磁性壳聚糖复合微粒固定化SOD的研究

制备了磁性壳聚糖复合微粒(Fe3O4/CS),并以Fe3O4/CS为载体固定了超氧化物歧化酶(Superoxide Dismutase,SOD)。研究了温度、pH、储存时间、操作次数等对固定化SOD和游离SOD活性的影响。研究结果表明,固定化SOD的热稳定性、pH稳定性、储存稳定性等性能明显优于游离SOD,固定化SOD的操作稳定性良好。研究了固定化SOD和游离SOD的半衰期t1/2和动力学性质。固定化SOD的半衰期t1/2,1=35.7 d,游离SOD的半衰期t1/2,2=8.8 d;固定化SOD的米氏常数Km,1=0.04 mmol/L,最大反应速率Vm,1=40.19 mmol/min,游离SOD的米氏常数Km,2=0.19 mmol/L,最大反应速率Vm,2=85.76 mmol/min。     

酵母菌与糖化酶的共固定化研究

以海藻酸铝为载体,用包埋法共固定酵母菌和糖化酶,探讨了此双酶体系的储存稳定性和重复使用稳定性,研究了共固定化酶的双酶比例及pH和温度对酒精发酵的影响,结果表明,酵母菌和糖化酶的最佳加入量分别为2.5 g和300 U/g;固定化酶重复使用10次后,残余活力仍能保持最初活力的83.47%;共固定化酶的半衰期可达205.1 d;pH为4.0~4.5、温度为37℃时,其活性可达最高。     

猪骨基多孔碳固定化酶的研究

本文以猪骨基多孔碳为载体,采用明胶包埋与戊二醛交联结合的方法,制备固定化过氧化氢酶,并对固定化酶制备条件的优化,酶作用最适条件及其稳定性进行研究。结果表明:以猪骨基多孔碳为载体吸附2h、1%明胶包埋、1%戊二醛交联过氧化氢酶制备的固定化酶,在最适的缓冲液浓度、溶液pH和体系温度下,虽然催化效率有所下降,但其贮存稳定性和操作稳定性都有所增加,重复使用8次后,活力仍保持在初始活力的90%以上。这对于过氧化氢酶进一步在食品工业和纺织工业的推广可能有潜在的应用价值。     

明胶膜固定化脲酶的制备及性质

以明胶为载体,戊二醛为交联剂,采用包埋-交联联用法制备了明胶膜固定化脲酶,其酶活力为6 07U/g载体,酶活力收率为66 1%。最优固定化条件是包酶量为10mg酶/g明胶,ρ(明胶)=100g/L,φ(戊二醛)=0 5%。研究了固定化酶的性质,并与游离酶作了比较,游离酶的最适pH=7 0,固定化酶的最适pH=6 5;游离酶的最适温度为60℃,固定化酶的最适温度升至70℃;固定化酶与游离酶的米氏常数Km分别为11 7mM和12 4mM;固定化酶在80℃下180min仍保留初始活力的10%,而游离酶几乎完全失活。固定化酶重复使用20次其活力仅下降15%,4℃下贮存35d后仍保持初始活力的55%。     

磁性硅基壳聚糖微球固定化柚苷酶水解柚皮苷的研究

本文在磁性Fe3O4外包覆一层SiO2,再在其外包裹壳聚糖制备出磁性硅基壳聚糖微球（MSC）,对MSC进行环氧基修饰后用于柚苷酶的固定化研究,并对磁性硅基壳聚糖微球固定化柚苷酶水解柚皮苷的pH、温度、操作和储藏稳定性进行了考察。通过单因素实验,确定了环氧基修饰的磁性硅基壳聚糖微球（MSCE）固定化柚苷酶的最佳工艺条件为：pH 3.0,温度30 ℃,时间4 h、给酶量57.48 U/mL。在此条件下,MSCE固定化柚苷酶的载酶率、酶活回收率和酶比活力分别为31.29%、88.92%和409.33 U/g。与游离柚苷酶相比,MSCE固定化柚苷酶用于水解柚皮苷具有良好的pH稳定性和温度稳定性,重复使用8次后仍具有53.36%的相对酶活力,4 ℃条件储存一个月后仍具有80.97%的相对酶活力。     

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%。     

明胶多孔支架固定化过氧化氢酶

以明胶多孔支架为载体,戊二醛为交联剂成功地制备了固定化过氧化氢酶,并对其固定化酶的性质进行了研究。固定化过氧化氢酶活力回收率可高达51.1%。与游离酶相比较,虽然催化效率有所降低,但是其固定化酶在65℃保存的稳定性有明显提高。固定化酶可重复使用10次后,活力仍保持在初始活力的60%以上,其具有良好的操作稳定性。     

琼脂包埋法固定化α-淀粉酶特性的研究

以琼脂作为载体材料,采用包埋法固定化α-淀粉酶,并对其特性进行了研究.结果表明,该固定化酶最适pH 值为7.5、最适温度为55～ 58℃,具有较好的贮存稳定性和反应稳定性,18 d后该固定化酶的残余活力仍保留原酶活的71.6%左右,重复使用7次,酶活力下降不大,其酶活回收率达到78.8%.     

多酚氧化酶的改性蛭石固定化及其催化特性研究

以马铃薯为原料,提取其中的多酚氧化酶,采用改性蛭石为载体,对其进行固定化,研究游离与固定化酶的最适催化温度、pH值,固定化酶的储存稳定性、重复使用稳定性及对苯酚的清除性能。结果表明,改性后蛭石出现了较多沟壑状孔隙,颗粒质感疏松,有利于固定化酶分子;最佳固定化条件为:改性蛭石含量0.4 g,戊二醛浓度2.0%,搅拌时间2 h,在此条件下,酶活力回收率可达到60.67%;游离与固定化酶的最适催化温度均为50℃,最适催化pH值均为7.0;固定化酶4℃下储存28 d后酶活力可保留52.1%,具有较好的储存稳定性;重复使用5次后,仍能保持61.0%的初始活性,说明固定化酶构象较稳定,固定化马铃薯多酚氧化酶对苯酚具有较好的清除性能。     

多酚氧化酶的改性蛭石固定化及其催化特性研究

以马铃薯为原料,提取其中的多酚氧化酶,采用改性蛭石为载体,对其进行固定化,研究游离与固定化酶的最适催化温度、pH值,固定化酶的储存稳定性、重复使用稳定性及对苯酚的清除性能。结果表明,改性后蛭石出现了较多沟壑状孔隙,颗粒质感疏松,有利于固定化酶分子;最佳固定化条件为:改性蛭石含量0.4 g,戊二醛浓度2.0%,搅拌时间2 h,在此条件下,酶活力回收率可达到60.67%;游离与固定化酶的最适催化温度均为50℃,最适催化pH值均为7.0;固定化酶4℃下储存28 d后酶活力可保留52.1%,具有较好的储存稳定性;重复使用5次后,仍能保持61.0%的初始活性,说明固定化酶构象较稳定,固定化马铃薯多酚氧化酶对苯酚具有较好的清除性能。     

氨基树脂固定胃蛋白酶的方法及性质研究

采用氨基树脂作为载体,戊二醛作为交联剂,对胃蛋白酶的固定化进行了研究,并对固定化条件和固定化胃蛋白酶的部分酶学性质进行了分析。确定固定条件为：戊二醛浓度为5%,载体处理温度为室温（25℃）,处理时间为5h,m（胃蛋白酶）：m（氨基树脂）为1：25,pH为3.0,固定时间为12h。此条件下固定化的胃蛋白酶活力为30U/g,酶的活力回收率为60%。与非固定化相比最适水解温度由50℃升高到60℃,最适pH值由2.0升高到4.0,游离酶米氏常数3.08g/L,固定化酶米氏常数1.2g/L,固定化胃蛋白酶的储存半衰期约为25天。对珠蛋白的操作半衰期为9天。     

固定化γ-谷氨酰转肽酶催化合成γ-L-谷氨酰-L-半胱氨酸

以环氧基树脂Eupergit C250L为载体对B.subtilis NX-2 GGT进行了共价固定化.固定化酶的最适作用pH为9.0,最适作用温度为60℃.固定化酶的热稳定性和贮存稳定性均较游离酶有显著的提高,经100 d 20个批次转化后,固定化残余酶活仍能保持初始值的80%左右.以固定化酶为催化剂,在反应条件为L-谷氨酰胺(Gln)20 mmol/L、S-苄基-半胱氨酸(S-Bzl-cys)20 mmol/L、酶浓度0.0375 U/mL和pH 9.0条件下,40℃水浴反应22 h,转肽产物S-苄基-y-L-谷氨酰-L-半胱氨酸(S-Bzl-GGC)得率为4.3 mmol/L,较游离酶提高了11.96%.S-Bzl-GGC经酸解脱除保护基后可得γ-L-谷氨酰-L-半胱氨酸,产物纯度可达94.1%.     

<Emphasis Type="Italic">Penicillium</Emphasis> sp. ZD-Z1 chitosanase immobilized on DEAE cellulose by cross-linking reaction

Chitosanase obtained fromPenicillium sp.ZD-Z1 was immobilized on DEAE cellulose with glutaraldehyde by cross-linking reaction. The optimal conditions of immobilization were as follows: 0.1 g DEAE cellulose was treated with 5 ml 5% glutaraldehyde solution; then 2.3 mg chitosanase was immobilized on the carrier. The optimal temperature and pH was 60 °C and 4.0, and the K _m value was 18.87 g/L. Under optimal conditions, the activity of immobilized enzyme is 1.5 U/g, and the recovery of enzyme activity is 81.3%. After immobilization, the optimal temperature and K _m value increased (from 50 °C to 60 °C, from 2.49 g/L to 18.87 g/L), whereas the optimal pH was reduced (from 5.0 to 4.0). The enzyme activity loss was less than 20% after 10 times batch reaction; the immobilized enzyme showed good operation stability.     

Immobilization of organophosphorus hydrolase enzyme by covalent attachment on modified cellulose microfibers using different chemical activation strategies: Characterization and stability studies

The plant cellulose powder was activated by two different methods using 1,4-butanediol diglycidyl ether(BTDE)and 1,1′-Carbonyldiimidazole(CDI) as the chemical coupling agents.Organophosphorus hydrolase(OPH) from Flavobacterium ATCC 27551 was immobilized on any of activated support through covalent bonding.The optimal conditions of affecting parameters on enzyme immobilization in both methods were found, and it was demonstrated that the highest activity yields of immobilized OPH onto epoxy and CDI treated cellulose were 68.32%and 73.51%, respectively.The surface treatment of cellulose via covalent coupling with BTDE and CDI agents was proved by FTIR analysis.The kinetic constants of the free and immobilized enzymes were determined, and it was showed that both immobilization techniques moderately increased the Kmvalue of the free OPH.The improvements in storage and thermal stability were investigated and depicted that the half-life of immobilized OPH over the surface of epoxy modified cellulose had a better growth compared to the free and immobilized enzymes onto CDI treated support.Also, the pH stability of the immobilized preparations was enhanced relative to the free counterpart and revealed that all enzyme samples would have the same optimum pH value for stability at 9.0.Additionally, the immobilized OPH onto epoxy and CDI activated cellulose retained about 59% and 68% of their initial activity after ten turns of batch operation, respectively.The results demonstrated the high performance of OPH enzyme in immobilized state onto an inexpensive support with the potential of industrial applications.     

聚乙烯亚胺/多巴胺改性氧化硅固定碳酸酐酶

利用聚乙烯亚胺（PEI）/多巴胺（DA）共沉积法改性氧化硅，并以此为载体固定化碳酸酐酶（CA）。考察了PEI/DA质量比、沉积时间对沉积率的影响，用傅里叶红外光谱（FTIR）和扫描电子显微镜（SEM）对改性前后的微球进行了表征；研究了沉积率、载体用量、酶浓度及戊二醛（GA）浓度对固定化酶活回收率的影响；考察了固定化酶的储存稳定性和重复利用性。结果表明，随PEI/DA质量比增加，沉积率先增加后降低，质量比为1∶1时最大；随沉积时间增加，沉积率线性增长，10 h后PEI/DA体系沉积率为单独DA沉积改性的2.66倍，但沉积时间对N元素含量和酶活回收率影响不大；酶固定化时载体用量存在饱和值，CA和GA浓度的最优值分别为0.8 mg/ml和0.1%(质量)，此时酶活回收率可达78.8%。在25℃下储存30 d后，固定化酶的保留活性为77.2%，而游离酶只有12%；重复使用10次后，固定化酶仍能保持88.3%的相对活性。     

仿生合成氧化锆固定化漆酶处理孔雀石绿

以溶菌酶作为诱导剂,仿生合成了ZrO₂固定化漆酶纳米颗粒,其酶活回收率达59%,采用场发射扫描电子显微镜（FESEM）、能谱仪（EDS）、热重分析仪（TGA）等手段对ZrO₂纳米颗粒及ZrO₂固定化漆酶颗粒进行表征,结果表明漆酶可成功固定到ZrO₂颗粒中,同时还证明了溶菌酶既作为诱导剂催化ZrO₂的形成,又作为生物模板同酶一起包埋在ZrO₂颗粒中。固定化漆酶的最适pH为3,最适温度为70℃,相比于游离酶,其pH、温度稳定性都有明显提高;固定化漆酶纳米颗粒在4℃下储存30d,活性为初始酶活的95%,重复使用5次,固定化酶的残余酶活力仍有60%。此外,固定化漆酶在6h内对孔雀石绿染料的脱色率高达95%以上,通过紫外-可见吸收光谱分析（UV-vis）可知,固定化漆酶对孔雀石绿染料的处理是由吸附和降解联合作用引起的脱色。     

Preparation and characterization of polymer-coated mesoporous silica nanoparticles and their application in Subtilisin immobilization

The preparation and characterization of polymer-coated mesoporous silica nanoparticles (MSNs) and their application in Subtilisin (Alcalase®) immobilization were investigated. For the synthesis of polymer-coated MSNs, acrylic acid (AA) and chitosan (CS) mixture were blended as poly(acrylic acid) (PAA) and CS polymer layer onto MSNs via in-situ polymerization technique. Then, both uncoated MSNs and polymer-coated mesoporous silica nanoparticles (CS-PAA/MSNs) were characterized by taking into account properties such as morphologic pattern, size distribution, surface charge of the particles as well as thermogravimetric stability with SEM, TEM, Zetasizer and TGA analyses. Subtilisin was immobilized onto polymer-coated mesoporous silica nanoparticles via adsorption technique. For optimizing the enzyme immobilization process, the percent enzyme loading depending on the matrix amount, immobilization time and pH were investigated. Then, the activity values of immobilized enzyme and free enzyme were compared at various pH and temperature values. The maximum enzyme activity was achieved at pH 9.0 for both immobilized and free enzyme. Immobilized enzyme showed more stability at higher temperatures compared with free enzyme. Furthermore, the operational and storage stability of immobilized enzyme were determined. The activity of immobilized enzyme was reduced from 100% to 45.83% after five repeated uses. The storage stability of immobilized enzyme was found to be higher than that of free enzyme. The activity of immobilized enzyme was reduced from 100% to 60% after 28 days of storage time. We concluded that the polymer-coated MSNs were a suitable matrix for Subtilisin immobilization compared to uncoated MSNs.     

磁性壳聚糖微球的制备及其用于甲酸脱氢酶的固定化

分别采用乳化交联法和共沉淀法制备磁性壳聚糖微球载体,并对形貌结构进行比较,结果表明,采用共沉淀法制备的磁性壳聚糖微球负载Fe3O4的效果好,故将其作为载体固定甲酸脱氢酶。最佳固定化条件:添加酶量9 U.g-1,pH=7.0,固定化时间5 h。游离酶和固定化酶的最适宜反应温度分别为50℃和30℃;游离酶的最适宜pH=7.0,固定化酶的最适宜pH=6.0;将游离酶和固定化酶分别置于60℃恒温水浴放置180 min后,游离酶和固定化酶的相对酶活力分别为0.78%和40.39%;将游离酶和固定化酶置于不同pH的缓冲液中保存1 h后,在强酸(pH=2.0)和强碱(pH=10.0)条件下,固定化酶的相对酶活力分别为11.03%和38.43%,游离酶已全部失活;固定化酶重复使用6次后,相对酶活力为73.53%,表明固定化酶具有较好的热稳定性、酸碱稳定性和操作稳定性。     

醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸（PAA）改性的聚乙烯（PE）膜为载体,研究了醇脱氢酶（ADH）的两种固定化路线,并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺（PEI）进一步改性,使用戊二醛（GA）固定化ADH。最优固定化pH为6.0,温度为5～15℃,酶浓度为1.0 mg/ml,GA浓度为0.01%(质量);固定化酶的最适反应pH为6.5,温度为15～30℃,反应速率最高为9.6 μmol/(L·min);重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体,用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）为活化剂,固定化ADH。EDC和NHS最优摩尔比为1∶0.5,固定化时间为24 h;固定化酶的最适反应pH为6.5,温度为20～37℃,反应速率为15.58 μmol/(L·min);重复利用10次后可保持53.8%的活性。     

Immobilization of alcohol dehydrogenase on the surface of polyethylene membrane

Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles.