脂肪酶仿生固定化及性质

将仿生钛化过程用于脂肪酶固定化,研究了该过程中工艺条件对脂肪酶固定化的影响及固定化脂肪酶的性质.结果表明：0.5 mL浓度8 mg/mL精蛋白诱导剂、0.5 mL浓度6 mg/mL脂肪酶与1 mL,0.25 mol/L钛前驱体（Ti-BALDH）在pH 7.5,0.05 mol/L磷酸盐缓冲液为反应介质的条件下,脂肪酶...     

交联烯丙基葡聚精凝胶固定化脂肪酸的研究

本文研究了烯丙基葡聚糖和N、N＇－亚甲基双丙烯酰胺经反相悬浮聚合包埋固定化脂肪酶的方法。考察了悬浮介质、交联度对固定化酶的影响，得出了包埋固定化脂肪酶的最佳条件；并测定了固定化脂肪酶的催化性能，确定了该固定化酶催化橄榄油水解的最适条件。     

肝素亲和层析介质制备方法及应用研究进展

肝素亲和层析是利用生物大分子与层析介质表面的肝素特异性结合而进行选择性分离的一种技术,使用条件温和,纯化过程中能较好保持目标蛋白的生物活性。本文在阐述肝素与层析介质化学反应机理的基础上,系统论述了溴化氰活化及偶联法、环氧活化及偶联法、席夫碱法和碳二亚胺缩合法四种肝素亲和层析介质制备方法,深入分析了各制备方法的优缺点,总结了肝素亲和层析在血液制品、病毒纯化方面的应用。最后,进一步分析了肝素亲和层析在蛋白分离纯化领域应用存在的问题,并展望了其未来发展趋势,以期为肝素亲和层析介质的制备方法提供新思路。     

疏水层析蛋白质动力学与平衡过程的考察

疏水层析是分离生物大分子的常用技术之一，但对疏水层析中蛋白质吸附动力学和平衡过程的研究并不多见．本文对蛋白质疏水吸附动力学和平衡过程作了基本假设，并用实验进行了验证。制备了两种不同丁基密度的疏水琼脂糖介质，用其吸附牛血清白蛋白(BSA)以验证对疏水吸附动力学与平衡过程作的假设，考察了盐浓度及配基密度对蛋白质疏水吸附的影响．还对三种疏水性不同的蛋白质：核糖核酸酶、卵清蛋白和牛血清白蛋白的混合体系进行了分离性能的研究，获得了满意的分离效果．实验表明，蛋白质在疏水介质上的吸附动力学和平衡过程与所作假设相符，在实验条件下等温吸附线符合Langmuir吸附等温方程：研制的丁基琼脂糖疏水介质具有优良的使用性能。     

有机相中固定化脂肪酶催化合成植物甾醇酯

酶法合成植物甾醇酯具有反应条件温和、产物纯度和产量高等优点,但非水相酶催化的活性和稳定性普遍较低.本文以大孔树脂固定化脂肪酶为催化剂,并在催化过程中添加乳糖的类似物,构建了有机相高效合成植物甾醇酯的工艺过程.以酯化率为考察指标,对脂肪酶和反应溶剂进行筛选,对酯化条件进行优化,同时考察了糖的种类及添加量对酶催化性能的影响.结果表明,大孔树脂NKA吸附固定化的褶皱假丝酵母(Candida rugosa)脂肪酶(NKA-CRL)为最适宜的催化剂,以正己烷为反应介质,在酸醇摩尔比为2和添加酶蛋白质量7.5%的海藻糖的条件下,40°C反应10 h,酯化率达到96.6%.连续6次催化后,植物甾醇的酯化率仍维持在85.0%以上.     

从酵母细胞中分离纯化醇脱氢酶

醇脱氢酶(ADH)用于食品工业，还是酶法测定乙醇或乙醛含量的工具酶。沉淀法和层析法至今仍然广泛用于分离蛋白质和酶。用离子交换层析、亲和层析等方法经过4～5个纯化步骤后，分离纯化ADH和其它蛋白质，纯化倍数可达100多倍。我们从酵母细胞中分离出醇脱氢酶，并通过硫酸铵分段盐析、凝胶层析以及离子交换层析对该酶进行纯化，用较少的步骤得到了相对高的纯化倍数。     

脂肪酶催化缩聚法合成可完全降解的聚羟基丙酸酯(英文)

以3-羟基丙酸甲酯为聚合单体,建立了以固定化脂肪酶Novozym 435为催化剂的酶催化缩聚反应体系,合成可完全降解的高分子聚酯聚羟基丙酸酯,考察了反应条件和介质对反应性能的影响,结果表明,纯度大于95%的单体即可在温和条件下合成聚羟基羧酸酯;降低反应压力可有效提升产物产率和分子量.通过选择合适的有机溶剂介质和表面活性剂,可使产物分子量提升至13000(Mw)以上.脂肪酶催化剂重复利用能力优异,经6批次反应后,其相对活性保持在95%以上.     

尼龙6纳米纤维膜的制备及其对己烷雌酚的吸附性能研究

采用静电纺丝设备制备尼龙6纳米纤维膜。用静、动态吸附方法,研究了尼龙6纳米纤维膜对己烷雌酚的吸附性能,考察了吸附时间、初始浓度及介质p H值对尼龙6纳米纤维膜吸附己烷雌酚的影响。结果表明,初始浓度为2mg/L时,尼龙6纳米纤维膜吸附己烷雌酚的过程符合准一级动力学模型;当初始浓度在5~20mg/L范围内,吸附过程符合准二级动力学模型。尼龙6纳米纤维膜吸附己烷雌酚的过程符合Freundlich等温吸附模型。     

离子液体中脂肪酶催化酯类合成的新进展

作为21世纪最具发展潜力的绿色溶剂,离子液体用于酶促合成的反应介质具有得天独厚的优势.与传统的有机溶剂相比,离子液体能够提高脂肪酶的稳定性和选择性,减少有机合成中的副反应和有毒气体的产生,后处理简单,可重复利用.参考10年来的文献,从脂肪酶催化酯类合成的常用离子液体种类、过程因素及场/反应器强化等方面进行了综述,同时展望了离子液体在未来酯类酶促合成领域的发展趋势.     

水饱和离子液体中萘普生的酶法拆分

 考察了五种水饱和离子液体反应介质中固定化脂肪酶催化外消旋萘普生甲酯的水解. 结果表明,这些具有不同阴阳离子组合的咪唑基离子液体作为反应介质都能促进水解反应的有效进行,但在水饱和1-正丁基-3-甲基咪唑六氟磷酸盐(［bmim］PF6)中,反应进行得更快,转化率更高. 这些离子液体对脂肪酶均有一定的溶解作用并使溶解的脂肪酶失活. 脂肪酶在离子液体中的溶解度与其剩余活性之间有一定的相关性. 为了解决脂肪酶在离子液体中的溶解问题,使用非极性、高比表面积的无定形多孔硅胶YWG-C6H5对脂肪酶进行了固定化. 在水饱和［bmim］PF6中使用固定化脂肪酶催化反应,反应72 h的转化率为28.3%, 产物的对映体过量值为98.2%, 继续进行反应,转化率将增加,但产物的对映体过量值明显下降. 利用离子液体有别于传统有机溶剂的特性,对离子液体的循环使用、产物的回收和水的补充方法进行了研究. 在反复批式反应中,固定化脂肪酶连续使用五次,活性仅略微下降.     

柱状假丝酵母脂肪酶同工酶的一种高效液相色谱分离新方法

建立一种“疏水界面亲和色谱”分离柱状假丝酵母脂肪酶同工酶的高效液相色谱新方法。将商品化的CRL经离子交换色谱分离为两个同工酶组分 (CRLA和CRLB) ,在极低离子强度下 ,根据同工酶活性中心周围处于“开放”构象的疏水腔具亲疏水界面的特性 ,用疏水界面亲和色谱在NucleosilC4 (10 μ ,3 0 0 ,2 5 0× 4.60mm)柱上将CRLA和CRLB都分离为 4种同工酶组分。疏水界面亲和色谱非常适用于分离这种结构差异轻微的同工酶组分     

Catalytic activity of lipase immobilized onto ultrathin films of cellulose esters

Ultrathin (approximately 2.0 nm) films of cellulose acetate (CA), cellulose acetate propionate (CAP), and cellulose acetate butyrate (CAB) supported on Si wafers have been prepared by adsorption and characterized by means of ellipsometry, atomic force microscopy (AFM), and contact angle measurements. CA, CAP, and CAB ultrathin films were characterized in air just after their formation and after annealing under reduced pressure at temperature higher than the corresponding melt temperature. Upon annealing, CA, CAP, and CAB ultrathin films became smoother and more hydrophobic, evidencing molecular reorientation at the solid-air interface. CA, CAP, and CAB films were used as supports for the immobilization of lipase. The adsorption of lipase onto annealed films was more pronounced than that onto untreated films, showing the strong affinity of lipase for the more hydrophobic substrates. Enzymatic activity was evaluated by a standard procedure, namely, (spectrophotometric) measurement of p-nitrophenol, the product formed from the hydrolysis of p-nitrophenyl dodecanoate (p-NPD). Lipase immobilized onto hydrophobic films exhibited higher activity than that of free lipase and could be recycled three times while retaining relatively high activity (loss of ca. 30% of original enzymatic activity). The effect of storing time on the activity of immobilized lipase was studied. Compared with free lipase, that immobilized onto more hydrophobic films retained 70% activity after 1 month. More importantly, the latter level of activity is similar to that of free lipase. However, lipase immobilized onto more hydrophilic films retained 50% and 30% activity after 20 and 30 days, respectively. These results are explained in terms of surface wettability and the contribution of the interactions between the polar residues of lipase and the glucopyranosyl moieties of cellulose ester to maintain the natural conformation of immobilized enzyme.     

纤维素金属螯合物及其质谱分析在研究血清蛋白质与多肽中的应用

用纤维素做固相支持物,通过碱处理、环氧活化、偶联螯合剂、固定金属离子等方法制成纤维素金属螯合物,并用合成的纤维素金属螯合物处理健康人血清,然后通过基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF-MS)检测蛋白质和多肽以确定其分离效果。确定了最佳的合成方法、螯合剂、金属离子和缓冲体系。再利用普通的纤维素制成了性能优良的纤维素金属螯合物,它能较好地分离血清中的蛋白质和多肽。     

Lipase bound cellulose nanofibrous membrane via Cibacron Blue F3GA affinity ligand

Cellulose (Cell) nanofibrous membranes were prepared by nucleophilic reaction of the cellulose hydroxyl with the triazinyl chloride of Cibacron Blue F3GA (CB) ligand and studied as affinity membranes for lipase enzyme. Cell nanofibrous membranes containing fibers with 200 nm average diameters were prepared by electrospinning of cellulose acetate (CA), followed by alkaline hydrolysis. The CB capacity of the Cell nanofibrous membranes was optimized by lengthening the nucleophilic reaction time and increasing CB concentration and ionic strength. The equilibrium adsorption isotherms of CB on the Cell nanofibrous membranes followed a typical Langmuir monolayer adsorption behavior. At 242 mg CB/g of Cell, the maximum lipase adsorption capacity (q_m) and the dissociation constant (K_d) values were 41.02 mg/g and 0.25 mg/mL, respectively. Optimal lipase adsorption capacity was obtained at pH 4.0, its isoelectric point, with added NaCl on Cell membranes 86 mg CB capacity per g of Cell. A facile lipase loading capacity of 16.21 mg/g of CB–Cell was achieved under moderated conditions and could be optimized to reach at least 150 mg/g. The CB–Cell bound lipase had similar catalytic rate and retained 86.2% activity as in its free form. These findings clearly show that the CB bound Cell nanofibrous membrane is a highly efficient ultra-high specific porous support for lipase enzyme and is potentially versatile for immobilizing other enzymes and as affinity membrane for proteins.     

The effect of carbon sources on the single cell proteins and extracellular enzymes production byChrysonilia sitophila (TFB 27441 strain)

The single cell protein and extracellular enzyme production from a lignocellulolytic fungus,Chrysonilia sitophila, using different carbon sources were evaluated. The mycelial dry mass composition showed a high protein (39.2%) and low nucleic acid content (3.3%), as well as carbohydrate, fatty acid, fiber, and ash levels comparable with single cell proteins currently studied. Mycelial protein showed amino acid content similar to or higher than FAO standard requirements. The amino acid, fatty acid, and carotenoid composition, as well as mycelial mass yield and enzyme production, were dependent on the carbon source used. Glucose, saccharose, cellobiose, cellulose, microcrystal-line cellulose, lactose, and rice hull as carbon sources were studied.     

稻壳吸附剂提高啤酒稳定性的研究

利用稻壳良好的吸附特性,制备成吸附剂用于提高啤酒的稳定性。稻壳粉碎与稀硫酸混合,240℃密封干馏,再以高温灼烧活化,得到对单宁有较强吸附能力的稻壳吸附剂。以此吸附剂去除啤酒中的部分单宁,以提高酒体的胶体稳定性,试验表明,每100ml啤酒以0．4g吸附剂在15℃下搅拌吸30min,可使酒中单宁量下降16．7％,从而减缓了引起啤酒混浊的缔合反应,使酒体稳定性明显提高。与聚乙烯聚吡咯烷酮比较,具有吸附速度快、吸附单宁更强及成本低廉的优点。     

Application of heparinized cellulose affinity membranes in recombinant adeno-associated virus serotype 2 binding and delivery

The microporous affinity membrane based on cellulose matrices offers minimal mass-transfer effects in membrane chromatography with low nonspecific adsorption. In this research, we tested a novel application of the microporous, heparinized cellulose membranes for their affinity toward recombinant adeno-associated virus serotype 2 (rAAV2, which uses heparan sulfate proteoglycans as the primary cellular receptor) to develop a controlled, substrate-mediated viral vector delivery. We conjugated rAAV2 to an epoxy-crosslinked heparin cellulose membrane, which led to vector transduction upon cellular adhesion. When adhered, human fibroblasts exhibited proliferation kinetics similar to those on the standard polystyrene tissue-culture surface. Using fluorescent proteins as the reporter, we showed that the heparin-bound rAAV2 particles remained active and that the rAAV2-heparin binding was reversible and capable of mediating transgene delivery in cell culture. In addition, we applied the affinity membrane to adsorb unpurified rAAV2 from the crude lysate of packaging cells via the ligand–receptor binding, avoiding the use of conventional ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work explores a new application of affinity cellulose membranes in substrate-mediated viral vector delivery, which can be a useful tool in developing protocols for localized gene transfer.     

两种固定化金属螯合复合亲和膜色谱介质制备

以纤维素滤纸为基质,通过碱处理、环氧活化、偶联亚氨基二乙酸二钠、固定化Ｃｕ＾２＋后制得了大孔纤维素亲和膜。另外,在活化后的膜上通过共价交联覆盖上琼脂糖,制得了具有类似“三明治”结构且性能优于的复合亲和膜,装柱后分别制得固定化金属螯合亲和膜色谱柱。对两种亲和膜进行牛血清白蛋白等温吸附测定显示,两者的最大吸附量分别为１．１７ｍｇ／ｃｍ＾２和１．３０ｍｇ／ｃｍ＾２,与传统的琼脂糖凝胶类介质吸附量相当,表     

分散固相萃取-高效液相色谱-质谱联用法测定水稻秸秆、糙米、稻壳和土壤中戊唑醇残留

建立了分散固相萃取-液相色谱-质谱法测定水稻秸秆、糙米、稻壳及土壤中戊唑醇残留量。方法以乙腈为萃取溶剂,N-丙基-乙二胺(PSA)为吸附剂,实现样品快速制备;在Zorbax Eclipse XDB-C18色谱柱上,以甲醇-5 mmol乙酸铵缓冲溶液(70/30,V/V)为流动相,采用电喷雾质谱检测器,选择离子监测模式,以m/z308为定量检测离子,戊唑醇保留时间在9.5 min左右,在0.005~2 mg/L范围内浓度与峰面积呈良好的线性关系,相关系数为0.9999。在4种样本中添加不同浓度戊唑醇其平均回收率在73.6%~105.8%,相对标准偏差(n=5)小于15%,检出限(S/N=5)为2.5×10-11g。该方法简单、快速、灵敏、准确。     

Affinity chromatography of porcine pepsin on different types of immobilized 3,5-diiodo-L-tyrosine

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.