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。     

Extraction optimisation,antioxidant activity and inhibition on α-amylase and pancreatic lipase of polyphenols from the seeds of Syzygium cumini

The objectives of this study were to determine the optimal extraction conditions of polyphenols from Syzygium cumini seeds by response surface methodology and investigate their antioxidant activity and inhibition on α-amylase and pancreatic lipase. As results, the optimal extraction conditions in the ultrasonic extraction process which maximised total polyphenols content, minimised the IC₅₀ values of α-amylase and pancreatic lipase were determined as follows: extraction time 60 min, ethanol concentration 63% and solvent/solid ratio 44 mL g⁻¹. The main phenolic compounds in partially purified fraction of Syzygium cumini seeds were catechin, epicatechin, kaempferol, gallic, 5-caffeoylquinic, caffeic and ferulic acids. In addition, the partially purified fraction inhibited 87.66 ± 5.55 and 86.61 ± 3.15% of α-amylase and pancreatic lipase, respectively. The results suggested that Syzygium cumini seeds could be explored as a natural antioxidant and could be used as a source of highly antidiabetic and anti-obesity bioactive compounds.     

板栗壳黄酮结构分析及其对胰脂肪酶活力的抑制作用

在分析板栗壳黄酮结构的基础上，研究其对胰脂肪酶的抑制作用及类型。采用醇提法提取板栗壳中的黄酮，并用AB-8大孔吸附树脂进行纯化，冻干后得到的黄酮含量为（107.50±1.00）mg/g，提取得率为（5.66±0.05）%。利用超高效液相色谱-四极杆/静电场轨道阱质谱联用技术对板栗壳黄酮进行结构分析，鉴定出8 种黄酮类化合物。采用对硝基苯酚法测定胰脂肪酶活力，分别考察板栗壳黄酮对胰脂肪酶的抑制效果及不同反应条件下板栗壳黄酮对胰脂肪酶活性的影响，结果表明，板栗壳黄酮对胰脂肪酶有较好的抑制作用，半抑制浓度（half maximal inhibitory concentration，IC50）为0.074 mg/mL，且使胰脂肪酶最适pH值向碱性偏移。通过Lineweaver-Burk双倒数作图法确定其抑制类型为非竞争性抑制，抑制常数Ki＝53.19 mg/mL。由此可见，板栗壳黄酮是一种效果良好的胰脂肪酶抑制剂。     

新型嗜热耐碱脂肪酶的纯化表征及应用

将来源于解脂嗜热互营杆菌（Thermosyntropha lipolytica）的脂肪酶（TlLipA）基因tll1导入大肠杆菌BL21（DE3）中表达，通过热处理和镍柱亲和层析获得纯酶，并对其酶学性质进行研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示TlLipA分子量为53×10³，其最适反应温度为65℃，最适反应pH为8.0。在55~65℃范围内酶活较高且比较稳定；在pH^{7.0~11.0于室温保存1 h后，残留相对酶活仍达80%以上。1 mmol/L 金属离子Zn2+、Fe3+和试剂SDS，0.05%（质量分数）Tween 80，对酶活力具有强烈的抑制作用，残留相对酶活皆低于15%；1 mmol/L Mg2+、Mn2+对酶活力表现出轻微的激活作用。由底物专一性实验可得，该酶对辛酸对硝基苯酯（C8）和癸酸对硝基苯酯（C10）偏好明显。以棕榈酸对硝基苯酯（p-NPP）为底物，该酶动力学参数K_m值为0.23 mmol/L，V_max为33.50 mmol/(L·min)，k_cat为22.83 _S-1。以重组脂肪酶为催化剂在无溶剂体系中制备生物柴油，含水率20%，酶加量200 U/g油，醇油比为4∶1的条件下，在55℃催化大豆油反应48 h，收率可达91.75%。}     

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).     

一株产低温脂肪酶酵母菌的鉴定及酶学性质

低温脂肪酶已成为工业低温工艺良好候选物，在生物质能源、食品、皮制品、废水处理等领域发挥重要作用。本文从实验室保藏的55株产低温脂肪酶酵母菌株中筛选出一株高产菌株NYNU 19160，通过形态学、生理生化以及ITS和26S rDNA序列分析，将该菌株鉴定为Papiliotrema fonsecae。经过硫酸铵分级沉淀、透析、浓缩将该脂肪酶纯化后，对酶学性质进行了研究。结果表明，该脂肪酶最适反应温度为20℃，最适作用pH为7.5，属于低温碱性脂肪酶；Cu²⁺显著促进该酶的水解活性，而Li⁺表现为显著抑制作用；有机溶剂乙腈、甲醇、乙酸对酶活性有较强的促进作用，而苯和正己烷则抑制了该酶的活性；该酶对对硝基苯酚丁酸脂（pNPC₄）底物表现出较强特异性。