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2 h。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。     

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。     

环氧化物水解酶交联细胞聚集体催化合成（R）-环氧氯丙烷

利用聚乙烯亚胺(PEI)絮凝、戊二醛(GA)交联对环氧化物水解酶全细胞进行了交联细胞聚集体(CLCAs)制备,考察了PEI浓度、GA浓度及硅藻土载体用量对CLCAs活力回收率的影响,结果表明PEI浓度、GA浓度及硅藻土载体用量最优值分别为3%(体积)和1%(体积)和6 g/L,此时CLCAs活力回收率可达88.4%。以CLCAs作为催化剂,以外消旋环氧氯丙烷((R,S)-ECH)为底物,在异辛烷/磷酸盐缓冲液两相体系中催化合成(R)-环氧氯丙烷((R)-ECH)。结果表明,在异辛烷与缓冲液的体积比3∶7,底物浓度800 mmol/L,CLCAs加入量18 g/L,缓冲液pH 8.0,温度35℃条件下,(R)-环氧氯丙烷的摩尔产率达到45.2%,产物光学纯度为99.1%ee。考察了CLCAs在两相体系中的操作稳定性,重复使用9个批次活力基本保持不变,显示了良好的操作稳定性。     

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。     

磁性磷脂酶D交联酶聚集体的制备及其酶学性质

以磁性Fe3O4为载体,采用吸附-聚集-交联的方法固定来源于Streptomyces chromofuscus的磷脂酶D （scPLD）。该方法制备的磁性磷脂酶D交联酶聚集体（MCLEA）酶活回收率可达72.89%,酶活为（437±6.60）U/g。与游离酶相比,MCLEA在不同温度和pH下的稳定性都得到了一定程度的提升,但由于与底物的亲和力降低,需要更高浓度的磷脂酰胆碱（PC）和Ca2+作为反应底物与MCLEA结合。另外,还研究了MCLEA的有机溶剂耐受性,发现乙醇、四氢呋喃、叔丁醇、乙酸乙酯、乙醚和甲苯等有机溶剂对MCLEA的酶促反应具有促进作用,此发现为scPLD催化转磷脂酰作用生成磷脂酰丝氨酸（PS）过程中构建双相体系时有机溶剂的选择提供了参考。MCLEA在进行连续13次酶促反应后,酶活仍可保留在76%以上,其半衰期为331.67 min,说明所选择的固定化方案可以提高酶的稳定性,从而提高其在催化反应过程中的重复利用性。     

特殊反应体系中交联β-葡萄糖苷酶聚集体催化合成红景天苷

采用具有较高操作稳定性的交联b-葡萄糖苷酶聚集体,在离子液体-混合有机溶剂-缓冲溶液的非常规反应体系中酶催化直接糖基化合成红景天苷,筛选反应体系的组成和各成分的比例. 结果表明,反应体系的组成为(%, j)：1,4-二氧六环42.5, 离子液体C4MIm×PF6 8.5, 乙酸乙酯34, pH 6.0柠檬酸-磷酸氢二钠缓冲溶液15.0. 该体系在水含量高达15%(j)的条件下水活度为0.66,适合直接糖基化反应. 底物b-D-葡萄糖100 g/L、对羟基苯乙醇200 g/L时,在50℃及250 r/min下反应120 h达到平衡,红景天苷最大浓度可达25.32 g/L.     

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。     

海藻酸铝固定化糖化酶特性研究

采用海藻酸铝固定化糖化酶,对固定化酶和游离酶的特性进行了比较。结果表明,固定化酶米氏常数Km为13.72 mg/mL,最适温度为65℃,半衰期为100.7 d,固定化糖化酶的活力回收率达61.8%。     

戊二醛交联糖化酶的条件探索及协同实验教学分析

介绍了在生物工程实验中探索利用分组协同创新模式进行实验教学的成果。将一个教学自然班分成12个实验小组,每组同学3人。以可能影响实验结果的四个变量分解成四个独立又互相联系的实验,每个变量分别由三组学生负责研究,研究过程通过定期协调沟通对实验结果进行分析总结并不断优化实验设计,得出了优化实验工艺,同时也培养了学生独立思考和协作配合的精神。实验研究了戊二醛交联糖化酶的优化条件,考察了p H、戊二醛浓度、温度和时间四个变量对交联结果的影响。综合分析各实验小组的实验结果显示:酸性条件交联有利于酶活力及稳定性的保持;使用0.5%或更低的戊二醛浓度有利于酶活力的保持,但其稳定性提升不明显;较高温度下交联不利于酶活力的保持及稳定性的提升;随着交联时间的增加酶活力损失逐渐变大,但其使用稳定性却有所提升。     

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。     

Immobilization of glucoamylase on magnetic poly(styrene) particles

Magnetic poly(styrene) particles including active groups were prepared for enzyme immobilization without any activation process. Glucoamylase, which is widely used in industry, was immobilized onto these particles. The effects of pH, buffer concentration, and temperature on immobilization were investigated; moreover, the effect of immobilization temperature on immobilized glucoamylase activity was determined for the hydrolysis of maltose. The acetate buffer with the concentration of 6 × 10⁻⁴ M at pH 4 and 20–30°C was found as the most suitable medium for the immobilization of the glucoamylase. The amount of bound protein is 8 mg/g particle with the immobilization yield of 70%. The maximum activity obtained with immobilized glucoamylase is approximately 70% of the free one. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 69–73, 1999     

Cross-linked enzyme aggregates of recombinant cyclodextrin glycosyltransferase for high-purity β-cyclodextrin production

The cover image is based on the Research Article Cross-linked enzyme aggregates of recombinant cyclodextrin glycosyltransferase for high-purity β-cyclodextrin production by Jianguo Zhang et al., DOI: 10.1002/jctb.5912 .

     

改性磁性壳聚糖微球的制备、表征及性能研究

以(NH4)2Fe(SO4)2.6H2O、NH4Fe(SO4)2.12H2O和壳聚糖为原料,经羟丙基化、胺基化,采用一步包埋法制备了一种新型的多胺基化磁性壳聚糖微球。通过正交实验法确定了磁性微球的最佳制备条件,即搅拌速度1200r/min,壳聚糖用量3.0g,环氧氯丙烷用量2.5mL,乙二胺用量2.5mL。并用IR、TG、XRD和SEM对其结构及形貌进行了表征。结果表明,Fe3O4磁性粒子已包埋了一层胺基化壳聚糖。磁性微球胺基含量为2.302mmol/g;呈较规则的球形,平均粒径为209nm,且具有顺磁性和良好的耐酸性。     

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.     

Cu(Ⅱ)螯合壳聚糖磁性微球的制备、表征及性能研究

以(NH4)2Fe(SO4)2.6H2O、NH4Fe(SO4)2.12H2O和壳聚糖为原料,经羟丙基化、Cu(Ⅱ)螯合,采用一步包埋法制备了一种Cu(Ⅱ)螯合壳聚糖磁性微球。通过正交实验法确定了磁性微球的最佳制备条件,即搅拌速度1 200 r/min,壳聚糖用量3.0 g,环氧氯丙烷用量1.0 mL,CuCl2.2H2O为0.010 mol。并用IR、TG、XRD和SEM对其结构及形貌进行了表征,结果表明,Fe3O4磁性粒子已包埋了一层Cu(Ⅱ)螯合壳聚糖,呈较规则的球形,平均粒径为240 nm,且具有顺磁性。     

Preparation and characterization of a novel injectable in situ cross-linked hydrogel

A novel injectable in situ cross-linked hydrogel was prepared from α,β-poly(N-hydroxyethyl)-dl-aspartamide (PHEA), which had excellent biocompatibility and biodegradability. PHEA was modified by acryloyl chloride (AC) via the reaction between hydroxyl groups and acryl groups to introduce reactable double bonds into the chain. Two macromers with different degrees of derivatization were prepared. Through NMR and FT-IR characterizations, the structures of the polymers were proposed. Hydrogels were prepared by covalent cross-linking between double bonds with a transition from liquid to gel at body temperature. The effect factors on gelation time, swelling ratio and gel content were investigated in detail. It was found that the grafting ratios of AC, concentrations of macromer and initiator had great influence on the gelation time. And it could be adjusted to meet the requirements of an injectable material.