United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 North University Street Peoria, IL 61604, United States.
J Biotechnol. 2012 May 31;159(1-2):69-77. doi: 10.1016/j.jbiotec.2012.01.025. Epub 2012 Feb 11.
A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads® EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.
通过自动化 PCR 生成了编码合成的截断假丝酵母脂肪酶 B（CALB）的基因，并在酿酒酵母中表达。Western blot 分析检测到五种截断的 CALB 变体，表明从六个框内 ATG 密码子有多个翻译起始点。最长的开放阅读框，对应于成熟脂肪酶的 35-317 个氨基酸，似乎表达量最大。将截断的 CALB 固定在 Sepabeads® EC-EP 树脂上，并用于从粗玉米油和精炼大豆油中生产乙酯和丁酯。与使用相同底物的商业可用脂肪酶树脂（Novozym 435）相比，从玉米油中获得的乙酯产量增加了 4 倍，从大豆油中获得的乙酯产量分别增加了 36%和 50%。当乙醇与玉米油的比例为 5:1（v/v）时，产量分别增加了 3.7 倍和 8.4 倍，而乙醇与玉米油的比例分别为 15:1 和 30:1。对于玉米油，丁酯的产量比乙酯的产量高 56%。与仅使用 CALB 树脂相比，在 CALB 树脂之前添加离子催化树脂步骤可使玉米油中乙酯的产量增加 53%。结果表明，固定化的截断 CALB 在使用生物催化剂生产生物柴油方面具有潜在的应用。
