木聚糖酶和甘露聚糖酶在毕赤酵母中的共表达及产酶分析
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摘要
木聚糖酶和甘露聚糖酶是两种重要的半纤维素酶,也是两种重要的饲用酶制剂,通过毕赤酵母表达系统中的体外串联表达盒构建多拷贝的方法构建了木聚糖酶DSB和甘露聚糖酶Man A共表达重组质粒p PICZαA/DSB-ManA,将该重组质粒电转化至宿主菌毕赤酵母X33中获得共表达两种酶的重组菌X33/DSB-ManA,实现了两种酶的共分泌表达,经诱导表达后木聚糖酶和甘露聚糖酶的酶活分别为273. 6 U/ml和256. 8 U/ml,为单独表达重组菌X33/DSB和X33/Man A酶活的30. 4%和73. 4%。酶学性质的分析显示DSB和Man A的最适反应温度均为75℃,在45℃～75℃范围内具有较好的温度稳定性,酶活可保持最高酶活的60%以上; DSB最适pH为6. 5,Man A最适pH为6. 0,在pH 3. 0、40℃条件下,Man A处理1h能保持最高酶活的80%以上,DSB处理1 h时能保持最高酶活的50%以上; DSB和Man A对多种金属离子和化学试剂(浓度为1 mM)具有较好的耐受性,均可保留60%以上的酶活力。通过单一菌株成功完成了不同酶的共表达,为复合酶饲料添加剂的生产和应用研究提供了一定的理论依据。
Xylanase and mannanase are two important hemicellulases and feed enzyme preparations.Construction method of in vitro multimers in Pichia pastoris expression system has been used to construct recombinant plasmid pPICZαA/DSB-ManA which contained both DSB gene and ManA gene. Then,pPICZαA/DSB-ManA was transformed to host cells Pichia pastoris X33 by electroporation to obtain the co-expression recombinant strain X33/DSB-ManA. In shake-flask cultivation,xylanase activity and mannanase activity in the supernatant were 273. 6 U/ml and 256. 8 U/ml that reached 30. 4% and 73. 4% activity compared to X33/DSB and X33/ManA,respectively. Properties of the mixed enzyme suggested that the optimal reaction temperature for DSB and ManA is 75℃. Both xylanase and mannanase showed more than 60% activity across temperature ranges45°C to 75° C. The optimal reaction pH value of DSB and ManA were 6. 5 and 6. 0,respectively. After incubation 1 hour under the pH3. 0 and 40℃ treatment condition,mannanase activity of ManA displayed greater than 80% activity and xylanase activity of DSB revealed more than 50% activity. Meanwhile,DSB and ManA have shown good tolerance to a variety of metal ions and chemical reagents. Both enzyme activities could keep over 60% activity when the treatment concentration of different metal ions and chemical reagents was 1 millimole.Xylanase DSB and mannanase ManA have been co-expressed in Pichia pastoris successfully. The successful expression of the two enzymes of the recombinant strain could offer the theory basis for production and application of compound enzyme in the animal feed.
Xylanase and mannanase are two important hemicellulases and feed enzyme preparations.Construction method of in vitro multimers in Pichia pastoris expression system has been used to construct recombinant plasmid pPICZαA/DSB-ManA which contained both DSB gene and ManA gene. Then,pPICZαA/DSB-ManA was transformed to host cells Pichia pastoris X33 by electroporation to obtain the co-expression recombinant strain X33/DSB-ManA. In shake-flask cultivation,xylanase activity and mannanase activity in the supernatant were 273. 6 U/ml and 256. 8 U/ml that reached 30. 4% and 73. 4% activity compared to X33/DSB and X33/ManA,respectively. Properties of the mixed enzyme suggested that the optimal reaction temperature for DSB and ManA is 75℃. Both xylanase and mannanase showed more than 60% activity across temperature ranges45°C to 75° C. The optimal reaction pH value of DSB and ManA were 6. 5 and 6. 0,respectively. After incubation 1 hour under the pH3. 0 and 40℃ treatment condition,mannanase activity of ManA displayed greater than 80% activity and xylanase activity of DSB revealed more than 50% activity. Meanwhile,DSB and ManA have shown good tolerance to a variety of metal ions and chemical reagents. Both enzyme activities could keep over 60% activity when the treatment concentration of different metal ions and chemical reagents was 1 millimole.Xylanase DSB and mannanase ManA have been co-expressed in Pichia pastoris successfully. The successful expression of the two enzymes of the recombinant strain could offer the theory basis for production and application of compound enzyme in the animal feed.
引文
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[11]张巍,王亚伟,陈丰,等.一株全基因合成耐热甘露聚糖酶的表达及酶学性质分析.中国生物工程杂志,2014,34(8):41-46.Zhang W,Wang Y W,Chen F,et al.Gene synthesis,expression and characterization of a thermostable endo-β-1,4-mannanase.China Biotechnology,2014,34(8):41-46.
[12]田军辉.β-甘露聚糖酶对不同蛋白饲料的体外酶解效果及其在断奶仔猪低能量日粮中的应用研究.河南:河南农业大学,2014.Tian J H.The effects ofβ-Mannanase on protein feed stuffs in vitro and study on weaned piglets in low energy diets.Henan:Henan Agricultural University,2014.
[13]郭玉光,杜红方,王敏,等.饲粮中添加复合酶对仔猪生长性能,血液生化指标和养分消化率的影响.中国畜牧杂志,2018,54(2):65-68.Guo Y G,Du H F,Wang M,et al.Effects of compound enzymes added to diet on growth performance,blood biochemical indexes and nutrient digestibility of piglets.Chinese Journal of Animal Science,2018,54(2):65-68.
[14]周雪梅,杨在宾,姜淑贞,等.复合酶制剂对蛋鸡生产性能,蛋品质,肠道健康微生物及养分利用率的影响.家禽科学,2017(11):7-12.Zhou X M,Yang Z B,Jiang S Z,et al.Effects of compound enzyme preparation on production performance,egg quality,intestinal health microorganism and nutrient utilization rate of laying hens.Journal of Poultry Science,2017(11):7-12.
[15]谭子超,刘浩民,薛梅,等.小麦-豆粕型饲粮添加复合酶制剂对肉鸡肠道微生物菌群和影响.中国饲料,2018(9):19-24.Tan Z C,Liu H M,Xue M,et al.Effects of different levels of compound enzyme preparation added in wheat-soybean meal diet on broiler’s gastrointestinal microbial flora and chyme acidity.China Feed,2018(9):19-24.
[16]Cozannet P,Meur M,Batut H,et al.Enzyme complex improves performance of broilers fed wheat-and soybean-based diets with graded density//Optimum definition,Australian Poultry Science Symposium.Sydney:Poultry Research Foundation,2013:68-71.
[17]Beauchemin K A,Colombatto D,Morgavi D P.A rationale for the development of feed enzyme products for ruminants.Canadian Journal of Animal Science,2004,84(1):23-36.
[18]刘高磊,胡蝶,邬敏辰,等.内切葡聚糖酶与木聚糖酶在毕赤酵母中的共分泌表达.食品与生物技术学报,2015,34(3):253-259.Liu G L,Hu D,Wu M C,et al.Co-expression of endoglucanase and xylanase in Pichia pastoris.Journal of Food Science and Biotechnology,2015,34(3):253-259.
[19]马蓉,徐昊,丁锐,等.大肠杆菌多基因共表达策略.中国生物工程杂志,2012,32(04):117-122.Ma R,Xu H,Ding R,et al.The strategy of gene coexpression in Escherichia Coli.China Biotechnology,2012,32(4):117-122.
[2]Pollet A,Delcour J A,Courtin C M.Structural determinants of the substrate specificities of xylanases from different glycoside hydrolase families.Critical Reviews in Biotechnology,2010,30(3):176-191.
[3]Le Nours J,Anderson L,Stoll D,et al.The structure and characterization of a modular endo-β-1,4-mannanase from Cellulomonas fimi.Biochemistry,2005,44(38):12700-12708.
[4]Couturier M,Roussel A,Rosengren A,et al.Structural and biochemical analyses of glycoside hydrolase families 5 and 26beta-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharides catalysis.Journal of Biological Chemistry,2013,288(20):14624-14635.
[5]魏川子,王兆斌,李燕蒙,等.半纤维素酶在畜禽饲料中的应用及研究进展.畜牧与饲料科学,2015,36(12):44-46.Wei C Z,Wang Z B,Li Y M,et al.Application and research progress of hemicellulases in animal feed.Animal Husbandry and Feed Science,2015,36(12):44-46.
[6]黄生平,汪昌丽,张桂敏,等.植酸酶和甘露聚糖酶双功能毕赤酵母工程菌的构建和产酶分析.微生物学报,2007,47(2):280-284.Huang S P,Wang C L,Zhang G M,et al.Construction of a double functional recombinant strainof Pichia pastoris coexpressing phytase and mannanase and the enzymatic analyses.Acta microbiologica Sinica,2007,47(2):280-284.
[7]吴振芳,唐自钟,陈惠,等.毕赤酵母中植酸酶和内切葡聚糖酶共表达菌株的构建及其高效表达.生物工程学报,2010,26(5):616-622.Wu Z F,Tang Z Z,Chen H,et al.Construction of Pichia pastoris recombinant strain capable of over-expressing phytase and endoglucanase.Chinese Journal of Biotechnology,2010,26(5):616-622.
[8]Damasceno L M,Huang C J,Batt C A.Protein secretion in Pichia pastoris and advances in protein production.Applied Microbiology and Biotechnology,2012,93(1):31-39.
[9]Rabert C,Weinacker D,Pessoa A Jr,et al.Recombinants proteins for industrial uses:Utilization of Pichia pastoris expression system.Brazilian Journal of Microbiology,2013,44(2):351-356.
[10]Yu H Y,Sun Y M,Wang W,et al.Purification and properties of Bacillus subtilis SA-22 Endo-1,4-β-D-mannanase.Chinese Journal of Biotechnology,2003,19(3):327-331.
[11]张巍,王亚伟,陈丰,等.一株全基因合成耐热甘露聚糖酶的表达及酶学性质分析.中国生物工程杂志,2014,34(8):41-46.Zhang W,Wang Y W,Chen F,et al.Gene synthesis,expression and characterization of a thermostable endo-β-1,4-mannanase.China Biotechnology,2014,34(8):41-46.
[12]田军辉.β-甘露聚糖酶对不同蛋白饲料的体外酶解效果及其在断奶仔猪低能量日粮中的应用研究.河南:河南农业大学,2014.Tian J H.The effects ofβ-Mannanase on protein feed stuffs in vitro and study on weaned piglets in low energy diets.Henan:Henan Agricultural University,2014.
[13]郭玉光,杜红方,王敏,等.饲粮中添加复合酶对仔猪生长性能,血液生化指标和养分消化率的影响.中国畜牧杂志,2018,54(2):65-68.Guo Y G,Du H F,Wang M,et al.Effects of compound enzymes added to diet on growth performance,blood biochemical indexes and nutrient digestibility of piglets.Chinese Journal of Animal Science,2018,54(2):65-68.
[14]周雪梅,杨在宾,姜淑贞,等.复合酶制剂对蛋鸡生产性能,蛋品质,肠道健康微生物及养分利用率的影响.家禽科学,2017(11):7-12.Zhou X M,Yang Z B,Jiang S Z,et al.Effects of compound enzyme preparation on production performance,egg quality,intestinal health microorganism and nutrient utilization rate of laying hens.Journal of Poultry Science,2017(11):7-12.
[15]谭子超,刘浩民,薛梅,等.小麦-豆粕型饲粮添加复合酶制剂对肉鸡肠道微生物菌群和影响.中国饲料,2018(9):19-24.Tan Z C,Liu H M,Xue M,et al.Effects of different levels of compound enzyme preparation added in wheat-soybean meal diet on broiler’s gastrointestinal microbial flora and chyme acidity.China Feed,2018(9):19-24.
[16]Cozannet P,Meur M,Batut H,et al.Enzyme complex improves performance of broilers fed wheat-and soybean-based diets with graded density//Optimum definition,Australian Poultry Science Symposium.Sydney:Poultry Research Foundation,2013:68-71.
[17]Beauchemin K A,Colombatto D,Morgavi D P.A rationale for the development of feed enzyme products for ruminants.Canadian Journal of Animal Science,2004,84(1):23-36.
[18]刘高磊,胡蝶,邬敏辰,等.内切葡聚糖酶与木聚糖酶在毕赤酵母中的共分泌表达.食品与生物技术学报,2015,34(3):253-259.Liu G L,Hu D,Wu M C,et al.Co-expression of endoglucanase and xylanase in Pichia pastoris.Journal of Food Science and Biotechnology,2015,34(3):253-259.
[19]马蓉,徐昊,丁锐,等.大肠杆菌多基因共表达策略.中国生物工程杂志,2012,32(04):117-122.Ma R,Xu H,Ding R,et al.The strategy of gene coexpression in Escherichia Coli.China Biotechnology,2012,32(4):117-122.