1887

Abstract

The structural gene encoding the extracellular lipase of MCC-2 was cloned and found to be expressed in using its own promoter. When the cloned gene () was expressed in minicells, an 80 kDa protein was identified. Subcellular fractionation of carrying the gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79.9 kDa protein with an estimated pl of 10.36. The deduced protein contains two putative signal peptide cleavage sites; one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [H]palmitate was incorporated into the Lip protein when expressed in . The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both and recombinant . In hydrolysis of -nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C to C for ester hydrolysis and C to C for triacylglycerol hydrolysis.

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1997-03-01
2021-08-05
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