响应面法优化热泉菌Bacillus sp.Lc50-1的发酵产卡拉胶酶条件

采用响应面法对1株高产卡拉胶酶的印度尼西亚热泉菌Bacillus sp.Lc50-1的发酵条件进行优化。分别以培养温度、pH值、C源、N源、金属离子及接种量作为唯一变量进行单因素实验,筛选出对酶活有显著影响的单因素取值范围;参考单因素实验结果,再利用Box-Behnken设计及响应面分析法进行回归分析以确定最佳发酵条件。结果表明,培养温度、卡拉胶浓度(C源)、蛋白胨浓度(N源)、KCl浓度(金属离子)与酶活存在着显著的相关性,通过求解回归方程得到Bacillus sp.Lc50-1的最佳发酵条件为:培养温度47.5℃、蛋白胨0.25%、卡拉胶0.3%、KCl 21.55mmol·L-1,优化后发酵上清液的酶活达到8.9U·mL-1,比优化前提高了1.5倍。

Isolation of Cr(Ⅵ) reducing bacteria from industrial effuents and their potential use in bioremediation of chromium containing wastewater

The present study was aimed to assess the ability of Bacillus sp.JDM-2-1 and Staphylococcus capitis to reduce hexavalent chromium into its trivalent form.Bacillus sp.JDM-2-1 could tolerate Cr（Ⅵ）（4800 μg/mL） and S.capitis could tolerate Cr（Ⅵ）（2800 μg/mL）.Both organisms were able to resist Cd^2＋（50 μg/mL）,Cu^2＋（200 μg/mL）,Pb^2＋（800 μg/mL）,Hg^2＋（50 μg/mL） and Ni2＋（4000 μg/mL）.S.capitis resisted Zn^2＋ at 700 μg/mL while Bacillus sp.JDM-2-1 only showed resistance up to 50 μg/mL.Bacillus sp.JDM-2-1 and S.capitis showed optimum growth at pH 6 and 7,respectively,while both bacteria showed optimum growth at 37°C.Bacillus sp.JDM-2-1 and S.capitis could reduce 85% and 81% of hexavalent chromium from the medium after 96 h and were also capable of reducing hexavalent chromium 86% and 89%,respectively,from the industrial effuents after 144 h.Cell free extracts of Bacillus sp.JDM-2-1 and S.capitis showed reduction of 83% and 70% at concentration of 10 μg Cr（Ⅵ）/mL,respectively.The presence of an induced protein having molecular weight around 25 kDa in the presence of chromium points out a possible role of this protein in chromium reduction.The bacterial isolates can be exploited for the bioremediation of hexavalent chromium containing wastes,since they seem to have a potential to reduce the toxic hexavalent form to its nontoxic trivalent form.

β-甘露聚糖酶基因在毕赤酵母中的高效表达

目的:构建高效表达β-甘露聚糖酶的毕赤酵母工程茵,研究重组菌的产酶水平。方法:从魔芋地土壤中筛选出1株高产β-甘露聚糖酶的菌株;生理生化鉴定及16S rDNA测序结果为芽孢杆菌,命名为Bacillus sp.QYW-1(GenBank登录号JX524224);以Bacillus sp.QYW-1基因组DNA为模板,克隆获得一段1 084 bp的β-甘露聚糖酶基因manW(GenBank登录号JX869490),其编码360个氨基酸,分子量40 kD,属糖苷水解酶家族26;SignalP4.0分析含一段25个氨基酸的信号肽,将优化设计的β-甘露聚糖酶基因序列插入表达栽体pPIC9K中,获得重组质粒pPIC9K-manW;BglⅠ酶切线性化后电转化Pichia pastorisGS115中。结果:经大量筛选,获得高效表达的茵株manW1,摇瓶发酵结果:该酶最适pH 6.5,最适温度37℃,具有良好的热稳定性和耐酸性,酶活达1 180 U/mL。结论:重组酶具有良好的应用前景。