Cloning and expression of a gene with phospholipase B activity from Pseudomonas fluorescens in Escherichia coli
2012
Jiang, Fangyan | Huang, Shen | Imadad, Kaleem | Li, Chun
A gene from Pseudomonasfluorescens BIT-18 encoding a protein with phospholipase B activity (Pf-PLB) was cloned in E. coli BL21 (DE3). The open reading frame consists of 1272bp and potentially encodes a protein of 423 amino acid residues with a calculated molecular mass of 45.8kDa. The nucleotide sequence of Pf-PLB is 45%, 42%, 41%, 40%, 33%, and 31% identical to that of Bifidobacteriumanimals, Mycobacteriumparascrofulaceum, Acidobacteriumcapsulatum, Lactobacillusjohnsonii, Moraxellabovis, and Moraxellacatarrhalis, respectively. The His-tagged protein was purified by affinity chromatography and the eluted protein hydrolyzed both the 1- and 2-ester bond of phosphatidylcholine. The recombinant Pf-PLB had optimal activity at pH 6.0 and 30°C, and it showed 20.1% higher efficiency in the conversion rate of the phosphorus content than the wild-type.
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