海藻酸裂解酶基因在毕赤酵母中的表达及重组酶性质的初步研究

岳明; 丁宏标; 乔宇

海藻酸裂解酶基因在毕赤酵母中的表达及重组酶性质的初步研究

中国农业科学院, 饲料研究所, 北京, 100081

基金项目: 国家973计划课题(2004CB117500);国家863计划课题(2005AA246010);国际科技合作重点项目计划课题(2005DFA31070)联合资助

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出版历程
- 收稿日期: 2006-03-10
- 修回日期: 2006-07-16

Secreted expression of Azotobacter chroococcum alginate lyase gene(algL) in Pichia pastoris and primary analysis of enzymic properties

Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

摘要

摘要: 褐球固氮菌(Azotobacter chroococcum)是一种海藻酸降解菌.本实验采用PCR的方法,以褐球固氮菌基因组DNA为模板,克隆出约1.05 kb的海藻酸裂解酶基因algL的成熟蛋白编码序列,并将其插入巴斯德毕赤酵母(Pichia pastoris)表达载体pPIC9K中,获得重组质粒pPIC9K-algL.重组质粒线性化后用聚乙二醇(PEG)法导入毕赤酵母菌株GS115中,获得高效分泌表达海藻酸裂解酶的毕赤酵母工程菌株.用甲醇诱导培养基进行摇瓶发酵,表达得到43 kDa的目的蛋白,酶活力可达1 400 U/cm³左右.经测定,该重组酶的最适反应pH为8.5,最适反应温度为40℃,并且在20~55℃,pH2.0~11.0具有较好的稳定性.另外,10 mmol/cm³的Cu²⁺,Fe²⁺,Co²⁺,Mn²⁺和Ca²⁺对酶有不同程度的抑制作用.
- 海藻酸裂解酶 /
- 毕赤酵母 /
- 海藻寡糖 /
- 诱导表达
Abstract: An alginate lyase gene of Azotobacter chroococcum, called algL, has been cloned with PCR technique.The sequence encoding the mature peptide of alginate lyase of Azotobacter chroococcum was added with EcoRI and NotI.Then the gene was inserted into the Pichia pastoris vector pPIC9K and introduced into the host Pichia pastoris GS115 by PEG method.After screen, the recombinant Pichia pastoris strain was obtained and induced in 25mL methylot rophic culture medium.Expression of the algL gene in P.pastor is cells resulted in the expression of alginate lyase activity (1 400 U/cm³) and the appearance of a new protein of 43 kDa detected on SDS-PAGE.To characterize the recom binantenzyme, it was found that the optimum temperature is 40℃ at pH 8.5 and that activity of alginate lyase decreased rapidly when analyzed above 60℃.The activity of recom binant AlgL decreased by over 50% in the presence of 10 mmol/L Cu²⁺ or Fe²⁺, furthermore, 10mmol/L Co²⁺, Mn²⁺, Ca²⁺ or 1mmol/L Fe²⁺ decreased it by 20% respectively.Other cations including K⁺, Na⁺, Zn²⁺ did not affect significantly the enzymatic activity.
- alginate lyase /
- Pichia pastoris /
- algao ligosaccharides /
- expression induced

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