当前位置： SCI文献检索 > BIOTECHNOLOGY AND BIOENGINEERING期刊下所有文献 > Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms.

Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms.

Abstract：

:In this study, an analysis of the transition between the inactive ("closed") and active ("open") conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphates. It is shown that >90% inhibition of the enzyme activity toward water-soluble substrates (esterolytic activity) can be achieved with as little as 0.3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsified substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, which even exceeds that of the untreated control. These experimental observations suggest that the inhibitors interact predominantly, if not exclusively, with the open form of the enzyme and that any transitions occurring between the two conformers of the enzyme in solution, in the absence of an interface, are extremely slow. This conclusion is verified by separating the open and closed forms of the enzyme by hydrophobic interaction column chromatography on phenyl-sepharose. Fractions enriched with the respective conformations of the enzyme are further purified using gel-permeation chromatography. On the basis of the elution pattern from this step, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in the absence of interface) form of the lipase is found to be present in solution as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triolein.

journal_name

Biotechnol Bioeng

journal_title

Biotechnology and bioengineering

authors

Turner NA,Needs EC,Khan JA,Vulfson EN

doi

10.1002/1097-0290(20010105)72:1<108::aid-bit14>3.0

subject

Has Abstract

pub_date

2001-01-05 00:00:00

pages

108-18

issue

eissn

0006-3592

issn

1097-0290

pii

10.1002/1097-0290(20010105)72:1<108::AID-BIT14>3.0

journal_volume

pub_type

杂志文章

在线工具

Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms.