脂解麻疯树油脂肪酶产生菌的筛选鉴定及其催化特性

[目的]分离筛选具有脂解麻疯树油能力的脂肪酶产生菌株,为以麻疯树油为原料酶法生产生物柴油奠定基础.[方法]以麻疯树油为唯一碳源,从麻疯树种子粉末处理过的土壤中分离筛选出1株具有脂解疯树油能力的脂肪酶产生菌,考察该菌株及其脂肪酶对有机溶剂耐受性以及脂肪酶催化酯化和转酯反应的能力,并通过生理生化特征和16S rDNA序列分...     

脂肪酶假单胞菌的分离培养及最佳产酶条件研究

以麻疯树油为唯一碳源,从以粉碎的麻疯树种子处理过的土壤中分离筛选出1株脂肪酶活性较高的菌株,初步鉴定为假单胞菌属(Pseudomonas).实验观察了碳源、氮源、无机盐及发酵工艺对产酶的影响,摇瓶发酵结果表明.该菌株最适产酶培养基的组成是(%,w/v):橄榄油2,酵母膏0.5,(NH4)2SO4 0.5,MgCl2·6H2O 0.5,最适产酶温度为30℃,最佳产酶pH为6.5,转速180r/min,发酵培养36h酶活达到最高,为14.17U/mL.本研究为以麻疯树油为原料酶法生产生物柴油奠定了一定的基础.     

脂肪酶二步法催化鱼油下脚料富集DHA

采用Pseudomonas sp．脂肪酶水解,Geotrichum sp．脂肪酶选择性酯化的两步法显著提高游离脂肪酸（FFA）中二十二碳六烯酸（DHA）的含量。通过筛选合适的底物醇,优化水解和选择性酯化反应条件达到富集DHA的目的。结果表明月桂醇为选择性酯化的最适底物醇。确定的最佳水解条件：4g反应底物,m（水）／m（粗鱼油）=1,1000 U Pseudomonas sp．脂肪酶,40℃,搅拌速度200r／min,反应24h;最佳酯化条件：3g反应底物,n（FFA）／n（醇）=1／2,1000 U固定化Geotrichum sp．脂肪酶,1g正己烷,30℃,200r／min搅拌,反应20h。经过水解和一次选择性酯化反应后,DHA含量从原料粗鱼油中的18．9％提纯到72．8％;经二次选择性酯化后,DHA含量上升到92％。脂肪酶水解-选择性酯化的两步法是富集DHA的有效方法。     

思茅松毛虫肠道产脂肪酶菌株的筛选鉴定及酶学性质初步研究

利用纯培养和筛选培养,从思茅松毛虫幼虫肠道中分离得到7株产脂肪酶的菌株.通过提取基因组DNA并进行16S rDNA序列测定,构建产酶菌株的系统发育树,初步鉴定结果显示:菌株D2、D12、D19属于假单胞菌属(Pseudomonas sp.),菌株D7、D17属于芽胞杆菌属(Bacillus sp.),菌株D9、D16属于克雷伯氏菌(Klebsiella sp.).初步研究所产脂肪酶的酶学性质,确定这些酶的最适作用温度30～40℃、最适作用pH值8.0～9.0,为中温碱性脂肪酶.     

UV与DES复合诱变选育高产脂肪酶菌株

以产脂肪酶菌株BaciUus sp CS-4为出发菌株,进行了UV与硫酸二乙酯(DES)复合诱变处理.筛选出一株高酶活的目的菌株,命名为Bacillus spDE-8.其酶活为每毫升14.85U,比出发菌株提高48.2%.传代实验证明,其遗传性能稳定.     

产脂肪酶嗜碱细菌的筛选及酶学性质研究

目的:筛选产脂肪酶嗜碱细菌,并研究其酶学性质.方法:以豆油为唯一碳源的固体平板筛选产酶菌株,16S rDNA同源性分析确定微生物菌属,单因素实验优化产酶条件、研究酶学性质.结果:筛选出1株产脂肪酶的嗜碱菌株,鉴定为假单胞菌(Pseudomonas),命名为Pseudomonas sp.C-36.菌株产酶的最佳培养条件为:甘油2%(V/V).蛋白胨0.7%(W/V),酵母提取物0.5%(W/V),K2HPO4 0.2%(W/V),MsS04·7H2O 0.05%(W/V),NaCl 0.3%(W/V),Triton X-100 0.01%(W/V),pH 9.5,转速180r/min,37℃下培养24h,产酶量为2.782 IU/ml.该酶的最适温度和pH分别为40℃和9.0,50℃时酶活半衰期为2h.Ca2+等金属离子对该酶酶活具有促进作用,而Zn2+对酶活的抑制作用明显.有机溶剂的耐受性实验表明,该酶在疏水性有机溶剂中稳定性良好.结论:筛选得到1株嗜碱的脂肪酶产生菌C-36,并鉴定为Pseudomonas.     

豆粕水解液为氮源发酵产脂肪酶的研究

研究了以豆粕水解液作为氮源,假丝酵母Candida sp．99—125发酵生产脂肪酶的过程。分析水解时间对于产酶的影响,对比豆粕水解前后作为氮源发酵时的产酶规律。在30L发酵罐中批次发酵酶活最高可达6000IU／mL,采用豆油反馈流加之后,发酵脂肪酶活力可达8500IU／mL。     

酵母菌促进铅和镉胁迫下麻疯树生长的研究

为了筛选对铅和镉具有抗性和吸附性的酵母菌,构建麻疯树根系-酵母菌联合修复体系,促进高浓度铅和镉胁迫下麻疯树的生长。该研究分别从麻疯树的根段、珙桐的茎段、珙桐的根段分离到3株具有铅、镉抗性的酵母菌,分别命名为Jc、Di1、Di2,测定了三者对铅、镉的抗性和吸附性,并将筛选出的2株能吸附铅、镉的酵母菌菌株接种到麻疯树幼苗,研究接种两种酵母菌的麻疯树植株对铅、镉胁迫的响应。结果表明:经形态学和生理生化特征观察,Jc初步鉴定红酵母属(Rhodotorula sp.),Di1为假丝酵母属(Candida sp.),Di2为德巴利酵母属(Debaryomyces sp.)。三种酵母菌对铅、镉都有一定的抗性,其抗性能力的大小为JcDi2Di1。Di1和Jc对铅和镉都具有一定的吸附性将其用于接种麻疯树幼苗。与不接种酵母菌(CK)的麻疯树植株相比,接种Di1和Jc的麻疯树植株在根、茎、叶、全株干重方面显著增加,叶绿素、全株氮、全株磷浓度显著增加,SOD、POD、CAT的活性提高,丙二醛(MDA)浓度显著下降。从综合接种效应来看,Jc、Di1作为铅、镉的钝化剂,是铅、镉胁迫下促进麻疯树生长的备选菌株,这对于提高麻疯树对铅、镉污染土壤修复效率具有重要的意义。     

Burkholderia sp.ZYB002中lipA基因的敲除对细胞脂肪酶活性的影响

Burkholderia sp.ZYB002菌株不仅能产生大量的胞外脂肪酶,还能产生大量的细胞结合脂肪酶。对细胞结合脂肪酶的种类进行定性研究,将为开发全细胞脂肪酶催化剂奠定基础。通过分析与Burkholderia sp.ZYB002菌株具有较高同源性的Burkholderia cepacia J2315菌株的脂肪酶基因家族,推测潜在的细胞结合脂肪酶编码基因。通过同源重组插入失活Burkholderia sp.ZYB002菌株的lipA基因,筛选出突变体转化子;测定突变体菌株细胞结合脂肪酶的活性,并与野生型菌株比较。试验结果表明,lipA基因插入失活的Burkholderia sp.ZYB002-ΔlipA菌株的细胞结合脂肪酶活性降低了42%。     

渤海沉积物产脂肪酶细菌的筛选及其多样性分析

【目的】从渤海沉积物中分离筛选产脂肪酶细菌,分析其物种多样性,增加人们对渤海生态系统中产脂肪酶菌多样性的认识,获取高效产脂肪酶菌株,为海洋产脂肪酶微生物的挖掘提供菌群资源。【方法】分别将8个渤海沉积物样品梯度稀释涂布至吐温-80筛选平板和三丁酸甘油酯筛选平板,选择性分离产脂肪酶细菌;分析基于16SrRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;利用对硝基苯酚法测定胞外脂肪酶活性,筛选出高效产脂肪酶菌株。【结果】从8个渤海沉积物样品中分离获得51株产脂肪酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria和Firmicutes三个门的8个属,其中Pseudoalteromonas(35.2%)、Marinobacter(23.5%)和Sulfitobacter(17.6%)是优势菌群;脂肪酶酶活性实验表明所有测定菌株都能够分泌脂肪酶,菌株70623分泌的脂肪酶酶活最高,为42.4 U/m L。【结论】渤海沉积物中可培养产脂肪酶细菌类群较为丰富,Pseudoalteromonas、Marinobacter和Sulfitobacter菌株是优势菌群,测定菌株所产胞外脂肪酶能力不同,获得了一株高效产脂肪酶菌株Marinobacter sp.70623。     

Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect

The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.     

Rational strategy for the production of new crude lipases from <Emphasis Type="BoldItalic">Candida rugosa</Emphasis>

Candida rugosa was cultured using different inducers (oleic acid, olive oil, sunflower oil, n-dodecanol and glycerol) as the only carbon source in batch conditions, as well as in several fed-batch fermentations (oleic acid as inducer) at variable feed rate conditions. The N-terminal analysis of each crude lipase revealed that, while the isoenzymes Lip2 and Lip3 are always secreted (at different proportions depending on the inducer), Lip1 was produced only using n-dodecanol (batch conditions) or oleic acid (fed-batch at high feed rate). The nature of the inducer controls the isoenzyme percentage; when this is fixed, as well as the feed rate in fed-batch fermentation, the isoenzymatic profile remained unaltered and the samples differed only in the activity of the lipases, as determined by heptyl oleate synthesis.     

微球菌的脂肪酸合成调节

本文介绍了微球菌（Micrococcussp）1504胞外脂肪酶的代谢调节模式。发现在发酵培养基中,通过补加长链脂肪酸及其油酯,能提高脂肪酶的生产水平,而中、短链脂肪酸及其酯抑制产酶能力;高浓度的葡萄糖抑制产酶能力;补加不同浓度的橄揽油,适宜补加时间也应不同。水洗细胞脂肪酶的合成受橄榄油和油酸诱导,受葡萄糖和甘油阻遏,并发现脂肪酶的分解代谢阻遏主要发生在转录水平上。     

Enzymatic transesterification of Jatropha oil

Background  

Transesterification of Jatropha oil was carried out in t-butanol solvent using immobilized lipase from Enterobacter aerogenes. The presence of t-butanol significantly reduced the negative effects caused by both methanol and glycerol. The effects of various reaction parameters on transesterification of Jatropha oil were studied.     

Studies on Lipase in Rat Brain

Lipase activity was measured in homogenates of rat cerebral hemispheres using radioactive glycerol trioleate emulsified with Triton X-100 as substrate. The labeled oleic acid was separated from the ester with a methanol-chloroform-heptane mixture. Under these assay conditions, the activity showed pH optima at about 5.5 and 7.5. The final products of these lipase activities were suggested to be free fatty acid and glycerol.     

Lipase catalyzed methanolysis to produce biodiesel: Optimization of the biodiesel production

A lipase from Candida sp., suitable for transesterification of fats and oils to produce fatty acid methyl ester (FAME), was immobilized on a cheap cotton membrane, in this paper. The conversion ratio of salad oil to biodiesel could reach up to 96% with the optimal reaction conditions. Continuous reaction in a fixed bed reactor was also investigated. A three-step transesterification with methanol (methanolysis) of oil was conducted by using a series of nine columns packed with immobilized Candida sp. 99–125 lipase. As substrate of the first reaction step, plant or waste oil was used together with 1/3 molar equivalent of methanol against total fatty acids in the oil. Mixtures of the first- and second-step eluates and 1/3 molar equivalent of methanol were used for the second- and third-reaction steps. A hydrocyclone was used in order to on-line separate the by-product glycerol after every 1/3 molar equivalent of methanol was added. Petroleum ether was used as solvent (3/2, v/v of oil) and the pump was operated with a flow rate of 15 L/h giving an annual throughput of 100 t. The final conversion ratio of the FAME from plant oil and waste oil under the optimal condition was 90% and 92%, respectively. The life of the immobilized lipase was more than 10 days. This new technique has many strongpoints such as low pollution, environmentally friendly, and low energy costs.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

Synthesis citric and isocitric acids by Yarrowia lipolytica during yeasts growth on rapeseed oils

The native strain Yarrowia lipolytica VKM Y-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapeseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.