面向生物能源的酶固定化的计算机模拟

利用酶固定化技术来对生物质发酵获取生物能源已显得日益重要。酶与表面间的相互作用强烈影响固定化酶的取向,进而影响催化效率。本文采用并行退火蒙特卡洛（PTMC）方法对三种生物能源用酶（脂肪酶、纤维素酶和氢化酶）在不同的带电表面和离子强度下的吸附取向进行了模拟研究。模拟结果发现三种酶的吸附主要由静电吸引力主导,并且很大程度上与蛋白表面带电氨基酸的分布和溶液离子的静电屏蔽有关。脂肪酶和氢化酶在带负电表面上吸附,其活性位和电子转移通道分别为朝向溶液和靠近表面,而纤维素酶则在带正电表面上取得较优的吸附取向。本文研究结果可为工业用酶以合理的取向在载体材料表面固定化提供一定的指导。     

Hydrogenases as catalysts for fuel cells: Strategies for efficient immobilization at electrode interfaces

Hydrogenases are the key enzymes for hydrogen metabolism in many microorganisms. Due to the high efficiency they develop for H₂ oxidation, research in the last five years has aimed towards their use as biocatalysts for H₂/O₂ biofuel cells to replace platinum-based chemical catalysts. We report in this review the major issues that have been addressed in view of the future development of such a novel biotechnological device. This includes enhancing the stability of either the enzyme itself or its immobilization onto conductive supports, increasing the amount of electrically connected enzymes and, finally, controlling hydrogenase orientation at the electrode surface, and hence the electron transfer process. We specifically focus on a particular [NiFe] membrane-bound hydrogenase purified from the hyperthermophilic and microaerophilic bacterium Aquifex aeolicus. This enzyme resists to O₂, CO, and high temperatures making it potentially efficient as a biocatalyst. Recent progress in these domains strengthens the credibility of a viable H₂/O₂ biofuel cell and opens new avenues for biofuel cell design.     

磁纳米颗粒固定化多能硫碱弧菌(T. versutus D301)生物脱硫

采用共沉淀法合成油酸修饰的Fe3O4纳米颗粒,用其固定化Thioalkalivibrio versutus D301细胞,考察了盐碱环境下纳米颗粒固定化细胞的工艺条件,比较了固定化细胞和游离细胞的硫氧化活性,研究了固定化细胞的重复利用性能. 结果表明,最佳固定化条件为Na+浓度0.6 mol/L,pH值9.5,固定化温度20℃,吸附时间10 min. 固定化细胞硫氧化速率是游离细胞的81%. 固定化细胞具有很好的硫氧化活性,可重复使用至少6次.     

负载型功能化离子液体的催化性能

用MCM-41为载体,通过化学法合成了负载羟基功能化的咪唑类离子液体催化剂HIILsBr/MCM-41,考察了在碳酸丙烯酯（PC）合成中的催化作用。考察了负载温度、负载时间、离子液体与载体的配比、溶剂等负载反应条件对催化剂催化性能的影响。结果表明： 在115 ℃,2.0 MPa,4 h条件下,PC产率为89.9%,选择性为99.5%。     

细胞固定化条件对紫草细胞生产紫草色素的影响

以海藻胶做包埋剂,研究了固化液构成、细胞包埋量和胞龄对紫草色素合成的影响。结果表明：固定化细胞合成紫草色素的合适固化液为含有０．１ｍｏｌ／ＬＣａＣｌ２的紫草色素生产培养基,该固化条件比较温和,并可长久保持固定化细胞活性;最适宜的细胞包埋质量分数为１０％２０％;用于细胞包埋的最佳细胞生长时间为１７ｄ。对紫草细胞固定化培养生产紫草色素过程的动力学特征进行了分析,建立了基质消耗和色素合成的动力学模型,并用该模型对实验数据进行了回归分析,实验数据与理论值之间具有比较令人满意的一致性     

羧基修饰的超顺磁纳米粒子直接固定化罗伊氏乳杆菌

利用共沉淀法结合高锰酸钾氧化制备所得表面羧基修饰的超顺磁性纳米粒子吸附于罗伊氏乳酸杆菌表面,在磁场协助下实现细胞的固定化。吸附机理分析表明小尺寸相互作用和静电相互作用是磁性纳米粒子与细胞之间的主要作用。分别考察了菌体/磁性粒子质量比、pH、温度、时间等对固定化罗伊氏乳酸杆菌的影响,确定最佳固定条件为菌体与磁性纳米粒子相对质量比为2.25,在pH=3、温度25℃的条件下固定化0.5 h,可实现91%的细胞固定化。最后,对固定化后的细胞进行再培养,与游离细胞相比,两者表现出类似的代谢特征,证实细胞经固定化后仍具有活性。因此,羧基修饰的超顺磁性纳米粒子可成功用于细胞固定化,在不影响细胞活性的情况下,通过磁分离实现细胞的重复利用。     

用固定化L-赖氨酸脱羧酶细胞制备1,5-戊二胺

研究了4种固定化蜂房哈夫尼菌(H.alveiAS1.1009)菌体细胞的材料和方法,包括海藻酸钙包埋法、半透膜透析袋法、海藻酸钙-明胶交联包埋法和明胶包埋法。其中海藻酸钙包埋法稳定性最好,该方法最优转化条件为:在ρ(海藻酸钠)=30g/L的水溶液中,最适菌体质量浓度为ρ(菌体)=30.8g/L,100mL转化液中海藻酸钙球体体积为30mL,转化最适温度为37℃,最适pH=5.0。用该方法转化测得比酶活可达1028.9U。重复性佳:第一批固定化细胞酶活可达游离菌体的98.62%,第四批可达第一批酶活的38.68%。     

表面活化甲壳素固定化Gibberella intermedia生物转化制备异烟酸

甲壳素及其衍生物资源来源广泛、具有生物安全性,在材料、食品、化工等领域拥有良好的应用前景。本研究首次尝试采用经表面活化的甲壳素为包埋材料对产腈水解酶的G. intermedia游离细胞进行固定化,对固定化条件进行了初步优化,选取甲壳素质量分数3%,三聚磷酸钠质量分数7%,固定化时间5 h,固定菌体浓度为20g/L时催化活力达到最高。对固定化细胞的催化应用特性进行了表征,结果表明固定化细胞对4-氰基吡啶转化反应的最适反应温度为50℃,最适pH值为7.0;最适底物浓度为125mmol/L,最大产物耐受浓度为400mmol/L,均明显优于游离细胞。探索了将制备的固定化细胞直接应用于4-氰基吡啶生物转化合成异烟酸,固定化细胞可重复利用14批次,而相应游离细胞仅为3次。     

Immobilization of mortierella vinacea cells by radiation polymerization

Immobilization of Mortierella vinacea cells, which contain active α-galactosidase, by radiation polymerization at low temperatures was studied. The durability of the enzymatic activity of the immobilized cells was examined by repeating the batch enzyme reaction. The enzymatic activities of the immobilized cells obtained with hydrophilic monomers was affected by the concentrations of the cells and monomer in which optimum conditions were observed. The enzymatic activity of the immobilized cells obtained with hydrophilic monomer was compared to that of hydrophobic monomers. Michaelis constants of the immobilized cells varied with monomer concentration. The effect of addition of porous solid substances on the immobilization of the cells was studied.     

Immobilization of proteolytic enzymes in carboxymethylchitin films and sponges (review)

The possibility of immobilization of the proteolytic enzymes collagenase and terrilytin in chitin carboxymethyl ester films and sponges was demonstrated and some characteristics of this process were investigated. It was found that the optimum pH for immobilization of collagenase and terrilytin lies in the range of 6.5–7.5, which approximately corresponds to the optimum pH of the effect of native enzymes. According to the data from in vitro experiments, the activity of the immobilized enzymes at the optimum pH of immobilization is 75–50% for collagenase and 80–90% for terrilytin. An increase in the molecular weight of carboxymethylchitin in the range of 60–600 kilodaltons significantly strengthens the films and simultaneously decreases the activity of the immobilized enzymes, probably due to the stronger binding of the molecules of the enzyme in the matrix of higher molecular weight. In immobilization of enzymes in sponges, the molecular weight of the polymer matrix has no effect on the activity of the immobilized enzymes. Changing the degree of substitution of carboxymethylchitin in the 0.7–1.3 range has almost no effect on the activity of the enzymes immobilized in the films and sponges.Translated from Khimicheskie Volokna, No. 5, pp. 34–37, September–October, 1995.     

氧化葡萄糖酸杆菌的固定化及在鼓泡式反应器中制备乙醇酸

考察了包埋法固定氧化葡萄糖酸杆菌的过程,并且研究了利用该固定化细胞在鼓泡塔生物反应器中转化乙二醇制备乙醇酸。固定化的最优条件为:聚乙烯醇的最佳质量分数为8%,海藻酸钠为0.8%,最适湿细胞包埋量为每毫升凝胶0.27 g。固定化细胞对酸碱的抗性和热稳定性均较游离细胞有所提高。利用固定化细胞在鼓泡式反应器中转化乙二醇制备乙醇酸,在乙二醇质量浓度70 g/L的条件下,首批转化率达到92.6%,并且可以实现连续转化6个批次,平均转化率在85%以上。     

以层柱黏土为载体固定辣根过氧化物酶

引 言 辣根过氧化物酶(HRP)能在较宽的温度、pH值、污染物浓度和盐度范围内将多种芳香性化合物催化氧化为酚氧自由基,它们之间可聚合生成溶解性较差的聚合物从溶液中沉淀出来,便于采用混凝法对其进行去除[1-2].许多学者将该酶用于含酚废水处理中,取得了较好的效果.然而,传统酶催化方法中使用的大多是溶液酶,不仅不能循环使用以节省费用,而且易受废水中其他污染物的影响,稳定性差,加上酶的费用较高,因此酶法在废水处理中尚未得到有效推广应用[3].国内外学者研究发现酶固定化后稳定性大大提高,可重复或连续使用,这样不仅降低了废水处理的成本,而且还避免了蛋白质的污染等问题[4].     

New hypotheses for hydrogenase implication in the corrosion of mild steel

The influence of [Fe]-hydrogenase from Clostridium acetobutylicum was studied on the anaerobic corrosion of mild steel. Two short-circuited mild steel electrodes were exposed to the same solution and hydrogenase was retained on the surface of only one electrode thanks to a dialysis membrane. The galvanic current and the electrode potential were measured as a function of time in order to monitor the difference in electrochemical behaviour induced by the presence of hydrogenase. A sharp potential decrease of around 500 mV was controlled by the deoxygenating phase. When hydrogenase was introduced after complete deoxygenation, significant heterogeneous corrosion was observed under the vivianite deposit on the electrode in contact with hydrogenase, while the other electrode only showed the vivianite deposit, which was analysed by MEB and EDX. The effect of hydrogenase was then confirmed by monitoring the free potential of single coupons exposed or not to the enzyme in a classical cell after complete deoxygenating. In both phosphate and Tris-HCl buffers, the presence of hydrogenase increased the free potential around 60 mV and induced marked general corrosion. It was concluded that [Fe]-hydrogenase acts in the absence of any final electron acceptor by catalysing direct proton reduction on the mild steel surface.     

Pectinlyase immobilization on epoxy supports for application in the food processing industry

The immobilization of a pectinlyase (PL, EC 4.2.2.3) contained in a commercial enzymic preparation was studied in view of its use for fruit pulp and juice processing. Two epoxy supports were tested for immobilization. These included Eupergit C (Rohm), a synthetic polymer, and γ-alumina functionalized with γ-glycidoxypropyltrimethoxysilane. In both biocatalysts, the catalytic response was not found to be high. The highest response in terms of immobilization yield and immobilized PL activity, however, was reported for Eupergit C (approx. 115 unit g^?1 at optimum pH).     

Immobilization of laccase from Myceliophthora thermophila on functionalized silica nanoparticles: Optimization and application in lindane degradation

This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003? laccase) on amino modified fumed nano-silica(AFNS) and the possible use in bioremediation. Hereby, for the first time, factors affecting the immobilization of Novozym 51003? laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization. The highest specific activity(13.1 IU·mg~(-1) proteins) was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield, reaching68.3% after the equilibrium time, at optimum pH 5.0, was obtained. Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min, following pseudo-first-order kinetics. The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane. Within 24 h, lindane concentration was reduced to 56.8% of initial concentration and after seven repeated reuses it retained 70% of the original activity.     

Effect of the polymer matrix on the immobilization of lipase by radiation polymerization

Summary Effect of the hydrophobicity of the polymer matrix on the immobilization of lipase by radiation polymerization was studied. The enzymatic activity of the immobilized enzyme composites was affected by hydrophobicity, and optimum hydrophobicity existed for the enzyme reaction at the surface of the immobilized enzyme composites. Furthermore, the enzymatic activity was affected by monomer concentration. In the immobilization using polyethyleneglycole diacrylate monomers, the enzymatic activity increased with increasing number of ethyleneglycole unit in the monomer molecule.     

氧化石墨烯固定化α-淀粉酶

以石墨粉为原料,使用改进Hummer法制备氧化石墨烯,采用沉积法固定化α-淀粉酶,并对固定条件进行优化。分别采用NaOH-CH₂ClCOOH法和HNO₃-H₂SO₄法制备羧基化氧化石墨烯,并将其用于固定化α-淀粉酶。结果表明,沉积法固定化酶的最适宜温度为65 ℃,最适宜pH=7.0。连续催化反应9次后,固定化酶活力仍能保持初始固定化酶活力的47.81%。经比较发现,NaOH-CH₂ClCOOH法更有利于羧基化氧化石墨烯的制备,该方法制备的羧基化氧化石墨烯产率为HNO₃-H₂SO₄法的1.2倍。     

PRODUCTION OF ACETIC ACID FROM HYDROGEN AND CARBON DIOXIDE BY CLOSTRIDIUM SPECIES ATCC 29797

Clostridium sp. ATCC 29797 converts H₂ and CO₂ to acetic acid in stationary phase in almost 100% yield. The resting cells are stable for up to 300 h and produce acetate to a final product concentration of 221 mM. The specific productivity of acetate during the stationary phase appears limited by the specific activity of hydrogenase. A high pressure fermentor was constructed to investigate the use of pressure, as a general strategy, for increasing the rates of microbial syngas conversions. Fermentations were conducted at pressures up to 1000 psig using various mixtures of H₂ and CO₂. The use of high pressure proved to be an effective method for eliminating gas transfer resistances and increasing the volumetric productivity. Hydrostatic pressure had little effect on the specific productivity of acetate, however, high partial pressures of hydrogen caused the specific productivity and total acetate accumulated to decrease. High pressures of hydrogen also caused a decrease in the specific activity of hydrogenase. These results suggest that Clostridium sp. ATCC 29797 attempts to control the input of reducing power by regulating the activity of hydrogenase.     

介孔分子筛SBA-16固定化木瓜蛋白酶性质的研究

以介孔分子筛SBA-16为载体采用物理吸附的方法对木瓜蛋白酶进行了固定化,研究了固定化条件对酶的相对活性的影响及在不同pH值下游离酶和固载酶的pH稳定性。实验结果表明当1 g载体的給酶量为30 mg,固定化时间为2.5 h,pH值为7.0时,固定化木瓜蛋白酶的相对活性最好。与游离酶相比,固定化酶的pH稳定性有明显改善。     

Urease immobilized on chitosan membrane: Preparation and properties

Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94% of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the free enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low pH values was improved by immobilization; temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potential for practical application.