CTAB混合反胶团萃取工业脂肪酶

研究了CTAB与Tween、含氧有机物形成的混合反胶团对工业脂肪酶进行萃取分离的效果. 实验表明,CTAB-Tween85和CTAB-含氧有机物混合反胶团的萃取率高于单一CTAB反胶团;反萃时CTAB-含氧有机物混合反胶团的反萃率与单一CTAB反胶团的反萃率相似,CTAB-Tween60和Tween40混合反胶团的反萃效果优于单一CTAB反胶团. 通过测定反萃水相的酶活,发现CTAB-TRPO混合反胶团的效果最好,酶活回收率最高,可以达到70%.     

Preparation of regioisomers of structured TAG consisting of one mole of CLA and two moles of caprylic acid

TAG (MLM) with medium-chain FA (MCFA) at the 1,3-positions and long-chain FA (LCFA) at the 2-position, and TAG (LMM) with LCFA at the 1(3)-position and MCFA at 2,3(1)-positions are a pair of TAG regioisomers. Large-scale preparation of the two TAG regioisomers was attempted. A commercially available FFA mixture (FFA-CLA) containing 9-cis, 11-trans (9c, 11t)- and 10t,12c-CLA was selected as LCFA, and caprylic acid (C₈FA) was selected as MCFA. The MLM isomer was synthesized by acidolysis of acyglycerols (AG) containing two CLA isomers with C₈FA: A mixture of AG-CLA/C₈ FA (1∶10, mol/mol) and 4 wt% immobilized Rhizomucor miehei lipase was agitated at 30°C for 72 h. The ratio of MLM to total AG was 51.1 wt%. Meanwhile, LMM isomer was synthesized by acidolysis of tricaprylin with FFA-CLA: A mixture of tricaprylin/FFA-CLA (1∶2, mol/mol) and 4 wt% immobilized R. miehei lipase was agitated at 30°C for 24 h. The ratio of LMM to total AG was 51.8 wt%. MLM and LMM were purified from 1,968 and 813 g reaction mixtures by stepwise short-path distillation, respectively. Consequently, MLM was purified to 92.3% with 49.1% recovery, and LMM was purified to 93.2% with 52.3% recovery. Regiospecific analyses of MLM and LMM indicated that the 2-positions of MLM and LMM were 95.1 mol% LCFA and 98.3 mol% C₈ FA, respectively. The results showed that a process comprising lipase reaction and short-path distillation is effective for large-scale preparation of high-purity regiospecific TAG isomers.     

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。     

聚合物接枝对脂肪酶活性及稳定性的影响

酶的稳定性和活性一直是困扰其工业应用的主要瓶颈，因此改造酶分子以提升其催化性能是当前研究的重要课题。本工作引入甲基丙烯酸异丁酯(IBMA)和甲基丙烯酸乙酯(EMA)两种疏水性不同的单体分子，通过原子转移自由基聚合分别接枝于褶皱假丝酵母脂肪酶(CRL)表面，合成聚合物接枝脂肪酶pIBMA-g-CRL和pEMA-g-CRL。酶学实验结果显示，聚合物接枝脂肪酶的催化活性和稳定性显著提升。pIBMA-g-CRL和pEMA-g-CRL的催化效率分别达到了野生型CRL的4.39倍和4.68倍。在50℃条件下孵育6 h后，pIBMA-g-CRL和pEMA-g-CRL仍分别保留51%和92%的活性，而野生型CRL的活性则仅剩14%。同样，在pH=9条件下孵育3 h后pIBMA-g-CRL和pEMA-g-CRL能够分别保留52%和83%的活性，并在pH=4~9范围内显示出更好的耐受性。光谱学实验进一步证实了聚合物接枝脂肪酶催化性能的提升与其二级结构和三级结构的变化密切相关。本研究表明，IBMA和EMA的接枝对脂肪酶的稳定性和活性的提升效果显著，是改造脂肪酶的优良材料。     

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%。}     

丙酮中麦芽糖月桂酸单酯的酶法选择性合成

为选择性地合成麦芽糖月桂酸单酯,该文研究了脂肪酶催化合成条件对麦芽糖月桂酸酯浓度和产率的影响,确定了麦芽糖月桂酸单酯的最佳合成参数。当c(麦芽糖)=25 mmol/L、c(月桂酸)=75 mmol/L、ρ(脂肪酶)=20 g/L、ρ(3A分子筛)=60 g/L时,在丙酮中50℃反应72 h,麦芽糖月桂酸单酯的产率和浓度分别可达93%和23.35 mmol/L。     

Selective Enrichment of Conjugated Linoleic Acid Isomers in Their Mixtures Using Combined Chemical and Enzymatic Methods

The aim of this study was to selectively enrich t10,c12-conjugated linoleic acid (t10,c12-CLA) and c9,t11-CLA in commercial CLA mixtures using a combination of urea crystallization and lipase-catalyzed esterification. The objective of the urea fractionation is to remove saturated and monounsaturated fatty acids (FA) from the CLA mixtures. CLA-enriched free FA (FFA) mixtures containing 53.8 wt% t10,c12-CLA and 39.1 wt% c9,t11-CLA were produced from the CLA mixtures containing ~34 wt% each of the two CLA isomers by a urea crystallization using methanol and the urea-to-FA weight ratio of 2.5:1. The CLA-enriched FFA mixtures were partially esterified with dodecan-1-ol in a recirculating packed-bed reactor using an immobilized lipase from Candida rugosa to further enrich the t10,c12-CLA and c9,t11-CLA in an FFA fraction and an FA dodecyl ester fraction, respectively, under the optimal conditions, i.e., temperature, 20 °C; FA-to-dodecan-1-ol molar ratio, 1:1; water content, 2 wt% of total substrates; residence time, 5 min; and reaction time, 24 h (for t10,c12-CLA enrichment) and 12 h (for c9,t11-CLA enrichment). After the reaction, an FFA fraction with 72.6 wt% t10,c12-CLA was obtained. Another FFA fraction with 62.0 wt% c9,t11-CLA was recovered after the saponification of the FA dodecyl ester fraction. The yields of t10,c12-CLA and c9,t11-CLA in the FFA fractions were 43.6 and 21.5 wt%, respectively, based on their initial weights in the CLA mixtures.     

三种脂肪酶活力测定方法的比较及改进

比较了碱滴定法、对硝基苯酚法和铜皂法等三种常用的脂肪酶活力测定方法的测定时间、准确性和重复性.为对硝基苯酚法找到了有效的终止反应方法,改进的对硝基苯酚法具有检测快捷、结果稳定的特点.对铜皂法进行改进,用甲苯代替了苯,改进的铜皂法具有毒性小、结果稳定、重复性好的优点.     

脂肪酶催化动力学拆分大分子手性化合物的研究进展

脂肪酶已广泛用于制备手性化合物的动力学拆分中,但大多数酶催化拆分外消旋化合物,特别是大分子手性化合物的对映选择性不很理想。目前,提高脂肪酶对映选择性的研究主要在改造脂肪酶蛋白结构、优化体系的反应条件、改善反应过程以及对映选择性抑制等方面进行。本文主要介绍了几种改善脂肪酶催化动力学拆分大分子手性化合物对映选择性的合理方法。