磁性聚乙烯醇缩丁醛微球固定化α-淀粉酶

制备出磁性聚乙烯醇缩丁醛微球,并用该微球做载体,采用共价交联法固定α 淀粉酶。最佳固定化工艺条件为:pH=6 07,激活和交联时戊二醛的质量分数分别为4%和0 025%。在最佳固定化条件下所制磁性固定化酶的活力为25426 3U/g微球,蛋白载量为187 2mg/g微球,比活为135 8U/mg蛋白,活性回收率为36 9%。磁性固定化酶的理化性质为:磁性固定化酶的最适温度(60℃)比自由酶(50℃)高,最适pH(6 97)与自由酶相同,磁性固定化酶Km(米氏常数)值(5 7×10-4kg/L)较自由酶Km值(5 0×10-4kg/L)大,热稳定性、pH稳定性及操作稳定性均比自由酶有所提高。     

mPEG-PLGA多孔微球的制备及其对降钙素的吸附行为

以聚乙二醇-聚(乳酸-乙醇酸)共聚物(mPEG-PLGA)为材料,采用溶剂挥发法与快速膜乳化法制备了尺寸均一的mPEG-PLGA多孔微球,用其吸附降钙素,考察了降钙素浓度、吸附时间、离子浓度、温度及pH值及微球性质对吸附的影响. 结果表明,优化的吸附条件为：降钙素浓度1.0 mg/mL, pH 7.4, NaCl浓度0.2 mol/L, 14℃下吸附8 h,该条件下,微球吸附量为48.9 mg/g;吸附量与微球比表面积正相关.     

Cu(Ⅱ)螯合壳聚糖磁性微球固定化胃蛋白酶的制备及性能研究

将Cu(Ⅱ)螯合壳聚糖磁性微球用作固定化胃蛋白酶的载体,讨论了固定化时间、pH值和给酶量对酶固定化的影响.确定最适固定化条件为:固定化时间1.0 h、pH值4～5、给酶量150 mg·(g载体)-1.与自由酶比较,固定化胃蛋白酶的催化特性和稳定性均令人满意.     

乙肝表面抗原在微球表面的吸附行为、活性和结构变化

采用快速膜乳化技术结合溶剂挥发法制备了尺寸均一的聚乳酸(PLA)微球,平均粒径为800 nm左右,并采用PLA微球对乙肝表面抗原(HBsAg)进行了吸附研究,考察了pH值、盐浓度、微球用量、HBsAg浓度及吸附温度对HBsAg吸附率、活性和结构的影响. 结果表明,在pH 6.0的磷酸缓冲溶液中,NaCl浓度为20 g/L、微球用量为20 mg/mL、HBsAg浓度为10 mg/mL、吸附温度为37℃的条件下,HBsAg吸附率可达60%左右. 4℃下,pH值为8.0及NaCl浓度为1 g/L、微球用量为2 mg/mL及HBsAg浓度为300 mg/mL时,HBsAg活性保留可达98%以上.     

梳状亲水性环氧基载体固定化Pseudomonas stutzeri LC2-8脂肪酶

以氯乙酰化聚苯乙烯微球(PS-acyl-Cl)为引发剂,亲水性丙烯酰胺(AM)和甲基丙烯酸缩水甘油酯(GMA)为单体,CuCl及联吡啶(Bipy)为催化体系,通过原子转移自由基聚合反应(ATRP)制备得到梳状亲水性环氧基柔性载体(PS-acyl-P(AM-co-GMA)),通过改变AM与GMA配比,使载体环氧基含量和亲水性得到控制.用该柔性载体固定Pseudomonas stutzeri LC2-8脂肪酶,优化了固定化条件,并对柔性固定化酶性质进行考察.结果显示,当n(AM)∶n(GMA)=20∶60,固定化时间为24 h,固定化温度为30℃,固定化pH为7.0时,固定化酶活力达到最高,为24.1U·g-1.固定化酶的最适pH为8.0,最适温度为30℃,其热稳定性比游离酶高,重复使用8次,剩余酶活力80％左右.以上表明,以ATRP法合成的载体PS-acyl-P(AM-co-GMA)可成功用于脂肪酶的固定化,有效提高脂肪酶的稳定性和实用性.     

青霉素酰化酶在新型复合载体上的固定化研究

在反应温度为45℃、反应时间为6 h、丙烯酰胺(AM)质量浓度为40 g/L、引发剂用量为单体质量1%的条件下制得PAM-SiO2复合载体,利用红外光谱表征了其化学结构,热失重法测定其接枝量为21%。在所制PAM-SiO2复合载体上固载青霉素酰化酶,其固载最适条件为:戊二醛用量0.1 mmol/L,pH值7.64,给酶量1%,温度42℃,时间11 h~12 h。此条件下所得固定化酶的活力为34000 U/mL;测得所制固定化酶的最适pH值为7.82,最适温度为45℃。     

固定化硝化菌去除污水中氨氮工艺参数优化

为优化固定化硝化菌去除氨氮的工艺条件,采用正交试验方法,考察了固定化微球投加量、通气速率、反应温度和pH值4个因素对氨氮去除效果的影响,获得固定化细菌对模拟废水中氨氮的最优去除条件。结果表明:当固定化微球投加量为200 g/L,反应温度为40℃,体系pH值为9.0,通入空气表观气速为1.5 L/(min·L)时,氨氮去除率最高。4种因素的影响程度依次为pH值固定化微球投加量反应温度表观气速。在此最优条件下,当初始氨氮质量浓度为100 mg/L时,可使其去除率达97%以上。     

sol-gel法固定化漆酶及其性质

采用sol-gel法固定化漆酶,最佳固定化条件为:聚乙二醇分子量PEG600;聚乙二醇添加量1.5%;酶液浓度15mg/mL;水/前驱体质量比1:6;缓冲液pH值4.5。固定化漆酶活性保持在游离漆酶的50%以上,最适反应温度为60℃,最适pH值为pH4.5。同时,热稳定性、酸碱稳定性和贮存稳定性都有明显的提高。当以ABTS为底物时,固定化漆酶的K_m值(122.8μmol/L)比游离漆酶的K_m值(32.9μmol/L)高,与底物的亲和力有所降低。     

中间相炭微球基活性炭对Fe~(3+)吸附性研究

为了研究含铁离子废水的净化方法和扩展中间相碳微球活性炭的应用领域,本文以中间相碳微球基活性炭(MCMB)为吸附剂,对模拟含铁离子废水进行吸附性实验研究。结果表明,MCMB对Fe~(3+)吸附的最佳参数:Fe~(3+)初始浓度300.00 mg/mL,吸附时间80 min,吸附温度50℃,Fe~(3+)平衡浓度17.3 mg/mL,溶液PH为1;最适工艺参数下,最大吸附量1308 mg/g。     

磁性壳聚糖微球的合成及其在固定化血管紧张素转化酶的应用

以化学共沉淀法合成Fe3O4纳米粒子为磁核,采用乳化交联法制备磁性壳聚糖微球,并对其形貌、结构和磁饱和强度等性质进行了表征。以磁性壳聚糖微球作为载体,固定化猪肺粗提物中的血管紧张素转化酶,并对固定化条件进行研究。结果表明,固定化血管紧张素转化酶的最佳条件为：pH值为8.3,最佳温度为50 ℃,最佳时间为1.5 h,最佳酶溶液蛋白浓度为6 mg/mL,此时固定化酶活力最高为0.048 U/g微球。与游离酶相比,固定化酶的pH值稳定性和热稳定性均得到提高。固定化酶重复使用10次,仍然保持40%以上相对活力,说明磁性壳聚糖微球是固定化血管紧张素转化酶的良好载体。     

温度对温敏性固定化酶的相对活力影响研究

以N 异丙基丙烯酰胺 (PNIPAM)均聚凝胶和N 异丙基丙烯酰胺与丙烯酰胺 [P(NIPAM -AM) ]共聚凝胶为载体制备了四种温敏性固定化酶。其相对活力 (f)随温度的升高而降低 ,在凝胶低临界溶解温度LCST(32 0℃ )附近骤降 (均聚枯草杆菌蛋白酶的f在 32 0℃前后由 86 2 %降至 18 8% ) ,即高温下 (LCST以上 )酶从凝胶中释放出来 ,说明温敏性凝胶可用作生物固定化催化剂的功能性载体。     

Dual thermo- and pH-sensitive poly(N-isopropylacrylamide-co-acrylic acid) hydrogels with rapid response behaviors

Novel dual temperature- and pH-sensitive comb-type grafted poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AAc)) hydrogels were successfully prepared by grafting PNIPAM chains with freely mobile ends onto the backbone of a cross-linked P(NIPAM-co-AAc) network. The prepared comb-type grafted P(NIPAM-co-AAc) hydrogels exhibited a more rapid deswelling rate than normal-type P(NIPAM-co-AAc) hydrogels in ultrapure water in response to abrupt changes from 25 °C to 60 °C. The same was true in buffer solution with a pH jump from 7.4 to 2.0 at 25 °C. Unexpectedly, the comb-type grafted P(NIPAM-co-AAc) hydrogels showed abnormal shrinkage behaviors in a buffer solution when the temperature increased from 25 °C to 60 °C with a pH value fixed at 7.4 or 2.0. In a buffer solution of pH 7.4, when the environmental temperature jumped from 25 °C to 60 °C, the grafted comb-type hydrogels shrank slower than the normal-type hydrogels, while at pH 2.0, the gels shrank faster than the normal-type gels in the beginning, which was followed by a slower shrinking. Interestingly, the much quicker shrinkage of the comb-type grafted P(NIPAM-co-AAc) hydrogels was observed because of the cooperative thermo-/pH-responses when the simultaneous temperature and pH stimuli met from pH 7.4/25 °C to pH 2.0/60 °C. The results of this study provide valuable information regarding the development of dual stimuli-sensitive hydrogels with fast responsiveness.     

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.     

Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water‐soluble low‐molecular‐weight chitosan

A nonspecific chitosan hydrolytic enzyme, cellulase, was immobilized onto magnetic chitosan microspheres, which was prepared in a well spherical shape by the suspension crosslinking technique. The morphology characterization of the microspheres was carried out with scanning electron microscope and the homogeneity of the magnetic materials (Fe₃O₄) in the microspheres was determined from optical micrograph. Factors affecting the immobilization, and the properties and stabilities of the immobilized enzyme were studied. The optimum concentration of the crosslinker and cellulase solution for the immobilization was 4% (v/v) and 6 mg/mL, respectively. The immobilized enzyme had a broader pH range of high activity and the loss of the activity of immobilized cellulase was lower than that of the free cellulase at high temperatures. This immobilized cellulase has higher apparent Michaelis–Menten constant K_m (1.28 mg/mL) than that of free cellulase (0.78 mg/mL), and the maximum apparent initial catalytic rate V_max of immobilized cellulase (0.39 mg mL^?1 h^?1) was lower than free enzyme (0.48 mg mL^?1 h^?1). Storage stability was enhanced after immobilization. The residual activity of the immobilized enzyme was 78% of original after 10 batch hydrolytic cycles, and the morphology of carrier was not changed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1334–1339, 2006     

Immobilization strategy of accessible transmission for trypsin to catalyze synthesis of dipeptide in mesoporous support

Immobilized trypsin in mesoporous silica foams was used to catalyze dipeptide synthesis in hydrophilic organic solvent instead of soluble form. The area surface of nano support was measured. The catalytic activity, coupled yield and kinetic characterization of immobilized trypsin were examined. Bz-Arg-OEt was chosen as the acyl donor with Lys-OH as the nucleophile. The trypsin-catalyzed synthesis condition was optimized, such as catalytic temperature, pH, reaction time, physical properties and content of organic solvents, together with the added enzyme amount. The immobilized trypsin showed 112.8% of residual activity with 91.9% of coupled yield, and the kinetic parameters exhibited accessibility for transmission. The product yield of 5.8% was reached at the optimum conditions for enzymatic synthesis of dipeptide: 800 mg of wet immobilized trypsin (200 mg/g support) was used in Tris-HCl buffer (0.1 mol/L, pH 8.0) containing 80% (v/v) ethanol solvents for 6 h of reaction time at 35 °C. This attempt of immobilized strategy for trypsin in nanopores renders the possibility of wide application of inorganic nano-sized support in catalytic synthesis process, which can avoid usage of large amounts of organic solvents in washing steps by chemical methods and reduce the tedious purification process of its soluble form.     

Fluorescence analysis for thermo-sensitive hydrogel microspheres

Poly(N-isopropylacrylamide) (PNIPAM) hydrogel microspheres were prepared by precipitation polymerization above the polymer's lower critical solution temperature (LCST) in water. The hydrodynamic size of PNIPAM microspheres exhibited a thermo-sensitive change around the LCST. In order to obtain information about the interface of the hydrogels, contact angle measurement, protein adsorption, and fluorescence analysis were performed. Contact angle measurement and protein adsorption showed a gradual change around the LCST similar to the change of the hydrodynamic size. However, fluorescence study exhibited a sharp change at the LCST. In addition, a quenching experiment was employed to elucidate the location of the fluorescent probes. In the PNIPAM latex, the probes were located where they were less quenchable as compared to the PNIPAM solution. It seems probable that the probes can penetrate into the crosslinked PNIPAM hydrogel and might reflect the inner environment of the hydrogel. The effect of salt on thermo-sensitive behaviour was also detected by fluorescence analysis. On the other hand, the hydrogel microsphere prepared from poly(acryloyl pyrrolidine) (PAPr) showed a gradual change in fluorescence with temperature.     

类人胶原蛋白的聚集行为与固定化方法

类人胶原蛋白(HLC)具有特殊的结构特点,能够和多种生物分子发生相互作用,将其固定化作为介质在分离纯化中具有一定的实用价值。通过考察不同的溶液组成和环境因素对HLC之间聚集行为的影响,使用环氧氯丙烷将HLC固定在Sepharose微球表面制成具有一定分离纯化功能的介质。结果表明:在固定化过程中,HLC浓度的影响较小,而温度、pH值影响较大,葡萄糖、CaCl2对于HLC的自聚合表现出较强的抑制作用。最终确定在温度为45℃,pH=9.0的环境条件下,每1 mL Sepharose微球添加15 mg HLC,葡萄糖质量浓度15%,CaCl2浓度0.3 mol/L的反应体系中,能够有效实现HLC的固定化。