一种酸性木聚糖酶在毕赤酵母中的分泌表达及体外活性评价研究
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摘要
通过构建硫色曲霉酸性木聚糖酶基因xynA串联双拷贝工程菌,提高酶的发酵活力。10 L发酵罐中,双拷贝菌株发酵活力达3 574 U/mL。重组木聚糖酶的最适催化温度为50℃,最适反应pH为2.2～3.4,在pH 1.7的酸性缓冲液下保持100 min,酶活残留70%以上。重组木聚糖酶对胃蛋白酶和胰蛋白酶也具有较好的耐受性。该木聚糖酶在低pH条件下的高催化活性、耐酸性及蛋白酶耐受性,使之具有较好的应用前景。通过体外模拟猪胃肠道环境以快速评价重组酶的作用效果。试验以小麦型日粮作为基础日粮,设计4个处理组,重组酶的添加量分别为0 U/kg、2 000 U/kg、4 000 U/kg和6 000 U/kg,每个处理5个重复,使用SDS-Ⅱ单胃动物仿生消化系统模拟猪的胃肠道环境消化试验日粮,消化后的残渣烘干后测定其总能、粗蛋白质、中性洗涤纤维含量以计算其消化率。结果表明,添加重组酸性木聚糖酶显著提高了总能、粗蛋白质(线性和二次;P<0.01)和中性洗涤纤维的消化率(线性和二次;P<0.05)。重组酶的最适宜添加量为4 000 U/kg。
In this study,engineering Pichia pastoris containing double copies of Aspergillus sulphureus xylanase gene xynA was constructed in order to earn a high expression level and low production cost.In a 10 L fermentor,doublecopy recombinant strain yielded the enzyme activity of 3 574 U / mL.The recombinant xylanase exhibited optimal activity at 50℃ and pH 2.2 ～ 3.4.Residual activity of the raw recombinant xylanase was over 70%,when fermentation broth was pretreated with Na 2 HPO 4-citric acid buffer of pH 1.7 for 100 min.And the recombinant xylanase exhibited strong resistance to pepsin and trypsin.High level of catalytic activity in low pH and acid resistance suggested that the recombinant xylanase has a prospective application in feed industry as an additive.Then the recombinant enzyme's action effect was rapidly evaluated by in vitro simulating swine gastrointestinal environment.The experiment was conducted to determine the optimal supplementation level of recombinant enzyme in wheat-based diet preliminarily.In this experiment,wheat-based diet was divided into 4 treats containing 0,2 000 U / kg,4 000 U / kg or 6 000 U / kg recombinant enzyme,with 5 repetitions per treat.Then SDS-Ⅱ monogastric animal bionic digestive system was used to digest experimental diet by imitating gastrointestinal tract environment of swine.Increasing the level of recombinant acidic β-1,4-xylanase in the diet increased the apparent digestibility of gross energy(GE),crude protein(CP)(linear and quadratic effect;P < 0.01) and neutral detergent fibre(NDF)(linear and quadratic effect;P <0.05).The optimal supplementation level of recombinant enzyme was determined to be 4 000 U / kg.
In this study,engineering Pichia pastoris containing double copies of Aspergillus sulphureus xylanase gene xynA was constructed in order to earn a high expression level and low production cost.In a 10 L fermentor,doublecopy recombinant strain yielded the enzyme activity of 3 574 U / mL.The recombinant xylanase exhibited optimal activity at 50℃ and pH 2.2 ～ 3.4.Residual activity of the raw recombinant xylanase was over 70%,when fermentation broth was pretreated with Na 2 HPO 4-citric acid buffer of pH 1.7 for 100 min.And the recombinant xylanase exhibited strong resistance to pepsin and trypsin.High level of catalytic activity in low pH and acid resistance suggested that the recombinant xylanase has a prospective application in feed industry as an additive.Then the recombinant enzyme's action effect was rapidly evaluated by in vitro simulating swine gastrointestinal environment.The experiment was conducted to determine the optimal supplementation level of recombinant enzyme in wheat-based diet preliminarily.In this experiment,wheat-based diet was divided into 4 treats containing 0,2 000 U / kg,4 000 U / kg or 6 000 U / kg recombinant enzyme,with 5 repetitions per treat.Then SDS-Ⅱ monogastric animal bionic digestive system was used to digest experimental diet by imitating gastrointestinal tract environment of swine.Increasing the level of recombinant acidic β-1,4-xylanase in the diet increased the apparent digestibility of gross energy(GE),crude protein(CP)(linear and quadratic effect;P < 0.01) and neutral detergent fibre(NDF)(linear and quadratic effect;P <0.05).The optimal supplementation level of recombinant enzyme was determined to be 4 000 U / kg.
引文
[1]国春艳,刁其玉,乔宇,等.酸性木聚糖酶产生菌株的筛选和酶学性质分析[J].中国农业科学,2010,43(7):1524-1530.Guo C Y,Diao Q Y,Qiao Y,et al..Isolation of microbialstrains producing acidic xylanase and characterization of theenzyme[J].Sci.Agric.Sin.,2010,43(7):1524-1530.
[2]李春华,李翔,马立新.酸性木聚糖酶基因的克隆及其在毕赤酵母中的分泌表达[J].微生物学通报,2005,32(6):89-95.Li C H,Li X,Ma L X.Cloning of acidic xylanase gene and itssecretion expression in Pichia pastoris[J].Microbiol.China,2005,32(6):89-95.
[3]Annison G.Polysaccharide composition of Australian wheatsand the digestibility of their starches in broiler chicken diets[J].Aust.J.Exp.Agric.,1990,30:183-186.
[4]石宁,刘卫国,李绍钰.木聚糖酶的研究与应用[J].饲料研究,2009,11:10-12.Shi N,Liu W G,Li S Y.Research and application of xylanase[J].Feed Res.,2009,11:10-12.
[5]曹钰,陆健,李胤.酸性木聚糖酶的研究进展[J].工业微生物,2005,35(4):41-44.Cao Y,Lu J,Li Y.Overview of acidic xylanase[J].Ind.Microbiol.,2005,35(4):41-44.
[6]Cao Y H,Qiao J Y,Li Y H,et al..De novo synthesis,constitutive expression of Aspergillus sulphureusβ-xylanase genein Pichia pastoris and partialenzymic characterization[J].Appl.Microbiol.Biotechnol.,2007,76:579-585.
[7]全国饲料工业标准化技术委员会.GB/T 6432-1994饲料中粗蛋白测定方法[S].北京:中国标准出版社,1994.
[8]中国农业科学院畜牧研究所,浙江大学,内蒙古畜牧科学院.GB/T 20806-2006饲料中中性洗涤纤维(NDF)的测定[S].北京:中国标准出版社,2006.
[9]ISO 9831-1998动物饲料,动物产品和粪便或尿液-总热值的测定-弹式量热计法[S].中国标准化研究院,1998.
[10]Romanos M.Advances in the use of Pichia pastoris for high-level gene expression[J].Curr.Opin.Biotechnol.,1995,6:527-533.
[11]Cregg J,Tschopp J,Stillman C,et al..High-level expressionand efficient assembly of hepatitis B surface antigen inmethylotrophic yeast,Pichia pastoris[J].Biotechnology,1987,5:479-485.
[12]Thill G,Davis G,Stillman C,et al..Positive and negativeeffects of multicopy integrated expression vectors on proteinexpression in Pichia pastoris[A].In:Proceedings of the sixthInternational Symposium on Genetics of IndustrialMicroorganisms[C].France,1990,477-490.
[13]Luo H Y,Wang Y R,Li J,et al..Cloning,expression andcharacterization of a novel acidic xylanase,XYL11B,from theacidophilic fungus Bispora sp.MEY-1[J].Enzyme Microbiol.Technol.,2009,45(2):126-133.
[14]Liu Z,Zhao X Q,Bai F W.Production of xylanase by analkaline-tolerant marine-derived Streptomyces viridochromogenesstrain and improvement by ribosome engineering[J].Appl.Microbiol.Biotechnol.,2013,97:4361-4368.
[15]Jiang X B,Lin J C,Liang S Z,et al..High-efficientexpression and pilot scale fermentation of Streptomyces xylanasefrom a constitutive Pichia pastoris vector[J].FoodBiotechnol.,2013,27:54-65.
[16]Do T T,Quyen D T,Dam T H.Purification andcharacterization of an acid-stable and organic solvent-tolerantxylanase from Aspergillus awamori VTCC-F312[J].Sci.Asia,2012,38:157-165.
[17]Mavromichalis I,Hancock J D,Senne B W,et al..Enzymesupplementation and particle size of wheat in diets for nurseryand finishing pigs[J].J.Animal Sci.,2000,78(12):3086-3095.
[18]Lunen T A V,Schulze H.Influence of Trichodermalongibrachiatum xylanase supplementation of wheat and cornbased diets on growth performance of pigs[J].Can.J.AnimalSci.,1996,76(2):271-273.
[19]Barrera M,Cervantes M,Sauer W C,et al..Ileal amino aciddigestibility and performance of growing pigs fed wheat-baseddiets supplemented with xylanase[J].J.Animal Sci.,2004,82(7):1997-2003.
[20]Thacker P A,Campbell G L,Groot W J.The effect of enzymesupplementation on the nutritive value of rye-based diets forswine[J].Can.J.Animal Sci.,1991,71(2):489-496.
[21]Omogbenigun F O,Nyachoti C M,Slominski B A.Dietarysupplementation with multienzyme preparations improvesnutrient utilization and growth performance in weaned pigs[J].J.Animal Sci.,2004,82(4):1053-1061
[2]李春华,李翔,马立新.酸性木聚糖酶基因的克隆及其在毕赤酵母中的分泌表达[J].微生物学通报,2005,32(6):89-95.Li C H,Li X,Ma L X.Cloning of acidic xylanase gene and itssecretion expression in Pichia pastoris[J].Microbiol.China,2005,32(6):89-95.
[3]Annison G.Polysaccharide composition of Australian wheatsand the digestibility of their starches in broiler chicken diets[J].Aust.J.Exp.Agric.,1990,30:183-186.
[4]石宁,刘卫国,李绍钰.木聚糖酶的研究与应用[J].饲料研究,2009,11:10-12.Shi N,Liu W G,Li S Y.Research and application of xylanase[J].Feed Res.,2009,11:10-12.
[5]曹钰,陆健,李胤.酸性木聚糖酶的研究进展[J].工业微生物,2005,35(4):41-44.Cao Y,Lu J,Li Y.Overview of acidic xylanase[J].Ind.Microbiol.,2005,35(4):41-44.
[6]Cao Y H,Qiao J Y,Li Y H,et al..De novo synthesis,constitutive expression of Aspergillus sulphureusβ-xylanase genein Pichia pastoris and partialenzymic characterization[J].Appl.Microbiol.Biotechnol.,2007,76:579-585.
[7]全国饲料工业标准化技术委员会.GB/T 6432-1994饲料中粗蛋白测定方法[S].北京:中国标准出版社,1994.
[8]中国农业科学院畜牧研究所,浙江大学,内蒙古畜牧科学院.GB/T 20806-2006饲料中中性洗涤纤维(NDF)的测定[S].北京:中国标准出版社,2006.
[9]ISO 9831-1998动物饲料,动物产品和粪便或尿液-总热值的测定-弹式量热计法[S].中国标准化研究院,1998.
[10]Romanos M.Advances in the use of Pichia pastoris for high-level gene expression[J].Curr.Opin.Biotechnol.,1995,6:527-533.
[11]Cregg J,Tschopp J,Stillman C,et al..High-level expressionand efficient assembly of hepatitis B surface antigen inmethylotrophic yeast,Pichia pastoris[J].Biotechnology,1987,5:479-485.
[12]Thill G,Davis G,Stillman C,et al..Positive and negativeeffects of multicopy integrated expression vectors on proteinexpression in Pichia pastoris[A].In:Proceedings of the sixthInternational Symposium on Genetics of IndustrialMicroorganisms[C].France,1990,477-490.
[13]Luo H Y,Wang Y R,Li J,et al..Cloning,expression andcharacterization of a novel acidic xylanase,XYL11B,from theacidophilic fungus Bispora sp.MEY-1[J].Enzyme Microbiol.Technol.,2009,45(2):126-133.
[14]Liu Z,Zhao X Q,Bai F W.Production of xylanase by analkaline-tolerant marine-derived Streptomyces viridochromogenesstrain and improvement by ribosome engineering[J].Appl.Microbiol.Biotechnol.,2013,97:4361-4368.
[15]Jiang X B,Lin J C,Liang S Z,et al..High-efficientexpression and pilot scale fermentation of Streptomyces xylanasefrom a constitutive Pichia pastoris vector[J].FoodBiotechnol.,2013,27:54-65.
[16]Do T T,Quyen D T,Dam T H.Purification andcharacterization of an acid-stable and organic solvent-tolerantxylanase from Aspergillus awamori VTCC-F312[J].Sci.Asia,2012,38:157-165.
[17]Mavromichalis I,Hancock J D,Senne B W,et al..Enzymesupplementation and particle size of wheat in diets for nurseryand finishing pigs[J].J.Animal Sci.,2000,78(12):3086-3095.
[18]Lunen T A V,Schulze H.Influence of Trichodermalongibrachiatum xylanase supplementation of wheat and cornbased diets on growth performance of pigs[J].Can.J.AnimalSci.,1996,76(2):271-273.
[19]Barrera M,Cervantes M,Sauer W C,et al..Ileal amino aciddigestibility and performance of growing pigs fed wheat-baseddiets supplemented with xylanase[J].J.Animal Sci.,2004,82(7):1997-2003.
[20]Thacker P A,Campbell G L,Groot W J.The effect of enzymesupplementation on the nutritive value of rye-based diets forswine[J].Can.J.Animal Sci.,1991,71(2):489-496.
[21]Omogbenigun F O,Nyachoti C M,Slominski B A.Dietarysupplementation with multienzyme preparations improvesnutrient utilization and growth performance in weaned pigs[J].J.Animal Sci.,2004,82(4):1053-1061
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