固定化木瓜蛋白酶载体的研究

本文主要概述了近年来国内外对于固定化木瓜蛋白酶载体的研究情况。木瓜蛋白酶是100％纯天然产物，也是一种有着广泛用途的重要生化试剂，在我国有着诱人的开发应用前景。目前，被用做固定木瓜蛋白酶的载体已从有机高分子载体、无机载体发展到了复合载体。用这些载体对木瓜蛋白酶进行固定化大多取得了较好的效果。相信对固定化木瓜蛋白酶载体的研究将会加快木瓜蛋白酶的工业化应用进程。     

壳聚糖-海藻酸钠微球固定化木瓜蛋白酶的工艺研究

以壳聚糖和海藻酸钠为原材料,采用乳化交联法制备空白微球,通过戊二醛固定木瓜蛋白酶,以固定化木瓜蛋白酶的活性回收率作为最终测定指标,并以星点设计-效应面法优化实验条件。通过一系列的实验,结果表明最优条件为:固定化时间是4 h,壳聚糖与海藻酸钠的质量配比(m/m)为5∶5、加酶量为700 U/mL、戊二醛浓度为1. 0%、固定化温度为40℃。木瓜蛋白酶的固定化效果良好,木瓜蛋白酶活性回收率为68. 9%。     

超大孔微球固定木瓜蛋白酶制备酵母抗氧化肽

文中选用新型超大孔聚甲基丙烯酸缩水甘油酯微球偶联木瓜蛋白酶,用于催化酵母蛋白水解制备抗氧化肽。木瓜蛋白酶在微球上负载量为66.5 mg/g,并且均匀分布于微球内外表面;固定化酶比活力为137.5 U/mg,活力回收率达60.6%,高于商品化介孔微球固定化酶的活力。在固定化木瓜蛋白酶的可控催化作用下,酵母蛋白水解产物的最高抗氧化活力为81.2 mol TE/g,远高于蔬果的抗氧化活力。此外,固定化木瓜蛋白酶重复使用20次后仍剩余35%的初始活力。     

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。     

壳聚糖载体柔性固定化木瓜蛋白酶

用酶柔性固定化模型,以壳聚糖为载体,双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化. 通过对固定化条件的优化,得出选用壳聚糖、双醛淀粉制得的柔性载体(Chitosan-DAS50)在酶用量为14.4 mg/g(酶/干球)、pH 8的条件下,固定木瓜蛋白酶18 h,所得的固定化酶活力回收率达72%,相当于采用壳聚糖-戊二醛(Chitosan-GA)手臂载体的3倍. 结果表明,酶的柔性固定化模型可以改善传统共价结合法固定化及手臂固定化酶活力回收率不高的缺陷.     

壳聚糖及其衍生物作为固定化蛋白酶的载体

介绍了以壳聚糖及其衍生物为载体的木瓜蛋白酶、微生物蛋白酶和胰蛋白酶的固定化条件和固定化方法;强调了酶活力回收率、稳定性的重要性;指出了壳聚糖及其衍生物作为固定化蛋白酶的载体具有环保、来源丰富和可以通过多种方法进行固定化处理的优点。     

氧化竹纤维固定木瓜蛋白酶

通过高碘酸钠-甲醛法对几种竹纤维氧化改性,制备了良好的竹纤维固定化酶载体,并对木瓜蛋白酶进行固定,考察了固定化酶固定条件、最适微环境以及稳定性。结果表明,在4℃、pH=7.5,给酶量为15 g/L条件下固定24 h,改性竹纤维固定化木瓜蛋白酶具有较高催化活性,活力可达805.5 U/g,其最适催化条件为50℃、pH=7.5,较游离酶有更好的耐热性和耐碱性,重复使用6次后活性仍能达到初始活力的76.9%。     

大分子拥挤下介孔中木瓜蛋白酶的微波辅助固定化

为了增强酶的固定化效果,通过添加大分子试剂,在微波辐射作用下将木瓜蛋白酶固定在介孔泡沫硅的孔道中. 结果表明,在加酶量为400 mg/g时,微波辐射下木瓜蛋白酶与牛血清白蛋白(BSA)共固定化制得的固定化酶催化效果最好. 当BSA含量为加酶量的5%(w)时,固定化酶表观活力高达419.1 U/mg,相对活力和酶活回收率分别为126.0%和119.1%. 影响固定化酶活力的主要因素依次为加酶量、BSA含量、微波功率和固定化pH. 该固定化酶的最适反应pH为7.0,最适反应温度为75℃,热稳定性优于游离酶和未加入BSA的固定化酶,80℃下热处理3 h,剩余活力仍为初始活力的88.2%.     

改性陶粒固定化酶的研究

探索了以改性陶粒为载体固定化木瓜蛋白酶,通过对比实验确定了偶联剂种类和最佳固定化方案:采用硅烷(KH-570)和戊二醛共同偶联固定化酶。通过正交实验确定了各因素浓度的最优组合:盐酸浓度为0.20 mol/L、硅烷质量分数为0.392%、戊二醛质量分数为0.25%。同时,测得该活化条件下固定化酶的活力回收和相对活力分别为7.16%和9.90%。     

壳聚糖/卵磷脂复合微球固定木瓜蛋白酶的研究

以壳聚糖和卵磷脂为材料,采用乳化-交联法制备壳聚糖/卵磷脂复合微球,并用光学显微镜和红外光谱对微球进行表征;再以此微球作为载体固定木瓜蛋白酶,以固定率为指标,应用正交试验法优选固定化酶的制备工艺,并对固定化酶的半衰期、米氏常数(Km)、操作稳定性进行研究.结果表明,制备的壳聚糖/卵磷脂复合微球呈完整的圆球形或椭球形;固定化酶的优化制备工艺为:m(壳聚糖)=250 mg,m(壳聚糖):m(卵磷脂)=1:2,V(戊二醛水溶液)=300 μL,m(木瓜蛋白酶)=20 mg,此时制备的固定化酶的固定率达61.94％,半衰期为86.27 h,米氏常数为6.37 mg/mL,固定化酶有很好的操作稳定性.     

银/二氧化硅复合载体固定木瓜蛋白酶的研究

以沉积了银纳米粒子的二氧化硅微球为载体,戊二醛为交联剂共价固定了木瓜蛋白酶。研究了戊二醛用量以及载体中银纳米粒子质量分数对酶负载率、相对活力和酶活回收率的影响,考察了温度和pH值对固定酶的影响。结果表明,戊二醛的适宜质量分数为5%;当载体的银负载量(质量分数)为0.68%时,固定木瓜蛋白酶活回收率最高,比使用未负载银载体提高了131%;固定酶的最适反应温度为75℃,与游离酶基本相同;最适pH值为7.5,与游离酶相比向碱性方向移动了1个单位。     

Study on a carboxyl‐activated carrier and its properties for papain immobilization

BACKGROUND: Most enzymes, including protease, play a key role in biotechnology, but their use is quite limited due to poor recovery, limited reusability and instability. Immobilized enzymes offer advantages over free enzymes. This paper reports a simple method for the preparation of immobilized papain, an endolytic cysteine protease (EC: 3.4.22.2), on carboxyl‐activated silica nanoparticles. RESULTS: The carboxyl‐activated carriers produced reactive carboxyl groups which then react with the free amino groups of enzyme to give peptide bonds (? CO? NH? ). The results showed that the thermal and pH stabilities of the immobilized papain were higher than those of free enzyme. And the apparent K_m value of the immobilized papain was 1.26 times higher than that of free enzyme. Moreover, the immobilized papain retained more than 45% of the original activity after ten reuses continuously. CONCLUSION: The results indicated that papain was successfully immobilized on the surface of the activated carriers. The immobilized papain had not only higher activity recovery, but also better stability, reusability and environmental adaptability. Copyright © 2012 Society of Chemical Industry     

Papain Adsorption on Chitosan-Coated Nylon-Based Immobilized Metal Ion (Cu2+, Ni2+, Zn2+, Co2+) Affinity Membranes

A novel immobilized metal affinity membrane was prepared for papain adsorption in this article. Higher papain adsorption capacity between 43-67 mg/g was observed and the adsorption isotherm fitted the Freundlich equation. Experimental data were analyzed using two adsorption kinetic models. The pseudo-second-order kinetic model provided better correlation to the experimental results. A significant amount of the adsorbed papain was eluted by 1.0 M NaSCN at pH 5.0 for all affinity membranes. It was concluded that the novel chitosan-coated nylon-based immobilized metal ion affinity membrane could be applied for the large-scale isolation of papain without resulting in enzyme denaturation.     

Chemical modification and immobilization of papain

Papain, an endolytic cysteine protease (EC: 3.4.22.2), from Carica papaya latex has been chemically modified using succinic anhydride. This reagent reacts with the amino group of the lysine residues in the enzyme, thereby changing its net charge from positive to negative. The resultant enzyme had its optimum pH shifted from pH 6 to 8 and there was no change in the temperature optima of 70 °C. The modified papain had a specific activity of about 62.8 IU mg^?1 of protein at pH 8.0 at 30 °C, whereas for the native enzyme it was 46.57 IU mg^?1 under same conditions. Stability of the modified papain was further increased by entrapping in alginate/starch beads. The immobilized papain retained its activity even after six cycles of hydrolysis. The wet beads, when dried at 50 ± 2 °C, increased the storage stability of the immobilized enzyme. The succinylated papain is active in various organic solvents and hence can be successfully used in biotransformations as well as being used as a proteolytic component in detergents. Copyright © 2004 Society of Chemical Industry     

胺化聚苯乙烯载体柔性固定化木瓜蛋白酶

提出酶的“柔性固定化”模型,并以Mannich反应得到担载量为0.4～6.0 mmol NH₂•g^-1的胺化聚苯乙烯树脂为载体,以双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化,酶活回收率可达40%～50％,相当于手臂固定化酶活力回收率的1.8～2.4倍,且柔性固定化酶稳定性较好.该结果说明,酶的“柔性固定化”模型可以改善传统共价结合固定化酶及手臂固定化酶活力回收率不高的缺陷.     

二氧化硅表面银诱导的木瓜蛋白酶的高效固定化和催化

Complexation and interaction between silver and amino group were applied to induce an efficient immobilization of papain on silica spheres.Tbe silver nanoparticles were deposited on the silica spheres before p apainwas coupled to the silica spheres. The silica spheres with silver nanoparticles were characterized by high resolution transmission electron microscopy （HR-TEM）, Fournier transform infrared spectroscopy （FT-IR）, and UV-Vis scanning spectrometer. FT-IR spectrum was also used to characterize the immobilized and free papain. Effect of some factors on the activities of the immobilized papain was investigated. It was observed that the coupled yield and relative activity of the papain on Ag/SiO2 were 1.17 and 1.86 times of those on the bare SiO2, respectively. At an optimum concentration of silver, theobserved activity of the immobilized papain was 2.1 timesof that on the bare.silica.In addition, the maximum specific activity of papain immobilized on Ag/SiO2 was 819.9 U·mg·^-1, which is slightly lower than that of the free papain, 906.2 U·mg^-1 . Stability of the immobilized papain was also examined. The resuits indicate that the silver nanoparticles successfully induce a fine immobilization of papain.     

Immobilization of Papain in Biosilica Matrix and Its Catalytic Property

A novel method was developed for papain immobilization through a biomimetic silicification process induced by papain. By incubating papain in a silica precursor solution, the papain-silica composite formed rapidly and papain was encapsulated. The encapsulation efficiency and the recovery activity were 82.60% and 83.09%, respectively. Compared with enzymes and biomolecules immobilized in biosilica matrix in the presence of additional silica-precipitating species, this papain encapsulation process, a biomimetic approach, realized high encapsulation efficiency by its autosilification activity under mild conditions (near-neutral pH and ambient temperature). Furthermore, the encapsulated papain exhibits enhanced thermal, pH, recycling and storage stabilities. Kinetic analysis showed that the biomimetic silica matrix did not significantly hinder the mass transport of substrate or the release of product.     

金属亲和膜色谱法纯化木瓜蛋白酶条件优化

建立了一种快速、简便的分离木瓜蛋白酶的方法。尼龙膜经壳聚糖改性后,以金属镍离子N i2+为配基制备了一种新型的蛋白质分离材料,并将该亲和膜应用于木瓜蛋白酶的分离纯化,在对洗脱液、上样速度、洗脱速度、洗脱液的pH值和洗脱液的离子强度进行优化的基础上,成功地分离纯化出木瓜蛋白酶,纯化倍数为20.59倍。