丙酮酸在增强丙酮酸脱羧酶对苯甲醛稳定性中的作用。

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde.

作者信息

Rosche Bettina, Breuer Michael, Hauer Bernhard, Rogers Peter L

机构信息

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

出版信息

J Biotechnol. 2005 Jan 12;115(1):91-9. doi: 10.1016/j.jbiotec.2004.08.002.

DOI:10.1016/j.jbiotec.2004.08.002

PMID:15607228

Abstract

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.

摘要

在低缓冲液浓度（20 mM MOPS）下，产朊假丝酵母丙酮酸脱羧酶（PDC）催化苯甲醛和丙酮酸转化为（R）-苯基乙酰甲醇（PAC）的反应，通过添加乙酸维持中性pH得以增强。PDC在这种缓冲液中非常稳定（6℃下半衰期为138小时），然而400 mM的苯甲醛乳液会导致其迅速失活。加入2M甘油并不能保护PDC免受苯甲醛的失活作用，但初始反应速率提高了50%，最终PAC水平从40 g l⁻¹提高到51 g l⁻¹。无论有无2M甘油，都会产生少量副产物乙醛（0.1 - 0.15 g l⁻¹）和乙偶姻（1.1 - 1.3 g l⁻¹）。有趣的是，当存在丙酮酸时，PDC对苯甲醛更稳定：在生物转化的第一个小时内没有活性损失（2M甘油，苯甲醛浓度从400 mM降至345 mM，丙酮酸从480 mM降至420 mM），但在没有丙酮酸的情况下，当暴露于相同浓度的苯甲醛时，PDC在不到30分钟内就完全失活。因此，催化作用中的酶比静止酶更稳定。