Cloning, overexpression, and characterization of a novel organic solvent-tolerant lipase from Paenibacillus pasadenensis CS0611

We found a novel lipase gene in the Paenibacillus pasadenensis CS0611 strain.The lipase gene sequence was cloned into the pET-28a expression vector to construct a recombinant lipase protein containing 6×His tags at the C-and N-termini,respectively.High-level expression of the lipase in E.coli BL21(DE3)was obtained upon induction with IPTG at 20°C.The recombinant lipase activity was approximately 1631-fold higher than the wild type.His-tagged recombinant lipase was purified rapidly and efficiently by using Ni-charged affinity chromatography with 63.5%recovery and a purification factor of 10.78.The purified lipase was stable in a broad range of temperatures and pH values,with the optimal temperature and pH being 50°C and 7.0,respectively.Its activity was stimulated to different degrees in the presence of metal ions such as Ca2+,Mg2+,and some non-ionic surfactants.In addition,the purified lipase was activated by a series of water-miscible organic solvents such as some short carbon chain alcohols and was highly tolerant to some water-immiscible organic solvents.

Stability of Surfactant-coated Candida Rugosa Lipase in Isooctane

The stability of Candida rugosa lipase coated with glutamic acid didodecyl ester ribitol amide was investigated taking esterification of lauryl alcohol and lauric acid in isooctane as a model reaction. At 30C, the half-life of the activity of the coated lipase was ca 10 h, the enzyme activity became less changed after 12 h and the residual activity was 39% of the initial value. The coated lipase obeyed a first-order deactivation model with a deactivation energy of 29.9J.mol-1.

Kinetics of Lipase Catalyzed Enantioselective Esterification of Racemic Ibuprofen in Isooctane

The kinetics of Candida rugosa lipase catalyzed esterification of racemic ibuprofen with n-butanol in isooctane was studied. The kinetic study was carried out with the addition of 0.1% and 2% (by volume) of water for enzyme activation respectively when celite was added into isooctane for enzyme dispersion. The specific initial rate for S-ibuprofen can be fitted with the Ping Pong Bi Bi mechanism with dead-end competitive inhibition by the alcohol. The time courses of the enantioselective esterification of the two ibuprofen enantiomers with different initial substrate concentrations and water contents were simulated with a model in which both effects of enzyme inactivation by long term reaction and reversed hydrolytic reaction under high water content were taken into consideration.

Optimization of technological conditions for one-pot synthesis of (S)-α-cyano-3-phenoxybenzyl acetate in organic media

Optically active form of α-cyano-3-phenoxybenzyl (CPB) alcohol, building block of pyrethroid insecticides, was synthesized as its acetate by the combination of anion-exchange resin (D301)-catalyzed transcyanation between m-phenoxybenzaldehyde (m-PBA) and acetone cyanohydrin (AC), and lipase (from Alcaligenes sp.)-catalyzed enantioselective transesterification of the resulting cyanohydrin with vinyl acetate. Through optimizing technological conditions, the catalyzing efficiency was improved considerably compared to methods previously reported. Concentrations of CPB acetate were determined by gas chromatograph. The enantio excess (e.e.) values of CPB acetate were measured by NMR (nuclear magnetic resonance) method. Effects of solvents and temperatures on this reaction were studied. Cyclohexane was shown to be the best solvent among the three tested solvents. 55 °C was the optimal temperature for higher degree of conversion. External diffusion limitation was excluded by raising the rotational speed to 220 r/min. However, internal diffusion could not be ignored, since the catalyst (lipase) was an immobilized enzyme and its particle dimension was not made small enough. The reaction rate was substantially accelerated when the reactant (m-PBA) concentration was as high as 249 mmol/L, but decreased when the initial concentration of m-PBA reached to 277 mmol/L. It was also found that the catalyzing capability of recovered lipase was high enough to use several batches. Study of the mole ratio of AC to m-PBA showed that 2:1 was the best choice. The strategy of adding base catalyst D301 was found to be an important factor in improving the degree of conversion of the reaction from 20% to 80%. The highest degree of conversion of the reaction has reached up to 80%.

脂肪酶固定化常用载体及方法的研究进展

脂肪酶被广泛应用于生物传感器、精细化工和生物催化等诸多领域,在此情形下脂肪酶固定化是提高其重复利用率和催化效率的重要手段。文章按照微生物属、海洋属、动植物属的分类,分别介绍了脂肪酶的不同来源,重点总结了金属有机框架、介孔氧化硅和凝胶等常用的新型酶固定化载体,并讨论了吸附法、共价交联法、包埋法、复合法等脂肪酶固定化常用方法的优缺点,以期为后续研究提供理论依据和参考。

酶促酯交换反应中金属有机框架固定化脂肪酶研究进展

酶促油脂转化为脂肪酸酯能同时满足食品安全和“双碳”战略的需求,然而游离脂肪酶性质脆弱、回收难、成本高等弊端限制了其大规模工业化应用。利用多孔材料为载体固定化脂肪酶能够克服上述问题,合理设计载体结构和功能是脂肪酶固定化的关键。金属有机框架(metal-organic frameworks,MOFs)因孔径可调、易修饰、生物相容性好等优势成为固定脂肪酶的优良载体。该文首先介绍了MOFs的种类及其不同的合成方式,并论述其优缺点,再详细阐明脂肪酶在MOFs上的固定化方法,最后对MOFs-脂肪酶作为催化剂在酯交换反应中的应用进行讨论与总结归纳,为固定化酶及油脂转化提供理论和技术支撑。

Resolution of DL-phenylalanine by porcine pancreas lipase catalyzed transesterification in monophasic organic solvent

With recent development of enzyme engineering, the use of enzyme in organic solvents is rapidly increasing. The remarkable stereoselectivity and regioselectivity of reaction catalyzed by enzyme were used to resolve many racemic alcohols and esters. A number of results have also been reported about the resolution of amino acids in organic solvents. For example, DL-phenylalanine methyl ester was resolved by α-chymotrypsin in toluene/water biphasic system; in fact, this reaction was carried, out in water;transesterification of DL-phenylalanine propyl ester and methanol was catalyzed by α-chymotrypsin in methanol, and the enzyme used was dissolved in water and the

一株产耐有机溶剂脂肪酶伯克霍尔德氏菌的分子鉴定及酶学性质分析

为筛选具有潜在应用价值的产脂肪酶菌株,采用三丁酸甘油酯固体培养基从黄山松树林土壤中筛选产脂肪酶菌株,利用16S rDNA测序对筛选的菌株进行鉴定,并对其产脂肪酶酶活力参数以及酶学性质进行分析。结果表明:筛选的产脂肪酶菌株HSU-7为伯克霍尔德氏菌(Burkholderia sp. HSU-7);菌株HSU-7所产脂肪酶的最适反应温度为45℃,最适pH为5,为一种耐酸性的中温脂肪酶;菌株HSU-7发酵5 d,所产脂肪酶酶活力最大,为72.22 U/mL。同时,K^(+)、Ca^(2+)和Mg^(2+)对菌株HSU-7所产脂肪酶酶活力具有显著的促进作用,而Ni^(2+)、Cu^(2+)、Fe^(3+)和Zn^(2+)具有一定的抑制作用,该脂肪酶可耐受有机溶剂甲醇、乙醇、异丙醇和二甲基亚砜,但Triton X-100可显著抑制其酶活力;此外,该脂肪酶能耐受65℃的高温。综上,菌株HSU-7可高产脂肪酶,且该脂肪酶有较强的热稳定性,可耐受多种有机溶剂,具有很好的工业应用价值。

蚜虫莫氏黑粉菌脂肪酶的酶学性质研究

为明确蚜虫莫氏黑粉菌脂肪酶的应用价值,研究了反应温度、pH值、金属离子、表面活性剂、抑制剂和有机溶剂等对脂肪酶活性的影响。结果表明:蚜虫莫氏黑粉菌脂肪酶的最适反应温度为70℃、pH值为8,具有较好的温度稳定性和pH耐受性,是一种常温发酵合成的耐高温碱性脂肪酶;Zn2+对其活力具有显著的抑制作用;吐温-20、吐温-80、曲拉通-100等表面活性剂和甲醇、乙醇、正丙醇、异丙醇、丙酮等有机溶剂对脂肪酶活力均表现出明显的促进作用;该脂肪酶对三油酸甘油酯的水解无位置特异性;水解对硝基苯酚棕榈酸酯(p-NPP)的米氏常数Km为1.14 mmol/L,最大反应速率vmax为119.05 mol/min。蚜虫莫氏黑粉菌脂肪酶在高温、弱碱性、有机溶剂等工业反应条件下具有良好的应用前景。

Preparation of hollow spherical covalent organic frameworks via Oswald ripening under ambient conditions for immobilizing enzymes with improved catalytic performance

The hollow spherical covalent organic frameworks(COFs)have a wide application prospect thanks to their special structures.However,the controllable synthesis of uniform and stable hollow COFs is still a challenge.We herein propose a self-templated method for the preparation of hollow COFs through the Ostwald ripening mechanism under ambient conditions,which avoids most disadvantages of the commonly used hard-templating and soft-templating methods.A detailed time-dependent study reveals that the COFs are transformed from initial spheres to hollow spheres because of the inside-out Ostwald ripening process.The obtained hollow spherical COFs have high crystallinity,specific surface area(2,036 m^(2)·g^(−1)),stability,and single-batch yield.Thanks to unique hollow structure,clear through holes,and hydrophobic pore environment of the hollow spherical COFs,the obtained immobilized lipase(BCL@H-COF-OMe)exhibits higher thermostability,polar organic solvent tolerance,and reusability.The BCL@H-COF-OMe also shows higher catalytic performance than the lipase immobilized on non-hollow COF and free lipase in the kinetic resolution of secondary alcohols.This study provides a simple approach for the preparation of hollow spherical COFs,and will promote the valuable research of COFs in the field of biocatalysis.

金属有机骨架固定化脂肪酶的构筑及其催化制备生物柴油的研究进展

金属有机骨架(MOF)具有多样性、高比表面积、化学稳定性和易于后修饰等优势,是固定化酶的优良载体。为了促进生物柴油的发展,针对MOF固定化脂肪酶的方法及其在生物柴油生产中的应用进展进行了综述。MOF固定化脂肪酶的方法有物理吸附法、共价结合法、原位包埋法等,采用MOF固定脂肪酶生产生物柴油,在一定程度上降低了生产成本和提高了产率。开发新型的脂肪酶固定化方法及寻求合适的固定化载体是生物柴油发展的关键。

有机相中脂肪酶催化糖酯合成的研究

研究了有机相中脂肪酶催化以单糖和乙酸乙烯酯或乙酸酐为底物的糖酯合成反应。建立了定性、定量检测糖酯的方法，考察了７种脂肪酶催化糖酯合成的活力，发现来自假单孢菌属的ＰＳＬ１的活力最高。研究了ＰＳＬ１对不同单糖底物的选择性，发现对甘露醇的选择性最好，转化率可达９５％，研究了反应体系中的含水量对糖酯合成的影响，探讨了酶浓度和温度对反应的影响。

红曲霉胞外脂酶催化己酸乙酯合成研究

己酸乙酯是浓香型曲酒的主体香味成分,也是曲酒优质品率低的主要限制因素。60年代Iwai发现在适当条件下脂酶可直接催化酸和醇合成酯。Langrand比较了各种脂酶合成短碳链羧酸酯的能力。我们在对酒窖生香微生物的研究中发现红曲霉、根霉具有较强的己酸乙酯合成能力,属首次报道。本文主要报道利用红曲霉的脂酶合成己酸乙酯条件试验的结果。

有机介质中酶催化合成己酸乙酯的研究

本实验研究了有机介质中CRL脂肪酶催化己酸与无水乙醇合成己酸乙酯的酯化反应条件,并利用红外光谱、GC-MS、气相色谱等现代方法对合成的产物进行了表征。结果显示,合成的产物为己酸乙酯,其纯度为96.91%,分子量144。酯化反应在以正庚烷为反应介质,反应温度为311K,己酸与无水乙醇的摩尔比为1:1.3,己酸浓度为0.2mol/L的条件下,反应24h后,转化率达到94.6%。

有机相脂肪酶催化合成技术在食品及相关领域的应用

对有机相脂肪酶催化合成技术的研究及其在食品、化妆品、医药、精细化工领域的应用进行了综述,表明有机相脂肪酶催化合成技术具有直接利用底物、反应条件温和、反应选择性高、产品容易分离纯化、产品具有绿色环保概念等优点。

超声辐照对有机相脂肪酶催化酯化反应的促进

探讨了超声辐照对有机相中脂肪酶催化有机硅醇与脂肪酸酯化反应的促进作用 ,系统地研究了超声功率、超声作用方式、底物脂肪酸的碳链长、反应体系的水活度等对超声辐照促进有机相脂肪酶催化酯化反应的影响 .研究表明 ,适宜的超声辐照可显著提高有机相脂肪酶催化酯化反应的速度 ;底物的碳链越长、反应体系的水活度越大 。

有机溶剂中固定化脂肪酶催化硅醇的酯化反应

固定化Ｍｕｃｏｒｍｉｅｈｅｉ脂肪酶可催化有机硅醇和脂肪酸的酯化反应．对固定化酶用量、脂肪酸链长、不同有机硅醇底物、有机溶剂极性和水含量等影响因素进行了初步研究．

脂肪酶改性对其在有机相中催化特性的影响

有机相酶催化近年来吸引了众多研究者的关注 .本文着重从酶的修饰 ,酶分子的生物印迹 ,酶晶体的交联 ,酶的纯化方式与预处理过程 。

耐有机溶剂脂肪酶产生菌的筛选及其粗酶酶学性质

以体积分数2%苯为筛选压力,利用罗丹明B平板显色法和摇瓶发酵法,从采集的花生地土壤样品中分离筛选得到1株中度耐热、耐碱脂肪酶产生菌,编号为H2。通过形态观察、生理生化特性实验及其16S rDNA基因序列对菌种进行鉴定。结果表明,H2菌株与短小芽孢杆菌(Bacillus pumilus)的亲缘关系最紧密。通过研究得到该菌株的摇瓶发酵条件:产酶培养基为:蛋白胨3%、酵母膏1%、NaCl 0.5%、橄榄油1%,pH7.0,摇瓶发酵温度为28℃,摇床转速为180r/min,发酵周期为48～60h。所产脂肪酶在40℃、pH9.0时酶活性最高,对pH值和温度的适应范围较宽,pH6.0～10.0比较稳定,35～50℃具有较高酶活性。

产有机相催化酯合成活性的脂肪酶菌株的筛选

通过添加10g/L的甲苯作为唯一碳源进行预培养,然后以透明圈平板筛选法从土壤样品中成功地筛选到了一株耐有机溶剂的产脂肪酶的酵母菌A213,初步鉴定为耶罗威亚酵母(Yarrowia sp．)。摇瓶实验表明,A213适宜的产酶培养基为(g/L):酵母膏40g,橄榄油10g,MgSO^4·7H^2O 1g,KH_2PO_45g,最佳培养条件为27℃、初始pH 6．5。脂肪酶活力最高可达67．8 IU/mL;该酶最适作用温度为40℃,最适作用pH为6．5,在70℃以下, pH 5．5～8．5范围内稳定,能直接在叔戊醇溶剂中催化合成L-抗坏血酸棕榈酸酯。