全细胞生物转化法制备γ-氨基丁酸
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摘要
γ-氨基丁酸(γ-aminobutyrate,GABA)是广泛存在于自然界的一种非蛋白质的功能性氨基酸.它是哺乳动物中枢神经中一种重要的抑制性神经递质.为了高效廉价制备GABA,采取谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)催化L-谷氨酸生成GABA的工艺.从K12来源的大肠杆菌(E. coli)基因组中PCR扩增得到GAD基因片段,并克隆到载体p ET-23a中,得到重组质粒p ET-23a-K12gad,并将其转化至大肠杆菌E. coli BL21(DE3)中,获得了能够重组表达GAD活性的工程菌株.从反应温度、p H、菌体量这三个方面对催化工艺进行了优化.为了进一步降低成本,通过化学法将谷氨酸钠转化为谷氨酸作为催化底物,提高转化速率的同时简化了纯化工艺.
γ-aminobutyric acid( GABA) is a non-protein functional amino acid widely found in nature. It is an important inhibitory neurotransmitter in mammalian central nervous system. In order to efficiently and cheaply produce GABA,glutamic acid decarboxylase( GAD) was used to synthesize the GABA from Lglutamic acid in this experiment. The GAD gene fragment amplified by PCR from Escherichia coli K12 genome was cloned into the vector pET-23 a to obtain the recombinant plasmid p ET-23 a-K12 gad which was transformed into Escherichia coli BL21( DE3),successfully constructing an engineered strains with GAD activity. We optimized the catalytic process from three aspects of reaction temperature,p H and microbial biomass. In order to further reduce costs,we chemically convert sodium glutamate to glutamate as a catalytic substrate,which improved the conversion rate and simplified the purification process.
γ-aminobutyric acid( GABA) is a non-protein functional amino acid widely found in nature. It is an important inhibitory neurotransmitter in mammalian central nervous system. In order to efficiently and cheaply produce GABA,glutamic acid decarboxylase( GAD) was used to synthesize the GABA from Lglutamic acid in this experiment. The GAD gene fragment amplified by PCR from Escherichia coli K12 genome was cloned into the vector pET-23 a to obtain the recombinant plasmid p ET-23 a-K12 gad which was transformed into Escherichia coli BL21( DE3),successfully constructing an engineered strains with GAD activity. We optimized the catalytic process from three aspects of reaction temperature,p H and microbial biomass. In order to further reduce costs,we chemically convert sodium glutamate to glutamate as a catalytic substrate,which improved the conversion rate and simplified the purification process.
引文
[1]郭晓娜,朱永义.生物体内γ-氨基丁酸的研究[J].氨基酸和生物资源,2003,25(2):70-72.
[2]Manyam B V,Katz L,Hare T A,et al.Isoniazid-induced elevation of CSF GABA levels and effects on chorea in Huntington’s disease[J].Annals of Neurology,2010,10(1):35-37.
[3]Watanabe M,Maemura K,Kanbara K,et al.GABA and GABA receptors in the central nervous system and other organs[J].International Review of Cytology,2002,213(4):1-47.
[4]Huang J,Mei L H,Xia J.Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA[J].Biotechnology&Bioengineering,2007,96(5):924-931.
[5]Diana M,Quílez J,Rafecas M.Gamma-aminobutyric acid as a bioactive compound in foods:a review[J].Journal of Functional Foods,2014,10(3):407-420.
[6]Tijs M Lammens,Maurice C R Franssen,Elinor L Scott,et al.Synthesis of biobased N-methylpyrrolidone by one-pot cyclization and methylation ofγ-aminobutyric acid[J].Green Chemistry,2010,12(8):1430-1436.
[7]Saskiawan I.Biosynthesis of polyamide 4,a biobased and biodegradable polymer[J].Microbiology Indonesia,2008,2(3):119-123.
[8]Kawasaki N,Nakayama A,Yamano N,et al.Synthesis,thermal and mechanical properties and biodegradation of branched polyamide 4[J].Polymer,2005,46(23):9987-9993.
[9]Dhakal R,Bajpai V K,Baek K H.Production of gaba(γ-aminobutyric acid)by microorganisms:a review[J].Braz JMicrobiol,2012,43(4):1230-1241.
[10]Kang T J,Ho N A,Pack S P.Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli[J].Enzyme&Microbial Technology,2013,53(3):200-205.
[11]Ke C,Yang X,Rao H,et al.Whole-cell conversion of L-glutamic acid into gamma-aminobutyric acid by metabolically engineered Escherichia coli[J].Springer Plus,2016,5(1):591-599.
[12]许建军,江波,许时婴.比色法快速测定乳酸菌谷氨酸脱羧酶活力及其应用[J].微生物学通报,2004,31(2):66-71.
[2]Manyam B V,Katz L,Hare T A,et al.Isoniazid-induced elevation of CSF GABA levels and effects on chorea in Huntington’s disease[J].Annals of Neurology,2010,10(1):35-37.
[3]Watanabe M,Maemura K,Kanbara K,et al.GABA and GABA receptors in the central nervous system and other organs[J].International Review of Cytology,2002,213(4):1-47.
[4]Huang J,Mei L H,Xia J.Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA[J].Biotechnology&Bioengineering,2007,96(5):924-931.
[5]Diana M,Quílez J,Rafecas M.Gamma-aminobutyric acid as a bioactive compound in foods:a review[J].Journal of Functional Foods,2014,10(3):407-420.
[6]Tijs M Lammens,Maurice C R Franssen,Elinor L Scott,et al.Synthesis of biobased N-methylpyrrolidone by one-pot cyclization and methylation ofγ-aminobutyric acid[J].Green Chemistry,2010,12(8):1430-1436.
[7]Saskiawan I.Biosynthesis of polyamide 4,a biobased and biodegradable polymer[J].Microbiology Indonesia,2008,2(3):119-123.
[8]Kawasaki N,Nakayama A,Yamano N,et al.Synthesis,thermal and mechanical properties and biodegradation of branched polyamide 4[J].Polymer,2005,46(23):9987-9993.
[9]Dhakal R,Bajpai V K,Baek K H.Production of gaba(γ-aminobutyric acid)by microorganisms:a review[J].Braz JMicrobiol,2012,43(4):1230-1241.
[10]Kang T J,Ho N A,Pack S P.Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli[J].Enzyme&Microbial Technology,2013,53(3):200-205.
[11]Ke C,Yang X,Rao H,et al.Whole-cell conversion of L-glutamic acid into gamma-aminobutyric acid by metabolically engineered Escherichia coli[J].Springer Plus,2016,5(1):591-599.
[12]许建军,江波,许时婴.比色法快速测定乳酸菌谷氨酸脱羧酶活力及其应用[J].微生物学通报,2004,31(2):66-71.