A NOVEL CHOLESTEROL ESTER HYDROLASE FROM PSEUDOMONASSPEC

Abstract

The genefor a cholesterol ester hydrolase (CE) [E.C.3.1.1.13] of a Pseudomonas spec. strain was cloned in pBR322 by screening a plasmid library with synthetic oligonucleotides derived from tryptic fragments ofthe purified enzyme. Subsequent sequence analysis of two overlapping clones showed a large open reading frame of 0.9 kb predicting a protein with a molecular weight of 30.700 kD. It contains a consensus-like signal peptide (SP) of 24 amino acids and a conserved sequence around the putative active center which is homologousto that of some other prokaryotic as well as eukaryotic esterases. No enzymatic activity as well as no immunological reaction could be detected in E. coli cells harbouring the CE-gene with 1.5 kb of its own upstream sequence on high copy plasmids. In order to improve gene expression the CE-gene was cloned behind strong E. coli promoters, the translation initiation region was optimized and several modifications of the SP-sequence were tested. The amount of CE after induction was 5 to 10 % oftotal protein but all of the enzyme was present in inclusion bodies. Again no enzymatic activity could be detected neither inside the E. coli cells nor in the culture medium. As the production of an enzymatically active CE in E. coli was not feasible, the whole expression cassette including promoter, SP and CE-gene was cloned into mobilizing vector pBT306-1, a vector system compatible for gram-negative organisms. Transconjugants with this vector in a CEnegative Pseudomonas strain showed fivefold higher CE-activity than the original strain. Using the same plasmid no activity could be observed in Pseudomonas putida strain 2440. As CE is a lipoprotein we assumethat specific lipids are necessary for enzymatic activity.