高纤维素降解放线菌的筛选与功能鉴定
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摘要
本研究从不同生境中筛选得到一株高纤维素降解放线菌SD1-4,通过形态、生理和分子研究,初步鉴定为链霉菌(Streptomyces sp.)。
对SD1-4液态发酵产酶条件进行优化,结果表明:初始pH为8.0,最适温度为39℃,最适氮源为酵母膏,最佳无机盐添加量为60mgKCI和15mg CaCl2,最佳灭菌时间为40min、最适装液量为50ml/250ml;0.15%Triton X-100抑制产酶,0.05%Triton X-100促进产酶,PEG8000和吐温-20两种浓度均呈促进作用,SDS均呈抑制作用;第9天产酶达到高峰。
对天然碳源(菌草)的研究表明:象草叶为最适碳源;筛选能源草的具体指标为高纤维素、低直链淀粉、低葡萄糖、低果糖、高蔗糖、高蛋白质含量;筛选种质资源的统计学工具为相关分析、主成分分析、回归分析和聚类分析,而灰色关联分析和通径分析不适合。象草营养成分含量(可溶性糖为9.04%、可溶性蛋白质为3.37%时,淀粉含量为5.95%时,粗纤维素含量为65.95% )为象草发酵的最佳成分含量。
采用Box-Bohnken设计,以最终的纤维素酶产量作为研究目标,构建纤维素酶产量(内切酶、外切酶和糖苷酶)和7个因子(温度、pH、颗粒大小、蛋白胨浓度、湿度、接种量和时间)之间的关系模型,比较了GA-ANN、ANN和RSM在优化能力上的差异,优化条件下为0.869、0.906和0.867(R值),相比不优化条件的0.796、0.881和0.840,表明GA-ANN在捕捉系统非线性行为的独特优势。
通过PEG浓缩、硫酸铵沉淀、乙醇洗涤和Sephadex G-200、DEAE A-50、Sephadex G-200,从SD1-4胞外粗酶液中分离得到一种内切葡聚糖酶,经SDS-PAGE检测为单一条带,分子量约为38KD,能水解CMC-Na,最适温度为60℃和pH值为7.0,Vmax、Km分别为22.57U·ml-1·min-1和0.14%;0.2mM Co2+对该酶有较强的促进作用,0.8 mM Co2+对该酶有弱抑制作用。
In this study, a cellulose-degrading Actinomyces called SD1-4 screened from different habitats was identified as Streptomyces (Streptomyces sp.) based on the result of morphological, physiological and molecular studies.
Liquid fermentation conditions of the strain SD1-4 were optimized for enzyme production, the results show that the initial pH was 8.0,, and optimum temperature was set at 39℃, and the optimum nitrogen source was yeast extract, the best salt addition level was 60 mgKCI and 15mg CaCl2, the best Sterilization time 40 min, the optimum liquid volume 50ml/250ml. The result also indicated that 0.15% Triton X-100 inhibit the enzyme production, reverse was true at 0.05% Triton X-100 treatment , PEG8000 and Tween -20 at two treatment concentrations showed promotion on the enzyme, however, SDS showed inhibitory effect on it; for enzyme production reached to the peak.detected at the first 9 days
The result also showed Pennisetum purpereum Schumach was the most suitable carbon source; the index of selection energy grass was high in the contents of fiber, sugar , and proteins but low in the contents of amylose, glucose and fructose. were relatation analysis, principal component analysis, regression analysis and cluster analysis were used as the optimal statistical tools, but gray correlation analysis and path analysis were not suitable for screening. The nutrient content (9.04% soluble sugar, soluble protein was 3.37%, the starch content of 5.95%, the crude fiber content of 65.95%) of Pennisetum purpereum Schumach was the best fermentation ingredients.
Designed by Box-Bohnken a related model has been constructed between cellulase production (endogulucanase, exoglucanase and glucosidase) and seven factors (temperature, pH, particle size, peptone concentration and humidity, inoculum and time). The difference of three models (GA-ANN, ANN and RSM) in optimization was compared. The R values were 0.869, 0.906 and 0.867 in the optimum conditions, compared to the R values of 0.796, 0.881 and 0.840 in non-optimal conditions ,, indicating that GA-ANN showed thw best in capturing nonlinear behavior.
A kind of endoglucanase from the extracellular crude enzyme of SD1-4 was purified and concentrated by PEG, ammonium sulfate precipitation, ethanol washing, Sephadex G-200, DEAE A-50 and Sephadex G-200. The enzyme was monomer with a molecular weight, about 38KD and visuated in a band in SDS-PAGE. It could hydrolyze CMC-Na, and the optimum temperature was 60℃and pH 7.0. Vmax and Km, were 22.57 U·ml-1·min-1 and 0.14%. respectively. It wass found that 0.2 mM Co2+ added to media had a strong role in promoting enzyme,activity, but 0.8 mM Co2+ showed inhibitory effect on the enzyme activity.
对SD1-4液态发酵产酶条件进行优化,结果表明:初始pH为8.0,最适温度为39℃,最适氮源为酵母膏,最佳无机盐添加量为60mgKCI和15mg CaCl2,最佳灭菌时间为40min、最适装液量为50ml/250ml;0.15%Triton X-100抑制产酶,0.05%Triton X-100促进产酶,PEG8000和吐温-20两种浓度均呈促进作用,SDS均呈抑制作用;第9天产酶达到高峰。
对天然碳源(菌草)的研究表明:象草叶为最适碳源;筛选能源草的具体指标为高纤维素、低直链淀粉、低葡萄糖、低果糖、高蔗糖、高蛋白质含量;筛选种质资源的统计学工具为相关分析、主成分分析、回归分析和聚类分析,而灰色关联分析和通径分析不适合。象草营养成分含量(可溶性糖为9.04%、可溶性蛋白质为3.37%时,淀粉含量为5.95%时,粗纤维素含量为65.95% )为象草发酵的最佳成分含量。
采用Box-Bohnken设计,以最终的纤维素酶产量作为研究目标,构建纤维素酶产量(内切酶、外切酶和糖苷酶)和7个因子(温度、pH、颗粒大小、蛋白胨浓度、湿度、接种量和时间)之间的关系模型,比较了GA-ANN、ANN和RSM在优化能力上的差异,优化条件下为0.869、0.906和0.867(R值),相比不优化条件的0.796、0.881和0.840,表明GA-ANN在捕捉系统非线性行为的独特优势。
通过PEG浓缩、硫酸铵沉淀、乙醇洗涤和Sephadex G-200、DEAE A-50、Sephadex G-200,从SD1-4胞外粗酶液中分离得到一种内切葡聚糖酶,经SDS-PAGE检测为单一条带,分子量约为38KD,能水解CMC-Na,最适温度为60℃和pH值为7.0,Vmax、Km分别为22.57U·ml-1·min-1和0.14%;0.2mM Co2+对该酶有较强的促进作用,0.8 mM Co2+对该酶有弱抑制作用。
In this study, a cellulose-degrading Actinomyces called SD1-4 screened from different habitats was identified as Streptomyces (Streptomyces sp.) based on the result of morphological, physiological and molecular studies.
Liquid fermentation conditions of the strain SD1-4 were optimized for enzyme production, the results show that the initial pH was 8.0,, and optimum temperature was set at 39℃, and the optimum nitrogen source was yeast extract, the best salt addition level was 60 mgKCI and 15mg CaCl2, the best Sterilization time 40 min, the optimum liquid volume 50ml/250ml. The result also indicated that 0.15% Triton X-100 inhibit the enzyme production, reverse was true at 0.05% Triton X-100 treatment , PEG8000 and Tween -20 at two treatment concentrations showed promotion on the enzyme, however, SDS showed inhibitory effect on it; for enzyme production reached to the peak.detected at the first 9 days
The result also showed Pennisetum purpereum Schumach was the most suitable carbon source; the index of selection energy grass was high in the contents of fiber, sugar , and proteins but low in the contents of amylose, glucose and fructose. were relatation analysis, principal component analysis, regression analysis and cluster analysis were used as the optimal statistical tools, but gray correlation analysis and path analysis were not suitable for screening. The nutrient content (9.04% soluble sugar, soluble protein was 3.37%, the starch content of 5.95%, the crude fiber content of 65.95%) of Pennisetum purpereum Schumach was the best fermentation ingredients.
Designed by Box-Bohnken a related model has been constructed between cellulase production (endogulucanase, exoglucanase and glucosidase) and seven factors (temperature, pH, particle size, peptone concentration and humidity, inoculum and time). The difference of three models (GA-ANN, ANN and RSM) in optimization was compared. The R values were 0.869, 0.906 and 0.867 in the optimum conditions, compared to the R values of 0.796, 0.881 and 0.840 in non-optimal conditions ,, indicating that GA-ANN showed thw best in capturing nonlinear behavior.
A kind of endoglucanase from the extracellular crude enzyme of SD1-4 was purified and concentrated by PEG, ammonium sulfate precipitation, ethanol washing, Sephadex G-200, DEAE A-50 and Sephadex G-200. The enzyme was monomer with a molecular weight, about 38KD and visuated in a band in SDS-PAGE. It could hydrolyze CMC-Na, and the optimum temperature was 60℃and pH 7.0. Vmax and Km, were 22.57 U·ml-1·min-1 and 0.14%. respectively. It wass found that 0.2 mM Co2+ added to media had a strong role in promoting enzyme,activity, but 0.8 mM Co2+ showed inhibitory effect on the enzyme activity.
引文
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