南极假丝酵母脂肪酶B在黑曲霉中的分泌表达及其硅藻土固定化应用
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摘要
为提高南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)表达量,根据黑曲霉(Aspergillus niger)密码子偏好性设计合成CALB基因,以PnaII/tpi为启动子构建CALB表达载体并转化到黑曲霉HL-1中表达,摇瓶发酵120 h后上清液中重组CALB活力为171 U/mL。研究重组CALB的酶学性质,最适反应温度和pH值分别为50℃和8.0,在pH 6.0～9.0和45℃以下具有较高的稳定性,10 mmol/L Cu~(2+)、Zn~(2+)和0.1 g/100 mL十二烷基硫酸钠强烈抑制酶活力,而1 mmol/L Ca~(2+)、0.1 g/100 mL山梨醇对酶活力具有非常明显的激活作用。此外,以硅藻土为载体固定CALB,固定化酶活力为187 U/g。将制备的硅藻土-CALB固定化酶用于生物合成己酸乙酯,经反应条件优化后在无溶剂体系中己酸乙酯产率达91%。
In order to increase the expression level of Candida antarctica lipase B(CALB), the CALB gene was designed and synthesized according to the codon preference of Aspergillus niger, and then the CALB expression vector was constructed with Pna II/tpi as promoter and transformed into A. niger HL-1. The enzymatic activity of recombinant CALB was 171 U/m L after shaking flask fermentation for 120 h. Then, the enzymatic properties of recombinant CALB were studied. The optimum reaction temperature and pH for the enzyme were 50 ℃ and 8.0, respectively. The enzyme was very stable at pH 6.0–9.0 and temperature below 45 ℃. Its activity was strongly inhibited by 10 mmol/L Cu~(2+), Zn~(2+) and 0.1 g/100 mL SDS but was potently activated by 1 mmol/L Ca~(2+) and 0.1 g/100 mL sorbitol. Furthermore, the CALB was immobilized using diatomite as the carrier, and the activity of immobilized enzyme was 187 U/g under the optimal immobilization conditions. The yield of ethyl hexanoate in a solventless system was up to 91% when biosynthesized using immobilized CALB under the optimized reaction conditions.
In order to increase the expression level of Candida antarctica lipase B(CALB), the CALB gene was designed and synthesized according to the codon preference of Aspergillus niger, and then the CALB expression vector was constructed with Pna II/tpi as promoter and transformed into A. niger HL-1. The enzymatic activity of recombinant CALB was 171 U/m L after shaking flask fermentation for 120 h. Then, the enzymatic properties of recombinant CALB were studied. The optimum reaction temperature and pH for the enzyme were 50 ℃ and 8.0, respectively. The enzyme was very stable at pH 6.0–9.0 and temperature below 45 ℃. Its activity was strongly inhibited by 10 mmol/L Cu~(2+), Zn~(2+) and 0.1 g/100 mL SDS but was potently activated by 1 mmol/L Ca~(2+) and 0.1 g/100 mL sorbitol. Furthermore, the CALB was immobilized using diatomite as the carrier, and the activity of immobilized enzyme was 187 U/g under the optimal immobilization conditions. The yield of ethyl hexanoate in a solventless system was up to 91% when biosynthesized using immobilized CALB under the optimized reaction conditions.
引文
[1]LIU Z Q,ZHENG X B,ZHANG S P,et al.Cloning,expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters[J].Microbiological Research,2012,167(8):452-460.DOI:10.1016/j.micres.2011.12.004.
[2]SMITH W P.Hydroxy acids and skin aging[J].Cosmet Toilet,1994,109:41-48.DOI:10.1159/000029808.
[3]任春梅,周玲妹,柳志强.南极假丝酵母脂肪酶B的克隆,表达及发酵条件优化[J].发酵科技通讯,2014,43(3):6-10.DOI:10.16774/j.cnki.issn.1674-2214.2014.03.008.
[4]SVENDSEN A,PATHAR S A,HANSEN M T,et al.C.antarctica lipase variants:U.S.Patent 6,074,863[P].2000-06-13.
[5]UPPENBERG J,HANSEN M T,PATKAR S,et al.The sequence,crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica[J].Structure,1994,2(4):293-308.DOI:10.1016/S0969-2126(00)0003 1-9.
[6]李迅,邓若冰,王飞.南极假丝酵母脂肪酶B在毕赤酵母中的分泌型表达及酶学性质初探[J].四川师范大学学报(自然科学版),2014(6):91 1-916.DOI:10.3969/j.issn.1001-8395.2014.06.025.
[7]JUN C,JEON B W,JOO J C,et al.Thermostabilization of Candida antarctica lipase B by double immobilization:adsorption on a macroporous polyacrylate carrier and R,silaffin-mediated biosilicification[J].Process Biochemistry,2013,48(8):1 181-1 187.DOI:10.1016/j.procbio.2013.06.0 10.
[8]MONTEIRO C M,LOURENCO N M T,AFONSO C A M.Separation of secondary alcohols via enzymatic kinetic resolution using fatty esters as reusable acylating agents[J].Tetrahedron:Asymmetry,2010,21(8):952-956.DOI:10.10 1 6/j.tetasy.2010.05.024.
[9]BARBOSA O,ORTIZ C,TORRES R,et al.Effect of the immobilization protocol on the properties of lipase B from Candida antarctica in organic media:enantiospecifc production of atenolol acetate[J].Journal of Molecular Catalysis B:Enzymatic,201 1,71(3/4):124-132.DOI:10.1016/j.molcatb.201 1.04.008.
[10]HOEGH I,PATKAR S,HALKIER T,et al.Two lipases from Candida antarctica:cloning and expression in Aspergillus oryzae[J].Canadian Journal of Botany,1995,73(S1):869-875.DOI:10.1 139/b95-333.
[11]LIU D,SCHMID R D,RUSNAK M.Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm:a screeningsystem for a frequently used biocatalyst[J].Applied Microbiology and Biotechnology,2006,72(5):1024-1032.DOI:10.1007/s00253-006-0369-7.
[12]HAN S Y,PAN Z Y,HUANG D F,et al.Highly efficient synthesis of ethyl hexanoate catalyzed by CALB-displaying Saccharomyces cerevisiae whole-cells in non-aqueous phase[J].Journal of Molecular Catalysis B:Enzymatic,2009,59(1/2/3):168-172.DOI:10.1016/j.molcatb.2009.02.007.
[13]孙金鹏,钱圣一,敬科举,等.南极假丝酵母脂肪酶B在毕赤酵母的表达及酶学性质研究[J].厦门大学学报(自然科学版),2011,50(4):765-771.
[14]BHARDWAJ K K,GUPTA R.Synthesis of chirally pure enantiomers by lipase[J].Journal of Oleo Science,2017,66(10):1073-1084.DOI:10.5650/jos.ess17114.
[15]ROBERT J M,LATTARI F S,MACHADO A C,et al.Production of recombinant lipase B from Candida antarctica in Pichia pastoris under control of the promoter PGK using crude glycerol from biodiesel production as carbon source[J].Biochemical Engineering Journal,2017,1 18:123-131.DOI:10.1016/j.bej.2016.1 1.018.
[16]KATO M,FUCHIMOTO J,TANINO T,et al.Preparation of a wholecell biocatalyst of mutated Candida antarctica lipase B(mCALB)by a yeast molecular display system and its practical properties[J].Applied Microbiology and Biotechnology,2007,75(3):549-555.DOI:10.1007/S00253-006-0835-2.
[17]LIU F,WANG B,YE Y,et al.High level expression and characterization of tannase tan7 using Aspergillus niger SH-2 with low-background endogenous secretory proteins as the host[J].Protein Expression and Purification,2018,144:71-75.DOI:10.1016/j.pep.2017.1 1.003.
[18]李远锋,靳珅,王登刚,等.黑曲霉组成型表面展示南极假丝酵母脂肪酶B及其发酵调控[J].生物工程学报,2018,34(7):1189-1196.DOI:10.13345/j.cjb.170477.
[19]SARKARI P,MARX H,BLUMHOFF M L,et al.An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger[J].Bioresource Technology,2017,245:1327-1333.DOI:1 0.10 16/j.biortech.20 17.05.004.
[20]潘志友,韩双艳,林影,等.南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成[J].生物工程学报,2008,24(4):673-678.DOI:10.13345/j.cjb.2008.04.014.
[21]XU D G,XIAO L S,CAI Y,et al.Research of lipase-catalyzed synthesis of ethyl hexanoate in nonaqueous conditions[J].Fine Chemicals,2004,21(4):279-281.DOI:10.13550/j.jxhg.2004.04.010.
[22]刘媛媛,郑永杰,田景芝.改性硅藻土对脂肪酶固定化研究[J].化学工程师,2014(3):14-17.DOI:10.16247/j.cnki.23-1171/tq.2014.03.001.
[23]PAN Z Y,YANG Z M,PAN L,et al.Displaying Candida antarctica lipase B on the cell surface of Aspergillus niger as a potential foodgrade whole-cell catalyst[J].Journal of Industrial Microbiology&Biotechnology,2014,41(4):711-720.DOI:10.1007/s 10295-014-1410-y.
[24]章素平,柳志强,郑裕国.南极假丝酵母脂肪酶B的修饰与改造[J].基因组学与应用生物学,2011,30(2):243-250.DOI:10.3969/gab.030.000243.
[25]张熙.黑曲霉表面展示CALB的发酵条件研究及其应用[D].广州:华南理工大学,2016.
[26]CAVALLARO V,ERCOLI D R,TONETTO G M,et al.Simple and economical CALB/polyethylene/aluminum biocatalyst for fatty acid esterification[J].Polymers for Advanced Technologies,2018,29(2):1002-1006.DOI:10.1002/pat.4189.
[27]YEH Y R,TZENG Y J,TSAI S W.Quantitative improvements and insights into CALB-catalyzed resolution of trans-and cis-2-phenylcyclopropyl azolides[J].ChemistrySelect,2018,3(19):5353-5360.DOI:10.1002/slct.201800578.
[28]LOOSER V,LUTHY D,STRAUMANN M,et al.Effects of glycerol supply and specific growth rate on methanol-free production of CAL1B by P.pastoris:functional characterisation of a novel promoter[J].Applied Microbiology and Biotechnology,2017,101(8):3163-3176.DOI:10.1007/s00253-017-8123-x.
[29]JAYACHANDRAN C,ATHIYAMAN B P,SANKARANARAYANAN M.Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase[J].PLoS ONE,2017,12(7):e0181370.DOI:10.1371/journal.pone.018 1370.
[2]SMITH W P.Hydroxy acids and skin aging[J].Cosmet Toilet,1994,109:41-48.DOI:10.1159/000029808.
[3]任春梅,周玲妹,柳志强.南极假丝酵母脂肪酶B的克隆,表达及发酵条件优化[J].发酵科技通讯,2014,43(3):6-10.DOI:10.16774/j.cnki.issn.1674-2214.2014.03.008.
[4]SVENDSEN A,PATHAR S A,HANSEN M T,et al.C.antarctica lipase variants:U.S.Patent 6,074,863[P].2000-06-13.
[5]UPPENBERG J,HANSEN M T,PATKAR S,et al.The sequence,crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica[J].Structure,1994,2(4):293-308.DOI:10.1016/S0969-2126(00)0003 1-9.
[6]李迅,邓若冰,王飞.南极假丝酵母脂肪酶B在毕赤酵母中的分泌型表达及酶学性质初探[J].四川师范大学学报(自然科学版),2014(6):91 1-916.DOI:10.3969/j.issn.1001-8395.2014.06.025.
[7]JUN C,JEON B W,JOO J C,et al.Thermostabilization of Candida antarctica lipase B by double immobilization:adsorption on a macroporous polyacrylate carrier and R,silaffin-mediated biosilicification[J].Process Biochemistry,2013,48(8):1 181-1 187.DOI:10.1016/j.procbio.2013.06.0 10.
[8]MONTEIRO C M,LOURENCO N M T,AFONSO C A M.Separation of secondary alcohols via enzymatic kinetic resolution using fatty esters as reusable acylating agents[J].Tetrahedron:Asymmetry,2010,21(8):952-956.DOI:10.10 1 6/j.tetasy.2010.05.024.
[9]BARBOSA O,ORTIZ C,TORRES R,et al.Effect of the immobilization protocol on the properties of lipase B from Candida antarctica in organic media:enantiospecifc production of atenolol acetate[J].Journal of Molecular Catalysis B:Enzymatic,201 1,71(3/4):124-132.DOI:10.1016/j.molcatb.201 1.04.008.
[10]HOEGH I,PATKAR S,HALKIER T,et al.Two lipases from Candida antarctica:cloning and expression in Aspergillus oryzae[J].Canadian Journal of Botany,1995,73(S1):869-875.DOI:10.1 139/b95-333.
[11]LIU D,SCHMID R D,RUSNAK M.Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm:a screeningsystem for a frequently used biocatalyst[J].Applied Microbiology and Biotechnology,2006,72(5):1024-1032.DOI:10.1007/s00253-006-0369-7.
[12]HAN S Y,PAN Z Y,HUANG D F,et al.Highly efficient synthesis of ethyl hexanoate catalyzed by CALB-displaying Saccharomyces cerevisiae whole-cells in non-aqueous phase[J].Journal of Molecular Catalysis B:Enzymatic,2009,59(1/2/3):168-172.DOI:10.1016/j.molcatb.2009.02.007.
[13]孙金鹏,钱圣一,敬科举,等.南极假丝酵母脂肪酶B在毕赤酵母的表达及酶学性质研究[J].厦门大学学报(自然科学版),2011,50(4):765-771.
[14]BHARDWAJ K K,GUPTA R.Synthesis of chirally pure enantiomers by lipase[J].Journal of Oleo Science,2017,66(10):1073-1084.DOI:10.5650/jos.ess17114.
[15]ROBERT J M,LATTARI F S,MACHADO A C,et al.Production of recombinant lipase B from Candida antarctica in Pichia pastoris under control of the promoter PGK using crude glycerol from biodiesel production as carbon source[J].Biochemical Engineering Journal,2017,1 18:123-131.DOI:10.1016/j.bej.2016.1 1.018.
[16]KATO M,FUCHIMOTO J,TANINO T,et al.Preparation of a wholecell biocatalyst of mutated Candida antarctica lipase B(mCALB)by a yeast molecular display system and its practical properties[J].Applied Microbiology and Biotechnology,2007,75(3):549-555.DOI:10.1007/S00253-006-0835-2.
[17]LIU F,WANG B,YE Y,et al.High level expression and characterization of tannase tan7 using Aspergillus niger SH-2 with low-background endogenous secretory proteins as the host[J].Protein Expression and Purification,2018,144:71-75.DOI:10.1016/j.pep.2017.1 1.003.
[18]李远锋,靳珅,王登刚,等.黑曲霉组成型表面展示南极假丝酵母脂肪酶B及其发酵调控[J].生物工程学报,2018,34(7):1189-1196.DOI:10.13345/j.cjb.170477.
[19]SARKARI P,MARX H,BLUMHOFF M L,et al.An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger[J].Bioresource Technology,2017,245:1327-1333.DOI:1 0.10 16/j.biortech.20 17.05.004.
[20]潘志友,韩双艳,林影,等.南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成[J].生物工程学报,2008,24(4):673-678.DOI:10.13345/j.cjb.2008.04.014.
[21]XU D G,XIAO L S,CAI Y,et al.Research of lipase-catalyzed synthesis of ethyl hexanoate in nonaqueous conditions[J].Fine Chemicals,2004,21(4):279-281.DOI:10.13550/j.jxhg.2004.04.010.
[22]刘媛媛,郑永杰,田景芝.改性硅藻土对脂肪酶固定化研究[J].化学工程师,2014(3):14-17.DOI:10.16247/j.cnki.23-1171/tq.2014.03.001.
[23]PAN Z Y,YANG Z M,PAN L,et al.Displaying Candida antarctica lipase B on the cell surface of Aspergillus niger as a potential foodgrade whole-cell catalyst[J].Journal of Industrial Microbiology&Biotechnology,2014,41(4):711-720.DOI:10.1007/s 10295-014-1410-y.
[24]章素平,柳志强,郑裕国.南极假丝酵母脂肪酶B的修饰与改造[J].基因组学与应用生物学,2011,30(2):243-250.DOI:10.3969/gab.030.000243.
[25]张熙.黑曲霉表面展示CALB的发酵条件研究及其应用[D].广州:华南理工大学,2016.
[26]CAVALLARO V,ERCOLI D R,TONETTO G M,et al.Simple and economical CALB/polyethylene/aluminum biocatalyst for fatty acid esterification[J].Polymers for Advanced Technologies,2018,29(2):1002-1006.DOI:10.1002/pat.4189.
[27]YEH Y R,TZENG Y J,TSAI S W.Quantitative improvements and insights into CALB-catalyzed resolution of trans-and cis-2-phenylcyclopropyl azolides[J].ChemistrySelect,2018,3(19):5353-5360.DOI:10.1002/slct.201800578.
[28]LOOSER V,LUTHY D,STRAUMANN M,et al.Effects of glycerol supply and specific growth rate on methanol-free production of CAL1B by P.pastoris:functional characterisation of a novel promoter[J].Applied Microbiology and Biotechnology,2017,101(8):3163-3176.DOI:10.1007/s00253-017-8123-x.
[29]JAYACHANDRAN C,ATHIYAMAN B P,SANKARANARAYANAN M.Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase[J].PLoS ONE,2017,12(7):e0181370.DOI:10.1371/journal.pone.018 1370.