Malherbe D F, du Toit M, Cordero Otero R R, van Rensburg P, Pretorius I S
Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, 7600 Stellenbosch, South Africa.
Appl Microbiol Biotechnol. 2003 Jun;61(5-6):502-11. doi: 10.1007/s00253-002-1208-0. Epub 2003 Feb 11.
There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.
消费者对酒精和化学防腐剂含量较低的葡萄酒的需求日益增长。本研究的目的是在酿酒酵母中表达黑曲霉编码葡萄糖氧化酶（GOX；β - d - 葡萄糖：氧氧化还原酶，EC 1.1.3.4）的基因，并评估转化体在发酵过程中降低酒精产量以及抑制葡萄酒变质微生物（如醋酸菌和乳酸菌）的能力。将黑曲霉结构葡萄糖氧化酶（gox）基因克隆到一个整合载体（YIp5）中，该载体包含酵母交配信息素α - 因子分泌信号（MFalpha1(S)）以及磷酸甘油酸激酶 - 1基因启动子（PGK1(P)）和终止子（PGK1(T)）。将PGK1(P) - MFalpha1(S) - gox - PGK1(T)盒（命名为GOX1）导入酿酒酵母的实验室菌株（Sigma1278）。在选择性琼脂平板和液体试验中分析酵母转化体产生生物活性葡萄糖氧化酶的情况。结果表明，重组葡萄糖氧化酶具有活性，在指数生长期早期开始产生，并在稳定期达到稳定水平。酵母转化体在平板试验中对乳酸菌和醋酸菌也表现出抗菌活性。这可能是因为GOX酶促反应的最终产物是过氧化氢，一种已知的抗菌剂。用实验室酵母转化体进行小规模酿酒，得到的葡萄酒酒精含量降低了1.8 - 2.0%。这可能是由于GOX将葡萄糖转化为d - 葡萄糖酸 - δ - 内酯和葡萄糖酸所致。这些结果为开发用于生产化学防腐剂和酒精含量降低的葡萄酒的葡萄酒酵母起始培养菌株铺平了道路。
