一种来源于酒曲地衣芽孢杆菌溶菌酶基因在毕赤酵母中的诱导表达及活性分析
|
摘要
利用梯度稀释法和划线纯化法对酒曲中的微生物进行分离纯化,将所得微生物进行液体培养并检测其溶菌酶活性。通过分子生物学方法进行菌种鉴定,得到一株具有溶菌酶活性的地衣芽孢杆菌。提取地衣芽孢杆菌全基因组,设计特异性引物,克隆基因序列Lyz并连接至毕赤酵母分泌型表达载体pPICZαA中,构建重组表达载体pPICZαA-Lyz。重组载体经电转化整合入甲醇型毕赤酵母菌株X33基因组中进行重组表达。摇瓶发酵结果显示,甲醇诱导72 h后,发酵液中溶菌酶活性为1 360 U/m L。酶学性质分析结果显示,重组地衣芽孢杆菌溶菌酶在pH3.0~9.0范围内可以保持较高的相对活性,具有良好的热稳定性。抑菌试验结果显示地衣芽孢杆菌溶菌酶对革兰氏阴性菌有抑菌作用。
We used the gradient dilution and streak plate methods to isolate and purify microorganisms from fermented grains.The microorganisms were cultured in liquid medium and the lysozyme activity were detected. Through observation of the morphological characteristics and molecular biological characterization,the bacterial strain with lysozyme activity was identified as Bacillus licheniformis. After extracting the bacteria genome DNA and designing primer,the lysozyme gene Lyz was cloned and inserted into the inducible yeast expression vector pPICZαA,constructed the recombinant expression plasmid pPICZαA-Lyz and to be transformed into Pichia pastoris X33. Shake flask fermentation indicated that the activity of the lysozyme of multi-copy transformant reached 1 360 U/m L after cultivation in bufferedmethanol-complex medium(BMMY) for 72 h. The lysozyme was very stable in a broad pH range(3. 0-9. 0) and thermostable. The antimicrobial activity of lysozyme showed that it had antimicrobial activity against several gram-positive strains.
We used the gradient dilution and streak plate methods to isolate and purify microorganisms from fermented grains.The microorganisms were cultured in liquid medium and the lysozyme activity were detected. Through observation of the morphological characteristics and molecular biological characterization,the bacterial strain with lysozyme activity was identified as Bacillus licheniformis. After extracting the bacteria genome DNA and designing primer,the lysozyme gene Lyz was cloned and inserted into the inducible yeast expression vector pPICZαA,constructed the recombinant expression plasmid pPICZαA-Lyz and to be transformed into Pichia pastoris X33. Shake flask fermentation indicated that the activity of the lysozyme of multi-copy transformant reached 1 360 U/m L after cultivation in bufferedmethanol-complex medium(BMMY) for 72 h. The lysozyme was very stable in a broad pH range(3. 0-9. 0) and thermostable. The antimicrobial activity of lysozyme showed that it had antimicrobial activity against several gram-positive strains.
引文
[1]FLEMING A.On a remarkable bacteriolytic element found in tissues and secretions[J].Proc Royal Soc London,1922,93:306-317.
[2]SALTON M R J,GHUYSEN J M.Acetylhexosamine compounds enzymically released from Micrococcus lysodeikticus cell walls:III.the structure of di-and tetra-saccharides released from cell walls by lysozyme and Streptomyces F1 enzyme[J].Biochim Biophys Acta,1960,45:355-363.
[3]PADGETT T,HAN I Y,DAWSON P L.Incorporation of foodgrade antimicrobial compounds into biodegradable packaging films[J].J Food Prot,1998,61(10):1330-1335.
[4]PLSS-SUARD C,PANNATIER A,RUFFIEUX C,et al.Changes in the use of broad-spectrum antibiotics after cefepime shortage:a time series analysis[J].Antimicrob Agents Chemother,2012,56(2):989-994.
[5]SIM Y C,LEE S G,LEE D C,et al.Stabilization of papain and lysozyme for application to cosmetic products[J].Biotechnol Lett,2000,22(2):137-140.
[6]吴晓英,林影,陈慧英.溶菌酶的研究进展[J].工业微生物,2002,32(4):55-58.
[7]谷绒,车振明,万国福.溶菌酶在食品工业中的应用[J].乳业科学与技术,2006(6):264-266.
[8]赵龙飞,徐亚军.鸡蛋清中溶菌酶的应用性研究[J].食品工业,2006(3):19-20.
[9]CEGIELSKA-RADZIEJEWSKA R,LENIEROWSKI G,KIJOWSKI J.Properties and application of egg white lysozyme and its modified preparations:a review[J].Polish J Food Nutr Sci,2008,58(1):5-10.
[10]程光胜.中国酒曲微生物利用的发展现状[J].酿酒科技,2014(3):122-124.
[11]陈林.酱香型白酒发酵过程中微生物群落结构分析[D].北京:北京林业大学,2012.
[12]续丹丹,温赛,王凤寰,等.柞蚕溶菌酶真核表达系统的构建及重组蛋白纯化分析[J].饲料研究,2016(10):31-37.
[13]张双燕,廖永红,纪南,等.基于高通量测序技术分析北京清香型大曲微生物多样性[J].中国酿造,2016,35(11):49-53.
[14]赵爽,杨春霞,窦灿,等.白酒生产中酿酒微生物研究进展[J].中国酿造,2012,31(4):5-10.
[15]谭映月,胡萍,谢和.应用PCR-DGGE技术分析酱香型白酒酒曲细菌多样性[J].酿酒科技,2012(10):107-111.
[16]ZHOU X,YU Y,TAO J,et al.Production of LYZL6,a novel human c-type lysozyme,in recombinant Pichia pastoris,employing high cell density fed-batch fermentation[J].J Biosci Bioeng,2014,118(4):420-425.
[17]ZHU D,CAI G,WU D,et al.Comparison of two codon optimization strategies enhancing recombinant Sus scrofa lysozyme production in Pichia pastoris[J].Cell Mol Biol,2015,61(2):43-49.
[18]WANG T,XU Y,LIU W,et al.Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris[J].Protein Exp Purif,2011,77(1):20-25.
[19]WU D,WEN C G,HU B Q,et al.Expression analysis of i-type lysozyme gene from Cristaria plicata,and enzymatic activity analysis[J].Fish Shellfish Immunol,2013,34(6):1746-1747.
[20]BYRNE B.Pichia pastoris as an expression host for membrane protein structural biology[J].Curr Opin Struct Biol,2015,32:9-17.
[21]IBRAHIM H R,HIGASHIGUCHI S,JUNEJA L R,et al.A structural phase of heat-denatured lysozyme with novel antimicrobial action[J].J Agric Food Chem,1996,44(6):1416-1423.
[22]DESANTIS G,JONES J B.Chemical modification of enzymes for enhanced functionality[J].Curr Opin Biotechnol,1999,10(4):324-330.
[23]IBRAHIM H R,KATO A,KOBAYASHI K.Antimicrobial effects of lysozyme against gram-negative bacteria due to covalent binding of palmitic acid[J].J Agric Food Chem,1991,39(11):2077-2082.
[24]ELLISON R T 3rd,GIEHL T J.Killing of gram-negative bacteria by lactoferrin and lysozyme[J].J Clin Invest,1991,88(4):1080-1091.
[25]KUMARESAN V,BHATT P,GANESH M R,et al.A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica[J].Mol Immunol,2015,68(2):421-433.
[26]ZHAO H,TANG J,CAO L,et al.Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system[J].Enzyme Microb Technol,2015,77(3):61-67.
[27]YANG D,WANG Q,CAO R,et al.Molecular characterization,expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum[J].Dev Compar Immunol,2017,73:109-118.
[28]ZHU D,CAI G,LI X,et al.Enhancing the antimicrobial activity of Sus scrofa lysozyme by N-terminal fusion of a sextuple unique homologous peptide[J].J Biotechnol,2017,243:61-68.
[2]SALTON M R J,GHUYSEN J M.Acetylhexosamine compounds enzymically released from Micrococcus lysodeikticus cell walls:III.the structure of di-and tetra-saccharides released from cell walls by lysozyme and Streptomyces F1 enzyme[J].Biochim Biophys Acta,1960,45:355-363.
[3]PADGETT T,HAN I Y,DAWSON P L.Incorporation of foodgrade antimicrobial compounds into biodegradable packaging films[J].J Food Prot,1998,61(10):1330-1335.
[4]PLSS-SUARD C,PANNATIER A,RUFFIEUX C,et al.Changes in the use of broad-spectrum antibiotics after cefepime shortage:a time series analysis[J].Antimicrob Agents Chemother,2012,56(2):989-994.
[5]SIM Y C,LEE S G,LEE D C,et al.Stabilization of papain and lysozyme for application to cosmetic products[J].Biotechnol Lett,2000,22(2):137-140.
[6]吴晓英,林影,陈慧英.溶菌酶的研究进展[J].工业微生物,2002,32(4):55-58.
[7]谷绒,车振明,万国福.溶菌酶在食品工业中的应用[J].乳业科学与技术,2006(6):264-266.
[8]赵龙飞,徐亚军.鸡蛋清中溶菌酶的应用性研究[J].食品工业,2006(3):19-20.
[9]CEGIELSKA-RADZIEJEWSKA R,LENIEROWSKI G,KIJOWSKI J.Properties and application of egg white lysozyme and its modified preparations:a review[J].Polish J Food Nutr Sci,2008,58(1):5-10.
[10]程光胜.中国酒曲微生物利用的发展现状[J].酿酒科技,2014(3):122-124.
[11]陈林.酱香型白酒发酵过程中微生物群落结构分析[D].北京:北京林业大学,2012.
[12]续丹丹,温赛,王凤寰,等.柞蚕溶菌酶真核表达系统的构建及重组蛋白纯化分析[J].饲料研究,2016(10):31-37.
[13]张双燕,廖永红,纪南,等.基于高通量测序技术分析北京清香型大曲微生物多样性[J].中国酿造,2016,35(11):49-53.
[14]赵爽,杨春霞,窦灿,等.白酒生产中酿酒微生物研究进展[J].中国酿造,2012,31(4):5-10.
[15]谭映月,胡萍,谢和.应用PCR-DGGE技术分析酱香型白酒酒曲细菌多样性[J].酿酒科技,2012(10):107-111.
[16]ZHOU X,YU Y,TAO J,et al.Production of LYZL6,a novel human c-type lysozyme,in recombinant Pichia pastoris,employing high cell density fed-batch fermentation[J].J Biosci Bioeng,2014,118(4):420-425.
[17]ZHU D,CAI G,WU D,et al.Comparison of two codon optimization strategies enhancing recombinant Sus scrofa lysozyme production in Pichia pastoris[J].Cell Mol Biol,2015,61(2):43-49.
[18]WANG T,XU Y,LIU W,et al.Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris[J].Protein Exp Purif,2011,77(1):20-25.
[19]WU D,WEN C G,HU B Q,et al.Expression analysis of i-type lysozyme gene from Cristaria plicata,and enzymatic activity analysis[J].Fish Shellfish Immunol,2013,34(6):1746-1747.
[20]BYRNE B.Pichia pastoris as an expression host for membrane protein structural biology[J].Curr Opin Struct Biol,2015,32:9-17.
[21]IBRAHIM H R,HIGASHIGUCHI S,JUNEJA L R,et al.A structural phase of heat-denatured lysozyme with novel antimicrobial action[J].J Agric Food Chem,1996,44(6):1416-1423.
[22]DESANTIS G,JONES J B.Chemical modification of enzymes for enhanced functionality[J].Curr Opin Biotechnol,1999,10(4):324-330.
[23]IBRAHIM H R,KATO A,KOBAYASHI K.Antimicrobial effects of lysozyme against gram-negative bacteria due to covalent binding of palmitic acid[J].J Agric Food Chem,1991,39(11):2077-2082.
[24]ELLISON R T 3rd,GIEHL T J.Killing of gram-negative bacteria by lactoferrin and lysozyme[J].J Clin Invest,1991,88(4):1080-1091.
[25]KUMARESAN V,BHATT P,GANESH M R,et al.A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica[J].Mol Immunol,2015,68(2):421-433.
[26]ZHAO H,TANG J,CAO L,et al.Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system[J].Enzyme Microb Technol,2015,77(3):61-67.
[27]YANG D,WANG Q,CAO R,et al.Molecular characterization,expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum[J].Dev Compar Immunol,2017,73:109-118.
[28]ZHU D,CAI G,LI X,et al.Enhancing the antimicrobial activity of Sus scrofa lysozyme by N-terminal fusion of a sextuple unique homologous peptide[J].J Biotechnol,2017,243:61-68.