理性设计提高华根霉脂肪酶对不饱和长链脂肪酸的底物特异性及其在大豆油水解中的应用

为了提高华根霉脂肪酶在大豆油水解制备脂肪酸中的应用效果,通过理性设计的方法改造脂肪酶的脂肪酸特异性。对突变酶的酶学性质进行了研究,并通过单因素实验优化脂肪酶催化水解大豆油的反应条件。结果表明:利用定点突变技术获得了一系列脂肪酸特异性改变的脂肪酶,其中突变酶HQL增强了对不饱和长链脂肪酸的特异性,并且对p-NPP(C16)的水解活力相比野生型提高了2.72倍;所有突变酶的最适温度和最适p H均为40℃和8.0,与野生型脂肪酶一致;得到脂肪酶催化水解大豆油的最适反应条件为反应时间24 h、水油质量比1∶1、加酶量500 U/g(以油质量计)、p H 8.0、温度40℃,在最适反应条件下,野生型酶催化大豆油水解率仅为80%,而突变酶HQL由于增强了对长链不饱和脂肪酸的底物特异性,对大豆油水解率提高到98%。     

碱性脂肪酶在正庚烷中催化合成乙酸乙酯的研究

用碱性脂肪酶在正庚烷中催化合成乙酸乙酯,探讨了加酶量、反应温度和反应时间以及采用不同的碱性脂肪酶等因素对乙酸转化率的影响。研究表明,当乙酸浓度为0.20mol/L、乙酸和乙醇的摩尔比为1∶1.25、摇床转速为150r/min、定时加入一定量的3A分子筛适当移走产物的水分时,最佳反应温度为36℃;产自扩展青霉碱性脂肪酶高产变株FS1884-1的酶A催化效果为最好;当酶A的加入量为0.30g/瓶(相当于碱性脂肪酶的加入量为1700u/g乙酸)、反应时间为48h,乙酸的转化率已基本趋于稳定,转化率达71.4%。     

微生物脂肪酶在工业中的应用及研究进展

脂肪酶是工业应用中很重要的一种酶。本文对目前微生物脂肪酶在工业中的应用进行了综述,包括食品工业、纺织和化工工业、洗涤添加剂、废水处理添加剂、医药、造纸行业以及手性药物的合成等,并展望了脂肪酶的研究方向及前景。     

复合酶酶解黄油制备天然奶油香精

采用由两种脂肪酶组成的复合酶体系酶解黄油制备天然奶油香精,结合酸价及感官评定,筛选出脂肪酶组合为Palatase 20000L、Lipase MER。经优化后确定复合酶酶解黄油的最佳工艺条件为:Palatase 20000L添加量0.18%(m/m),Lipase MER添加量0.09%(m/m),酶解温度40℃,时间9h,pH 7.0。通过GC—MS分析,风味成分主要由短中链脂肪酸、少量酮、内酯、酯等11种成分构成。经烘培应用评定,产品奶香厚重,口感饱满,未夹带苦涩味等不良后味,且留香持久。     

三液相酶催化体系水解橄榄油的研究

采用三液相酶催化体系水解橄榄油,考察构成三液相体系的物质对酶的影响;考察了硫酸铵的含量和PEG400的含量对酶的分配分数、回收率及催化效率的影响,研究了中下相与油的体积比对水解橄榄油的影响,并初步探究了三液相体系水解橄榄油的微观液滴结构。结果表明:在酶添加量142.5 U/g、硫酸铵含量15%、PEG400含量28%、中下相与油的体积比3∶1、转速200 r/min条件下,酶的分配系数达到28.28,酶的回收率达99.65%,水解24 h后,脂肪酸含量达98.17%,在对照(油水体系)中,相同条件下产生的脂肪酸含量仅为42.06%。显微镜下液滴观察结果表明,三液相体系中独特的液滴结构可能是该体系优于油水两相体系的原因。     

青钱柳多糖对高脂血症小鼠的降血脂作用及机制初探

为初步探究青钱柳多糖(CPP)对高脂血症小鼠降血脂作用和机制,将高脂血症小鼠随机分为模型对照组、辛伐他汀组、空白对照组以及青钱柳低、中、高剂量组,每组16组。模型对照组与空白对照组均喂以等量的无菌水,辛伐他汀组喂以4 mg/kg的辛伐他汀与CMC-Na(0.5%)的混悬液,青钱柳低、中、高剂量组分别喂以100 mg/kg、200 mg/kg和400 mg/kg的CPP水溶液,连续喂养4周。实验结束后,测定血清TC、TG、HDL-C、LDL-C、FFA水平和LPS活力,取肝脏做病理形态学观察,用RT-PCR方法测定HSL m RNA的表达量。结果表明,与模型对照组相比,CPP组小鼠血清TC、TG、LDL-C的水平和LPS的活力(P0.01)均显著降低,HDL-C和FFA的水平(P0.01)则显著增加;CPP组小鼠肝脂变程度较模型组有不同程度的改善;相比于模型对照组,CPP组的HSL m RNA的表达量均显著上升(P0.05)。综上,CPP可以明显降低高脂血症小鼠血脂水平,改善其因摄入过多脂质而导致肝脏脂肪变性。     

L-天冬氨酸离子液体联合猪胰脂肪酶催化油酸酯化工艺北大核心CSCD

为绿色高效制备生物柴油,利用L-天冬氨酸离子液体([Asp]HSO4)联合猪胰脂肪酶催化油酸制备油酸甲酯,采用单因素实验探究了反应时间、醇酸物质的量比、[Asp]HSO4用量、猪胰脂肪酶用量和反应温度对转化率的影响,在此基础上,采用正交实验进行优化,得到[Asp]HSO4联合猪胰脂肪酶催化油酸酯化反应的最优工艺条件为[Asp]HSO4用量6%、醇酸物质的量比5.5∶1、猪胰脂肪酶用量4%、反应时间21 h、反应温度45℃,在此条件下转化率可达91.91%。[Asp]HSO4可降低甲醇和温度对猪胰脂肪酶催化活性的影响,对猪胰脂肪酶催化油酸酯化具有协同效应。     

圆弧青霉产碱性脂肪酶的中试发酵

圆弧青霉PG3 7是碱性脂肪酶的高产菌 ,在摇瓶和 2 5L发酵罐小试的基础上进行 3M3发酵罐中试 .适合的发酵条件为 :发酵培养基 (% )　豆饼粉 3 .0 ,玉米浆 3 .0 ,磷酸氢二钾 1 .0 ,硫酸镁0 .1 ,大豆磷脂 0 .5,柠檬酸钠 0 .0 5,花生油 0 .2 ;发酵起始 pH值为 8.0 ,发酵温度 2 9± 1℃ ,接种量8%～ 1 0 % ,通风量 0 .8L/ (L·min) ,搅拌转速 2 4 0r/min ,发酵周期 72h ,期间于 3 0、4 2、54h分别各流加 0 .4 %花生油 .在上述条件下 ,圆弧青霉PG3 7发酵酶活达 2 0 0 0 μmol/ (min·mL)     

Amphiphilic Pentablock Copolymers Prepared from Pluronic and ε-Caprolactone by Enzymatic Ring Opening Polymerization

Amphiphilic copolymers are appealing materials because of their interesting architecture and tunable properties. In view of their application in the biomedical field, the preparation of these materials should avoid the use of toxic compounds as catalysts. Therefore, enzymatic catalysis is a suitable alternative to common synthetic routes. Pentablock copolymers (CUC) were synthesized with high yields by ring-opening polymerization of ε-caprolactone (ε-CL) initiated by Pluronic (EPE) and catalyzed by Candida antarctica lipase B enzyme. The variables to study the structure–property relationship were EPEs’ molecular weight and molar ratios between ε-CL monomer and EPE macro-initiator (M/In). The obtained copolymers were chemically characterized, the molecular weight determined, and morphologies evaluated. The results suggest an interaction between the reaction time and M/In variables. There was a correlation between the differential scanning calorimetry data with those of X-ray diffraction (WAXD). The length of the central block of CUC copolymers may have an important role in the crystal formation. WAXD analyses indicated that a micro-phase separation takes place in all the prepared copolymers. Preliminary cytotoxicity experiments on the extracts of the polymer confirmed that these materials are nontoxic.     

Evaluation of the Impact of Esterases and Lipases from the Circulatory System against Substrates of Different Lipophilicity

Esterases and lipases can process amphiphilic esters used as drugs and prodrugs and impact their pharmacokinetics and biodistribution. These hydrolases can also process ester components of drug delivery systems (DDSs), thus triggering DDSs destabilization with premature cargo release. In this study we tested and optimized assays that allowed us to quantify and compare individual esterase contributions to the degradation of substrates of increased lipophilicity and to establish limitations in terms of substrates that can be processed by a specific esterase/lipase. We have studied the impact of carbonic anhydrase; phospholipases A1, A2, C and D; lipoprotein lipase; and standard lipase on the hydrolysis of 4-nitrophenyl acetate, 4-nitrophenyl palmitate, DGGR and POPC liposomes, drawing structure–property relationships. We found that the enzymatic activity of these proteins was highly dependent on the lipophilicity of the substrate used to assess them, as expected. The activity observed for classical esterases was diminished when lipophilicity of the substrate increased, while activity observed for lipases generally increased, following the interfacial activation model, and was highly dependent on the type of lipase and its structure. The assays developed allowed us to determine the most sensitive methods for quantifying enzymatic activity against substrates of particular types and lipophilicity.