Aspergillus ficuum AF-98菊粉酶发酵条件的优化及酶的分离纯化研究
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摘要
菊粉酶(Inulinase)是一类能够水解β-2,1-D-果聚糖果糖苷键的水解酶,在一定温度下水解菊粉生成果糖或低聚果糖,目前果糖和低聚果糖已被广泛应用于食品和医药工业,因此近年来菊粉酶的研究受到越来越多的重视。
本论文对实验室保藏的一株无花果曲霉AF-98产菊粉酶的液体发酵条件进行了研究,通过优化培养基和培养条件提高酶产量,并对该菌株所产菊粉酶进行了分离纯化,以及对该酶的酶学性质进行了初步研究。论文的主要研究内容和结论如下:
1.对菌株产菊粉酶的液体发酵条件进行了研究.采用单因素试验初步确定影响Aspergillus ficuum AF-98产菊粉酶的各单因子的较优水平,在此基础上,利用Box-Behnken响应曲面法和正交试验分别对影响该菌株发酵产酶的培养基组分和培养条件进行进一步优化。优化后的发酵条件为:菊粉20.25g/L、眎蛋白胨21.20g/L、(NH_4)_2SO_4 5.3g/L、NaCl 5g/L、MgSO_4·7H_2O 0.5 g/L、ZnSO_4·7H_2O 0.1 g/L,初始pH6.5、接种量6%、装液量50mL/250mL三角瓶、培养温度30℃、培养时间48h。在此条件下菊粉酶酶活达到158.44U/mL。
2.菊粉酶粗酶液经硫酸铵分级沉淀、DEAE-Sephacel离子交换层析和SephadexG-100凝胶层析等分离纯化方法,得到电泳纯的菊粉酶。该酶被提纯了7.06倍,酶回收率为4.9%,酶活力达到376.78U/mg。经SDS-PAGE电泳检测该菊粉酶为单链蛋白,相对分子量约为48KDa。
3.对菊粉酶的酶学性质进行了研究。实验结果表明:菊粉酶反应最适温度为60℃,最适pH为4.0,在60℃以下保温1小时仍具有75%以上的活性,在pH3.0～7.0的范围内室温保存1小时具有80%以上的稳定性;以菊粉为底物,Mn~(2+)对酶活有微弱的促进作用,Co~(2+)、Cu~(2+)和Mg~(2+)对酶活影响不大;K~+对酶活有微弱的抑制作用,Ag~+、Zn~(2+)、Ca~(2+)、Ba~(2+)、Na~+和EDTA对酶活有明显的抑制作用;以菊粉为底物的K_m=35.72mg/mL,V_(max)=16.34μmol·min~(-1) mL~(-1);
Inulinases are 2,1-β-fructanohydrolases which convert inulin,a polymer of fructose inβ-2,1 linkage,to fructoses or fructooligosaccharides at proper temperature.Thus,the use of microbial inulinase has been proposed as the most promising approach to obtain fructoses and fructooligosaccharides.So the research on inulinase has been receiving more and more attention.
A strain of Aspergillus ficuum AF-98 which produces inulinase was stored in our laboratory.In this paper,we studied the optimization of fermentation parameters,the purification and properties of inulinase from Aspergillus ficuum AF-98.The main research findings are as follows:
1.Based on the results of single factor experiments,response surface methodology and orthogonal experiments have been used to optimize the cultivation medium and the fermentation parameters,respectively.The optimal levels oft he main factors are as follows: inulin 20.25g/L、protose-peptone 21.20g/L、(NH_4)_2SO_4 5.3g/L,NaCl 5 g/L、MgSO_4·7H_2O 0.5 g/L,ZnSO_4·7H_2O 0.1 g/L、initial pH 6.5、inoculum amount 6%、volume of medium 50mL/250mL Erlenmeyer flask、cultivation temperature 30℃with a fermentation time of 48h.Under this condition,the enzyme activity value was158.44 U/mL.
2.Inulinase was purified from the culture broth by ammonium sulphate precipitation, followed by successive chromatographies on DEAE-Sephacel and Sephadex G-100 columns. The enzyme was purified 7.06 fold with 4.9%activity yield from the starting culture broth and the specific activity reached 376.78U/mg.The molecular weight of the purified en-yme was estimated to be 48977KDa by SDS-PAGE.
3.The optimal reaction temperature and pH were 60℃and 4.0 respectively.The enzyme were stable under 60℃within pH3.0～7.0.The enzyme activity was enhanced with 5mM Mn~(2+),whereas the addition of Co~(2+),Cu~(2+) and Mg~(2+) almost had no effect on inulin hydrolytic activity;on the contratry the inulinase activity was significantly inhibited in the presence of Ag~+,Zn~(2+),Ca~(2+),Ba~(2+),Na~+ and metal chelating agent EDTA.The Michaelis constant(K_m) and maximum reaction velocity(V_(max)) were found to be 35.72mg/mLand 16.34μmol·min~(-1)mL~(-1),respectively.
本论文对实验室保藏的一株无花果曲霉AF-98产菊粉酶的液体发酵条件进行了研究,通过优化培养基和培养条件提高酶产量,并对该菌株所产菊粉酶进行了分离纯化,以及对该酶的酶学性质进行了初步研究。论文的主要研究内容和结论如下:
1.对菌株产菊粉酶的液体发酵条件进行了研究.采用单因素试验初步确定影响Aspergillus ficuum AF-98产菊粉酶的各单因子的较优水平,在此基础上,利用Box-Behnken响应曲面法和正交试验分别对影响该菌株发酵产酶的培养基组分和培养条件进行进一步优化。优化后的发酵条件为:菊粉20.25g/L、眎蛋白胨21.20g/L、(NH_4)_2SO_4 5.3g/L、NaCl 5g/L、MgSO_4·7H_2O 0.5 g/L、ZnSO_4·7H_2O 0.1 g/L,初始pH6.5、接种量6%、装液量50mL/250mL三角瓶、培养温度30℃、培养时间48h。在此条件下菊粉酶酶活达到158.44U/mL。
2.菊粉酶粗酶液经硫酸铵分级沉淀、DEAE-Sephacel离子交换层析和SephadexG-100凝胶层析等分离纯化方法,得到电泳纯的菊粉酶。该酶被提纯了7.06倍,酶回收率为4.9%,酶活力达到376.78U/mg。经SDS-PAGE电泳检测该菊粉酶为单链蛋白,相对分子量约为48KDa。
3.对菊粉酶的酶学性质进行了研究。实验结果表明:菊粉酶反应最适温度为60℃,最适pH为4.0,在60℃以下保温1小时仍具有75%以上的活性,在pH3.0～7.0的范围内室温保存1小时具有80%以上的稳定性;以菊粉为底物,Mn~(2+)对酶活有微弱的促进作用,Co~(2+)、Cu~(2+)和Mg~(2+)对酶活影响不大;K~+对酶活有微弱的抑制作用,Ag~+、Zn~(2+)、Ca~(2+)、Ba~(2+)、Na~+和EDTA对酶活有明显的抑制作用;以菊粉为底物的K_m=35.72mg/mL,V_(max)=16.34μmol·min~(-1) mL~(-1);
Inulinases are 2,1-β-fructanohydrolases which convert inulin,a polymer of fructose inβ-2,1 linkage,to fructoses or fructooligosaccharides at proper temperature.Thus,the use of microbial inulinase has been proposed as the most promising approach to obtain fructoses and fructooligosaccharides.So the research on inulinase has been receiving more and more attention.
A strain of Aspergillus ficuum AF-98 which produces inulinase was stored in our laboratory.In this paper,we studied the optimization of fermentation parameters,the purification and properties of inulinase from Aspergillus ficuum AF-98.The main research findings are as follows:
1.Based on the results of single factor experiments,response surface methodology and orthogonal experiments have been used to optimize the cultivation medium and the fermentation parameters,respectively.The optimal levels oft he main factors are as follows: inulin 20.25g/L、protose-peptone 21.20g/L、(NH_4)_2SO_4 5.3g/L,NaCl 5 g/L、MgSO_4·7H_2O 0.5 g/L,ZnSO_4·7H_2O 0.1 g/L、initial pH 6.5、inoculum amount 6%、volume of medium 50mL/250mL Erlenmeyer flask、cultivation temperature 30℃with a fermentation time of 48h.Under this condition,the enzyme activity value was158.44 U/mL.
2.Inulinase was purified from the culture broth by ammonium sulphate precipitation, followed by successive chromatographies on DEAE-Sephacel and Sephadex G-100 columns. The enzyme was purified 7.06 fold with 4.9%activity yield from the starting culture broth and the specific activity reached 376.78U/mg.The molecular weight of the purified en-yme was estimated to be 48977KDa by SDS-PAGE.
3.The optimal reaction temperature and pH were 60℃and 4.0 respectively.The enzyme were stable under 60℃within pH3.0～7.0.The enzyme activity was enhanced with 5mM Mn~(2+),whereas the addition of Co~(2+),Cu~(2+) and Mg~(2+) almost had no effect on inulin hydrolytic activity;on the contratry the inulinase activity was significantly inhibited in the presence of Ag~+,Zn~(2+),Ca~(2+),Ba~(2+),Na~+ and metal chelating agent EDTA.The Michaelis constant(K_m) and maximum reaction velocity(V_(max)) were found to be 35.72mg/mLand 16.34μmol·min~(-1)mL~(-1),respectively.
引文
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