Immobilization of Pseudomonas delafieldii with magnetic polyvinyl alcohol beads and its application in biodesulfurization

Pseudomonas delafieldii was immobilized in magnetic polyvinyl alcohol (PVA) beads using a hydrophilic magnetic fluid, which was prepared by a co-precipitation method. The beads had distinct super-paramagnetic properties and were compared with immobilized cells in non-magnetic PVA beads. Their desulfurizing activity was increased slightly from 8.7 to 9 mmol sulfur kg(-1) (dry cell) h(-1). The main advantages was that the magnetic immobilized cells maintain a high desulfurization activity and remain in good shape after 7 times of repeated use, while the non-magnetic immobilized cells could only be used for 5 times. Furthermore, the magnetic immobilized cells could be easily collected or separated magnetically from the biodesulfurization reactor.     

Immobilization of porcine pancreatic lipase on glycidyl methacrylate grafted poly vinyl alcohol

Graft copolymerization of glycidyl methacrylate (GMA) on to polyvinyl alcohol (PVA) using benzophenone (BP) as initiator was carried out. Grafted PVA was used as carrier for pancreatic lipase immobilization. The effects of GMA and BP concentrations as well as grafting reaction times on grafting yields and activities of the immobilized lipase were determined. The influence of enzyme concentrations was also studied. The optimal conditions for the grafting reaction were: 1 h at 15 mM BP and 2.3 M GMA, the optimum enzyme concentration for immobilization was 1 mg/ml. After optimization of the immobilization process a physical and chemical characterization of the immobilized enzyme was performed. Furthermore, the thermal, pH, storage and operational stability of the immobilized enzyme in comparison to the free form was tested.     

Physical crosslinking of lipase from Rhizomucor miehei immobilized on octyl agarose via coating with ionic polymers: Avoiding enzyme release from the support

Lipase from Rhizomucor miehei (RML) was immobilized on octyl-agarose (OC) at different loadings. Using low enzyme loadings (1/7 of the maximum loading), the incubation of the enzyme with polyethylenimine (PEI) increased the resistance to enzyme desorption in the presence of Triton X-100. However, more than 10% of the enzyme activity could be released from the OC-RML-PEI. The same treatment using fully loaded biocatalyst reduced the enzyme desorption to less than 5%. Further treatment with dextran sulfate (DS) of the PEI treaded immobilized enzyme fully avoids the enzyme desorption even in presence of a Triton X-100 concentration higher than that required for the complete enzyme release from OC-RML. This treatment produced a high stabilization of OC-RML in thermal or organic solvent inactivations, reducing the enzyme release under these drastic conditions. Nevertheless, the support could be recovered by incubation under adequate conditions, and reused in several adsorption/desorption cycles. Thus, the strategy permitted to avoid enzyme desorption, very likely by physical intermolecular crosslinking improving enzyme stability, while still maintaining the reversibility of the immobilization.     

聚乙烯醇脱氢酶的提取及检测

为解决结合在细胞上的可溶性蛋白聚乙烯醇脱氢酶（PVADH）的检测困难问题,从提取及检测两方面对该酶进行研究,并对检测方法进行改进。结果表明,非离子型表面活性剂Triton X-100对可溶性蛋白PVADH的提取效果优于离子型表面活性剂炕基苯磺酸钠（LAS）和溴化十六烷基吡啶（CPB）,酶活力比LAS和CPB提取后所得酶活力分别提高246．5％和831．3％。而非离子型表面活性剂中,Triton X-100与Tween80相比,所得最高酶活提高了101．4％。Triton X—100浓度和提取时间对测定有明显影响,以1％Triton X-100提取18h为宜,最高比酶活达14．9U／g。在PVADH检测体系中,加入电子受体启动反应比加入酶液与底物启动反应可使酶活性分别提高60．6％和126．5％;酶液与吡咯喹啉醌（PQQ）预先保温对检测该酶活性是十分重要的,可使酶活性提高59．1％.在检测系统中加入的KCN、CaCl2和PQQ的适宜浓度分别为1．ommol／L、0．5mmol／L和2μmol／L,可使测定酶活分别提高37．1％、38．7％和214．0％．     

Environmentally benign beta-stereoselective glycosidations of glycosyl phosphites using a reusable heterogeneous solid acid, montmorillonite K-10

Environmentally benign and stereoselective beta-glycosidations of glycopyranosyl phosphites and alcohols using a reusable heterogeneous solid acid, montmorillonite K-10, as an activator have been developed. By these glycosidations, beta-gluco-, 2-deoxy-beta-gluco-, and beta-mannopyranosides were selectively produced in good to high yields.     

Polyhydric alcohol protective effect on Rhizomucor miehei lipase deactivation enhanced by pressure and temperature treatment

The influence of polyhydric alcohols (sorbitol, xylitol, erythritol, glycerol) on the thermal stability of Rhizomucor miehei lipase has been studied at high hydrostatic pressure (up to 500 MPa). In the absence of additives, a protective effect (PE) (the ratio between the residual activities determined at 480 MPa for the enzyme in the presence or absence of polyhydric alcohols) of low-applied pressures (from 50 MPa to 350 MPa) against thermal deactivations (at 50°C and 55°C) has been noticed. In the presence of additives, a strong correlation between PE and the total hydroxyl group concentration has been obtained, for the first time, under treatments of combining denaturing temperatures and high hydrostatic pressures. This relationship does not seem to be dependent on the nature polyhydric alcohols as the same effect could be observed with 1 M sorbitol and 2 M glycerol. This PE, against thermal and high pressure combined lipase deactivation, increases with polyhydric alcohol concentrations, and when temperature increases from 25°C to 55°C.     

Environmentally benign and stereoselective formation of beta-mannosidic linkages utilizing 2,3-di-O-benzyl-4,6-O-benzylidene-protected mannopyranosyl phosphite and montmorillonite K-10

An environmentally benign and stereoselective beta-mannopyranosylation has been developed employing 4,6-O-benzylidene-protected mannopyranosyl diethyl phosphite as a glycosyl donor and montmorillonite K-10 as an activator.     

蜡样芽孢杆菌聚乙烯醇脱氢酶的表达及酶学特性

【背景】聚乙烯醇脱氢酶(polyvinyl alcohol dehydrogenase,PVADH)能够使聚乙烯醇(polyvinyl alcohol,PVA)氧化脱氢,在PVA的生物降解过程中起到重要作用。【目的】从PVA降解菌株蜡样芽孢杆菌DG01中获取pvadh基因,实现PVADH在毕赤酵母中的异源表达并探究其对不同型号PVA的降解特异性,为PVADH在PVA实际降解中的应用提供指导。【方法】通过反转录扩增技术获得长度为1 965 bp的pvadh基因片段,构建pPIC9K-cpvadh重组表达质粒并在毕赤酵母GS115中实现异源表达,甲醇诱导表达蛋白,进行分离纯化后对其酶学性质及降解特异性进行研究。【结果】最佳发酵条件下PVADH粗酶液酶活达到54.55 U/mL。经分离纯化后表达蛋白PVADH的比酶活为173.42 U/mg,分子量为67.1 kDa,等电点为6.06,该酶最适作用温度为41℃,最适作用pH值为7.5,在27-32℃、pH 7.0-8.0条件下酶的半衰期超过4 h,1 mmol/L的Ca2+对酶活力有激活作用。PVADH分别作用于PVA1788、PVA1799...     

Immobilization and activity of Rhizomucor miehei lipase. Effect of the matrix properties prepared from nonionic fluorinated surfactants

We report the immobilization of Rhizomucor miehei lipase (RmL) onto mesoporous silica materials, in particular the investigations concerning the effects of the level of silica condensation and of the pore size on the enzyme activity. The efficiency of the immobilization was revealed by FTIR spectroscopy. Infrared was also used to determine the quantity of adsorbed enzyme. Immobilization efficiency increased when the RmL concentration in the buffer solution was changed from 2 to 10 mg/mL. Nevertheless, while upon enzyme immobilization the mesopore ordering was sustained for the support recovered after hydrothermal treatment at 100 °C, a structure collapse occurred for the one prepared at 80 °C. The difference in behavior is attributed to the lower hydrothermal stability of this material, which reflects the lower level of silica condensation. The enzyme-containing mesostructured silica was effectively used to catalyze the model esterification reaction of lauric acid with 1-propanol, as the immobilized lipase retained its catalytic activity. A linear relationship was observed between the reaction rate and the amount of catalyst. RmL immobilized on mesoporous materials presented a satisfactory reusability, while the remaining activity of RmL after 4 months of storage was 47% of the initial one.     

海藻酸-SiO2杂化凝胶固定化洋葱伯克霍尔德茵脂肪酶的研究

利用四乙氧基硅烷（TEOS）原位水解法将SiO2掺杂于海藻酸（ALG）凝胶中,通过双交联制备出新型ALG—SiO2杂化凝胶以固定化洋葱伯克霍尔德菌脂肪酶。结果表明,固定化酶的最优条件：质量分数为2．0％的ALG、0．2mol／LCaCl2、V（ALG）／V（TEOS）为5、加酶量为1gALG加100mg酶粉、固定化60min、采用直径为0．8mm的针头滴定、真空冷冻干燥。在此条件下,酶蛋白的包埋率可达100％,酶活回收率可达91％。固定化酶的最适pH为8．0,最适作用温度为50℃,重复使用8次后,酶活性仍能保持80％以上。ALG—Si02杂化凝胶的场扫描电镜（FESEM）观察发现凝胶的整体构造仍然是海藻酸凝胶骨架;与ALG凝胶平滑的内部相比较,杂化凝胶仍具有完整的网络结构,但内部更为粗糙,结构更为致密。     

磁性纳米颗粒固定化乙醇脱氢酶的研究

本研究采用3-丙氨基三乙氧基硅烷(APTES)和戊二醛修饰包裹有SiO2磁性Fe3O4纳米颗粒表面,将其作为固定化载体固定化乙醇脱氢酶,研究固定化条件对固定化效率的影响,并对固定化酶性质进行分析。研究发现,当Fe3O4@SiO2纳米颗粒修饰上氨基和醛基后依然具有良好的水分散性和胶体稳定性,适合作为固定化载体。通过单因素优化,发现当最适给酶量为11. 3U/100 mg,搅拌转速为150 r/min,固定化p H和固定化温度分别控制在6. 5和5℃～15℃,固定化时长为45 min时,具有较好的固定化效果,固定化率可达到60. 2%。在此条件下制备得到的固定化酶与游离酶相比,固定化酶具有良好的耐高温和耐碱性。所得固定化乙醇脱氢酶在连续使用8次后,固定化率仍保留在57%左右,表明该固定化酶具有较好的操作稳定性,可为连续生产NADH提供技术依据。     

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.     

Synthesis of N-Octyl Oleate with Lipase from Mucor miehei Immobilized onto Polyethylene Based Graft Copolymers

Lpase from Mucor miehei was immobilized onto partially hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE/HEMA) via spacer arms of 1,6-diaminohexane and glutaraldehyde-. The PE/HEMA-lipase system was used for the enzymatic esterification of n-octanol with oleic acid in the absence of organic solvents. The influence of lipase' concentration, in the attachment solution, on the ester production profile and initial reaction rate was studied. It was found that very small amounts of lipase gave preparations which reached good degrees of conversion. The effect of the initial oleic acid concentration on that pseudo-first order reaction, as well as the presence of water in the reactional medium and the influence of temperature were evaluated. It was found that initial oleic acid concentrations lesser than 1.2 M did not inhibit the immobilized lipase activity; the presence of small amount of water (10–30μ) solubilized in the reaction mixture (6.5 cm³) increased the lipase activity and a maximum of activity of the immobilized lipase preparation was found at 55d`C. The operational stability of the preparation was determined at 37d`C in a BSTR type reactor and a half-life time of three days for the immobilized lipase was obtained.     

Variables that Affect Immobilization of Mucor Miehei Lipase on Nylon Membrane

Nylon membrane was used to immobilize Mucor miehei lipase. Variables that affect this immobilization procedure were studied by experimental design. A 2³ full factorial design was employed for this purpose. The protein retention and hydrolytic activity of the immobilized lipase were used as response variables. The rapid loss of enzyme activity was the main problem during repetitive use. Two strategies were used to improve the low operational stability: nylon treated with HCl and nylon coated with polyvinyl alcohol (PVA). Lipase-nylon-PVA was the best enzyme derivative, allowing performance of five consecutive assays, with a retained activity of 0.5 U mg of protein⁻¹ g of support⁻¹.     

Biocatalytic properties of thermolysin immobilized on polyvinyl alcohol cryogel

Preparations with different contents of thermolysin were obtained by the immobilization of the enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mixtures of polar organic solvents of different composition were investigated. The catalytic properties of the preparations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the concentrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic peptide synthesis catalyzed by immobilized thermolysin were determined. A series of peptides of the general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized enzyme was shown to retain substrate specificity in an organic medium.     

Immobilization of Rhizomucor miehei lipase on a polymeric film for synthesis of important fatty acid esters: kinetics and application studies

Bioprocess and Biosystems Engineering - The present work deals with the designing of biocompatible hybrid blend of cellulosic copolymers made of hydroxypropyl methylcellulose (HMC) and chitosan...     

Immobilization of Alcaligenes eutrophus using PVA crosslinked with sodium nitrate

A new cell immobilization technique using polyvinyl alcohol crosslinked with sodium nitrate was developed. This new technique can simultaneously eliminate the agglomeration of PVA beads and the toxicity of boric acid caused by the PVA-boric acid and PVA-orthophosphate methods. Alcaligenes eutrophus was immobilized using four different PVA entrapment processes. The stability, swelling, relative mechanical strength and denitrification activity of the PVA beads were compared in this study. © Rapid Science Ltd. 1998     

Stabilization of polyvinyl alcohol coating of polyester cloth for reduction of bacterial adhesion

Bacterial adhesion onto polyester cloth was greatly reduced by coating the cloth with polyvinyl alcohol (PVA). Under autoclaving conditions, the PVA coating was stable in water but not in 1% SDS. However, the treatment of the PVA-coated cloth with acidic 1% glutaraldehyde stabilized the coating to retain full resistance to bacterial adhesion even after autoclaving in 1% SDS.     

Improvement of the stability and selectivity of Rhizomucor miehei lipase immobilized on silica nanoparticles: Selective hydrolysis of fish oil using immobilized preparations

We report the effect of random and oriented immobilization of Rhizomucor miehei lipase (RML) on its functional properties. For this purpose, silica nanoparticles (MCM-41 and SBA-15) were prepared, characterized and functionalized by glycidyloxypropyl trimethoxysilane. Direct immobilization of RML on these supports was performed via the variety of amino acid residues on the surface of RML which promotes random immobilization. To perform oriented immobilization, partial modification of epoxy functionalized supports was carried out by introducing iminodiacetic acid groups followed by addition of Cu²⁺. In this way, immobilization is mainly directed via the most accessible histidine group, followed by intramolecular reaction of the other nucleophilic residues of the enzyme and the remaining epoxy groups on the support. The results showed higher thermal stability for immobilized derivatives compared to the soluble enzyme. Co-solvent stability of the derivatives was also studied in presence of six polar organic solvents (DMSO, THF, acetonitrile, 1-propanol, 2-propanol and dioxane). Influence of the immobilization procedure on activity and selectivity of the immobilized preparations was studied in selective hydrolysis of fish oil. All the derivatives discriminate between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. Remarkable improvement in selectivity was obtained using oriented immobilization of RML.     

Synthesis of tolerogenic monomethoxypolyethylene glycol and polyvinyl alcohol conjugates of peptides

Recent studies from this laboratory showed that tolerogenic peptide conjugates are very effective reagents for obtaining epitope-specific immunosuppression of antibody responses to immunopathogenic sites on multideterminant complex protein antigens. This paper describes the procedure for synthesis of well-defined conjugates of peptides to monomethoxypoly-ethylene glycol (mPEG) or to polyvinyl alcohol (PVA). The first step involves succinylation of the hydroxyl groups on the polymers by reaction with succinic anhydride. The polymer is then coupled via the carboxyl of the succinyl group to the -NH₂ of the completed peptide on the synthetic resin, while maintaining intact all the side-chain protecting groups on the peptide. The mPEG or PVA-peptide conjugates are cleaved from the resin and purified by standard procedures. This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is coupled to the N-terminal of an otherwise unaltered peptide molecule.