氨化大孔球状聚氯乙烯固定化木瓜蛋白酶的研究

以氨化大孔球状聚氯乙烯为载体,采用戊二醛载体交联的方法,将木瓜蛋白酶进行了固定化。以酪蛋白为底物,测定了固定化酶的活力回收。研究了固定化条件对固定化酶活力回收的影响。同时,对所得固定化酶的性质,如温度-活力关系、pH-活力关系、热稳定性以及重复使用性进行了考察。结果表明,所得固定化木瓜蛋白酶具有较好的稳定性和重复使用性。     

聚丙烯偕胺肟-聚乙烯醇大孔球状载体的合成及固定化嗜热菌蛋白酶的研究

以线型聚醋酸乙烯酯为致孔剂,由悬浮聚合制得了丙烯腈-醋酸乙烯酯-二乙烯基苯大孔树脂(MR-AV树脂),通过MR-AV树脂的化学改性,合成了聚丙烯偕胺肟-聚乙烯醇大孔球状载体(MR-AVa载体),用FT-IR、SEM、压汞仪和元素分析法研究了树脂及载体的结构与组成,结果表明MR-AVa载体含氮量较高,具有一定的亲水性,且平均孔径大于8,000(?),嗜热菌蛋白酶用戊二醛固定于所得载体之上。以酪蛋白为底物,考察了固定化嗜热菌蛋白酶的催化水解活性及其影响因素,与可溶酶比较,固定化嗜热菌蛋白酶的热稳定性和抗抑制能力显著提高,当载体孔径较大时,所得固定化酶显示出较好的催化活性。     

固定化木瓜蛋白酶的制备和性质研究

多孔硅球固定化木瓜蛋白酶具有热增活性 .本文在前文研究的基础上 ,用载体交联法制备了甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶 .考察了固定化pH值、戊二醛浓度和给酶量对固定化木瓜蛋白酶活力的影响 .研究了固定化木瓜蛋白酶的性质 ,特别是热稳定性和耐热性 ,并与溶液酶和多孔硅球固定化木瓜蛋白酶进行了比较 .所制得的甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶的最适反应温度均达到了 80℃ ;90℃温育 1h后固定化酶的活力保持在 95 %以上 ;70℃温育处理 5h和 6h后固定化酶的活力也仍能保持在 90 %以上 .固定化木瓜蛋白酶的热稳定性和耐热性得到了显著提高     

吸附-纤维素覆膜联合固定化酶

以食品工业中常用的木瓜蛋白酶为模式酶, 建立了吸附-纤维素覆膜联合固定化酶方法. 通过对吸附载体类别、 纤维素种类及溶剂、 保护剂种类及其浓度、 干燥方式及时间等的优化, 得到最佳的吸附-纤维素覆膜联合固定化酶工艺. 以硅藻土或HPD-417(大孔树脂)作为吸附载体, 甲基纤维素(分子量40000~50000)丙酮溶液作为覆膜溶液, 加入6%(质量分数)的聚乙二醇或麦芽糖作为覆膜保护剂, 于4 ℃干燥9 h, 制得固定化木瓜蛋白酶, 硅藻土吸附-纤维素覆膜固定化酶酶活回收率达到96.50%, HPD-417吸附-纤维素覆膜固定化酶酶活回收率达到93.92%. 对吸附-纤维素覆膜固定化酶的性质进行了研究, 发现纤维素覆膜后固定化酶具有良好的热稳定性, 于80 ℃下保存12 h后, 固定化酶活残余率仍然能保持90%左右; 在pH=4.5~9.5的范围内, 固定化酶的稳定性较好; 连续使用9次后, 固定化酶活残余率仍能保持95%左右.     

THERMAL ACTIVATION OF IMMOBILIZED PAPAIN

Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beadsby cross linking with glutaraldehyde. The thermal activation of this immobilized papainin aqueous system was found at a temperature range from 50 to 90℃. The higher thetemperature, the more active the immobilized papain will possess. At the same time,the durability of the immobilized papain on heating was greatly improved. The effect ofadditives and salts on the activity of the immobilized papain were also studied. The resultsshowed that the additives and some of the salts studied could markedly enhance the activityof the immobilized papain at elevated temperature.     

尼龙亲和膜色谱法纯化半胱氨酸蛋白酶抑制剂

建立了一种快速、简便的分离半胱氨酸蛋白酶抑制剂的方法,尼龙膜经壳聚糖改性后,以木瓜蛋白酶为配基制备了一种新型的蛋白质分离材料,并研究了此分离材料的物理及化学性能。所得到的亲和膜上固定化木瓜蛋白酶的热稳定性提高,最适催化温度为55℃,最适pH值为8.0,米氏常数为353.8 g/L;将该亲和膜应用于半胱氨酸蛋白酶抑制剂的分离纯化,在对洗脱液、洗脱速度进行优化的基础上,成功的分离纯化出半胱氨酸蛋白酶抑制剂,纯化倍数为7.25倍。     

化学修饰木瓜蛋白酶的固定化及性质研究

在底物保护和无底物保护下,用丁二酸酐对木瓜蛋白酶进行化学修饰,以三硝基苯磺酸法测定修饰酶的平均氨基修饰度,以棉布为载体,戊二醛为交联剂,对修饰前后的木瓜蛋白酶分别进行固定化.考察了温度、pH和表面活性剂SDS对化学修饰的固定化木瓜蛋白酶活力的影响,并与固定化天然木瓜蛋白酶进行了比较.研究表明,化学修饰固定化木瓜蛋白酶的最适反应温度为80℃;最适pH为9.0;在SDS浓度为20mg/mL时酶活也仍能保持在40%左右;米氏常数为187g/L.与天然的固定化酶相比,化学修饰的固定化木瓜蛋白酶的热稳定性、耐碱性和耐洗涤性得到了显著提高.     

Optimization of protease immobilization by covalent binding using glutaraldehyde

Immobilization of a protease, Flavourzyme, by covalent binding on various carriers was investigated. Lewatit R258-K, activated with glutaraldehyde, was selected among the tested carriers, because of the highest immobilized enzyme activity. The optimization of activation and immobilization conditions was performed to obtain high recovery yield. The activity recovery decreased with increasing carrier loading over an optimal value, indicating the inactivation of enzymes by their reaction with uncoupled aldehyde groups of carriers. The buffer concentrations for carrier activation and enzyme immobilization were optimally selected as 500 and 50 mM, respectively. With increasing enzyme loading, the immobilized enzyme activity increased, but activity recovery decreased. Immobilization with a highly concentrated enzyme solution was advantageous for both the immobilized enzyme activity and activity recovery. Consequently, the optimum enzyme and carrier loadings for the immobilization of Flavourzyme were determined as 1.8 mg enzyme/mL and 0.6 g resin/mL, respectively.     

Immobilization of Modified Papain with Anhydride Groups on Activated Cotton Fabric

Papain (EC 3.4.22.2) has been chemically modified using two novel reagents including different anhydrides of 1,2,4-benzenetricarboxylic and pyromellitic acids. Then, the modified papain was immobilized on the activated cotton fabric by a two-step method. The number of free amino groups in the modified protein was investigated through the 2,4,6-trinitrobenzenesulfonic acid method. Energy dispersive spectrometer was used to characterize papain immobilization. Some parameters of both modified and native papain immobilized on cotton fabric, such as optimum temperature, optimum pH, and the stabilities for reservation in various detergents were studied and compared. The resultant papain had its optimum pH shifted from 6.0 to 9.0. Compared with immobilized native papain, the thermal stability and the resistance to alkali and washing detergent of immobilized modified enzyme were improved considerably. When the concentration of detergent was 20 mg/ml, the activity of the immobilized pyromellitic papain retained about 40% of its original activity, whereas the native papain was almost inhibited. This work demonstrated that the cotton fabric immobilized modified papain has potential applications in the functional textiles field.     

Monolithic macroporous albumin/chitosan cryogel structure: a new matrix for enzyme immobilization

A novel monolithic macroporous material was developed by cross-linking hen egg albumin (HEA) and chitosan with glutaraldehyde at subzero temperatures. A macroporous cryogel structure allowed efficient mass transport of solutes within the material. In one application, albumin was partially replaced with active enzymes (glucose oxidase and horseradish peroxidase) resulting in the production of macroporous biocatalyst preparations suitable for flow-injection analysis of glucose in the low millimolar range. In another application, the proteolytic enzymes savinase and esperase were coupled to the macroporous structure via free amino groups on the pore walls using glutaraldehyde as cross-linker/spacer agent. The low hydraulic resistance of the matrix allowed for the development of a generic, high-performance online protein digestion system utilizing the wall-bound proteases.     

以HPD-750大孔树脂为载体材料固定化果胶酶

HPD-750树脂是中极性大孔吸附树脂,生物相容性好,机械性能稳定,具有较大的比表面积,可用于固定化酶载体材料。本文以HPD-750大孔树脂为载体固定化果胶酶,研究各因素对固定化酶的影响,并采用正交试验对固定化条件进行优化。结果表明,当pH为4.0、固定化温度为45℃、固定化时间为4h、加酶量为0.16g/mL时,固定化酶活力可达5146U/mg。以HPD-750大孔树脂为载体材料制备的固定化酶相较于游离酶具有更好的酸碱稳定性和热稳定性。在循环使用10次后,酶活力依然保留80%以上;4℃储藏25d之后,其酶活力仍保留60%以上。与D311大孔树脂、聚丙烯酰胺和海藻酸钠微球制备的固定化酶相比,HPD-750大孔树脂固定化酶的活性、操作稳定性、机械稳定性和储存稳定性都较好。该结果说明,HPD-750大孔树脂可作为固定化酶较好的载体材料。     

单分散磁性纳米粒子固定化猪胰脂肪酶的研究

借助溶热法制备了一种亲水及生物相容良好的Fe3O4磁性纳米粒子,用γ-氨丙基三乙氧基硅烷直接对所得磁性粒子表面改性,然后用戊二醛偶联法制得了固定化猪胰脂肪酶.表征研究显示,所得磁性粒子粒径约200 nm,具有良好的单分散性和磁响应性.考察了戊二醛浓度、给酶量和反应时间对脂肪酶固定化过程的影响,并通过游离酶与固定化酶的比...     

无机/金属纳米颗粒与木瓜蛋白酶组装的研究

运用氧化还原反应在二氧化硅表面沉积组装了银纳米粒子,并在该载体表面共价连接木瓜蛋白酶,试图组合超微载体与化学共价固定化酶的优点,进一步提高组装后的酶活性.初步研究了微球尺寸及银纳米粒子对组装酶活力影响.实验结果表明,在给酶量为33.3mg/g时,木瓜蛋白酶直接共价连接在二氧化硅表面时活力仅为402u/mg,而在组装有银的载体上时活力为536u/mg,催化活性提高30%左右.同时在高给酶量条件下,酶活力回收提高了60%左右,活力回收最大值达到了30.5%,明显优于戊二醛直接共价固定的文献报道值.     

Horseradish peroxidase immobilized in macroporous hydrogel for acrylamide polymerization

Thermosensitive hydrogel made up of poly(N‐isopropylacrylamide) (PNIPA)‐chitosan semi‐interpenetrating network (semi‐IPN) with ultrarapid responding rate was synthesized. Horseradish peroxidase (HRP) was then immobilized on this hydrogel that acted as an enzyme‐carrier by glutaraldehyde bridge. Polymerization of acrylamide was initiated by a redox system (hydrogen peroxide/acetylacetone (Acac)) and was catalyzed by the immobilized enzyme at room temperature. The attention was focused on the properties of the carrier‐enzyme systems. The hydrogel was proofed to be macroporous by environmental scanning electron microscope images. Swelling properties of the hydrogel such as swelling ratio and deswelling–reswelling kinetics were measured. The properties of the immobilized enzyme such as enzyme activity, storage stability, and thermostability were also studied. The immobilized enzyme could be used repeatedly. Gel permeation chromatography measurement of the resulted polyacrylamide (PAAm) showed that the molecular weight reduced as the repeated times of the immobilized enzyme catalysis increased. In conclusion, the macroporous hydrogel would be a suitable enzyme carrier for practical applications in future. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2222–2232, 2008     

Immobilization of a lactase onto a magnetic support by covalent attachment to polyethyleneimine-glutaraldehyde-activated magnetite

A magnetic immobilized lactase has been prepared using magnetite as the magnetic material. Magnetite was functionalized by treatment with polyethyleneimine and crosslinked with glutaraldehyde. Lactase was then covalently coupled to the activated magnetic matrix via the aldehyde groups. The conditions for optimal immobilization of enzyme are described. Eighty percent of the lactase activity was lost on immobilization and is thought to be owing to the orientation of enzyme binding to the matrix. The amount of protein coupled was 80% of that applied. The maximum lactase activity retained on the matrix following immobilization was 360 U/g matrix. The immobilized lactase showed optimal activity at pH 4.5 and 65 degrees C. The immobilized lactase was more heat stable than the free enzyme, and retained 83% of its original activity after 14 d at 55 degrees C. Galactose competitively inhibited the immobilized lactase preparation (Ki 20 m/M). The presence of high initial concentrations of galactose (10% w/v) did not prevent total hydrolysis of lactose. Glucose and calcium ions were activators of the immobilized enzyme. The immobilized enzyme hydrolyzed high concentrations of lactose (up to 25% w/v) to completion within 4-6 h in a stirred batch reactor at 55 degrees C. There was no evidence of substrate inhibition at high substrate concentrations. The efficiency of hydrolysis of lactose by the immobilized lactase was better than that of the free enzyme. The magnetic immobilized lactase was demonstrated to be suitable for use in the enzymatic hydrolysis of both pure, and cheese whey permeate, lactose.     

Immobilization of single-strand specific nuclease (S1 nuclease) fromAspergillus oryzae

S1 nuclease fromAspergillus oryzae (EC 3.1.30.1) was coupled to gelatin-alginate composite matrix using the residual free aldehyde groups on the surface of glutaraldehyde crosslinked matrix. The immobilized enzyme retained approximately 10% activity of the soluble enzyme. When partially purified enzyme was bound to the matrix, the immobilized preparation did not show any detectable enzyme activity. However, the activity could be restored when the coupling was carried out in the presence of a coprotein or substrate. The optimum pH of the immobilized S1 nuclease shifted to 3.8 from 4.3 for the soluble enzyme. Also, optimum temperature increased to 65°C after immobilization. Bound S1 nuclease showed increased pH and temperature stabilities. Immobilization brought about a twofold decrease in the Michaelis-Menton constant (K _m).     

一种简化的重氮化法制备固定化酶的载体合成方法

重氮化法是固定化酶时常用的一种方法。用多孔玻璃等无机物作载体时,一般是先用g-氨丙基三乙氧基硅烷与多孔玻璃等载体反应[1,2],生成烷基胺玻璃,然后与对硝基苯酰氯反应,产物经过还原,生成带有芳胺的衍生物,最后进行重氮化。本文通过烷基胺与对氨基苯甲酸反应,直接生成芳胺的衍生物,比常用的方法缩短了一步。通过在新合成的载体上对木瓜蛋白酶进行固定化,研究了固定化条件对酶活力回收的影响,最适固定化条件如下:pH为7.0,时间为6h,酶量为240mg/g载体,并比较了固定化酶和溶液酶的有关性质,考察了固定化酶的操作稳定性。结果表明,用这种方法合成的载体固定化酶,其对热稳定性、操作稳定性及产率都比较理想。     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g^?1 dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K _m) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.     

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.