巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高表达  

High Expression of Penicillin G Acylase Gene from Bacillus megaterium in Bacillus subtilis

作　　者：杨晟[1] 黄艳红[1] 黄晓冬[1] 李士云[1] 袁中一[1] 

机构地区：[1]中国科学院上海生物化学研究所,上海200031

出　　处：《生物化学与生物物理学报》1999年第5期601-603,共3页

摘　　要：用 Ｐ Ｃ Ｒ 方法从巨大芽孢杆菌的基因组 Ｄ Ｎ Ａ 中扩增到青霉素 Ｇ 酰化酶基因， 并装载到枯草杆菌表达质粒ｐ Ｐ Ｚ Ｗ１０３ 中， 将其转化到枯草杆菌 Ｄ Ｂ１０４ 中进行了分泌表达。重组菌株产酶无需苯乙酸诱导。在３７ ℃培养２４ ｈ ，菌液中酶活力可达６ ｕ／ｍｌ。１０ 天的连续传代实验表明重组菌株的稳定性很高。发酵液上清中的青霉素 Ｇ 酰化酶经 Ａｌ２ Ｏ３ 层析， 再经ｐｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ４ Ｂ 疏水层析， 将其浓缩百倍以上， 两步总得率为７９ ％ ，The penicillin G acylase gene ( pga ) amplified by PCR from Bacillus megaterium was subcloned into an expressing vector pPZW103 ( P 43 as promoter). The recombinant plasmid was transferred into Bacillus subtilis DB104. Penicillin G acylase production in the B. subtilis transformant was 3—6 u/ml, higher than that of published recombinant strains. Penicillin G acylase production was induced by phenylacetic acid in B. megaterium , whereas the enzyme was produced constitutively in the B. subtilis transformant carrying B. megaterium pga. The recombinant strain showed high stability in antibiotic free medium for 10 days. Enzyme in crude broth was purified by Al 2O 3 chromatography and phenyl Sepharose CL 4B hydrophobic chromatography and the total yield is 79%. The purified enzyme with specific activity of 52 u/mg can be directly immobilized for use.

关 键 词：巨大芽孢杆菌 青霉素G酰化酶 枯草芽孢杆菌 基因表达 

分 类 号：Q939.124[生物学—微生物学] TQ465.1[化学工程—制药化工]