弱碱性大孔树脂固定化氨基酰化酶的性能研究

对以弱碱性大孔阴离子树脂二乙胺基乙基羟乙酯(DEAE-E/H)为载体制备得到的固定化氨基酰化酶的性质进行系统研究。结果表明,DEAE-E/H固定化氨基酰化酶具有很好的热稳定性。与自由酶相比,固定化酶的最适反应温度分别由47℃变为57℃,pH值由7.0变为6.5,并且最适反应条件范围明显变宽。Co2+对固定化酶活力具有较强的激活作用,其最佳浓度为1×10-3mol/L。分别对其在间歇和连续操作过程巾的反应进行研究,结果表明,固定化酶具有较高的操作稳定性。     

DEAE-D/H树脂固定化氨基酰化酶

选用6种弱碱性大孔树脂固定化氨基酰化酶,结果发现DEAE-D/H（二乙基氨基乙基–二乙烯基苯聚合物）树脂固定化效果较好,固定化酶活1 356.41 U/g。考察了不同酶液浓度下的DEAE–D/H固定化酶活收率。研究发现：酶液浓度115 U/mL时,酶活收率最高,为59.49%。具体分析了此固定化酶拆分乙酰–DL–蛋氨酸的最佳操作条件。结果表明,最佳pH值、温度和Co2+浓度分别为6.5、65℃和5×10－4　mol/L。同时考察了固定化酶的稳定性、再生性。固定化酶连续拆分乙酰-DL-蛋氨酸30天,酶活降为最初的72.6%。失活的固定化酶经洗脱、再生后,酶活达1380.64 U/g,达到新树脂固定化效果,证明载体可重复使用。     

固定化黄孢原毛平革菌木素过氧化物酶的研究

用大孔吸附树脂进行黄孢原毛平革菌来源的木素过氧化物酶固定化试验,筛选出固定化效果较好的XAD7HP大孔树脂,研究了其固定化条件。结果表明,当树脂1.0g,酶液pH4.5,加酶量87.2U,吸附温度25℃,吸附4h,戊二醛质量分数0.2%,戊二醛处理时间120min,可获得最佳的固定化效果,固定化酶活力可达到16U/g(对载体)。     

D201大孔离子交换树脂固定糖化酶的研究

在单因素实验基础上,本文以D201大孔离子交换树脂为载体进行糖化酶的固定,以酶活回收率为评价指标,分析了固定化时间、料液比(酶与载体的比例)、给酶量等因素在糖化酶固定化中的影响.通过响应面法分析得到固定化最佳工艺为:固化时间27 min,料液比1:1,给酶量1800 U/g.此条件下的酶活回收率为60.66％,响应面优...     

固定酶法催化合成生物柴油Ⅰ.大孔树脂固定化脂肪酶的制备

研究了用于生物柴油酶催化的大孔树脂固定化脂肪酶的制备过程,考察和优化了脂肪酶固定化方法及条件。结果表明,采用大孔树脂D3520作载体,以载体涂布法固定化脂肪酶的最适固定化条件为:酶用量为酶∶树脂=0.16∶1(质量比),吸附时间1~3 h,pH值范围为9.0~9.4,固定化温度40℃。酶活力可达91.49 U/g,酶活回收率约为54%。     

纳米晶镁铝水滑石固定化脂肪酶性质研究

以阴离子型层状材料纳米晶镁铝水滑石为载体通过直接吸附对脂肪酶进行固定,考察了各因素对酶固定化的影响,优化了固定化条件.研究表明,脂肪酶的较优固定化条件为载体用量0.32 g/mL(720 U/mL酶液),30～35℃,pH值7.5,负载6～7 h,制得的固定化酶表观酶活达725 U/g.游离酶和固定化酶的水解活化能分别为5.45 kJ/mol和16.31 kJ/mol,游离酶和固定化酶的表观失活活化能分别为20.28 kJ/mol和29.02 kJ/mol,固定化酶较游离酶稳定.     

CALB脂肪酶的固定化及其拆分2-辛醇的研究

以AB-8、HZ-841、HZ-802三种大孔树脂做载体,采用物理吸附法,制备出固定化南极假丝酵母脂肪酶(CALB),并用其进行了拆分2-辛醇的研究。其中AB-8树脂做载体拆分效果最佳,其蛋白吸附量为37.94 mg/g树脂,吸附率94.86%,转酯化酶活3 000 U/g固定化酶,对映体选择性E=104。单因素优化实验得到的最佳拆分条件为:温度40℃,加酶量2.67 g/L,底物醇浓度3.76 mol/L。在该条件下,产物转化率可达50%,e.ep为97.8%。固定化pH在5.0~9.0内对拆分效果无显著影响。     

大孔树脂D380固定化硫酸软骨素裂解酶

目的以大孔树脂D380为载体,戊二醛为交联剂,进行硫酸软骨素裂解酶(ChSase)的固定化,并考察固定化酶的酶学性质。方法分别考察加酶量、吸附温度、吸附时间、吸附pH值、戊二醛交联浓度、交联时间及交联温度对ChSase固定化效果的影响,并分析该固定化酶的最适反应温度、最适反应pH值、米氏常数(Km)及其操作稳定性。结果ChSase的最佳固定化条件为:加酶量150U/g树脂,吸附温度15℃,吸附时间6h,吸附pH值7.0,戊二醛交联浓度0.01%,交联时间3h,交联温度4℃。以此条件制备的固定化酶,其酶结合效率可达79.1%。该固定化ChSase的最适反应温度为45℃;最适反应pH值为7.0;Km达1.46×10-1g/L,较游离酶高;具有较好的操作稳定性。结论以大孔树脂D380为载体固定化ChSase是可行的,所得固定化酶有较高的使用效率和稳定性,适合于工业化生产。     

二氧化硅/壳聚糖大孔复合材料固定化漆酶及其对2,4-二氯苯酚的降解

通过壳聚糖在块体Si O_2大孔材料孔壁上的吸附,制备二氧化硅/壳聚糖大孔复合材料(Si O_2/CS),经戊二醛交联后用于固定诺维信工业漆酶。优化漆酶固定化的实验条件并对比了游离漆酶和固定化漆酶的酶学性质。实验结果表明:在p H值为4.5、漆酶初始浓度为40 mg×m L~(-1)的条件下,固定化4 h效果最佳,固定化酶酶活回收率为85.5%;相对于游离漆酶,固定化漆酶的p H稳定性和热稳定性均得到明显提高,且具有良好的操作稳定性。应用固定化漆酶去除2,4-二氯苯酚(2,4-DCP),研究了降解时间、p H、温度和2,4-DCP初始浓度对其去除率的影响。在优化条件下,固定化漆酶对2,4-DCP(20 mg×L~(-1))的去除率为83.2%,固定化漆酶可重复使用,并且便于从反应体系中分离出来。     

树脂吸附法固定Candida rugosa脂肪酶

Candida rugosa脂肪酶具有优良的催化性能,对其进行固定化可以很方便地实现酶的回收和再利用。采用南开大学化工厂生产的4种阴离子交换树脂和4种大孔吸附树脂为载体,对来源于Candida rugosa的脂肪酶进行了吸附固定化,结果表明,以大孔吸附树脂AB-8为载体的固定酶比活性最高。固定化酶制备过程中缓冲液的最适宜pH值为7.2,最佳固定化时间为1 h,载体和酶的最佳质量配比为10∶1。与游离酶相比,固定化后酶活损失大约30%,但稳定性平均约提高60%。     

葡萄糖氧化酶固定条件的优化

以醋酸纤维素(CA)为原料,采用溶液浇铸法成膜。用物理包埋法将荧光指示剂固定CA膜中。以戊二醛为交联剂,将葡萄糖氧化酶固定于膜表面。研究了高碘酸钠、乙二胺、戊二醛、给酶量、pH值等固定化条件对酶活的影响,确定了优化的固定条件:在室温下与0.5 mol/L的高碘酸钠反应30 min;与0.04 mol/L的乙二胺反应150 min时;与溶度为1.5%(v/v)的戊二醛交联120 min;在4℃条件下,在pH6.5、给酶量35 mg/mL的酶液中交联21 h。利用SEM分析表明,荧光指示剂均匀分布膜中,成膜质量较好。通过优化固定条件,把荧光指示剂和葡萄糖氧化物酶(GOD)同时固定醋酸纤维素膜上,可以得到同时具有光敏感性和酶催化能力的复合敏感膜。     

氨基树脂表面活化提高固定化核酸酶P1连续催化性能

考察了固定化酶常用载体-氨基树脂几何结构及表面活化过程对固定化核酸酶P₁性能的影响,并进行了动力学和连续催化稳定性研究。FESEM、BET及FTIR等表征发现,氨基树脂具有大量酶可利用的孔,在固定化过程中核酸酶P₁主要利用的孔径范围为4～30 nm。相对游离的核酸酶P₁而言,所得固定酶耐酸、耐热性增强;米氏动力学研究表明各组固定酶对底物的亲和力下降,最大反应速率下降;后交联组的重复利用性相对物理吸附、化学交联组明显增强。优化的固定化条件为：酶和载体比例为3：20（质量比）,酶浓度为0.8 g·L^-1,酶液pH为6.0,固定时间为10 h,在优化条件下所得固定酶的单位载体酶活为10013 U·g^-1。同时设计及优化柱连续反应器操作条件,确定反应温度为65℃,进料流量为0.75 ml·min^-1,使得产品的核苷酸浓度维持30 g·L^-1（水解率为60%）以上的累计时长达120 h,有利于核酸酶P₁连续生产核苷酸的工业化应用。     

Preparation of Polyglycidylmethacrylate Macropore Beads and Application in Candida species 99–125 Lipase Immobilization

Polyglycidylmethacrylate beads with macroporous structure were synthesized by suspension polymerization and directly used for Candida species 99–125 lipase immobilization because of their rich surface epoxy groups. The properties of the synthesized polymer beads were characterized by scanning electron microscopy and FT‐IR spectroscopy. To increase the enzyme activity, divinyl benzene and toluene/heptane were used as cross‐linker and porogen, respectively, and the effects of cross‐linker content and porogen content on lipase immobilization were studied. After optimizing the synthesis conditions, the optimum lipase concentration for immobilization was determined as 10 mg mL^–1. The immobilized lipase showed a broader pH stability and higher temperature stability. The immobilized enzyme could be reused for ten batches with 62 % of residual activity.     

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。     

Adsorption of lipase from Candida rugosa on multi walled carbon nanotubes

In this work lipase from Candida rugosa was adsorbed on unmodified surface of multi walled carbon nanotubes (raw-MWCNT). The effects of immobilization time, initial enzyme concentration and buffer ionic strength on enzyme loading and activity of immobilized preparations were tested. High loadings are attained. The immobilized enzyme obtained at lowest initial enzyme concentration and high ionic strength retained 85% of initial enzyme activity. It is assumed that immobilization on hydrophobic surface led to conformational changes that resulted in the adsorption of lipase in active conformation. Immobilized preparations were characterized, with FT-IR spectroscopy, AFM, and cyclic voltammetry.     

Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water‐soluble low‐molecular‐weight chitosan

A nonspecific chitosan hydrolytic enzyme, cellulase, was immobilized onto magnetic chitosan microspheres, which was prepared in a well spherical shape by the suspension crosslinking technique. The morphology characterization of the microspheres was carried out with scanning electron microscope and the homogeneity of the magnetic materials (Fe₃O₄) in the microspheres was determined from optical micrograph. Factors affecting the immobilization, and the properties and stabilities of the immobilized enzyme were studied. The optimum concentration of the crosslinker and cellulase solution for the immobilization was 4% (v/v) and 6 mg/mL, respectively. The immobilized enzyme had a broader pH range of high activity and the loss of the activity of immobilized cellulase was lower than that of the free cellulase at high temperatures. This immobilized cellulase has higher apparent Michaelis–Menten constant K_m (1.28 mg/mL) than that of free cellulase (0.78 mg/mL), and the maximum apparent initial catalytic rate V_max of immobilized cellulase (0.39 mg mL^?1 h^?1) was lower than free enzyme (0.48 mg mL^?1 h^?1). Storage stability was enhanced after immobilization. The residual activity of the immobilized enzyme was 78% of original after 10 batch hydrolytic cycles, and the morphology of carrier was not changed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1334–1339, 2006     

融合蛋白CR2-GDH固定化及不对称还原制备(S)-4-氯-3-羟基丁酸乙酯

比较介孔分子筛材料SBA-15、MCM-41、海藻酸钙、改性二氧化硅4种载体固定化融合蛋白CR2-GDH其酶固载量和酶活回收率,选择SBA-15为固定化载体。研究固定化条件对固定化融合酶量的影响以及固定化酶的稳定性,固定化酶在双相体系催化不对称还原反应。结果表明,在pH值为5.5、酶浓度为1.4mg/mL、反应1h条件下,固定化酶量为27.7mg/g。加入25mmol/L的Ca²⁺,固定化酶的酶活回收率由58.6%提高到78.1%。与游离酶相比,固定化酶的热稳定性显著提高,40℃条件下酶活回收率提高19.1%。固定化酶水相中反复使用7批次后,剩余活性仍超过30%,具有较好的操作稳定性。与游离酶相比,固定化酶更耐受烷烃类有机溶剂。在水/有机溶剂双相反应体系中,Ca²⁺/SBA-15固定化酶和游离酶催化相比,产物得率提高23.8%。     

Preparation and characterization of polymer-coated mesoporous silica nanoparticles and their application in Subtilisin immobilization

The preparation and characterization of polymer-coated mesoporous silica nanoparticles (MSNs) and their application in Subtilisin (Alcalase®) immobilization were investigated. For the synthesis of polymer-coated MSNs, acrylic acid (AA) and chitosan (CS) mixture were blended as poly(acrylic acid) (PAA) and CS polymer layer onto MSNs via in-situ polymerization technique. Then, both uncoated MSNs and polymer-coated mesoporous silica nanoparticles (CS-PAA/MSNs) were characterized by taking into account properties such as morphologic pattern, size distribution, surface charge of the particles as well as thermogravimetric stability with SEM, TEM, Zetasizer and TGA analyses. Subtilisin was immobilized onto polymer-coated mesoporous silica nanoparticles via adsorption technique. For optimizing the enzyme immobilization process, the percent enzyme loading depending on the matrix amount, immobilization time and pH were investigated. Then, the activity values of immobilized enzyme and free enzyme were compared at various pH and temperature values. The maximum enzyme activity was achieved at pH 9.0 for both immobilized and free enzyme. Immobilized enzyme showed more stability at higher temperatures compared with free enzyme. Furthermore, the operational and storage stability of immobilized enzyme were determined. The activity of immobilized enzyme was reduced from 100% to 45.83% after five repeated uses. The storage stability of immobilized enzyme was found to be higher than that of free enzyme. The activity of immobilized enzyme was reduced from 100% to 60% after 28 days of storage time. We concluded that the polymer-coated MSNs were a suitable matrix for Subtilisin immobilization compared to uncoated MSNs.