Biocatalytic production of useful esters by two forms of lipase from Candida rugosa

Two distinct lipase forms were obtained from Candida rugosa lipase by Phenyl Sepharose hydrophobic interaction and DEAE Sepharose ion exchange chromatography, L1 at 45% yield and L2 at 4·7% yield. Both purified lipases were able to catalyse esterification of 1-butanol and oleic acid and trans-esterification of 2-ethyl-1-hexanol and rapeseed oil. Lipase L1 gave a 98% yield for esterification over 12 h and a 99% conversion of rapeseed oil for trans-esterification over 24 h. The minor fraction L2 gave a 97% yield for esterification over 30 h and only a 79% conversion for trans-esterification over 24 h. The superiority of fraction L1, especially in trans-esterification, could be clearly shown by reversed phased HPLC analysis. Sodium deoxycholate treatment of the purified main lipase L1 considerably improved the initial rate in both esterification and trans-esterification.     

Chemoenzymic synthesis of optically active 3-methyl- and 3-butylphthalides

Optically active (S)-3-methylphthalide was synthesized by the enzymic reduction of 2-iodoacetophenone using baker's yeast, followed by palladium-catalysed carbonylation under carbon monoxide. With enzymic reduction using baker's yeast, however, 2-bromo- or 2-iodovarelophenones were not reduced, even after 25 days. On the other hand, the enantioselective hydrolysis of α-alkylated 2-halobenzyl acylates using pig liver esterase, horse liver esterase or lipase from Candida rugosa resulted in the formation of (R)-α-alkylated 2-halobenzyl alcohol and (S)-acylate, and the following palladium-catalysed carbonylation of the products yielded the (R)- and (S)-3-alkylated phthalides, respectively.     

Enzymatic modification of evening primrose oil: Incorporation of n−3 polyunsaturated fatty acids

Immobilized lipase SP435 fromCandida antaractica was used as a biocatalyst for the modification of the fatty acid composition of evening primrose oil by incorporating n−3 polyunsaturated fatty acid (PUFA) and eicosapentaenoic acid (EPA). Transesterification (ester-ester interchange) was conducted in organic solvent or without solvent, with EPA ethyl ester (EEPA) as the acyl donor. Products were analyzed by gas-liquid chromatography (GLC). After 24-h incubation in hexane, the fatty acid composition of evening primrose oil was markedly changed to contain up to 43% EPA. The amount of 18:2n−6 PUFA was reduced by 32%, and the saturated fatty acid content was also reduced. The effects of incubation time, molar ratio, enzyme load, and reaction medium on mol% EPA incorporation were also studied. Generally, as the incubation time (up to 24 h), molar ratio, and enzyme load increased, EPA incorporation also increased. Evening primrose oil, containing EPA and γ-linolenic acid (18:3n−6) in the same glycerol backbone, was successfully produced and may be more beneficial for certain applications than unmodified oil.     

Enzymatic synthesis of trierucin from high-erucic acid rapeseed oil

The lipase fromCandida rugosa has been shown to discriminate against erucic acid. Advantage of this property has been taken to produce trierucin from high-erucic acid rapeseed (HEAR) oil. A method has been developed for extracting erucic acid from the oil as dierucin and subsequently enzymatically converting it to trierucin. Unrefined HEAR oil was hydrolyzed with lipase fromC. rugosa to produce a mixture of free fatty acids and dierucin. Precipitation and filtration from cold ethanol gave 73% pure dierucin, free of fatty acids. This dierucin was treated in two ways to produce trierucin. First, in the presence of an immobilized lipase and a known amount of water, some trierucin is produced by interesterification. Second, a more efficient route to trierucin utilizedRhizopus arrhizus lipase to completely hydrolyze dierucin to erucic acid, which was then combined with an appropriate amount of dierucin in the presence of an immobilized lipase to produce trierucin in a quantitative yield. Partly presented at the AOCS Annual Meeting held in Toronto, May 10–14, 1992.     

不同生长期的低毒大麻叶浸膏抑菌作用的研究

目的:研究不同生长期低毒大麻叶浸膏的抑菌作用.方法:采用乙醇作溶剂对大麻叶药用成分进行提取,并且对其浸膏进行抑菌实验研究.结果:4月份大麻叶浸膏(DML4)和9月份(DML9)大麻叶浸膏对金黄色葡萄球菌、大肠杆菌、绿脓杆菌有明显(p<0.001)的抑制作用,而DML4对白色念珠菌的抑制作用很微弱,DML9对白色念珠菌的抑制作用有所提高.结论:同浓度的DML9(成熟的)比DML4(未成熟)大麻叶具有明显(p<0.001)的抑菌作用.     

皱褶假丝酵母脂肪酶催化水解大豆油工艺优化及机制研究北大核心CSCD

为了降低脂肪酶催化水解植物油脂的应用成本,以皱褶假丝酵母脂肪酶(CRL)为催化用酶,对比了CRL催化水解不同植物油脂的效果,同时以大豆油为研究对象,采用单因素实验考察pH、反应温度、反应时间、水油体积比和酶用量对水解率的影响,并运用响应面法优化工艺条件,进而对CRL催化甘油三酯水解的机制进行了总结。结果表明:CRL催化水解C脂肪酸甘油酯的选择性较高;CRL催化大豆油水解的最优工艺条件为反应温度39.5℃、pH 6.7、水油体积比1∶2.2、酶用量0.6%(以大豆油质量计),在此条件下反应2 h水解率可达77.21%,反应12 h水解率可达96.33%;CRL催化水解甘油三酯的反应机制主要为亲核催化三联体中的丝氨酸对甘油三酯的羰基进行亲核反应以夺取脂肪酸前体,然后水分子对该前体进行亲核反应释放脂肪酸。CRL能够在温和条件下高效催化植物油脂生产脂肪酸,是一种具有广阔应用前景的生物催化剂。     

A comparative study on the transport of L(-)malic acid and other short-chain carboxylic acids in the yeast Candida utilis: Evidence for a general organic acid permease

Cells of the yeast Candida utilis grown in medium with short-chain mono-, di- or tricarboxylic acids transported L(-)malic acid by two transport systems at pH 3·0. Results indicate that probably a proton symport for the ionized form of the acid and a facilitated diffusion for the undissociated form were present. Dicarboxylic acids such as succinic, fumaric, oxaloacetic and α-ketoglutaric acids were competitive inhibitors of the malic acid for the high-affinity system, suggesting that these acids used the same transport system. In turn, competitive inhibition uptake studies of labelled carboxylic acid in the low-affinity range indicated that this system was non-specific and able to accept not only carboxylic (mono-, di- or tri-) acids but also some amino acids. Additionally, under the same growth conditions, C. utilis produced two mediated transport systems for lactic acid: a proton symport for the anionic form which appeared to be a common monocarboxylate carrier and a facilitated diffusion system for the undissociated acid displaying a substrate specificity similar to that observed for the low-affinity dicarboxylic acid transport. The mediated carboxylic acid transport systems were inducible and subjected to repression by glucose. In glucose-grown cells the undissociated dicarboxylic acids entered the cells slowly by simple diffusion. Repressed glucose-grown cells were only able to produce both transport systems if an inducer, at low concentration (0·5%, w/v), was present during starvation in buffer. This process was inhibited by the presence of cycloheximide indicating that induction requires de novo protein synthesis. If a higher acid concentration was used, only the low-affinity transport system was detectable, showing that the high-affinity system was also repressed by high concentrations of the inducer.     

A deviation from the universal genetic code in Candida maltosa and consequences for heterologous expression of cytochromes P450 52A4 and 52A5 in Saccharomyces cerevisiae

We demonstrate that serine instead of leucine is specified by the CUG codon in the yeast Candida maltosa. Evidence for this deviation from the universal genetic code was obtained by means of in vitro translation experiments. Depending on the cell-free system used, either serine, in the C. maltosa system, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG-containing mRNAs. Moreover, we were able to transfer the non-universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated from C. maltosa. This finding indicates the presence in C. maltosa of an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression in Saccharomyces cerevisiae of C. maltosa cytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the original C. maltosa genes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host cells.     

Factors affecting the acyltransfer activity of the lipase fromCandida parapsilosis in aqueous media

This study describes the influence of various factors on the hydrolysis and alcoholysis activities of the lipase fromCandida parapsilosis (Ashford), Langeron and Talice in aqueous media. Optimal activities were obtained at 45°C and pH 6–6.5. The influence of the nature of the substrate on the temperature activity profiles was observed. Total or partial recovery of the activities was obtained when methanol was added to the enzyme extract after thermal denaturation. A tyrosin residue appeared to be necessary for lipase function. Magnesium was a required metal cofactor. These activities were optimal in the presence of high amounts of water (water activity >0.9).