共查询到19条相似文献,搜索用时 78 毫秒
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采用(NH4)2SO4分级沉淀及Q-Sepharose HP柱层析两步纯化,首次从可高效降解聚乙烯醇(PVA)的混合菌系发酵产物中分离纯化获得了纯蛋白聚乙烯醇脱氢酶(PVADH),并对其酶学性质进行了研究. 结果表明,PVADH分子量为134.3 kDa,最适作用温度为35℃,最适作用pH值为7.5;PVADH以仲醇为底物时酶活性普遍高于以其他醇类为底物时,PVADH与PVA反应产物中检测到羰基化合物,证实PVADH对PVA有降解作用. 相似文献
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采用原位共聚法制备了磁性凝胶包埋固定化仲醇脱氢酶,并将其用于催化手性R-乙醇基吡啶的合成,通过SEM和VSM对其形貌和磁性进行了表征,并对其酶学性质进行了研究。结果表明,固定化仲醇脱氢酶具有较高的热稳定性和酸碱稳定性,最适反应温度为45℃,最适反应pH值为7.0;重复使用5次后的残留活性为41%;利用磁性凝胶包埋固定仲醇脱氢酶,方法简单,操作简便,且易于回收重复使用。 相似文献
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膜材料的亲疏水性对固定化脂肪酶的影响 总被引:5,自引:0,他引:5
用吸附法固定脂肪酶时,膜材料的亲疏水性对固定化酶的量、比活力和活力稳定性等有很大影响.今以柱状假丝酵母脂肪酶和猪胰脂肪酶为研究对象,选取了8种亲疏水性不同的膜材料(醋酸纤维素、聚丙烯腈、聚酰胺、聚砜、聚醚砜、聚偏二氟乙烯、聚丙烯和聚四氟乙烯)作为固定化载体,用吸附法制备了固定化脂肪酶膜.研究结果表明,强疏水性聚四氟乙烯和聚丙烯膜对两种酶的吸附量都比较大,且固定化酶的比活力和活力回收率比较高,聚四氟乙烯固定化柱状假丝酵母酶比游离态酶的半衰期提高了6倍以上.强亲水性醋酸纤维素膜对猪胰脂肪酶的吸附量比聚四氟乙烯高,但是固定化酶的比活力、活力回收率比强疏水性膜低,而接触角在40°~50°的聚酰胺膜和聚砜膜的吸附量最小.因此吸附法制备固定化脂肪酶膜,选择聚丙烯膜和聚四氟乙烯膜是合适的,制备的优化条件为吸附温度25℃,酶溶液的pH为7.5,吸附时间10 h. 相似文献
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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。 相似文献
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转录表达来源于Klebsiella.pl,3-丙二醇脱氢酶(PDH)基因片段的E.coli工程菌,在5L发酵罐、30℃、pH值7.0、200r/min(微氧)条件下培养20h;发酵菌体经破碎分离后所得样品通过HisTrap HP柱亲和层析一步纯化得到电泳纯的PDH,纯化倍数为2.94倍,活性回收率为50%,经SDS-PAGE电泳鉴定获得一条清晰条带,表达蛋白亚基相对分子质量约为41kDa。用改进的溶胶-凝胶法(杂化凝胶)包埋纯化酶,考察正硅酸乙酯(TEOS)浓度、海藻酸盐(ALG)浓度、CaCl2浓度及ALG与TEOS体积比等因素对酶固定化效果的影响,结果表明,较优包埋条件为:ρ(TEOS)=20g/L,ρ(ALG)=30g/L,ρ(CaCl2)40g/L,V(ALG):V(TEOS)=3:1;由此制备的PDH的ALG-SiO2杂化凝胶保藏60天后酶活仍保持80%,进行批次反应时,反应3批后酶活保持70.3%,反应第6批时酶活剩余29.8%。 相似文献
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生物催化剂的膜固定化 总被引:2,自引:0,他引:2
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。 相似文献
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转录表达来源于Klebsiella.pl,3-丙二醇脱氢酶(PDH)基因片段的E.coli工程菌,在5L发酵罐、30℃、pH值7.0、200r/min(微氧)条件下培养20h;发酵菌体经破碎分离后所得样品通过HisTrap HP柱亲和层析一步纯化得到电泳纯的PDH,纯化倍数为2.94倍,活性回收率为50%,经SDS-PAGE电泳鉴定获得一条清晰条带,表达蛋白亚基相对分子质量约为41kDa.用改进的溶胶-凝胶法(杂化凝胶)包埋纯化酶,考察正硅酸乙酯(TEOS)浓度、海藻酸盐(ALG)浓度、CaCl2浓度及ALG与TEOS体积比等因素对酶固定化效果的影响,结果表明,较优包埋条件为:p(TEOS)=20g/L,p(ALG)=30g/L,p(CaCl2)=40g/L,V(ALG):V(TEOS)=3:1;由此制备的PDH的ALG-SiO2杂化凝胶保藏60天后酶活仍保持80%,进行批次反应时,反应3批后酶活保持7013%,反应第6批时酶活剩余29.8%. 相似文献
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构建了无电荷超滤膜酶膜反应器,考察了影响辅酶截留率的多种因素.实验结果表明,连续超滤膜反应器中加入聚乙烯亚胺(PEI,50 kDa),NAD 能够有效地被超滤膜截留.在Tris-HCl缓冲液中,PEI/NAD 摩尔比为5:1时,达到较高的辅酶截留率(R=0.823).溶液离子强度的增加减少辅酶的截留率,但可增加超滤膜超滤流速.在含盐溶液中,添加0.5%或1.O%的牛血清白蛋白可增加NAD 截留率.本装置对NADH的截留率高于NAD 的截留率,因此PEI适用于辅酶再生体系.同时还考察了1,3-丙二醇酶催化体系中PEI对甘油脱氢酶和1,3-丙二醇氧化还原酶酶活力的影响. 相似文献
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Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles. 相似文献
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W. Richard Bowen Nigel Lambert Shirley Y. R. Pug Frank Taylor 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》1986,36(6):267-272
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. 相似文献
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M. G. Roig J. F. Bello M. A. De Moreno Vega J. M. Cachaza J. F. Kennedy 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》1990,49(2):99-113
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). 相似文献
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Protective effects of Lactococcus lactis expressing alcohol dehydrogenase and acetaldehyde dehydrogenase on acute alcoholic liver injury in mice 下载免费PDF全文
Yunbin Lyu Lei Zhong Yanan Liu Jing Lu Gisèle LaPointe Fengxia Lu Zhaoxin Lu 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2018,93(5):1502-1510
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W. Richard Bowen Shirley Y. R. Pugh Nicola J. D. Schomburgk 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》1986,36(4):191-196
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. 相似文献
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M Yakup Arca 《Polymer International》2000,49(7):775-781
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 Km values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the Vmax 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 相似文献
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脂肪酶固定化研究和应用 总被引:11,自引:0,他引:11
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。 相似文献
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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。 相似文献