醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸(PAA)改性的聚乙烯(PE)膜为载体,研究了醇脱氢酶(ADH)的两种固定化路线,并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺(PEI)进一步改性,使用戊二醛(GA)固定化ADH。最优固定化pH为6.0,温度为5～15℃,酶浓度为1.0 mg/ml,GA浓度为0.01%(质量);固定化酶的最适反应pH为6.5,温度为15～30℃,反应速率最高为9.6μmol/(L·min);重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体,用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为活化剂,固定化ADH。EDC和NHS最优摩尔比为1∶0.5,固定化时间为24 h;固定化酶的最适反应pH为6.5,温度为20～37℃,反应速率为15.58μmol/(L·min);重复利用10次后可保持53.8%的活性。     

The yeast alcohol dehydrogenase catalysed conversion of cinnamaldehyde to cinnamyl alcohol

The kinetics of the conversion of trans-cinnamaldehyde to trans-cinnamyl alcohol catalysed by yeast alcohol dehydrogenase (EC 1.1.1.1) have been characterised. Reaction with the free enzyme is curtailed after a short time as a result of inactivation by the substrate. It has been shown that immobilisation of the enzyme provides substantial protection against this inactivation. Use of the immobilised enzyme in a flow-through reactor further enhanced the enzyme lifetime, creating a viable synthetic process. The product cinnamyl alcohol may be recovered by continuous solvent extraction.     

聚乙烯亚胺/多巴胺改性氧化硅固定碳酸酐酶

利用聚乙烯亚胺（PEI）/多巴胺（DA）共沉积法改性氧化硅，并以此为载体固定化碳酸酐酶（CA）。考察了PEI/DA质量比、沉积时间对沉积率的影响，用傅里叶红外光谱（FTIR）和扫描电子显微镜（SEM）对改性前后的微球进行了表征；研究了沉积率、载体用量、酶浓度及戊二醛（GA）浓度对固定化酶活回收率的影响；考察了固定化酶的储存稳定性和重复利用性。结果表明，随PEI/DA质量比增加，沉积率先增加后降低，质量比为1∶1时最大；随沉积时间增加，沉积率线性增长，10 h后PEI/DA体系沉积率为单独DA沉积改性的2.66倍，但沉积时间对N元素含量和酶活回收率影响不大；酶固定化时载体用量存在饱和值，CA和GA浓度的最优值分别为0.8 mg/ml和0.1%(质量)，此时酶活回收率可达78.8%。在25℃下储存30 d后，固定化酶的保留活性为77.2%，而游离酶只有12%；重复使用10次后，固定化酶仍能保持88.3%的相对活性。     

Liver alcohol dehydrogenase immobilized on polyvinylidene difluoride

A physical method for immobilization of liver alcohol dehydrogenase (ADH) by hydrophobic adsorption onto a supporting membrane of polyvinylidene difluoride (PVDF) was performed. Simultaneously, a physicochemical characterization of the immobilized enzyme regarding its kinetic behaviour was performed. The activity/pH profile observed points to an effect of pH on activity that is completely different from the case of ADH in solution. The disturbance in the typical bell-shaped profile owing to the fact that the enzyme was immobilized is explained on the basis of a potent limitation to the diffusion of the protons in the support. The findings of the present work also reveal the existence of an effect that limits free external diffusion of the substrate towards and/or the product from the support; this effect seems to be the determinant of the overall rate of the enzymatic reaction and is thus of great importance in the effective kinetic behaviour (v([S])) of immobilized ADH, whose kinetic behaviour is complex (non-Michaelian), as may be seen from the lack of linearity observed in the corresponding double reciprocal and Eadie-Hofstee plots. By non-linear regression numerical analysis of the v([S]) data and application of the F-test for model discrimination, the minimum rate equation necessary to describe the intrinsic kinetic behaviour ofPVDF-immobilized ADH proved to be one of the polynomial quotient type of degree 2:2 (in substrate concentration).     

Inhibition of horse liver and yeast alcohol dehydrogenase by aromatic and aliphatic aldehydes

The reactivity of yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) towards 12 different aldehydes was tested. YADH was inhibited by pre-incubation with citral, citronellal, p-cuminaldehyde, p-anisaldehyde, m-tolualdehyde, trans-cinnamaldehyde, salicylaldehyde, p-hydroxybenzaldehyde and benzaldehyde, although of these aldehydes only trans-cinnamaldehyde acted as a substrate for the enzyme. HLADH was inhibited, to a much smaller extent, by pre-incubation with citral, salicylaldehyde, citronellal, p-anisaldehyde, piperonaldehyde, trans-cinnamaldehyde, p-hydroxybenzaldehyde and p-cuminaldehyde and all of these aldehydes acted as substrates for the HLADH. Immobilisation of the enzymes on CNBr-activated Sepharose 4B gave protection against inhibition by the aldehydes, suggesting a means of significantly extending the useful lifetime of the enzymes when they are used in industrial processes.     

Immobilization of polyphenol oxidase on carboxymethylcellulose hydrogel beads: preparation and characterization

Carboxymethylcellulose (CMC) beads were prepared by a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently attached to the CMC beads. Polyphenol oxidase (PPO) was then covalently immobilized onto CMC beads. The enzyme loading was 603 µg g⁻¹ bead and the retained activity of the immobilized enzyme was found to be 44%. The K_m values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the V_max values were found to be 1890 and 760 U mg⁻¹ for the free and the immobilized enzyme, respectively. The optimum pH was 6.5 for the free and 7.0 for the immobilized enzyme. The optimum reaction temperature for the free enzyme was 40 °C and for the immobilized enzyme was 45 °C. Immobilization onto CMC hydrogel beads made PPO more stable to heat and storage, implying that the covalent immobilization imparted higher conformational stability to the enzyme. © 2000 Society of Chemical Industry     

脂肪酶固定化研究和应用

采用硅藻土作载体,进行了脂肪酶的固定化。利用固定化酶选择性催化1-苯乙醇与乙酸乙烯酯的转酯化反应,得到R-乙酸苯乙酯,进行1-苯乙醇的拆分。实验考察了不同吸附方法固定化酶的效果,确定效果最好的固定方法为载体涂布法,并对该法的固定化条件进行了优化。制备的固定化酶的转酯比活比游离酶提高了14．3倍。固定化没有改变酶的选择性．对映体过剩值仍大于98％。初步探讨了固定化酶和游离酶的反应过程。     

Retention and reusability of trypsin activity by covalent immobilization onto grafted polyethylene plates

This study investigated the activity of trypsin that had been covalently immobilized onto acrylic acid (AA)– and methacrylic acid (MAA)–grafted polyethylene (PE) plates—PE–g–PAA and PE–g–PMAA—using a water‐soluble carbodiimide as a coupling agent, as a function of the immobilized amount, the grafted amount, the pH value on immobilization, and the pH value and temperature at the activity measurement. The activity of trypsin immobilized on the PE–g–PAA plates at pH 6.0 decreased with an increase in the immobilized amount because of the crowding of trypsin molecules in the vicinity of the surfaces of the grafted PAA layers. The increase in the grafted amount resulted in a decrease in the activity of immobilized trypsin because of a decrease in the diffusivity of BANA molecules caused by the formation of dense grafted PAA layers for the PE–g–PAA plates and led to the increased activity because of the increase in the hydrophilicity of the whole grafted layers for the PE–g–PMAA plates. The activity of trypsin immobilized on the PE–g–PAA and PE–g–PMAA plates at pH 6 increased with an increase in the pH value, probably because of the expansion of trypsin‐carrying grafted PAA and PMAA chains and the increased diffusivity of Nα‐benzoyl‐DL ‐arginine‐nitroanilide hydrochloride molecules in the grafted layers. The optimum temperature of the activity of immobilized trypsin shifted to 50°C from 30°C for native trypsin. Immobilized trypsin was reusable without any denaturation and isolation at temperatures ranging from 20°C to 60°C and pH values ranging from 6 to 10. Trypsin immobilized on a PE–g–PAA plate had 95% of the remaining activity in relation to native trypsin at 30°C after preservation in a pH 7.8 buffer at 4°C over 6 months. These results made clear that alkaline and thermal stability, reusability, and storage stability can be much improved by the covalent coupling of trypsin on PE–g–PAA and PE–g–PMAA plates. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 3574–3581, 2003     

手性醇脱氢酶与甲酸脱氢酶的融合蛋白体系的构建

为了在手性催化的同时实现辅酶的再生,利用源于红平红球菌Rhodococcus erythropolis的手性醇脱氢酶chiral alcohol dehydrogenase(READH)与来源于博伊丁假丝酵母Candida boidinii的甲酸脱氢酶formate dehydrogenase(CbFDH)构建了一系列具有双酶活性的融合蛋白体系。分别为：1）READH的N端与麦芽糖结合蛋白maltose binding protein(MBP)的C端融合;2）CbFDH的N端与MBP的C端融合;3）READH的N端与CbFDH的C端融合;4）READH的C端与CbFDH的N端融合。结果表明：在所有的融合策略中,READH的活性都受到了较大的影响。READH在N端融合在CbFDH的C端后活性最高,但是此时CbFDH不具有活性。相反,READH的C端与CbFDH的N端融合后,CbFDH具有最高的活性。在READH的C端与CbFDH的N端融合的策略下,两者都具有活性。表明通过合理设计具有双重功能的融合蛋白,有望能提高其催化效率,为新型辅酶再生系统的建立提供基础。     

Immobilization of soluble eggshell membrane protein on polyethylene film surface: Effect on the culture of NIH3T3 in vitro

Polyethylene (PE) film surface is modified by combining plasma treatment and soluble eggshell membrane protein (SEP) immobilization. Contact angle measurements, attenuated total reflectance infrared spectroscopy, and X‐ray photoelectron spectroscopy confirm that SEP adheres tightly to the PE film surface. Mouse 3T3 fibroblasts are used as model cells to evaluate the biocompatibility of PE film surfaces before and after modification. Plasma pretreatment can incorporate polar groups onto the surface, benefitting tight immobilization of SEP. The hydrophilicity of the PE film surface modified by combining plasma treatment and SEP immobilization is increased as evidenced by contact angle measurements, and the biocompatibility of the surface is greatly improved as shown by cell culture. The surface of the modified material can endure rinsing with 10% acetic acid (a good solvent of SEP), which would be an advantage for further application as a biomaterial. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 1340–1345, 2006     

转谷氨酰胺酶在聚丙烯微孔膜上的固定化

研究了转谷氨酰胺酶在聚丙烯微孔膜上的化学固定化的影响因素,确定了最佳固定化工艺条件,即为:第一步光照反应6 min,单体质量分数为20%,第二步光照时间25 min,接枝率最高可达35.2%;己二胺质量分数为25%,胺烷基化时间150 min,胺烷基化温度60℃;戊二醛质量分数3%,戊二醛作用时间45 min;酶液浓度10 mg/mL,固定化时间20 h,固定化温度4℃,固定化酶膜的活力最高可达游离酶的45%.并研究了温度、pH、金属离子对固定化酶膜的酶学性质的影响,其贮存性能和操作稳定性也做了初步研究.     

醇脱氢酶KpADH的127位点对催化活性和对映选择性的影响

醇脱氢酶可用于合成手性化合物,在医药、材料等领域应用广泛。来源于多孢克鲁维酵母（Kluyveromyces polysporus）的醇脱氢酶KpADH同时具有还原(4-氯苯基)-(吡啶-2-基)-甲酮（CPMK）和氧化异丙醇、1,4-丁二醇的活性。通过分子对接和结构分析,发现Y127是紧靠底物的关键位点。本文通过对127位点定点饱和突变来提高催化活性和对映选择性,突变体Y127V和Y127I还原CPMK的比活力达到95.0U/mg和84.0U/mg,分别是野生型的6.5倍和5.8倍,突变体Y127L催化CPMK生成(R)-(4-氯苯基)-(吡啶-2-基)-甲醇[(R)-CPMA]的e.e.值由82％提高到99.2％。同时,127位点的突变提高了对醇类底物的氧化活性,突变体Y127I对异丙醇的比活力是野生型的1.46倍,而Y127C更青睐于对1,4-丁二醇的催化氧化,其比活力是野生型的3.00倍。分子间作用力分析表明,Y127L与底物CPMK间增加的氢键和π-π作用力稳定了底物构象,进一步提高了还原CPMK的对映选择性。本文为醇脱氢酶KpADH的分子改造和机制解析提供了指导,提高了该酶的工业应用潜力。     

Extraction of yeast alcohol dehydrogenase using reversed micelles formed with CTAB

A large molecular weight protein, alcohol dehydrogenase (ADH, 141 kDa), has been purified from baker's yeast using reversed micelles formed with a cationic surfactant (cetyltrimethylammonium bromide, CTAB) using the phase transfer method. Various parameters, such as pH, ionic strength and contact time of the two phases in ADH forward and backward transfer were studied. The active ADH was successfully recovered after a full forward and backward extraction cycle. The recovery of ADH activity obtained was ∼90% and the purification factor was 3.1 for the overall process. © 2000 Society of Chemical Industry     

生物催化剂的膜固定化

合成膜以其优良的载体性能在生物催化剂的固定化中，得到愈来愈广泛的应用。本文就膜固定生物催化剂的方式和方法作了详细的阐述，并扼要地介绍了生物催化剂的膜固定化技术的发展方向。     

Immobilization of yeast alcohol dehydrogenase by entrapment and covalent binding to polymeric supports

Yeast alcohol dehydrogenase (YADH), which catalyzes oxidoreductions of a broad spectrum of substrates, was immobilized by entrapping it into a network of a poly(acrylamide‐co‐hydroxyethyl methacrylate) copolymer and was also covalently bound onto porous chitosan beads activated through glutaraldehyde. Maximum retention of YADH activity achieved was 90 and 24% for entrapment and covalent binding, respectively. The results obtained for thermal, storage, and operational stability of entrapped and covalently bound YADH were compared with free YADH. The immobilized enzyme showed improved thermal and storage stability. The immobilized enzymes also retained 50% activity after six and eight cycles. Enzyme‐catalyzed oxidation of ethanol was observed to be diffusion‐controlled through Lineweaver–Burk plots. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 1299–1305, 2001     

酶的固定化及化学修饰

综述了酶的固定化与化学修饰方法 ,对改善酶在工业应用时的各种性质 ,使酶制剂实现大规模工业应用具有积极的意义。     

乙酰胆碱酯酶固定化方法的研究

以戊二醛为交联剂,牛血清白蛋白(BSA)为保护剂,将乙酰胆碱酯酶(AchE)交联固定到商品载体上,制备固定化酶片。对影响酶固定化的重要因素进行了考察,获得了最佳固定化条件。实验结果表明,以孔径为0．45μm的硝酸纤维素滤膜作栽体,乙酰胆碱酯酶用量10U,5％(体积分数)戊二醛2μL,1％(质量分数)BSA10μL,配成70μL的酶溶液,3℃固定8h,可获得较好的固定化效果。不同批次制备的酶片,其活力值标准偏差为3.27％～5．03％,酶片在0．1mol／L pH8．5磷酸盐缓冲溶液中3℃下可保存60d。‘     

用于酶固定化的高分子载体材料研究进展

固定化酶是一种高效、高选择性和反应条件温和的生物催化剂。近年来,高分子材料作为酶固定化载体的研究越来越受到重视,相关的研究报道很多。对近十多年来用于固定化酶的高分子载体材料以及它们的优缺点进行了综述,并对用于酶固定化高分子载体材料的发展前景作了展望。