In:
The FEBS Journal, Wiley, Vol. 280, No. 16 ( 2013-08), p. 3780-3796
Abstract:
A novel metal ion‐independent phospholipase B ( PLB 684 ) from S treptomyces sp. strain NA684 was purified 264‐fold from the culture supernatant with 2.85% recovery (6330 U·mg protein −1 ). The enzyme functions as a monomer with a molecular mass of 38.9 kDa. Maximum activity was found at pH 8.4 and 50 °C. The substrate specificity was in the order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine 〉 phosphatidylserine 〉 phosphatidylinositol 〉 phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB 684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent K m , V max and k cat for hydrolysis of dimyristoyl phosphatidic acid were 14.5 m m , 15.8 mmol·min −1 ·mg protein −1 and 1.02 × 10 4 s −1 , respectively. The positional specificity of 1‐palmitoyl‐2‐oleoyl‐ sn ‐glycero‐3‐phosphocholine hydrolysis was investigated using GC . In the reaction equilibrium, the molar ratio of released fatty acids ( sn ‐1 : sn ‐2) was 45 : 55. The ORF of the gene is 1239 bp in length and codes for a 30‐amino acid signal peptide and a 382‐amino acid mature enzyme. The deduced amino acid sequence of PLB 684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001 (UniProt accession number: J1RQY0 ). The extracellular production of PLB 684 was achieved using a pUC702 expression vector and S treptomyces lividans as the host. Mutagenesis analysis showed that S er12 is essential for the catalytic function of PLB 684 and that the active site may include residues S er330 and H is332.
Type of Medium:
Online Resource
ISSN:
1742-464X
,
1742-4658
DOI:
10.1111/febs.2013.280.issue-16
Language:
English
Publisher:
Wiley
Publication Date:
2013
detail.hit.zdb_id:
2172518-4
SSG:
12
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