壳聚糖-精氨酸树脂固定化胰凝乳蛋白酶及其性质

以具柔性亲水手臂的壳聚糖-精氨酸树脂为载体,用戊二醛交联胰凝乳蛋白酶,获得壳聚糖-精氨酸树脂固定化胰凝乳蛋白酶. 最佳固定化条件为:m(酶)∶ m(载体)=20∶ 1 000、戊二醛体积分数为1.0%、pH=5.20、30 ℃交联60 min. 固定化酶活力达850 U/g,Km为1.83 mmol/L,比游离酶增大33.6%,比交联壳聚糖固定化酶低24.0%. 壳聚糖-精氨酸树脂固定化胰凝乳蛋白酶水解时间进程曲线与游离酶基本一致,均在反应30 min达到最大速率,最适温度为70 ℃,比游离酶升高10 ℃;在75 ℃时的半衰期可达6.0 h,比游离酶提高约4.3倍;最适pH值为5.92,比游离酶向酸性偏移2pH单位. 4 ℃贮存半衰期为49 d.     

磁性壳聚糖微球用于酶的固定化研究Ⅱ磁性壳聚糖微球对脲酶的吸附及其酶学性质的研究

本文用磁性壳聚糖作为载体用吸附法对脲酶进行固定化研究。结果表明,磁性壳聚糖对脲酶的固载量与磁性壳聚糖微球的粒径、交联度及酶溶液的离子强度成反比;固定化脲酶和自由酶的最适温度分别为80℃和70℃,固定化脲的最适合pH值变化不大,固定化脲酶和自由酶的米氏常数km分别为0.00546mol/L和0.19mol/L。     

壳聚糖固定化血管紧张素转化酶及其性质

以壳聚糖微球为载体, 戊二醛为交联剂固定化血管紧张素转化酶, 研究了酶固定化的最优条件和固定化酶的性质. 结果表明, 在戊二醛质量分数为2.5%、给酶量为8 mg/mL时, 固定化酶的比活性最大, 为0.085 U/g. 固定化酶在40~50 ℃, pH在7~9之间有最大活性, 其米氏常数Km为2.39 mmol/L. 同时, 固定化酶具有良好的稳定性, 可重复利用.     

壳聚糖–精氨酸树脂固定化胰凝乳蛋白酶及其性质

以自制的多孔、具柔性亲水手臂的壳聚糖–精氨酸树脂为载体,戊二醛为交联剂固定胰凝乳蛋白酶,确定了酶与载体的最佳比例为20 mg酶/g湿树脂,交联剂的最佳用量为10 mL 1.0%戊二醛/1.5 g湿树脂,交联时间为60 min,所得固定化酶的活力回收率达68.95%。固定化胰凝乳蛋白酶的Km为8.36 mg/mL,比游离酶增大1.52倍,其酶促反应10 min达到最大速率,具有接近游离酶的催化时间进程曲线;其最适温度为70 ℃,比游离酶升高10 ℃;其最适pH值为5.92,比游离酶酸性偏移2个pH值。此外,固定化胰凝乳蛋白酶具有良好的热稳定性和贮存稳定性,75 ℃时的半衰期为8 h,4 ℃时的半衰期为46天。     

壳聚糖固定化醇脱氢酶的制备及其酶学性质研究

报道了醇脱氢酶(ADH)的固定化和酶学性质研究。以壳聚糖作为载体,戊二醛作为交联剂。固定化ADH的最适条件为:以6%戊二醛将壳聚糖交联2 h,与ADH反应2.5 h。对游离和固定化ADH酶学性质的研究表明:酶促反应的最适pH均为8.2,最适温度分别为37℃和40℃,对乙醇的表观米氏常数Km分别为33.9 mmol/L和46.2 mmol/L。与游离酶相比,固定化酶具有良好的操作稳定性。     

固定化乙酰胆碱酯酶的制备及其特性研究

本研究以期研制出能重复使用的固定化乙酰胆碱酯酶(AChE),为天然产物复杂体系中AchE抑制剂筛选新方法的发展奠定基础.以氨基化硅胶(APS-Si)微球为载体,戊二醛为交联剂对乙酰胆碱酯酶进行交联固定化,并研究了酶的最佳固定化条件和固定化酶的性质.结果表明,0.05 g氨基化硅胶微球载体,用戊二醛溶液活化6 h后,在给酶量5 U,28℃固定16 h条件下,得到固定化酶的活性最大.固定化酶在常温(20～40℃),以及较宽pH范围内(pH 6～10)均具有较高的活性,并且具有良好的保存稳定性和可重复利用率,为基于固定化靶酶亲和-色谱质谱联用分析快速筛选乙酰胆碱酯酶抑制剂新方法的发展奠定了基础.     

固定化酶法测定微量组分

以壳聚糖为载体对醇脱氢酶(ADH)和脲酶(UASE)进行固定化。固定化的最适条件:ADH和UASE的偶联时间分别为2.5h和1.5h,交联剂戊二醛浓度为0.6%和0.5%,偶联pH值为6.8和5.0。对游离酶及固定化酶的性质研究表明,ADH酶促反应的最适宜pH为8.0和8.4,最适宜温度为33℃和35℃,米氏常数为48mmol/L和13mmol/L;UASE酶促反应的最适宜pH均为7.0,最适宜温度为60℃和72℃,米氏常数为0.4mmol/L和2mmol/L。固定化酶与游离酶相比在复用性上具有优势,用固定化酶测定了试样中尿素含量以及模拟样中银离子含量。     

固定化脲酶的制备及应用

以壳聚糖作为载体,戊二醛作为交联剂对脲酶进行固定化。固定化的最适条件为:酶的偶联时间60min,戊二醛浓度0.5%,pH值7.0。对游离及固定化脲酶的酶学性质研究表明,酶促反应的最适pH均为7.0,最适温度分别为33℃和70℃。米氏常数分别为29.8mmol/L和13.9mmol/L。与游离酶相比,固定化酶的热稳定性和贮存稳定性更佳。应用固定化酶测定了试样中的微量组分。     

壳聚糖固定化乳酸脱氢酶的制备及酶学性质研究

以磁性壳聚糖作为载体,戊二醛作为交联剂,对乳酸脱氢酶(LDH)进行固定化.固定化的最适条件为:戊二醛浓度6%,pH值7.5,酶的偶联时间2 h.对游离及固定化LDH酶学性质的研究表明,酶促反应的最适pH值为9.2,最适温度分别为37℃和50℃,对乳酸的表观米氏常数分别为1.6 mmol/L和0.9 mmol/L.游离酶和固定化酶在40℃放置150 min后,其活力分别为最初的56.5%和76.1%.固定化酶在4℃贮存4周后,活力仍保留50%以上.固定化酶在室温下与底物重复反应6次后,活力仍保留60%以上,说明固定化酶具有较好的热稳定性、贮存稳定性和复用性.     

哈茨木霉CGMCC 2979生物转化栀子中的京尼平苷制备京尼平

采用微生物直接转化药材的方法,将栀子中的京尼平苷转化为京尼平,无需糖苷酶和京尼平苷的制备. 在培养温度为30 ℃,pH 6.1以及栀子载量为80 g/L的条件下,48 h京尼平苷的转化率为97.8%. 转化后的京尼平通过XAD-16N大孔树脂偶联硅胶层析的方法,制备得到纯度大于95%的京尼平,收率为62.3%. 在催化、转化机制研究中,从哈茨木霉CGMCC2979的发酵液中分离得到了分子量为74.4 kDa的京尼平苷β-葡萄糖苷酶,该酶最优催化条件为50 ℃和pH 4.0-5.0. K_m和V_max分别为3.6 mmol/L和775 μmol/h/mg蛋白. 本文提供了一种简便、高效制备京尼平的新方法.     

Comparison of the Properties of Lipase Immobilized onto Mesoporous Resins by Different Methods

Genipin, a natural cross-linking agent, was used for the immobilization of lipase from Candida sp. 99-125 by cross-linking to two kinds of mesoporous resins. Under optimum conditions, the activity recovery of immobilized lipase on resin NKA-9 could reach up to 96.99% when the genipin concentration was 0.5%, and it could reach up to 86.18% for S-8 with a genipin concentration of 0.25%. Compared with using glutaraldehyde as a cross-linking agent, the immobilized lipase using genipin showed better pH and thermal stability, storage stability, and reusability. The residual activity of immobilized lipase using genipin as cross-linker remained more than 60% of its initial activity after six hydrolytic cycles, whereas only about 35% activity remained by using glutaraldehyde as cross-linker.     

Immobilization and Properties of Lipase from Candida rugosa on Electrospun Nanofibrous Membranes with Biomimetic Phospholipid Moities

Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly（acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine）s（PANCMPCs） were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile（PAN） nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%（molar fraction） in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.     

自组装磁性聚苯乙烯微球固定化木瓜蛋白酶

通过逐层自组装（Layer-By-Layer self-assembly）的方式在自制的经过磺化的聚苯乙烯微球上连接高分子电解质和磁性物质制备了磁性微球,并用此磁性微球固定化木瓜蛋白酶。研究了固定化酶的最优工艺条件（固定化率最高）为：0.5g液态分散磁球(固含量为7.9%),温度30℃,给酶量15mg,pH值为6.0,固定化时间1.0h,此时可达非常高的固定化率93.0%。并且研究了固定化酶的性质：最佳使用温度为50℃,最佳使用pH值8.0,使用3次或80℃保存1.5~2.0h活性大约降为一半。     

Preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization

Nanofibrous membrane with a fiber diameter of 80-150 nm was fabricated from mixed chitosan/poly(vinyl alcohol) (PVA) solution by an electrospinning process. Field emission scanning electron microscope and transmission electron microscope were used to characterize the morphology of the nanofibrous membrane. It was found that chitosan nanofibrous membrane with stabilized morphology could be prepared through removing most of PVA from the nascent one with 0.5 M NaOH aqueous solution. This treatment also resulted in an obvious decrease in fiber diameter. The stabilized chitosan nanofibrous membrane was explored as support for enzyme immobilization due to the characteristics of excellent biocompatibility, high surface/volume ratio, and large porosity. Lipase from Candida rugosa was immobilized on the nanofibrous membrane using glutaraldehyde (GA) as coupling reagent. The properties of the immobilized lipase were assayed and compared with the free one. Results showed that, the observed lipase loading on this nanofibrous membrane was up to 63.6 mg/g and the activity retention of the immobilized lipase was 49.8% under the optimum condition. The pH and thermal stabilities of lipase were improved after it was immobilized on the chitosan nanofibrous membrane. In addition, the experimental results of reusability and storage stability indicated that the residual activities of the immobilized lipase were 46% after 10 cycles and 56.2% after 30 days, which were obviously higher than that of the free one.     

铁氨基黏土纳米结构脂肪酶的构筑 及催化特性

以铁氨基黏土(Fe-aminoclay)为载体, 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)为共价交联剂, 构筑了铁氨基黏土纳米结构脂肪酶催化剂(Feclay-lipase). 利用X射线衍射(XRD)、 透射电子显微镜(TEM)和傅里叶变换红外光谱(FTIR)等技术对Feclay-lipase进行了表征, 并通过酶动力学对比研究了游离脂肪酶和Feclay-lipase的酶学特性. 结果表明, Fe-aminoclay的载酶量为414.4 mg/g, 固定化效率可达82.88%, Feclay-lipase的酶活较游离酶提高了3倍, 最适反应温度提高了10 ℃, 最适反应 pH向碱性偏移, 储存稳定性更好, 在4 ℃下贮存30 d后其酶活无明显减弱.