kattan Y.,Lin T.H.,Hutchison T.L.,Babu Y.S.,Bantia S.,Elliott A.J.,and Montgomery J.A.,2001,Systematic structure based design and stereoselective synthesis of novel multisubstitu tedcyclo-pentane derivatives with potent antiinfluenzaactivity,Med.Chem.,44(25):4379-4392
Abusco A.S.,Casadio R.,Tasco G.,Tasco G.,Giangiacomo L.,Giartosio A.,Calamia V.,and Marco S.,2014,Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature,Archaea-an International Microbiological Journal,1(6):411-423
Lagoja I.M.,and Clercq E.D.,2008,Anti-influenzavirus agents:synthesis and mode of action,Med.Res.Rev.,28(1):1-38
Simon H.,Bader J.,and Gunther H.,1985,Chiral compounds synthesized by biocatalyic reductions,Angewandte Chemie International Edition,24(7):539-553
Taylor S.J.C.,McCague R.,Wisdom R.,Lee C.,Dickson K.,Ruecroft G.,Littlechild J.,Bevan J.,Roberts S.,and Evans C.T.,19 93,Development of the biocatalytic resolution of2-azabicyclo[2.2.1]hept-5-en-3-one as an entry to singleeantiomer carbocyclic nucleosides,Tetrahedron Asymmetry,4(6):1117-1128
Toogood H.S.,Brown R.,Line K.,and Littlechild J.,2004,The use of a thermostable signature amidase in the resolution of the bicyclic synthon(rac)γ-lactam,Tetrahedron,60(3):711-716
Wang J.J.,Zhu J.G.,Min C.,and Wu S.,2014,CBD binding domain fusedγ-lactamase from Sulfolobus solfataricus is an efficient catalyst for(-)γ-lactam production,Bmc Biotechnology,14(1):1-8
Whitesides G.M.,and Wong C.H.,2010,Enzymes as catalysts in synthetic organic chemistry,New Synthetic Methods,24(8):617-638
Xiao L.,Wang Y.J.,and Cao Z.,Liu Z.Q.,and Zheng Y.G.,2013,Development of biocatalytic process for t-butyl 6-cyano-(3R,5R)-dihydroxylhexanoate,Shenwu Jiagong Guochen(Chinese Journal of Bioprocess Engineering),11(1):29-34(肖黎,王亚军,曹政,柳志强,郑裕国,2013,生物催化法合成6-氰基-(3R,5R)-二羟基己酸叔丁酯,生物加工过程,11(1):29-34)
Xie X.L.,Cheng J.S.,Wang X.M.,Mei X.H.,Liu L.,and Li J.,2014,One-Step preparation of biotinylated protein using prokaryotic expression system,Zhongguo Shengwu Gongcheng Zazhi(Journal of Chinese Biotechnology),34(1):36-41(谢小丽,程剑松,王晓敏,梅香寒,刘琳,李静,2014,利用原核表达系统一步法制备生物素标记蛋白质,中国生物工程杂志,34(1):36-41)
Zhang X.J.,Chi N.Y.,Wang Q.,Wang X.H.,Jin L.D.,and Zhang Q.F.,2016,Isolation and identification of a(+)γ-lactamases producing strain and preliminary study on its enzymatic properties,Zhongguo Ninagzao(China Brewing),35(5):52-55(张旭姣,迟乃玉,王强,王晓辉,金连豆,张庆芳,2016,(+)γ-内酰胺酶菌株的筛选鉴定及其部分酶学特性研究,中国酿造,35(5):52-55)