酶的寡聚结构与催化稳定性

酶的结构与催化稳定性是生物催化与转化过程中的研究热点之一。与单体酶相比,寡聚酶在进化过程中亚基之间的聚合使其在结构和功能上具有一定的优越性,然而寡聚酶独特的四级结构导致其在制备和应用中存在诸多问题,如制备效率低、催化位点利用率低、催化稳定性差等,其中亚基解离导致的催化稳定性问题在很大程度上限制了其工业化应用。目前,介质工程、多亚基固定化、亚基界面工程和融合蛋白策略被应用于寡聚酶的催化稳定性改造,而寡聚酶至单体酶的改造策略则试图从根本上解决寡聚酶的制备和应用问题,具有较好的应用前景。本文介绍了酶的寡聚结构演替所产生的新功能,总结了寡聚酶在制备和应用中存在的问题,重点阐述了提高寡聚酶制备效率和催化稳定性的策略。     

液体洗涤剂配方中复合酶的稳定性研究

简单介绍了衣用液体洗涤剂中3种酶（蛋白酶、脂肪酶和纤维素酶）以及这3种酶的复合物的稳定性体系。通过数据处理得出了酶存在的稳定性条件,通过Terg-o-tometer法研究了酶的性能,得出了酶稳定存在时衣用液体洗涤剂的优化配方。     

蛋白酶在洗衣液中的稳定性改良

以硼砂为对照,考察了16种柠檬酸盐、多元醇及酯系列稳定剂,以及温度、pH、稳定剂用量对3种蛋白酶在洗衣液中稳定性的影响;同时基于洗衣液的主要成分考察了表面活性剂AES,AEO和APG等对蛋白酶活的影响。实验结果表明,柠檬酸盐尤其是柠檬酸铵钙的稳定效果最佳,APG0810和APG1214的稳定效果与硼砂接近。当蛋白酶用量为0.5%时,以柠檬酸铵钙-APG0810-APG1214三元复合体系稳定蛋白酶,含有Purafect 4000L和Purafect Prime 4000L的复合稳定剂体系在pH=7和25 ℃下的3周酶活保持率分别可达到84.3%和71.5%;在pH=8和37 ℃下的3周酶活保持率分别为47.2%和30.4%;硼砂体系pH=7和37 ℃下的3周酶活保持率分别为2.87%和13.5%。此外,AES会较大幅度地降低酶活,而非离子表面活性剂AEO_7和AEO_9对其酶活无显著影响,但AEO和AES的混合并不能降低AES对酶活的损伤,可以考虑在液体加酶洗涤剂中增加APG和AEO的使用。     

酶分子稳定性改造研究进展

酶催化转化在食品、医药和精细化工等领域起着越来越重要的作用。然而,目前大多数酶反应需要在较温和的条件下进行以维持其正常活性,而在实际应用的逆境中(如高热、高酸、高盐等),酶的耐受性却较差、容易失活从而导致反应效率下降,极大地限制了其推广和应用。因此,对酶分子进行抗逆改造以提高其稳定性和催化活性,是当前研究的热点也是难点。本文从化学修饰和分子改造两个方面总结了酶分子稳定性改造的新进展,从定向进化、半理性设计、理性设计和糖基化修饰4个角度重点阐述了分子改造提高酶稳定性的方法,重点介绍了糖基化作为一种新的酶分子稳定性改造技术的思路。     

Biological effects of nanoparticles used as glidants in powders

Glidant nanoparticles used in the production of pharmaceuticals and foods may have biological effects. Such biological effects may affect organs of workers and consumers and may well be different for particles that have a similar performance as glidant.     

Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR

In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp. I-5 (CGTase I-5). A mutant CGTase[3-18] with the three mutations M234T, F259I and V591A was selected by agar plate assay. Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E. The cyclization activity of CGTase[3-18] was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold. These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization. The addition of CGTase[3-18] reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C. No cyclodextrin (CD) was detected in bread treated with CGTase[3-18], whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.     

利用嗜极微生物提高酶的稳定性

随着酶的大量使用,酶的稳定性正越来越受到重视。以嗜极细菌酶作为研究对象,采用基因工程、化学修饰、同定化等方法来实现酶的稳定性。为了找到合适的稳定途径,必须充分考虑酶和稳定方法之间的关系。     

Production and dispersion stability of nanoparticles in nanofluids

This paper presents an experimental study on the homogeneous dispersion of nanoparticles in nanofluids. In this study, various physical treatment techniques based on two-step method, including stirrer, ultrasonic bath, ultrasonic disruptor, and high-pressure homogenizer were systematically tested to verify their versatility for preparing stable nanofluids. Initially carbon black and silver nanoparticles dispersed in base fluids with the presence of surfactant were found to be highly agglomerated with the hydrodynamic diameter of 330 nm to 585 nm, respectively. After both CB and Ag nanofluids were treated by various two-step methods, stirrer, ultrasonic bath, and ultrasonic disrupter was found to do a poor performance in deagglomeration process for the initial particle clusters. However, the high-pressure homogenizer produced the average diameter of the CB and Ag particles of 45 nm and 35 nm, respectively, indicating that among various physical treatment techniques employed in this study, the high-pressure homogenizer was the most effective method to break down the agglomerated nanoparticles suspended in base fluids. In order to prepare another nanofluid with much smaller primary nanoparticles, we also employed a modified magnetron sputtering system, in which the sputtered nanoparticles were designed to directly mix with the running surfactant-added silicon oil thin film formed on a rolling drum (i.e. one-step method). We observed that Ag nanoparticles produced by the modified magnetron sputtering system were homogeneously dispersed and long-term stable in the silicon oil-based fluid, and the average diameter of Ag nanoparticles was found to be ~ 3 nm, indicating that the modified magnetron sputtering system is also an effective one-step method to prepare stable nanofluids.     

Influence of nanoparticle diameter on conjugated enzyme activity

Lactase conjugated to nanomaterials represents an area of significant potential to the food processing as a means to produce novel value-added products, reduce waste, and enable diagnostics. While it is recognized that, in general, matter exhibits unique properties when manipulated at the nanoscale, little is known about how reducing the size of the carrier to the nanoscale effects attached lactase. The purpose of this work is to investigate the influence of particle size on activity retention of lactase (Aspergillus oryzae) covalently conjugated to magnetic nanoparticles of varying sizes. Lactase was attached to carboxylic acid functionalized magnetic nanoparticles 18 nm, 50 nm, and 200 nm in diameter using carbodiimide chemistry. After attachment, activity retention was 73%, 39%, and 14% compared to the free enzyme for the 18 nm, 50 nm, and 200 nm conjugates, respectively. The apparent K_m was not significantly different as a function of particle size while the apparent k_cat decreased with increasing particle size. Reducing the particle size of magnetic nanoparticles can increase the activity retention of conjugated lactase. This work provides improved understanding of enzyme-nanoparticles systems and allows for enhanced design of lactase-conjugated materials.     

Evaluation of nanogels as supports for enzyme immobilization

Nanogels are hydrophilic polymers made up of crosslinked nano‐sized particles. These nanogels have large surface area that offers several functional groups as reserves for binding drugs, generating biosensors and as supports for enzyme immobilization. This mini‐review is an attempt to evaluate the recent developments in the use of nanogels as supports in enzyme immobilization. Emphasis is laid on the effect of nanogel structure and immobilization protocol on the property profile of the immobilized enzymes as compared with their free counterparts. The prospective applications of the nanogel‐immobilized enzymes are also evaluated. © 2014 Society of Chemical Industry     

可逆抑制剂对发酵生产洗涤用蛋白酶的影响

在微生物发酵生产蛋白酶过程中添加可逆抑制剂,通过抑制发酵液中蛋白酶活性,减少蛋白酶自身水解,提高储存稳定性.结果表明,可逆抑制剂的最佳添加方式为在发酵过程中连续流加,流加量为抑制蛋白酶活性40％最佳,最佳条件下蛋白酶产量提高49.1％.抑制剂的添加,不影响蛋白酶的提取,提取时发酵液中的可逆抑制剂可以通过超滤法减少或者去...     

Freeze‐drying of lignin peroxidase: influence of lyoprotectants on enzyme activity and stability

The effect of different lyoprotectants (sucrose, dimethyl‐succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran, mw 60 kDa) on the stability of the enzyme lignin peroxidase (LiP, EC number: 1.11.1.‐), both during the freeze‐drying process and storage were investigated. The shelf stability tests were performed at 4 °C and 27 °C. Both DMS buffer and sucrose showed a good protective action: the former was particularly effective during the process, while the latter improved the stability during storage. In contrast, mannitol and dextran had negative effects, reducing the activity also in the lignin peroxidase solution. BSA was discarded because, in the range of compatible concentrations with LiP, it does not confer a consistent structure to the freeze‐dried product. © 2002 Society of Chemical Industry     

Preparation and nanoencapsulation of l-asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study

This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene (ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.     

Enhanced stability of gold nanoparticles capped with double hydrophilic block copolymers synthesized by eATRP

In this study, the gold nanoparticles (Au NPs) are capped with a novel, well-defined double hydrophilic block copolymer, poly (ethylene glycols)-block-poly(mono-2-(methacryloyloxy) ethyl succinate) (PEG-b-PSEMA). The PEG-b-PSEMA, with long side chains and relatively narrow dispersity index, is successfully prepared by aqueous electrochemically mediated atom transfer radical polymerization using a PEG-Br macroinitiator (M_n≈2000 g mol⁻¹). By simply mixing PEG-b-PSEMA and Au precursor before adding reductants, coulombic interactions occur between the Au precursor and the carboxylate group of the PSEMA, as a result dense Au NPs are formed. This enables the Au NPs to remain dispersed even in the presence of temperature variations, pH adjustments, and incubation in organic solvents. Au NPs were endowed by PEG-b-PSEMA with more remarkable catalytic activity in organic solvents of 4-nitrophenol into 4-aminophenol than by sodium citrate (Au@citrate) and MeO-PEG-OH (Au@PEG). In addition, PEG-b-PSEMA-stabilized Au NPs (Au@PEG-b-PSEMA) were also tested for the dye degradation property against Rhodamine-B in only pH 10 basic solution, and over 99% of dye degradation can be achieved in 9 min. PEG-b-PSEMA induces coulombic interactions with Au NPs, providing outstanding stability for catalytic applications in organic or specific acidic-base environments.     

Improvement of enzyme electrocatalysis using substrate containing electroactive polymers. Towards limiting efficiencies of bioelectrocatalysis

Direct electrocatalysis by the enzymes in the absence of any freely diffusing mediators requires proper orientation of the protein macromolecule on the electrode surface. We report a conception on how to improve efficiency of bioelectrocatalysis using electroactive polymers containing analogues of the enzyme substrates (usually acting as artificial donors-acceptors). Indeed, substrate analogues having affinity to terminal redox groups of the enzymes can anchor them in an optimal orientation upon immobilization. The improvement of bioelectrocatalysis using such electroactive polymers is reported for different enzymes: hydrogenases and cellobiose dehydrogenase. Highly efficient electrocatalysis by hydrogenases was determined by thermodynamics of the catalyzed reaction confirming the direct bioelectrocatalysis. For hydrogenases immobilized over viologen substituted conducting polymers the limiting performance characteristics in bioelectrocatalysis have been achieved.     

溶菌酶结晶法提取及其酶活力的探讨

以湖光岩鹌鹑蛋为原料,采用结晶法从蛋清中分离提取溶菌酶。探讨不同条件对溶菌酶收率及活性的影响,确定溶菌酶最佳的分离提取条件:盐析Na Cl质量分数为5.1%,pH=10.7,盐析时间为120h,收率达到0.378%,酶活力达到13700 U/mg。     

Immobilization of α‐chymotrypsin for use in batch and continuous reactors

α‐Chymotrypsin from bovine pancrease (EC 3.4.21.1) was entrapped in Ca‐alginate gel particles to carry out hydrolysis of N‐acetyl‐L ‐phenylalanine methyl ester (APME) in batch as well as continuous fixed bed reactor. The enzyme was covalently modified with homo‐bifunctional polyethylene glycol derivatives in order to reduce its leakage from the beads; 85% modification of the ∈‐NH₂ groups of lysine residues caused reduction in the enzyme activity by 50%. However, this modification was helpful in a long run because it reduced both enzyme leakage and deactivation. Effective diffusivities and the distribution coefficients of the substrate and the product were determined experimentally, and later used in simulation of a batch experiment employing the beads. A continuous fixed bed reactor with the gel beads was operated to study the deactivation of the enzyme. During a 15‐day period, the enzyme showed about 15% loss in the conversion which occurred only during the first 5 days. After that the enzyme did not deactivate further which demonstrates that this method can be applied for continuous reactions. © 2000 Society of Chemical Industry     

The effect of operating conditions on the response of a differential enzyme thermistor

The performance of a low cost differential enzyme thermistor system was studied. Initial results from physical characterisations showed the interdependence of operating parameters (flow rate and ambient temperature) on detector sensitivity. Subsequent work demonstrated the detection of urea using an immobilised urease column.     

乙氧基化烷基硫酸钠稳定性的探讨

考察了乙氧基化烷基硫酸钠（简称AES）在不同因素下（包括储存温度、是否添加缓冲剂、起始pH值）产品的pH值随时间变化的情况,由pH值的变化表征AES的稳定性。结果表明,储存温度极大影响AES稳定性,AES适宜的储存温度应小于40℃;提高AES的起始pH值能有效延长AES储存时间;添加缓冲剂可延长的AES储存时间。