耐碱性β-甘露聚糖酶产生菌的分离鉴定及发酵条件优化
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摘要
研究旨在高产β-甘露聚糖酶饲用益生芽孢杆菌的筛选及培养条件优化。用透明圈法筛选得到一株产β-甘露聚糖酶活力较高的枯草芽孢杆菌(Bacillus subtilis)WMYB-2,通过单因素实验、Box-Behnken实验及响应面分析对该菌的产酶培养基及培养条件进行了优化,并对其酶学性质进行了初步研究。结果表明,该菌株产酶的最佳培养基及发酵条件为:魔芋微粉10.0 g/l,大豆蛋白胨10.0 g/l、CaCl_2 0.6 g/l、K_2HPO_4 0.8 g/l、MgSO_4·7H_2O 1.0 g/l,pH三角瓶,37℃、220 r/min发酵36 h,酶活力达355.75 U/ml,是初始酶活力的3.5倍。该酶的最适反应温度和pH值分别为55℃和5.5。在45～55℃内酶活力稳定,55℃保温30 min和3 h后其相对酶活力保留92%和57%;在pH值5.0～10.0内酶活力稳定,在pH值9.0和10.0的环境下处理2 h其相对酶活力保留95%和81%。Li~+和K~+对该酶具有一定的激活作用。该酶具有良好的耐碱特性及应用潜力。
The experiment was conducted to screen the highly β-Mannanase producing probiotic Bacillus sp. and optimization its culture conditions. A highly β-Mannanase producing Bacillus subtilis named WMYB-2, was screened by transparent circle method. Single-factor experiment and BoxBehnken experiment of response surface analysis were performed to obtain the optimal medium and fermentation conditions for β-Mannanase production, and the enzymatic properties were studied. The result showed that culture with an inoculation size of 2% for 36 h at 37 ℃ and 220 r/min in a 250 ml shaking flask filled with 40 ml liquid medium consisting of 10.0 g/l konjac powder, 10.0 g/l soybean peptone, CaCl_20.6 g/l, K_2HPO_4 0.8 g/l, MgSO_4·7 H_2O 1.0 g/l, at initial pH 7.51, the β-Mannanase activity reached 355.75 U/ml, which was 3.5 times higher than before the optimization. The optimum temperature and pH of the enzyme were 55 ℃ and 5.5. The enzyme was stable between temperature 45～55 ℃ and pH 5.0～10.0. The enzyme activity remained 92% at 55 ℃ for a treatment of 30 minutes and 57% after a treatment of 3 hours. The enzyme activity remained 95% and 81% after a treatment of 2 hours at pH 9.0 and 10.0. The enzyme was activated by Li~+ and K~+. The enzyme showed excellent alkali resistance characteristics, and had the potential for application.
The experiment was conducted to screen the highly β-Mannanase producing probiotic Bacillus sp. and optimization its culture conditions. A highly β-Mannanase producing Bacillus subtilis named WMYB-2, was screened by transparent circle method. Single-factor experiment and BoxBehnken experiment of response surface analysis were performed to obtain the optimal medium and fermentation conditions for β-Mannanase production, and the enzymatic properties were studied. The result showed that culture with an inoculation size of 2% for 36 h at 37 ℃ and 220 r/min in a 250 ml shaking flask filled with 40 ml liquid medium consisting of 10.0 g/l konjac powder, 10.0 g/l soybean peptone, CaCl_20.6 g/l, K_2HPO_4 0.8 g/l, MgSO_4·7 H_2O 1.0 g/l, at initial pH 7.51, the β-Mannanase activity reached 355.75 U/ml, which was 3.5 times higher than before the optimization. The optimum temperature and pH of the enzyme were 55 ℃ and 5.5. The enzyme was stable between temperature 45～55 ℃ and pH 5.0～10.0. The enzyme activity remained 92% at 55 ℃ for a treatment of 30 minutes and 57% after a treatment of 3 hours. The enzyme activity remained 95% and 81% after a treatment of 2 hours at pH 9.0 and 10.0. The enzyme was activated by Li~+ and K~+. The enzyme showed excellent alkali resistance characteristics, and had the potential for application.
引文
[1]李玉鹏,杨鸿雁,钟荣珍,等.棕榈仁粕在畜禽养殖中的应用研究进展[J].饲料研究,2018,2(4):15-18.
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[12]廖婷婷,翟磊,高成华,等.一株产甘露聚糖酶菌株的分离鉴定及酶的纯化与性质[J].微生物学报,2011,51(11):1521-1526.
[13]杨苗,卢晓华,王常高,等.产β-甘露聚糖酶菌株的筛选鉴定及产酶条件的优化[J].工业微生物,2015,45(4):51-57.
[14]张建新,韩科芳,刘起丽,等.产β-甘露聚糖酶内生菌最适发酵条件的优化[J].饲料工业,2012,33(14):53-57.
[15]成莉凤,冯湘沅,段盛文,等.一种耐热偏酸性β-甘露聚糖酶基因克隆与高效表达[J].微生物学通报,2015,42(11):2143-2150.
[16]张建新,赵丹丹,刘起丽,等.产β-甘露聚糖酶内生菌的筛选及酶学特性分析[J].微生物学通报,2011,38(8):1172-1178.
[17]吴华伟,蔚鑫鑫,陈雪秋.产甘露聚糖酶细菌的分离鉴定、酶的部分纯化及酶学性质研究[J].湖北农业科学,2014,53(15):3601-3605.
[18]赵梅,魏喜换,王春娟,等.耐热β-甘露聚糖酶基因的克隆与表达及酶学性质[J].食品与生物技术学报,2014,33(6):590-596.
[19]王梅,石璟,谭德水,等.枯草芽孢杆菌MSJ-5产β-甘露聚糖酶的性质研究[J].福建农业学报,2015,30(3):293-297.
[20]唐嘉婕,郭苏,王伟,等.短小芽孢杆菌耐碱性β-甘露聚糖酶基因的异源表达及其酶学特性[J].微生物学报,2015,55(11):1445-1457.
[21]陈智慧,严琳,莫海飞,等.产β-甘露聚糖酶内生菌的筛选、鉴定及酶学性质研究[J].中国畜牧兽医,2017,44(8):2496-2502.
[22]杨升,李立,付权,等.β-甘露聚糖酶产生菌的鉴定及其粗酶性质的研究[J].湖北大学学报(自然科学版),2010,32(2):214-216.
[2]Srivastava P K,Kapoor M.Production,properties,and applications of endo-β-mannanases[J].Biotechnol.Advances,2017,35(1):1-19.
[3]张蕊,朱虹,周峻沛,等.β-甘露聚糖酶分子生物学研究进展[J].中国农业科技导报,2018,20(5):34-46.
[4]Chauhan P,Puri N,Sharma P,et al.Mannanases:microbial sources,production,properties and potential biotechnological applications[J].Appl.Microbiol.Biotechnol.,2012,93(5):1817-1830.
[5]徐扬,刘起丽,聂国兴,等.β-甘露聚糖酶的研究进展[J].河南农业科学,2011,40(4):34-37.
[6]张文会,孙同韦,张会图,等.饲用耐酸抗蛋白酶β-甘露聚糖酶的筛选分离及酶学性质[J].天津科技大学学报,2015,30(6):7-11.
[7]蒙海林,张云开,凌敏,等.β-甘露聚糖酶产生菌的选育[J].现代食品科技,2006,22(2):73-75.
[8]Akino T,Nakamura N,Horikoshi K.Production ofβ-mannosidase andβ-mananase by an alkalophilic Bacillus sp.[J].Applied Microbiology and Biotechnology,1987,26(4):323-327.
[9]龚劲松,李恒,刘恒霞,等.碳氮源对枯草芽孢杆菌发酵产β-甘露聚糖酶的影响[J].食品与发酵工业,2015,41(10):334-339.
[10]唐艳斌,闫巧娟,江正强,等.枯草芽孢杆菌产β-1,3-1,4-葡聚糖酶的响应面优化[J].微生物学通报,2013,40(4):551-557.
[11]曲丽娜,王瑞明,肖静,等.β-甘露聚糖酶高产菌株发酵条件优化[J].食品与发酵工业,2012,38(12):364-369.
[12]廖婷婷,翟磊,高成华,等.一株产甘露聚糖酶菌株的分离鉴定及酶的纯化与性质[J].微生物学报,2011,51(11):1521-1526.
[13]杨苗,卢晓华,王常高,等.产β-甘露聚糖酶菌株的筛选鉴定及产酶条件的优化[J].工业微生物,2015,45(4):51-57.
[14]张建新,韩科芳,刘起丽,等.产β-甘露聚糖酶内生菌最适发酵条件的优化[J].饲料工业,2012,33(14):53-57.
[15]成莉凤,冯湘沅,段盛文,等.一种耐热偏酸性β-甘露聚糖酶基因克隆与高效表达[J].微生物学通报,2015,42(11):2143-2150.
[16]张建新,赵丹丹,刘起丽,等.产β-甘露聚糖酶内生菌的筛选及酶学特性分析[J].微生物学通报,2011,38(8):1172-1178.
[17]吴华伟,蔚鑫鑫,陈雪秋.产甘露聚糖酶细菌的分离鉴定、酶的部分纯化及酶学性质研究[J].湖北农业科学,2014,53(15):3601-3605.
[18]赵梅,魏喜换,王春娟,等.耐热β-甘露聚糖酶基因的克隆与表达及酶学性质[J].食品与生物技术学报,2014,33(6):590-596.
[19]王梅,石璟,谭德水,等.枯草芽孢杆菌MSJ-5产β-甘露聚糖酶的性质研究[J].福建农业学报,2015,30(3):293-297.
[20]唐嘉婕,郭苏,王伟,等.短小芽孢杆菌耐碱性β-甘露聚糖酶基因的异源表达及其酶学特性[J].微生物学报,2015,55(11):1445-1457.
[21]陈智慧,严琳,莫海飞,等.产β-甘露聚糖酶内生菌的筛选、鉴定及酶学性质研究[J].中国畜牧兽医,2017,44(8):2496-2502.
[22]杨升,李立,付权,等.β-甘露聚糖酶产生菌的鉴定及其粗酶性质的研究[J].湖北大学学报(自然科学版),2010,32(2):214-216.