以伴刀豆球蛋白为介质定向固定化脲酶的研究

将戊二醛将伴刀豆球蛋白(ConA)和壳聚糖载体交联,然后利用ConA与脲酶糖链的特异性结合作用,实现脲酶的定向固定化.定向固定化的最适条件为戊二醛浓度3.5%、ConA浓度1mg/mL、ConA溶液pH值7.0、脲酶浓度0.4mg/mL.定向固定化脲酶的最适pH 5.0～6.0、最适温度77℃,米氏常数Km11.76mmol/L,与游离酶及非定向固定化脲酶比较,定向固定化脲酶的最适pH向酸性范围发生了偏移并有更宽的pH适用范围,最适温度提高,与底物的亲和力较大,且有较好的操作稳定性.     

米曲霉F-81产中性蛋白酶的固定化条件及其特性

以海藻酸钠为载体,戊二醛为交联剂固定化米曲霉F-81产中性蛋白酶,研究了固定化条件及固定化酶的性质。结果表明,固定化的最佳条件为:固定化时间1 h、海澡酸钠浓度4%、戊二醛浓度9%、CaCl2浓度0.7 mol/L。在此条件下固定化的中性蛋白酶活力为游离酶活力的68%。固定化酶的最适作用温度为65℃,最适作用pH值为7.0。60℃下酶稳定性较好,80℃下处理60 min,粗酶中几乎检测不到酶活力;中性蛋白酶pH稳定范围为6.5-9.5。Km值为24.83 mg/mL,最大反应速率Vmax为0.043 12 mg/min。     

壳聚糖固定化木瓜蛋白酶的研究

以自制的壳聚糖作为载体,用戊二醛作交联剂,优化了固定化条件,研制成壳聚糖固定化木瓜蛋白酶。其活性回收率达到42—53％,操作半衰期达到一个月以上,对热、乙醇以及尿素的稳定性有很大的提高,Km值为0.67×10~2mg/mL,最适温度65—70℃,最适pH8.0,能使啤酒中的蛋白质浓度从56.5mg/L减少到2.7mg/L,可以消除啤酒的低温混浊现象。     

高分子膜载体固定化木瓜蛋白酶

在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0～7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。     

壳聚糖固定化德氏根霉脂肪酶的研究

研究了壳聚糖吸附和戊二醛交联对脂肪酶固定化条件,在室温条件下将0．4g酶粉溶于pH6．0缓冲液中,加入10g壳聚糖,摇匀,再加入浓度为0．6％戊二醛交联6h,得到固定化酶,酶活力回收率约为54．2％。固定化酶的半失活温度比游离酶的高,半失活温度由游离酶的47℃提高到100℃,最适反应温度由40℃上升至80℃,最适pH由6下降到5．5,固定化酶K’m值由游离酶的Km 50mg／mL增加到56mg／mL。该固定化脂肪酶用于酯的合成;在80℃条件下经过10批次连续水解植物油反应,固定化酶的活力仍保持在82．6％以上。     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

固定化黑曲霉β-葡萄糖苷酶制备染料木素活性苷元的研究

比较了以海藻酸钠为载体,用胶囊法、包埋-交联法、交联-包埋法三种不同方法固定化黑曲霉β-葡萄糖苷酶的效果,并研究了最佳固定化方法的固定化条件和固定化酶的部分性质。结果表明,交联-包埋法即β-葡萄糖苷酶与0.20%戊二醛交联后再用2.0%海藻酸钠包埋的固定化方法中酶结合效率和酶活力回收率最高。海藻酸钠浓度和戊二醛浓度对酶结合效率影响较大,戊二醛浓度和包埋颗粒直径大小对酶活力回收率影响显著。与游离酶相比,制备的固定化酶最适温度、最适pH值和Km值分别由50℃、4.5和2.57μg/mL下降到40℃、4.0和2.02μg/mL。固定化酶具有更强的耐酸性和稳定性。该固定化酶用于大豆异黄酮活性苷元染料木素的合成,重复使用6次后,固定化酶的活力仍保持84.94%,染料木苷转化率为56.04%。     

壳聚糖载体的制备及脲酶的固定化研究

以甲壳素为原料,制备出壳聚糖载体,并对脲酶进行固定化。通过测量悬挂醛基探讨了交联条件对载体性能的影响,优化了脲酶的固定化条件,研究了固定化酶的酶学性质,并与游离酶进行了比较。结果表明。制备载体的最优条件是将微球用6％的戊二醛活化2h,最佳联酶条件是载体与脲酶共反应1h。该固定化酶的最适温度为65℃,最适pH值为6．6,米氏常数为0．009mol／L,较游离酶均有较大改善。热稳定性较游离酶有很大的提高,且具有良好的操作稳定性。     

固定化黑曲霉β-葡萄糖苷酶制备染料木素活性苷元的研究

比较了以海藻酸钠为载体,用胶囊法、包埋-交联法、交联-包埋法三种不同方法固定化黑曲霉β-葡萄糖苷酶的效果,并研究了最佳固定化方法的固定化条件和固定化酶的部分性质。结果表明,交联-包埋法即β-葡萄糖苷酶与0.20%戊二醛交联后再用2.0%海藻酸钠包埋的固定化方法中酶结合效率和酶活力回收率最高。海藻酸钠浓度和戊二醛浓度对酶结合效率影响较大,戊二醛浓度和包埋颗粒直径大小对酶活力回收率影响显著。与游离酶相比,制备的固定化酶最适温度、最适pH值和K_m值分别由50℃、4.5和2.57 μg/mL下降到40℃、4.0和2.02 μg/mL。固定化酶具有更强的耐酸性和稳定性。该固定化酶用于大豆异黄酮活性苷元染料木素的合成,重复使用6次后,固定化酶的活力仍保持84.94%,染料木苷转化率为56.04%。     

脱乙酰壳聚糖固定化宇佐美曲霉木聚糖酶及固定化酶的性质和应用(英文)

研究壳聚糖吸附和戊二醛交联对木聚糖酶固定化条件 .将酶液加入到经醋酸溶液处理过的脱乙酰壳聚糖的pH 4 8的悬液中 ,加入浓度为 0 3%～ 0 4 %的戊二醛溶液 ,室温下 ,8h后得到固定化酶 .固定化酶的半失活温度比游离酶高 ,由 5 1℃升至 71℃ ,Km 值由游离酶的 1 2mg ml增加到1 5mg ml ,最适反应温度也由 5 5℃增加到 71℃ ,而最适反应pH由 4 6下降到 3 8.该固定化木聚糖酶可用于制造低聚木糖 .经过 10次连续应用实验后 ,该固定化酶的活力保持 81%     

Modification and characterization of cellulosic cotton fibers for efficient immobilization of urease

Cotton fibers were first grafted by polyacrylonitril in the presence of KMnO(4) and oxalic acid as a combined redox initiator. Moreover, modification of the grafted cotton fibers was done by changing the nitrile group (-CN) into hydrazidine group through the reaction with hydrazine hydrate, then the fibers were activated by glutaraldehyde to introduce free aldehyde groups which were able to react with amino groups of urease to form Schiff's base, and result in cotton fibers immobilized urease. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of enzyme before and after the immobilization of urease. The results showed that the optimum temperature of immobilized urease was 35°C, which was higher than that of the free enzyme (30°C), and the immobilized urease exhibited a higher relative activity than that of free urease over 35°C. The optimal pH for immobilized urease was 6.5, which was lower than that of the free urease (pH 7.0), and the immobilization resulted in stabilization of enzyme over a wider pH range. The kinetic constant value (K(m)) of immobilized urease was higher than that of the free urease. However, the thermal and operational stabilities of immobilized urease have been improved greatly.     

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

Urease Immobilization on Arylamine Glass Beads and its Characterization

Jack bean urease has been immobilized on arylamine glass beads (200–400 mesh size, 75–100 Å pore size) and its properties compared with soluble enzyme. The binding of urease was 13.71 mg per gram beads. The K_m for soluble and immobilized urease for urea was 4.20 mM and 8.81 mM, respectively. V_max values of urease decreased from 200 to 43.48 μmol of ammonia formed per min per mg protein at 37°C on immobilization. Both pH and buffer ions influenced the activities of soluble as well as immobilized urease. Soluble urease exhibited pH optima at 5.5 and 8.0. However, immobilized urease showed one additional pH optimum at 6.5. In comparison to phosphate buffer, citrate buffer was inhibitory to urease activity. Immobilization of urease on arylamine glass beads resulted in improved thermal, storage and operational stability. Because of inertness of support and stability of immobilized urease, the preparation can find applications in ‘artificial kidney’ and urea estimation in biological fluids viz., blood, milk etc.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

烟草多酚氧化酶的分离与固定化技术研究

多酚氧化酶属于氧化还原酶类,国际酶学委员会推荐名为儿茶酚氧化酶（ＥＣ１．１０．３．１ｐｏｌｙｐｈｅｎｏｌｏｘｉｄａｓｅ,ＰＰＯ）．该酶与食品工业、三废处理、医药卫生关系较为密切,因而研究较多．如近年来鸭梨［１］、蘑菇［２］、香蕉果肉组织［３］、荔枝果皮［４］等等中的多酚氧化酶均有研究报道．目前研究用固定化多酚氧化酶检测废水中酚类物质含量,进行环境检测;及其从工业废水中除去酚类,达到治理三废的目的．Ｍｏｓｂａｃｎ［５］（１９７６）研制成多酚氧化酶固定化酶柱,与氧电极检测器组合联用,可检测水中２０…     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。