β-甘露聚糖酶异源表达及甘露寡糖制备研究
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摘要
以产β-甘露聚糖酶(MAN)的枯草芽孢杆菌HD145为目的菌株,PCR扩增MAN基因片段,克隆到p HBM905A载体并转化至毕赤酵母,重组子利用甲醇进行诱导。纯化的MAN酶解槐豆胶与瓜尔豆胶,质谱检测水解产物。SDS-PAGE结果表明,MAN基因实现了表达,摇瓶酶活性为3 200 U/m L,酶最适温度与pH分别为55℃与6.0,pH 4~7时稳定性较好,在40、50、60℃的半衰期分别为165、105、42 min,Zn~(2+)、Ag~+、Co~(2+)对酶活性存在一定的抑制。槐豆胶与瓜尔胶经MAN酶解后得到二糖至十糖的寡糖。
A mannanase(MAN) DNA fragment was amplified from the total DNA of Bacillus subtilis HD145 by PCR,and cloned to plasmid p HBM905 A,then the recombinant plasmid was transformed into Pichia pastoris,and the recombinant strain was induced by methanol. The locust bean gum and guar gum were hydrolyzed by purified MAN,and the hydrolysate was detected by Mass. The SDS-PAGE results showed that MAN gene was expressed correctly,and the enzyme activity of flask culture was 3 200 U / m L. The optimum temperature and p H were 55 ℃ and 6.0,respectively. The p H stability was 4~7,and half-life times of 40 ℃,50 ℃ and 60 ℃ were 165 min,105 min and 42 min,respectively. Zn~(2+),Ag~+ and Co(2+)exhibited a preferred inhibition on enzyme activity. The enzymatic hydrolysis products of locust bean gum and guar gum were mannan oligosaccharides of dimer to decamer.
A mannanase(MAN) DNA fragment was amplified from the total DNA of Bacillus subtilis HD145 by PCR,and cloned to plasmid p HBM905 A,then the recombinant plasmid was transformed into Pichia pastoris,and the recombinant strain was induced by methanol. The locust bean gum and guar gum were hydrolyzed by purified MAN,and the hydrolysate was detected by Mass. The SDS-PAGE results showed that MAN gene was expressed correctly,and the enzyme activity of flask culture was 3 200 U / m L. The optimum temperature and p H were 55 ℃ and 6.0,respectively. The p H stability was 4~7,and half-life times of 40 ℃,50 ℃ and 60 ℃ were 165 min,105 min and 42 min,respectively. Zn~(2+),Ag~+ and Co(2+)exhibited a preferred inhibition on enzyme activity. The enzymatic hydrolysis products of locust bean gum and guar gum were mannan oligosaccharides of dimer to decamer.
引文
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[19]张娟,罗长财.耐酸性黑曲霉β-甘露聚糖酶的克隆及其在毕赤酵母中的表达分析[J].生物技术通讯,2011,22(2):182-187.
[20]聂金梅,李阳源,刘金山.两拷贝β-甘露聚糖酶基因在毕赤酵母中的表达[J].生物技术,2014,24(2):24-28.
[21]LIAO H P,LI S X,ZHENG H P,et al.A new acidophilic thermostable endo-1,4-β-mannanase from Penicillium oxalicum GZ-2:Cloning,characterization and functional expression in Pichia pastoris[J].BMC Biotechnol,2014,14(1):1-12.
[22]傅亮,何紫姣,李存芝,等.酶法制备瓜尔胶半乳甘露低聚糖的研究[J].食品与机械,2010,26(5):122-124.
[23]KONO H,OTAKA F,OZAKI M.Preparation and characterization of guar gum hydrogels as carrier materials for controlled protein drug delivery[J].Carbohydr Polym,2014,111:830-840.
[2]MALHERBE A R,ROSE S H,VILJOEN-BLOOM M,et al.Expression and evaluation of enzymes required for the hydrolysis of galactomannan[J].J Ind Microbiol Biotechnol,2014,41(8):1201-1209.
[3]李长影,孔雯,王家昕,等.甘露聚糖酶产生菌的分离鉴定和酶学性质[J].华中农业大学学报,2011,30(2):138-142.
[4]张云光,韦玉琴,卢丽萍,等.产β-甘露聚糖酶的海洋微生物的筛选[J].广西轻工业,2011,27(3):3-5.
[5]李慧玲,刘祖艳,赵敏.瓜尔胶降解菌诱变及甘露聚糖酶特性研究[J].安徽农业科学,2013,41(36):14041-14043,14046.
[6]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):64-69.
[7]单修环,刘钇君,刘新利.产β-甘露聚糖酶菌株的筛选及产酶条件优化的研究[J].齐鲁工业大学学报,2014,28(3):28-32.
[8]刘项羽,徐美娟,杨套伟,等.β-甘露聚糖酶基因在枯草芽孢杆菌中的克隆及表达[J].应用与环境生物学报,2012,18(4):672-677.
[9]GHOSH A,LU魱S A S,BR魣S J L,et al.Thermostable recombinantβ-(1→4)-mannanase from C.thermocellum:Biochemical characterization and manno-oligosaccharides production[J].J Agric Food Chem,2013,61(50):12333-12344.
[10]BAI X,HU H,CHEN H,et al.Expression of aβ-Mannosidase from Paenibacillus polymyxa A-8 in Escherichia coli and characterization of the recombinant Enzyme[J].PLo S One,2014,9(11):e111622.
[11]唐卫华,王瑞明,肖静,等.β-甘露聚糖酶基因在毕赤酵母中的高效表达[J].生物技术,2013,23(2):18-22.
[12]王志江,李致瑜,黄水华,等.不同魔芋葡甘聚糖降解物抑制肿瘤活性的比较研究[J].食品与机械,2011,27(5):72-74.
[13]徐丽萍,吴正勇,任富修,等.魔芋胶酶解工艺条件的优化[J].安徽农业科学,2011,3(21):13058-13059.
[14]倪玉佳,周旻昱,欧阳嘉,等.黑曲霉嗜热β-甘露聚糖酶在毕赤酵母中的克隆表达及其魔芋降解产物分析[J].生物技术通报,2014(6):182-187.
[15]KANG L X,CHEN X M,FU L,et al.Recombinant expression of chitosanase from Bacillus subtilis HD145 in Pichia pastoris[J].Carbohydr Research,2012,352:37-43.
[16]DEMO G,FLIEDROV魣B,WEIGNEROV魣L,et al.Crystallization and preliminary X-ray crystallographic analysis of recombinantβ-mannosidase from Aspergillus niger[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2013,69(3):288-291.
[17]LI R K,CHEN P,NG T B,et al.Highly efficient expression and characterization of aβ-mannanase from Bacillus subtilis in Pichia pastoris[J].Biotechnol Appl Biochem,2014.doi:10.1002/bab.1250.
[18]LV J,CHEN Y,PEI H,et al.Cloning,expression,and characterization ofβ-mannanase from Bacillus subtilis MAFIC-S11in Pichia pastoris[J].Appl Biochem Biotechnol,2013,169(8):2326-2340.
[19]张娟,罗长财.耐酸性黑曲霉β-甘露聚糖酶的克隆及其在毕赤酵母中的表达分析[J].生物技术通讯,2011,22(2):182-187.
[20]聂金梅,李阳源,刘金山.两拷贝β-甘露聚糖酶基因在毕赤酵母中的表达[J].生物技术,2014,24(2):24-28.
[21]LIAO H P,LI S X,ZHENG H P,et al.A new acidophilic thermostable endo-1,4-β-mannanase from Penicillium oxalicum GZ-2:Cloning,characterization and functional expression in Pichia pastoris[J].BMC Biotechnol,2014,14(1):1-12.
[22]傅亮,何紫姣,李存芝,等.酶法制备瓜尔胶半乳甘露低聚糖的研究[J].食品与机械,2010,26(5):122-124.
[23]KONO H,OTAKA F,OZAKI M.Preparation and characterization of guar gum hydrogels as carrier materials for controlled protein drug delivery[J].Carbohydr Polym,2014,111:830-840.