新型层状交联酶聚集体的制备条件优化与性质研究

以假丝酵母脂肪酶(Candida sp. lipase)为研究对象,开发了一种新型层状交联酶聚集体。用蛋白或氨基酸对纳米氧化锌粒子进行修饰,继以交联剂交联后作为核芯,酶分子再交联在纳米核表面形成层状结构。实验结果表明牛血清白蛋白(BSA)是纳米氧化锌适宜的修饰剂。并且对纳米芯层状交联酶聚集体(BSA-N-LCLEAs)其他制备条件进行了优化,优化后BSA-N-LCLEAs制备条件为:沉淀剂硫酸铵饱和度为58%,交联剂戊二醛浓度为3.5%,交联温度和时间分别为0℃和2h。BSA-N-LCLEAs酶活收率较传统CLEAs提高了196.5%。扫描电镜表征表明BSA-N-LCLEAs较传统CLEAs孔道大幅增加。纳米芯层状CLEAs的pH稳定性和热稳定性也都比传统CLEAs有所提高,并将该固定化酶用于催化维生素E琥珀酸酯的合成,反应五批次后反应产率还能达90%左右,说明该新型交联酶聚集体具有良好的催化活性和操作稳定性。     

双交联剂体系氨基酰化酶交联聚体的制备

采用酶交联聚集体技术（CLEAs）制备氨基酰化酶(EC 3.51.14)交联聚集体,优化了制备条件。90%（体积比）乙醇为沉淀剂,戊二醛和乙二醇二缩水甘油醚（EGDE）作为混合交联剂,以酶、戊二醛和EGDE的质量比为1.00∶0.75∶2.00,在30 ℃下交联12 h。制备所得氨基酰化酶CLEA酶活保留率为63.42%。氨基酰化酶经固定化后热稳定性得到增强,热失活过程由自由酶的一阶指数失活模型变为二阶指数失活模型,分子间亚基的结合力得到显著加强。自由酶在37 ℃下保存24 h后几乎损失了所有活力,而氨基酰化酶CLEA则保留了43%的酶活力,在57 ℃下保存24 h后仍有32%的酶活力保留。此外,氨基酰化酶CLEA重复使用性能得到明显提升,在重复水解底物10次后仍保留了72%的酶活力。     

磁响应交联糖化酶聚集体的制备及催化特性

提出了一种采用羧基磁性纳米粒子制备杂化磁响应交联酶聚集体（M-CLEAs）的方法。表面羧基修饰的约10 nm的磁性纳米粒子与酶分子表面的氨基位点通过静电相互作用,形成复合物,在磁场作用下可将磁性纳米粒子-酶复合物从溶液中分离,经戊二醛交联即形成M-CLEAs。传统的表面氨基修饰的磁性纳米粒子与酶需在沉淀剂作用下,从溶液中分离,而后采用戊二醛共交联,而本方法无须沉淀剂,过程更为简化。以糖化酶为对象,对该过程的影响因素（交联时间、pH、酶浓度、戊二醛浓度等条件）进行了探索,并对制得的M-CLEAs的酶学性质进行了较为详细考察。结果表明,最优制备条件为：酶浓度1 mg·ml^-1,磁流体浓度10 mg·ml^-1,戊二醛浓度0.25%（质量体积比）,在pH 6.0下交联反应6 h,最终载酶量可达80 mg·g^-1、比活为50 U·mg^-1。制得的固定化酶pH稳定性、热稳定性和储存稳定性均显著改善,可实现糖化酶重复使用10次,仍保留接近60%的酶活。     

交联肌酸激酶聚集体催化合成磷酸肌酸

从兔肌中分离纯化出肌酸激酶,经乙醇沉淀得到肌酸激酶聚集体,再以氧化葡聚糖为交联剂制备交联肌酸激酶聚集体,沉淀率为90%,相对酶活性为58%。交联肌酸激酶聚集体的pH稳定性和热稳定性显著高于游离酶。以交联肌酸激酶聚集体在碱性条件下催化肌酸和ATP合成磷酸肌酸,转化率(以ATP计)为74%,且可以使用12批次。     

乳糖酶交联酶聚体的制备及性能

以双醛淀粉(DAS)为交联剂,分别制备了乳酸克鲁维酵母和米曲霉来源的乳糖酶交联酶聚体(CLEAs),同时添加牛血清白蛋白(BSA)作为保护剂以提高CLEAs活性。当DAS质量分数为10%、氧化度为80%、BSA与酶蛋白质量比为1∶8时得到的酵母和曲霉乳糖酶CLEAs的活力保留分别为53.84%和55.25%。CLEAs的最适pH值较游离酶有所降低。曲霉乳糖酶CLEAs在60℃下具有较好的热稳定性,并且在37℃下重复使用5次(20 h)后活力可保留52%。     

基于乳化液体系脂肪酶球形酶的制备

在含有脂肪酶的油包水（W/O）乳化液体系中添加交联剂，经乳化交联后通过离心分离得到脂肪酶球形酶颗粒，研究了不同交联剂、交联时间和酶液质量浓度对球形酶活性的影响，得到制备脂肪酶球形酶的适宜条件为：以体积比1︰1的25%(v/v)戊二醛溶液与3%(v/v)聚乙烯亚胺溶液，在室温下反应5min而形成的Glu-PEI共聚物为交联剂，交联2h，酶质量浓度为50g/L时形成的球形酶酶活残留率最高达到86.3%。     

复合蛋白酶交联酶聚集体制备枸杞蛋白多肽

该研究利用交联酶聚体技术制备出复合蛋白酶交联酶聚体固定化酶,以酶活回收率为指标,通过单因素实验得到复合蛋白酶交联酶聚集体最佳制备条件：风味蛋白酶与木瓜蛋白酶比例为3∶1,沉淀剂硫酸铵浓度为60%,戊二醛浓度0.5%,交联时间为50分钟,交联pH为7.5。此时最大酶活回收率为80.32%。相比单一蛋白酶,复合蛋白酶交联酶聚集体具有更好的热稳定性和pH稳定性,同时表现出良好的连续多次水解枸杞蛋白的性能。以蛋白水解度为指标,优化后复合蛋白酶交联酶聚集体最佳水解条件为：温度为60℃,pH为6,酶浓度为10 mg/mL,水解时间为100 min,在此条件下枸杞蛋白水解度为41.21%,比游离复合蛋白酶提高了6.9%。     

磁响应交联糖化酶聚集体的制备及催化特性

提出了一种采用羧基磁性纳米粒子制备杂化磁响应交联酶聚集体(M-CLEAs)的方法。表面羧基修饰的约10 nm的磁性纳米粒子与酶分子表面的氨基位点通过静电相互作用,形成复合物,在磁场作用下可将磁性纳米粒子-酶复合物从溶液中分离,经戊二醛交联即形成M-CLEAs。传统的表面氨基修饰的磁性纳米粒子与酶需在沉淀剂作用下,从溶液中分离,而后采用戊二醛共交联,而本方法无须沉淀剂,过程更为简化。以糖化酶为对象,对该过程的影响因素(交联时间、pH、酶浓度、戊二醛浓度等条件)进行了探索,并对制得的M-CLEAs的酶学性质进行了较为详细考察。结果表明,最优制备条件为:酶浓度1 mg·ml-1,磁流体浓度10 mg·ml-1,戊二醛浓度0.25%(质量体积比),在pH 6.0下交联反应6 h,最终载酶量可达80 mg·g~(-1)、比活为50 U·mg~(-1)。制得的固定化酶pH稳定性、热稳定性和储存稳定性均显著改善,可实现糖化酶重复使用10次,仍保留接近60%的酶活。     

基于乳化液体系脂肪酶球形酶的制备

在含有脂肪酶的油包水(W/O)乳化液体系中添加交联剂,经乳化交联后通过离心分离得到脂肪酶球形酶颗粒,考察了不同交联剂、交联时间和酶液质量浓度对球形酶活性的影响,得到制备脂肪酶球形酶的适宜条件为:以体积比1∶1的体积分数25%戊二醛溶液与体积分数3%聚乙烯亚胺溶液,在室温下反应5 min而形成的Glu-PEI共聚物为交联剂,交联2 h,酶质量浓度为50 g/L时形成的球形酶酶活残留率最高达到86.3%。     

交联柚苷酶聚集体水解柚皮苷制备普鲁宁

为了提高普鲁宁的收率、简化分离提纯的工艺、得到较高纯度的普鲁宁,本实验采用交联柚苷酶聚集体水解柚皮苷制备普鲁宁,以普鲁宁浓度为优化指标,研究了底物浓度、p H、温度、加酶量、反应时间对制备普鲁宁的影响。利用正交设计对水解过程进行优化,确定最佳工艺条件为:柚皮苷浓度13.79 mmol/L、p H=8.0、温度65℃、加酶量18.0 mg/m L、反应时间12.0 h,优化后制备得到的普鲁宁浓度为13.36 mmol/L、收率达到96.87%。连续使用3个批次后,普鲁宁的收率可以达到91.29%,交联酶聚集体的稳定性较好。采用聚酰胺树脂对水解产物进行吸附,体积分数为40%~60%的异丙醇线性梯度洗脱,从0.36 g柚皮苷反应液中可以分离得到0.22 g普鲁宁。     

A facile technique to prepare cross-linked enzyme aggregates using <Emphasis Type="Italic">p</Emphasis>-benzoquinone as cross-linking agent

To obtain robust and thermo-stable enzyme aggregates, p-benzoquinone was used as cross-linker and bovine serum albumin (BSA) as crowding macromolecules to prepare cross-linked enzyme aggregates (CLEAs) of lipase. Effects of cross-linking time and cross-linker content on the activity, thermal stability and characteristics of enzyme aggregates were examined carefully. It was observed that when the content of p-benzoquinone was 5 mM and amount of BSA was 125% of that of lipase (w/w), the specific activity of cross-linked co-aggregates of lipase and BSA was 79.8 U mg⁻¹, 2.44-fold of that of cross-linked enzyme aggregates of lipase without BSA. Moreover, after heat treatment for 96 h at 50 °C, the CLEAs prepared with this facile routine kept 75.18% of their initial activity, 5.01-fold more than that of the just CLEAs using glutaraldehyde. Furthermore, BSA macromolecules in lipase CLEAs enhanced the catalytic efficiency of free and just lipase CLEAs without BSA by 1.45 and 2.83 times, respectively. The proposed crosslinking technique would rank among the potential strategies for efficiently preparing robust and thermo-stable enzyme aggregates.     

Characterization of Cross-Linked Lipase Aggregates

Commercially available microbial lipases from different sources were immobilized as cross-linked enzyme aggregates (CLEAs) using different precipitants and glutaraldehyde as cross-linkers. These CLEAs were assayed based on esterification between lauric acid and n-propanol in solvent-free systems. Precipitants were found to have a profound influence on both specific activities and total activity recovery of CLEAs, as exemplified by Candida antarctica lipase B (CALB). Among the CLEAs of CALB studied, those obtained using PEG600, ammonium sulfate, PEG200 and acetone as precipitants were observed to attain over 200% total activity recovery in comparison with acetone powder directly precipitated from the liquid solution by acetone. PEG200 precipitated CLEA gave the best specific activity (139% relative to acetone powder). The results of kinetic studies showed that V _max/K _m does not significantly change upon CLEA formation. This work presents a characterization of CLEAs based on an esterification activity assay, which is useful for exploring the synthetic application potential of CLEA technology with favorable perspectives.     

Preparation of stable cross-linked enzyme aggregates (CLEAs) of NADH-dependent nitrate reductase and its use for silver nanoparticle synthesis from silver nitrate

The NADH-dependent nitrate reductase from Fusarium oxysporum cell extract was directly immobilized as cross linked enzyme aggregates (CLEAs) and investigated for the synthesis of silver nanoparticles by a reduction of silver nitrate. The effects of precipitant type and cross-linking on activity recovery of enzyme in CLEAs were studied. After aggregation of enzyme with ammonium sulfate followed by cross-linking formed aggregates for 4 h with 8 mM glutaraldehyde, 93% activity recovery was achieved in CLEAs with enhanced thermal stability at 50 °C and 40 °C. Scanning electron microscopy analysis showed that immobilized NADH-dependent nitrate reductase was of spherical structure. CLEAs showed 90% catalytic yield even after 4 cycles of repeated use in silver nanoparticle synthesis at pH 7.2 and temperature 35 °C.     

Evaluation of cross-linked enzyme aggregates of Lactobacillus cell-envelope proteinases,for protein degradation

Enzymatic hydrolysis is a widely used approach to improve the functional, nutritional and physiological properties of food proteins. In this study, cross-linked enzyme aggregates (CLEAs) have been prepared from cell-envelope proteinases (CEPs) of Lactobacillus delbrueckii subsp. lactis 313 and their proteolytic properties have been evaluated using several food proteins. We have optimized cross-linking conditions including ammonium sulphate concentration, incubation temperatures, agitation speed, glutaraldehyde cross-linker concentration, reaction time and the addition of proteic feeders. Particularly, the presence of BSA improves retained activity of cross-linked CEP aggregates (CLCEPAs) from 21.5% to 40.9%. Blocking unreacted cross-linking groups on aggregates is important to enhance recyclability of CLCEPAs. CLCEPAs had attractive thermal stability at 50 °C and it showed enhanced catalytic activity over long-term storage after lyophilization. We have demonstrated that CLCEPAs has proteolytic properties on different food proteins including complex (chicken egg albumin, skimmed-milk protein), fractionated (bovine casein, whey protein isolate), and purified (bovine serum albumin) proteins. Being the first report of CLEAs from lactobacilli CEPs, this study demonstrates the feasibility of using LDL 313 CLCEPAs for degradation of various food proteins.     

Systemic Concocting of Cross-Linked Enzyme Aggregates of Candida antarctica Lipase B (Novozyme 435) for the Biomanufacturing of Rhamnolipids

In the present study, Candida antarctica lipase B was immobilized on amine-functionalized silica microspheres as cross-linked enzyme aggregates (CLEA) and utilized for the biomanufacturing of rhamnolipids (RL). Lipase CLEA synthesized under optimized conditions of 2.0:1.0 by volume of silica microsphere/enzyme concentration, a 1.0:2.5 (v/v) ratio of enzyme/2-propanol, 7 mM glutaraldehyde concentration, when incubated at pH 9.0 and 40 °C, for a cross-linking time of 30 min were observed to exhibit superior biocatalytic properties and a maximum enzyme load of 770 U g⁻¹. Lipase CLEA exhibited enhanced pH stability in acidic and alkaline media and increased temperature resistance as compared to free lipase. Both free and CLEA lipases were used to synthesize RL in different solvent systems. After 12 h, from initiation of the esterification, the degree of esterification (molar conversion yield) reached 46% and 71% in the batch mode. ¹H and ¹³C nuclear magnetic resonance (NMR) and high-performance liquid chromatographic (HPLC) analysis confirm RL production by CLEA lipase. The CLEA showed greater confrontation to enzyme-mediated bioprocess approach as compared to its soluble counterpart and exhibited excellent RL production and catalytic activity even after its tenth successive reuse.     

One-dimensional crosslinked enzyme aggregates in SBA-15: Superior catalytic behavior to conventional enzyme immobilization

α-Chymotrypsin (CT) and lipase (LP) were immobilized in SBA-15 mesoporous silica by crosslinking adsorbed enzymes. This simple approach resulted in one-dimensional crosslinked enzyme aggregates (CLEAs) in the linear pore channels of SBA-15, which was very effective in preventing the enzyme leaching and consequently improving the enzyme stability. Both CLEAs of CT and LP showed negligible activity decrease under harsh shaking condition for one week while the conventional approaches including adsorption and covalent attachment resulted in more than 50–90% enzyme inactivation under the same condition. This effective stabilization results from the bent pore structure of SBA-15 with a high aspect ratio, which prevents the leaching of one-dimensional CLEAs and thereby achieves the higher enzyme loading capacity. Along with the higher specific activity than that of adsorbed enzymes, this CLEA approach is much simpler than that of covalent attachment by obviating the tedious processes for silica functionalization and enzyme attachment.     

氨基硅烷功能化磁性材料固定化琼胶酶的性能

利用3-氨丙基三乙氧基硅烷(APTES)对制备的SiO₂包覆Fe₃O₄复合粒子(Fe₃O₄@SiO₂)进行改性，制备了氨基硅烷功能化磁性材料Fe₃O₄@SiO₂-NH₂，将Fe₃O₄@SiO₂-NH₂作为载体用于固定化琼胶酶。采用SEM、FTIR、VSM对Fe₃O₄@SiO₂-NH₂和固定化琼胶酶进行了表征，对琼胶酶的固定化条件进行了优化，评价了固定化琼胶酶的性能。结果表明，琼胶酶成功固定在载体上。Fe₃O₄@SiO₂-NH₂的磁化饱和强度为48.4 emu/g，固定化琼胶酶的磁化饱和强度为42.8 emu/g...     

海藻酸钠-明胶协同固定化S-腺苷甲硫氨酸合成酶的研究

以海藻酸钠和明胶为载体,对S-腺苷甲硫氨酸合成酶进行固定化。再用戊二醛对其进一步交联,增强固定化酶的稳定性。考察了海藻酸钠和明胶质量分数、CaCl2质量分数、酶和载体比例以及交联剂戊二醛体积分数等因素对固定化酶的影响。结果表明,最佳固定化条件为:海藻酸钠质量分数2.0%、明胶质量分数1.0%、CaCl2质量分数4.0%、固定化酶量为2.5 g/L凝胶、戊二醛体积分数0.6%。交联固定化酶热稳定性得到大幅度提高,在50℃下保温5 h仍保留72%的活力,而游离酶则完全失活。交联固定化酶在碱性溶液中的稳定性较高,在pH=8.0～9.0的缓冲液中4℃保温10 h酶活性仍保留87%以上。将交联固定化酶用于S-腺苷甲硫氨酸的合成,连续反应8批次后酶活性仍保留65%。