Immobilization of lipase onto metal–organic frameworks for enantioselective hydrolysis and transesterification

Enzyme immobilization enhances the catalytic activity and stability of the enzyme, and also improves reusability. Metal–organic frameworks (MOFs), which possess diversified structures and porosity, have been used as excellent carriers for enzyme immobilization. Pseudomonas fluorescens lipase (PFL) has been successfully immobilized onto MOFs by covalent cross-linking to obtain a series of immobilized lipase (PFL@MOFs). PFL@MOFs are used for catalytic enantioselective hydrolysis of 2-(4-hydroxyphenyl) propionic acid ethyl ester enantiomers (2-HPPAEE) in aqueous medium and transesterification of 4-methoxymandelic acid enantiomers (4-MMA) in organic medium. The experimental results indicated that PFL@Uio-66(Zr) exhibits excellent enzymatic catalysis performances and high enantioselectives. In addition, to improve catalytic activity and reusability, PFL is modified by the polyethylene glycol (PEG) to prepare PEG-modified lipase (PFL-PEG), then PFL-PEG is immobilized onto Uio-66(Zr) to prepare PFL-PEG@Uio-66(Zr), demonstrating better reusability and catalytic activity compared with PFL@Uio-66(Zr).     

生物酶法转化酵母油脂合成生物柴油

以一株高产油脂圆红冬孢酵母菌(Rhodosporidum toruloides Y4#)干菌粉为原料,利用酸热法提取了该酵母油脂,并对所得油脂进行了分析. 进一步利用该酵母油脂为原料分别研究了无溶剂体系中三步甲醇法及在叔丁醇介质体系中脂肪酶催化合成生物柴油,发现脂肪酶可以有效转化该酵母油脂制备生物柴油. 在优化反应条件下,生物柴油得率可达90%左右,略低于相同条件下利用精制大豆油合成生物柴油的得率.     

染料木黄酮抑制3T3-L1细胞成脂分化机制

目的：研究染料木黄酮是否通过雌激素受体介导影响3T3-L1前脂肪细胞成脂分化,并探讨其可能的机制。方法：以不同浓度染料木黄酮处理3T3-L1细胞,四甲基偶氮唑蓝（methyl thiazolyl tetrazolium,MTT）法测定细胞存活率;在3T3-L1前脂肪细胞成脂分化过程中分别加入不同浓度染料木黄酮、染料木黄酮和ICI182780（一种雌激素受体抑制剂）混合物及不同浓度雌二醇,油红染色观察分化结果,测定细胞甘油三酯（triglyceride,TG）含量;染料木黄酮、染料木黄酮和ICI182780混合物及不同浓度雌二醇处理诱导成熟后的脂肪细胞,测定培养液甘油含量;分别用染料木黄酮（30 μmol/L）、染料木黄酮（30 μmol/L）和ICI182780混合物干预细胞成脂分化,Western blotting法检测细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）、p-ERK、腺苷酸活化蛋白激酶（adenosine monophosphate activated protein kinase,AMPK）、p-AMPK、激素敏感性脂肪酶（hormone sensitive lipase,HSL）、脂肪酸合成酶（fatty acid synthetase,FAS）蛋白表达量。结果：3T3-L1细胞存活率随染料木黄酮浓度的升高而降低,50～200 μmol/L染料木黄酮能抑制细胞生长（P＜0.01）;染料木黄酮能负向调节成脂分化后细胞胞浆内TG含量,在0～50 μmol/L浓度范围内呈剂量依赖关系,高剂量雌二醇（100 nmol/L）能下调TG含量;染料木黄酮能促进成熟脂肪细胞脂解,提高细胞培养液甘油浓度;染料木黄酮（30 μmol/L）能上调p-AMPK、HSL蛋白表达,下调FAS蛋白表达（P＜0.01）,对ERK、p-ERK、AMPK表达量无明显影响（P＞0.05）。ICI182780（1 μmol/L）能部分阻断染料木黄酮（30 μmol/L）对p-AMPK的调节作用（P＜0.05）。结论：染料木黄酮能抑制3T3-L1细胞成脂分化,其机制可能是染料木黄酮一方面下调FAS蛋白表达,抑制脂肪合成,另一方面激活AMPK-HSL途径促进脂肪分解;染料木黄酮还可能通过非雌激素受体途径抑制3T3-L1细胞成脂分化。     

Characterization of Gram‐Negative Psychrotrophic Bacteria isolated from Italian Bulk Tank Milk

Eighty psychrotrophic bacterial strains, isolated from different northwest Italian bulk tank milks destined for Grana Padano cheese production, were identified by 16S rRNA gene amplification and partial sequence analysis of the rpoB gene. Pseudomonas spp. were the most commonly occurring contaminants, P. fluorescens being the predominant isolated species, along with Enterobacteriaceae, primarily Serratia marcescens. RAPD‐PCR was used to study genetic variability and distinguish closely related strains; a high degree of genetic heterogeneity among the strains was highlighted. All the strains were characterized for their ability to produce proteases, lipases and lecithinases at different temperatures (7, 22, and 30 °C). Forty‐one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all the incubation temperatures was found for lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities, and the enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains. The proteolytic psychrotrophic strains were screened for the presence of the aprX gene, coding for a heat‐resistant metalloprotease in Pseudomonas spp. The aprX gene was detected in 19 of 63 Pseudomonas strains, and was widespread in the P. fluorescens strains (14/19).     

油污土壤中脂肪酶产生菌的筛选

为了获得脂肪酶产生菌的菌种,进而获得高酶活菌株.通过添加橄榄油作为唯一碳源进行富集培养,然后以透明圈平板筛选法从富油土样中筛选出能够在溴甲酚紫培养基上形成黄色透明圈的菌落,再在罗丹明B的培养基上得到高活菌种以及水解圈大小,摇瓶复筛得高活菌株,进而测定酶的活力,经筛选有10株脂肪酶产生菌酶活力较高.     

超临界体系脂肪酶膜催化合成甘油二酯的研究

以醋酸纤维素和聚四氟乙烯为材料制备醋酸纤维素/聚四氟乙烯复合膜,采用吸附-交联相结合的固定化方法,用该复合膜固定化脂肪酶。并研究了在超临界CO2状态下,采用该固定化脂肪酶膜催化一级大豆油与甘油反应合成甘油二酯的工艺,以反应产物中甘油二酯的含量为指标,通过单因素与响应面法进行分析,确定最佳的工艺条件为:一级大豆油与甘油的物质的量比为2∶1,反应温度为64℃,时间为7 h,加酶量为3.2%,甘油含水量为1%。在此条件下,得到反应产物中甘油二酯的质量分数为66.3%。     

高强脉冲电场对脂肪酶的影响研究

采用高强脉冲电场对脂肪酶进行处理,研究了电场强度、脉冲数目、温度、介质环境等因素对酶活的影响.结果表明,高强脉冲电场可引起脂肪的失活,且失活率与电场强度和脉冲数目呈正比,但酶对电场的抑制比微生物要强,热效应和溶液离子浓度对酶活的影响很小;不同介质环境的影响表明酶在环境中的三维结构状态是影响电场处理效果的最可能的原因.     

Contribution of Response Surface Design to the Synthesis of Monoacylglycerols Catalyzed by Rhizopus sp. lipase

The partially purified lipase from Rhizopus sp was tested as a biocatalyst for the synthesis of monolaurin from lauric acid and glycerol. A central composite rotatable design was applied to optimize the substrate molar ratio and the percentage of molecular sieve for achieving maximum monolaurin yield. The best results were obtained when the molar ratio of substrates was 1:1, the medium contained 100% (w/w) molecular sieve, and the reaction was conducted at 50 °C and 200 rpm applying 2 mg of the partially purified lipase. The maximum yield in monolaurin (17.52%) was achieved after a 72‐h reaction.     

恒顺香醋对人低密度脂蛋白(LDL)氧化修饰的影响

本文研究了恒顺香醋浓缩物（HV），恒顺香醋提取物（HVE）及其各不同分了量组分（HF1、HF2、HF3、HF4）对低密度脂蛋白氧化修饰（LDL）的影响，并研究了恒顺香醋浓缩物对已受到氧化修饰的LDL的影响。在加入Cu^2＋与LDL一起温浴前，添加HV、HVE及HVE各分离组分均能够抑制丙二醛的产生。LDL与Cu^2＋一起温浴2h，然后添加HV，添加组与对照组相比，丙二醛的产生量有明显差别。结果表明恒顺香醋浓缩物（HV），恒顺香醋提取物（HVE）及其各不同分予量组分（HF1、HF2、HF3、HF4）能抑制人低密度脂蛋白氧化修饰，且HV对已受到氧化修饰的LDL的氧化有一定的阻断作用。