摘要
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。
Objective： This study aimed to construct Pichia pastoris which can highly expressed β - mannanase, and to study the enzyme - producing level. Method ： We identified a mannanase - producing strain isolated from konjac soil. The mannanase - producing strain were grown on selective plates （LB; 0.5% konjac glucomannan; 0.1% Congo Red; 2. 0% agar）. Sequence analysis of the 16S rDNA fragment of the strain was conducted , and the strain was identified as Bacilus sp, named Bacillus sp. QYW - 1 （ GenBank Accession Number JX524224）. Using degenerate polymerase chain reaction a new β- mannanase gene, denoted as manW（ GenBank Accession Number JX869490 ） , was obtained from Bacillus sp. QYW - 1 genome. An open reading frame （ORF） of 1 084 bp encoded a protein of 360 amino acids including a putative 25 - residue signal peptide and belonging to Glycosyl hydrolase family 26. The expressed enzyme had a molecular mass of approximately 40 kD determined by SDS - PAGE. A codon optimized β - mannanase gene from Bacillus sp. QYW - 1 cleaved by EcoR I and Not I was inserted into the expression vector pPIC9K,downstream of an α -factor signal peptide sequence, and transformed into Escherwhia coli DH5α. The resulting plasmid linearized with Bgl I was transformed into Pichia pustoris GS115. Result： After exten- sive screening, the recombinant strain manW1 that expresses the secretory protein at high level was obtained. Fermentation conditions and enzymatic characteristics were studied preliminarily. Optimum pH and temperature for the recombinant mannanase was 6. 5 and 37℃, re- spectively. The recombinant enzyme was stable at pH 5.0 - 7.4 and maintained over 40% of original activity after incubation at 70℃ for 20min. β -mannanase activity was assayed using 3,5 - dinitrosalicylic acid （DNS） method. The activity of the recombinant mannanase reached 1 180 U/mL. Conclusion：The recombinant β -mannanase has a better prospect.
出处
《生物技术》
CAS
CSCD
北大核心
2013年第2期18-22,共5页
Biotechnology
基金
国家高技术研究发展计划(863计划)项目("糖链修饰与糖衍生物的研制"
2012AA021504)资助
