Development of an improved in vitro activity assay for medium chain length PHA polymerases based on Coenzyme A release measurements

被引：18

作者：

de Roo, G ^{[1
]}

Ren, Q ^{[1
]}

Witholt, B ^{[1
]}

Kessler, B ^{[1
]}

机构：

[1] ETH Zurich, Swiss Fed Inst Technol, Inst Biotechnol, Honggerberg HPT, CH-8093 Zurich, Switzerland

来源：

JOURNAL OF MICROBIOLOGICAL METHODS | 2000年 / 41卷 / 01期

关键词：

PHA polymerase; in vitro activity assay; medium chain length polyhydroxyalkanoates; Pseudomonas oleovorans;

D O I：

10.1016/S0167-7012(00)00129-9

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

An improved activity assay for polyhydroxyalkanoate (PHA) polymerases from Pseudomonas oleovorans GPol was developed. The activity assay is based on the detection of released Coenzyme A (CoA) using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), a compound which specifically reacts with thiol groups. The formed adduct was measured spectrophotometrically with high sensitivity and accuracy. The assay was used to study the effect of several additives on the activity of granule-associated PHA polymerase. Mild non-ionic detergents such as Tween-20, Triton X-100, CHAPS and Hecameg all appeared to be strongly inhibitory. In contrast, bovine serum albumin (BSA) had a strong stimulatory effect on the activity and stability of the PHA polymerases. Using optimized conditions, activities up to 5.8 U/mg granule-bound polymerase have been measured. (C) 2000 Elsevier Science B.V. All rights reserved.

引用

页码：1 / 8

页数：8

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