磁性SiO2纳米粒子的制备及其用于漆酶固定化

以氨基修饰的磁性SiO2纳米粒子为载体,通过交联剂戊二醛固定漆酶,对固定化条件进行了优化,比较了固定化酶与游离酶的酶学性质. 结果表明,漆酶固定化的最佳条件为戊二醛浓度8%(w),固定化时间6 h,缓冲液pH值7.0,初始酶液浓度0.15 g/L. 固定化的漆酶的最适pH为4.0,最适温度为20℃. 在60℃条件下保温4 h,固定化漆酶仍能保持酶活力60.9%,在连续10次操作后,酶活力仍能保持55%以上,其热稳定性和操作稳定性均比游离酶高.     

漆酶在介孔分子筛MCM-41上的固定化研究

以介孔分子筛MCM-41作为漆酶固定化的载体,采用物理吸附法进行固定化,考察了时间、pH和给酶量对固定化效果的影响,并对固定化酶的活性及其稳定性进行了研究,讨论了影响固定化过程和固定化酶性质的主要原因.结果显示,在pH为3时,酶和载体比例为62.5 mg·g-1时吸附12 h固定化效果最好,固定化酶活性回收率为50%;与游离漆酶相比,MCM-41固定化漆酶的最适反应pH略有升高,最适温度没有变化,其pH稳定性和热稳定性都显著优于游离漆酶,固定化漆酶具有可重复操作的性质,与底物反应反复操作10批次后剩余活性为40%.以上表明,MCM-41作为固定化漆酶的一种新型载体材料,有利于改善漆酶的稳定性和实用性.     

利用酰胺活化聚乙烯醇作为载体固定漆酶的研究

交联聚乙烯醇经羧基化、酰胺化制成一种活性固定化酶的载体,并在温和的条件下对漆酶进行了固定。比较不同的固定化时间,pH值,温度和离子强度对固定化效果的影响,发现在12 h,40℃,pH值为3.2所制得的固定化酶的活力最高;在0.05~1.0 mol/L的范围内,随着作为固定化反应介质的缓冲溶液浓度的增加,所制得的固定化酶的活力有所下降。还发现固定化酶较游离酶的酶催化反应最适pH值有所升高。     

陶瓷-壳聚糖复合材料固定真菌漆酶酶学性质研究

以陶瓷为第一载体、壳聚糖为二次载体、戊二醛为交联剂,采用共价结合和吸附联用法制备固定化漆酶,并研究了固定化漆酶的性质.固定化酶最适pH为3.0,最适温度分别为25℃和50℃,均与游离酶相同.在pH 3.0,温度25℃时,固定化酶对ABTS的表观米氏常数为66.64 μmol/L.与游离酶相比,固定化酶的热稳定性明显提高,并具有良好的贮存和操作稳定性.     

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。     

固定化漆酶对二氯酚的脱氯作用

采用活性炭吸附与海藻酸钙凝胶包埋相结合的方法使Coriolus versicolor漆酶固定化.利用固定化漆酶对2,4-二氯酚进行脱氯反应,其最适pH值为4.5、最适温度为40℃.与游离酶相比,固定化酶反应的pH值和温度范围更宽,其稳定性得到了明显改善.使用柱式固定化酶反应器处理2,4-二氯酚,在批式反应工艺条件下,当底物浓度为1 mmol•L^-1、反应3～5 h, 2,4-二氯酚的去除率可达99.5%以上（脱除的氯离子浓度达0.5 mmol•L^-1）.连续8批反应的结果表明：固定化漆酶性能稳定、催化效率高,在环境污染废水治理方面具有良好的应用前景.     

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。     

SBA-15固定化漆酶及其稳定性研究

以大孔径介孔分子筛SBA-15为载体,用吸附法对漆酶进行固定化,以2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐为底物考察了漆酶的最适pH值以及固定化体系中给酶量对固定化漆酶活性及其稳定性的影响。结果表明,在pH值为3时,漆酶具有较好的活性;固定化漆酶的活性随着酶和载体质量比的升高先升高后降低,当酶和载体质量比为48 mg/g时,固定化漆酶的活性最高;与游离漆酶相比,固定化漆酶可以重复利用,经过8批次的反应后,固定化漆酶的剩余活性仍然达到85%以上,表现出良好的操作稳定性。     

微胶囊固定酶催化合成烷基糖苷的工艺优化

用微球截留固定化和酶催化有机合成技术对β-葡萄糖苷酶固定化及其催化合成烷基糖苷的工艺进行了研究.优化出酶同定化最优上艺为:壁材海藻酸钠4.0%、交联剂戊二醛0.2%、凝聚剂CaCl20.10 mol/L、酶用量140加mg.以固定酶为催化剂、葡萄糖和正丁醇为原料合成烷基糖苷,确定合成的最佳工艺为:正丁醇20 mL、糖醇比2.0:20(g/mL)、固定酶6 g、反应温度40℃,反应时间3 h.得到的固定酶活力比游离酶高,稳定性好,重复使用3次后仍有较好活性.     

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。     

整体型大孔/介孔PDA/SiO2复合材料固定化漆酶及其在染料降解中的应用

用硬模板法制得具有三维连续贯通孔道结构的整体型大孔/介孔SiO₂，通过多巴胺（DA）在大孔/介孔SiO₂孔道表面的原位氧化聚合，制得聚多巴胺（PDA）功能化修饰的整体型大孔/介孔复合材料（PDA/SiO₂）。应用SEM、BET、FTIR和TG等技术对修饰前后的材料进行表征。以PDA/SiO₂为载体固定诺维信工业级漆酶，系统研究了pH、固定化时间、漆酶初始浓度及温度对漆酶固定化的影响；以偶氮荧光桃红作为模拟污染物，研究了固定化漆酶对染料的催化降解性能。结果显示，在漆酶浓度为80mg/mL、pH为4.0、固定化时间为6h及固定化温度为25℃时，固定化漆酶酶活达到最高（348.9U/g）。在偶氮荧光桃红浓度为10mg/L、pH为7.0、温度为30℃、降解时间为8h时，固定化漆酶对偶氮荧光桃红脱色率 ≥ 99.9%，且固定化漆酶易从反应体系中分离，重复使用性能良好。     

Immobilization of Myceliophthora thermophila laccase on poly(glycidyl methacrylate) microspheres enhances the degradation of azinphos-methyl

Laccase enzymes are multicopper oxidases capable of oxidizing different compounds. However, to be able to use this biocatalyst for industrial applications, their immobilization is needed. The present work investigates the immobilization of Myceliophthora thermophila laccase (MtL) on monodisperse microspheres of poly(glycidyl methacrylate) (PGMA) to be used in azinphos-methyl degradation. The immobilization was optimized to achieve the highest activity of the immobilized enzyme. As result, the protein load obtained was 2.5 mg protein g⁻¹ carrier (35 U g⁻¹ of carrier). The immobilized enzymes showed a broadened pH and temperature range of optimum activity and significantly improved the storage and operational stability. Finally, the complete degradation of azinphos-methyl using immobilized laccase was achieved after 1 h of reaction. The collected data indicate that the immobilization of MtL on PGMA microspheres is an excellent alternative to improve biochemical properties in the enzyme and to allow their efficient use in pesticides degradation. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47417.     

Immobilization of laccase on carbon nanomaterials

Laccase from Trametes versicolor was readily immobilized on carbon nanomaterials including multiwalled carbon nanotubes (MWNTs), carboxylated multiwalled carbon nanotubes (MWNT-COOHs), and graphene oxides (GOs), by physical adsorption without using coupling agents. The immobilized amount of laccase strongly depends on the pH of the aqueous buffers of the immobilization mixture. As the pH of the aqueous buffer for immobilization increases, the immobilized amount of laccase decreases. The activity of the immobilized laccase on the three carbon nanomaterials exhibits a bell-shaped dependence on the pH of the immobilization solution with maximum activity at pH 6 or 7. When the immobilization solution becomes acidic or basic, the activity of the immobilized laccase declines significantly. The amount and the activity of immobilized laccase were maximum for graphene oxides as substrate material for immobilization.     

Immobilization of laccase from Myceliophthora thermophila on functionalized silica nanoparticles: Optimization and application in lindane degradation

This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003? laccase) on amino modified fumed nano-silica(AFNS) and the possible use in bioremediation. Hereby, for the first time, factors affecting the immobilization of Novozym 51003? laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization. The highest specific activity(13.1 IU·mg~(-1) proteins) was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield, reaching68.3% after the equilibrium time, at optimum pH 5.0, was obtained. Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min, following pseudo-first-order kinetics. The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane. Within 24 h, lindane concentration was reduced to 56.8% of initial concentration and after seven repeated reuses it retained 70% of the original activity.     

Fabrication of hydrophilic nanoporous PMMA/O-MMT composite microfibrous membrane and its use in enzyme immobilization

The electrospun PMMA/O-MMT microfibrous membranes were pretreated by oxygen plasma to create substrates with better adsorption capability. The amount of O-MMT and conditions of plasma treatment were optimized for maximum laccase immobilization on these pretreated surfaces of the microfibrous membranes. The surface morphology and chemistry of the composite microfibrous membranes after plasma treatment and laccase immobilization were investigated by SEM and FTIR. The immobilized laccase showed better resistance to pH and temperature changes than that of the free form laccase, and after 10 successive runs of repeated use, the immobilized laccase still retained 30 % of its initial activity. Reactive X-3B was successfully degraded by both free and immobilized laccase.     

Immobilization of Pycnoporus sanguineus laccase by metal affinity adsorption on magnetic chelator particles

BACKGROUND: Immobilized enzymes provide many advantages over free enzymes including repeated or continuous reuse, easy separation of the product from reaction media, easy recovery of the enzyme, and improvement in enzyme stability. In order to improve catalytic activity of laccase and increase its industrial application, there is great interest in developing novel technologies on laccase immobilization. RESULTS: Magnetic Cu²⁺‐chelated particles, prepared by cerium‐initiated graft polymerization of tentacle‐type polymer chains with iminodiacetic acid (IDA) as chelating ligand, were employed for Pycnoporus sanguineus laccase immobilization. The particles showed an obvious high adsorption capacity of laccase (94.1 mg g^?1 support) with an activity recovery of 68.0% after immobilization. The laccase exhibited improved stability in reaction conditions over a broad temperature range between 45 °C and 70 °C and an optimal pH value of 3.0 after being adsorbed on the magnetic metal‐chelated particles. The value of the Michaelis constant (K_m) of the immobilized laccase (1.597 mmol L^?1) was higher than that of the free one (0.761 mmol L^?1), whereas the maximum velocity (V_max) was lower for the adsorbed laccase. Storage stability and temperature endurance of the immobilized laccase were found to increase greatly, and the immobilized laccase retained 87.8% of its initial activity after 10 successive batch reactions. CONCLUSION: The immobilized laccase not only can be operated magnetically, but also exhibits remarkably improved catalytic capacity and stability properties for various parameters, such as pH, temperature, reuse, and storage time, which can provide economic advantages for large‐scale biotechnological applications of laccase. Copyright © 2007 Society of Chemical Industry     

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.