洋葱假单胞菌G63脂肪酶基因及其伴侣基因的克隆和同源高效表达

Cloning and homologous over expression of the lipase gene and its chaperone gene from Pseudomonas cepacia G63

从油污土壤中筛选到一株脂肪酶活性较高的洋葱假单胞菌（Pseudomonas cepacia）G63。运用PCR的方法从该菌株中克隆了脂肪酶基因（lip A）及其伴侣基因（lipB）。该脂肪酶基因开放式阅读框（ORF）为1092bp，编码364个氨基酸残基。经KpnI／HindiⅢ酶切，将其克隆到广泛宿主质粒pBBR1Tp载体上，构建成质粒pBBR1-lipAB。通过三亲杂交，在辅助质粒pRK2013的帮助下，转入原宿主菌P．cepaciaG63中，构建成洳A基因同源高效表达的基因工程菌。该菌株在连续转接5次，培养45h后质粒保持率为82．6％，适用于规模化发酵生产。摇床发酵表明，工程菌在60h时酶活达到150．63U／mL，较原始菌株提高3．6倍。

A lipase-producing strain （ Pseudomonal cepacia ） G63 was isolated from the oil polluted suil. The lipase gene lipA and its chaperone gene lipB uf P. cepacin G63 were cloned by PCR. The size the of open reading frame （ORF） uf lipA was 10921,p and encoded 364 amino acids. Digested with KpnⅠ anti HindⅢ, the lipA anti lipB were cluned into the broad host range plasmid pBBRITp, anti constructed the recombinant plasmids pBBR1-lipAB. The pBBR1-lipAB was transferred into P. cepacia G63 strain by a tri-parental mating, using the pRK2013 plasmid as a helper. After five times of trans-inoculation and 45 h culture, the stability of pBBRI-IipAB was 82.6%, indicating that the recumbinant strain is suitable for large scale lipase production. Fermenting in the 500 mL flasks for 6Oh, the enzyme activity of the recombinant P. cepacia G63＇ strain was 150.63 U/mL, 3.6-fold higher than that of the starting strain.