嗜热芽胞杆菌α-环糊精葡萄糖基转移酶在枯草芽胞杆菌中的表达
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摘要
【目的】构建分泌表达α-环糊精葡萄糖基转移酶(α-CGTase)的重组枯草芽胞杆菌,实现α-CGTase的安全高效表达。【方法】通过PCR法扩增嗜热地芽胞杆菌α-CGTase基因,运用EcoRI/Xho I分别对α-CGTase基因及pBES进行双酶切,然后将该基因片段插入到大肠杆菌-枯草芽胞杆菌穿梭载体pBES中,再电转化法转化枯草芽胞杆菌RIK1285;对重组枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT发酵条件进行探索。【结果】(1)利用发酵培养基分泌表达α-CGTase,重组枯草芽胞杆菌工程菌B.subtilis RIK1285/pBE-CGT发酵上清液产酶达到2.9U·mL~(-1)。(2)TB为最适发酵培养基(配方:甘油0.5%,蛋白胨1.2%,酵母粉2.4%,K_2HPO_4 1.64%,KH_2PO_40.23%);在初始pH 6.5,温度为37℃下,摇瓶发酵培养24h后,α-环糊精酶的环化活性达到5.3U·mL~(-1),是野生菌株嗜热地芽胞杆菌(0.66U·mL~(-1))的8倍。【结论】成功构建了枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT工程菌,并确定其最适发酵培养基和培养条件。
【Objective】To construct a vector for expressingα-cyclodextrin glycosyltransferase(α-CGTase)gene in Bacillus subtilis.【Method】Theα-CGTase gene from Gebacillius sp.CHB1 was amplified by PCR.The EcoRIdigested pBES and Xho I-digested α-CGT gene were connected and transformed into B.subtilis RIK1285.Subsequently,fermentation of the recombinant B.subtilis RIK1285/pBE-CGT was optimized.【Result】(1)Theα-CGTase gene was expressed in a fermentation medium to show an enzymatic activity of 2.9 U·mL~(-1) by B.subtilis RIK1285/pBE-CGT.(2)Medium TB with the formula of 0.5% glycerol,1.2% peptone,2.4% yeast extract,1.64% K_2HPO_4,and 0.23% KH_2PO_4 was found to be optimal for the fermentation.After fermentation in TB at37℃ for 24 h,theα-CGTase activity reached 5.3 U·mL~(-1),which was 8-fold of what the wild Gebacilliussp.CHB1 could generate.【Conclusion】The engineered B.subtilis RIK1285/pBE-CGT was successfully obtained and the fermentation process optimized.
【Objective】To construct a vector for expressingα-cyclodextrin glycosyltransferase(α-CGTase)gene in Bacillus subtilis.【Method】Theα-CGTase gene from Gebacillius sp.CHB1 was amplified by PCR.The EcoRIdigested pBES and Xho I-digested α-CGT gene were connected and transformed into B.subtilis RIK1285.Subsequently,fermentation of the recombinant B.subtilis RIK1285/pBE-CGT was optimized.【Result】(1)Theα-CGTase gene was expressed in a fermentation medium to show an enzymatic activity of 2.9 U·mL~(-1) by B.subtilis RIK1285/pBE-CGT.(2)Medium TB with the formula of 0.5% glycerol,1.2% peptone,2.4% yeast extract,1.64% K_2HPO_4,and 0.23% KH_2PO_4 was found to be optimal for the fermentation.After fermentation in TB at37℃ for 24 h,theα-CGTase activity reached 5.3 U·mL~(-1),which was 8-fold of what the wild Gebacilliussp.CHB1 could generate.【Conclusion】The engineered B.subtilis RIK1285/pBE-CGT was successfully obtained and the fermentation process optimized.
引文
[1]CHEN J,WU J,LI Z F,et al.Gamma-cyclodextrin:a review on enzymatic production and applications[J].Appl Microbiol Biotechnol,2007,77(2):245-255.
[2]金征宇,柏玉香,王金鹏.环糊精葡萄糖基转移酶的筛选及其定向改造[J].食品与生物技术学报,2012,31(2):113-123.JIN Z Y,BAI Y X,WANG J P.Screen and Modification of Cyclodextrin Glycosyltransferase[J].Journal of Food Science and Biotechnology,2012,31(2):113-123.(in Chinese)
[3]JARUNEE K,WANIDA P,VICHIEN R,et al.Expression and characterization of a fusion protein-containing cyclodextrin glycosyltransferase from Paenibacillus sp.A11[J].Journal of Basic Microbiology,2010,50(5):427-435
[4]PENKA P,ALEXANDRA T,KALOYAN P.Sequence analysis,cloning and extracellular expression of cyclodextrin glucanotransferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in Escherichia coli[J].Process Biochemistry,2012,47(12):2139-2145.
[5]LIU H,LI J H,DU G C,et al.Enhanced production ofα-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization[J].Journal of Industrial Microbiology&Biotechnology,2012,39(12):1841-1849.
[6]NAM S W,PARK H Y,KIM J H,et al.Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Sacchoromyces cerevisiae[J].Biotechnol Lett,2001,23(9):727-730.
[7]张佳瑜,吴丹,李兆丰,等.来源于软化芽孢杆菌的环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达[J].生物工程学报,2009,25(12):1948-1954.ZHANG J Y,WU D,LI Z F,et al.Expression of Paenibacillus macerans cyclodextrin glycosyltransferase in Pichia pastoris and Bacillus subtilis[J].Chinese Journal of Biotechnology,2009,25(12):1948-1954.(in Chinese)
[8]WONG S L.Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins[J].Curr Opin Biotechnol,1995,6(5):517-522.
[9]LIDIA W,HELGA W,WIM J Q.Bacillus subtilis as cell factory for pharmaceutical proteins:a biotechnological approach to optimize the host organism[J].Biochim Biophys Acta,2004,1694(1-3):299-310.
[10]陈龙军,陈济琛,林晓栩,等.嗜热芽孢杆菌CHB1环糊精酶基因优化及其在毕赤酵母中的表达[J].食品与生物技术学报,2018,37(9):994-999.CHEN L J,CHEN J C,LIN X X,et al.Codon Optimization And Expression Of Cyclodextrin Glycosyltransferase From Gebacillius sp.CHB1In Pichia pastoris.[J].Journal of Food Science and Biotechnology,2018,37(9):994-999.(in Chinese)
[11]陈龙军,陈济琛,林新坚,等.环糊精酶基因在毕赤酵母中的组成型表达[J].福建农业学报,2017,32(1):82-86.CHEN L J,CHEN J C,LIN X J,et al.Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris[J].Fujian Journal of Agricultural Sciences,2017,32(1):82-86.(in Chinese)
[12]蔡海松,林晓栩,郭永华,等.信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响[J].微生物学通报,2017(3):601-610.CAI H S,LIN X X,GUO Y H,et al.Effects of different signal peptides and chemical penetrators on extracellular production of recombinant cyclodextrin glycosyltransferase[J].Microbiology China,2017(3):601-610.(in Chinese)
[13]LEJEUNE A,SAKAPUCHI K,IMANAKA T.ASpectrophotometric Assay for the Cyclization Activity of Cyclomaltohexaose(α-Cyclodextrin)Glucanotransferase[J].Analytical Biochemistry,1989,181:6-11.
[14]叶学军.地芽孢杆菌CHB1产CGTase的分离纯化及基因的克隆表达[D].福州:福州大学,2014.YE X J.Purification And Expression Of Cyclodextrin Glycosyltransferase From Gebacillius sp.CHB1[D].Fuzhou:Fuzhou University,2014.(in Chinese)
[2]金征宇,柏玉香,王金鹏.环糊精葡萄糖基转移酶的筛选及其定向改造[J].食品与生物技术学报,2012,31(2):113-123.JIN Z Y,BAI Y X,WANG J P.Screen and Modification of Cyclodextrin Glycosyltransferase[J].Journal of Food Science and Biotechnology,2012,31(2):113-123.(in Chinese)
[3]JARUNEE K,WANIDA P,VICHIEN R,et al.Expression and characterization of a fusion protein-containing cyclodextrin glycosyltransferase from Paenibacillus sp.A11[J].Journal of Basic Microbiology,2010,50(5):427-435
[4]PENKA P,ALEXANDRA T,KALOYAN P.Sequence analysis,cloning and extracellular expression of cyclodextrin glucanotransferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in Escherichia coli[J].Process Biochemistry,2012,47(12):2139-2145.
[5]LIU H,LI J H,DU G C,et al.Enhanced production ofα-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization[J].Journal of Industrial Microbiology&Biotechnology,2012,39(12):1841-1849.
[6]NAM S W,PARK H Y,KIM J H,et al.Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Sacchoromyces cerevisiae[J].Biotechnol Lett,2001,23(9):727-730.
[7]张佳瑜,吴丹,李兆丰,等.来源于软化芽孢杆菌的环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达[J].生物工程学报,2009,25(12):1948-1954.ZHANG J Y,WU D,LI Z F,et al.Expression of Paenibacillus macerans cyclodextrin glycosyltransferase in Pichia pastoris and Bacillus subtilis[J].Chinese Journal of Biotechnology,2009,25(12):1948-1954.(in Chinese)
[8]WONG S L.Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins[J].Curr Opin Biotechnol,1995,6(5):517-522.
[9]LIDIA W,HELGA W,WIM J Q.Bacillus subtilis as cell factory for pharmaceutical proteins:a biotechnological approach to optimize the host organism[J].Biochim Biophys Acta,2004,1694(1-3):299-310.
[10]陈龙军,陈济琛,林晓栩,等.嗜热芽孢杆菌CHB1环糊精酶基因优化及其在毕赤酵母中的表达[J].食品与生物技术学报,2018,37(9):994-999.CHEN L J,CHEN J C,LIN X X,et al.Codon Optimization And Expression Of Cyclodextrin Glycosyltransferase From Gebacillius sp.CHB1In Pichia pastoris.[J].Journal of Food Science and Biotechnology,2018,37(9):994-999.(in Chinese)
[11]陈龙军,陈济琛,林新坚,等.环糊精酶基因在毕赤酵母中的组成型表达[J].福建农业学报,2017,32(1):82-86.CHEN L J,CHEN J C,LIN X J,et al.Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris[J].Fujian Journal of Agricultural Sciences,2017,32(1):82-86.(in Chinese)
[12]蔡海松,林晓栩,郭永华,等.信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响[J].微生物学通报,2017(3):601-610.CAI H S,LIN X X,GUO Y H,et al.Effects of different signal peptides and chemical penetrators on extracellular production of recombinant cyclodextrin glycosyltransferase[J].Microbiology China,2017(3):601-610.(in Chinese)
[13]LEJEUNE A,SAKAPUCHI K,IMANAKA T.ASpectrophotometric Assay for the Cyclization Activity of Cyclomaltohexaose(α-Cyclodextrin)Glucanotransferase[J].Analytical Biochemistry,1989,181:6-11.
[14]叶学军.地芽孢杆菌CHB1产CGTase的分离纯化及基因的克隆表达[D].福州:福州大学,2014.YE X J.Purification And Expression Of Cyclodextrin Glycosyltransferase From Gebacillius sp.CHB1[D].Fuzhou:Fuzhou University,2014.(in Chinese)
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