胺化聚苯乙烯载体柔性固定化木瓜蛋白酶

提出酶的“柔性固定化”模型,并以Mannich反应得到担载量为0.4～6.0 mmol NH₂•g^-1的胺化聚苯乙烯树脂为载体,以双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化,酶活回收率可达40%～50％,相当于手臂固定化酶活力回收率的1.8～2.4倍,且柔性固定化酶稳定性较好.该结果说明,酶的“柔性固定化”模型可以改善传统共价结合固定化酶及手臂固定化酶活力回收率不高的缺陷.     

Efficient Biocatalytic Cleavage and Recovery of Organic Substrates Supported on Soluble Polymers

The applicability of novel solution‐phase supports in combination with enzymes for biocatalytic transformations is reported. Ex novo designed styrene‐based copolymers, bearing a phenylacetic residue in variable loadings and linked as a pendant group to the macromolecular backbone, through a spacer of variable length, have been synthesized and characterized. These derivatives are compatible and can be used as soluble supports in combination with immobilized penicillin G acylase (PGA – EC 3.5.1.11) for the biocatalytic cleavage of the covalently anchored organic substrate in quantitative yields, in water or water/dimethylformamide solvent mixtures, with recovery of the immobilized enzyme with negligible losses in activity.     

Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N₂ adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. ²⁷Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N₂ adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. K _m was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.     

Immobilization of catalase on a novel polymer support,crosslinked polystyrene ethylene glycol acrylate resin: Role of the macromolecular matrix on enzyme activity

Crosslinked polystyrene ethylene glycol acrylate resin (CLPSER) was developed for the immobilization of the enzyme catalase by the introduction of a crosslinker, O,O′‐bis(2‐acrylamidopropyl) poly(ethylene glycol)₁₉₀₀, to styrene. The crosslinker was prepared by the treatment of acryloyl chloride with O,O′‐bis(2‐aminopropyl) poly(ethylene glycol)₁₉₀₀ in the presence of diisopropylethylamine. The resin was characterized with IR and ¹³C‐NMR spectroscopy. The catalytic activity of the catalase‐immobilized system of CLPSER was compared with divinylbenzene‐crosslinked polystyrene, ethylene glycol dimethacrylate crosslinked polystyrene, and 1,4‐butanediol dimethacrylate crosslinked polystyrene systems. Crosslink levels of 2, 8, and 20 mol % were evaluated. Among these crosslinked systems, the 2 mol % system was found to be most suitable to support catalytic activity. When a long flexible hydrophilic poly(ethylene glycol) crosslink, introduced between the polystyrene (PS) backbone and functional groups was used for immobilization, the extent of coupling and enzyme activity increased. Depending on the nature of the support, the catalytic activity of the system varied. The hydrophilic CLPSER support was most efficient for immobilization compared to the other PS‐based supports. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 8–19, 2005     

Covalent immobilization of porcine pancreatic lipase on amorphous AlPO4 and other inorganic supports

Porcine pancreatic lipase (PPL) was covalently immobilized through the formation of an aromatic Schiff's-base obtained by reaction of the ε-amino group of lysine residues in the enzyme and the aromatic aldehyde function of activated supports. The enzymatic activities of different immobilized PPL systems on amorphous AlPO₄ and several inorganic supports at different pH values and temperatures were determined by hydrolysis of ethyl acetate. The nature of the support material critically affects the efficiency of enzyme immobilization as well as enzyme stability. All the supports examined, with the exceptions of cellulose and natural sepiolite, exhibited good properties for enzyme attachment. AlPO₄ was the best support for enzyme immobilization, giving the highest percentage binding of enzyme (90%) and the highest retention of enzyme activity after immobilization (92·8%). © 1998 SCI     

Immobilization of active α‐chymotrypsin on RF‐plasma‐functionalized polymer surfaces

Various polymeric surfaces (polyester, polyethylene, polystyrene) were functionalized under oxygen and dichlorosilane‐RF‐cold‐plasma environments and were employed as substrates for further in situ derivatization reactions and immobilization of α‐Chymotrypsin. The nature and morphology of the derivatized substrates and the substrates with immobilized enzymes were analyzed using survey and high‐resolution X‐ray photoelectron spectroscopy, attenuated total reflectance‐fourier transform infrared (ATR‐FTIR), laser desorption fourier transform ion cyclotron resonance mass spectrometry, chemical derivatization, and atomic force microscopy (AFM) techniques. It was demonstrated that the tacticity of the polystyrene substrate did not notably influence the activity of the immobilized enzyme, however, spacer molecules intercalated between the polymeric substrates (e.g., polyethylene) and the enzyme significantly increased the enzyme activity (comparable with that of the free enzyme). Computer‐aided conformational modeling of the substrate‐spacer systems indicated that the longer the spacer chain, the greater the mobility of the enzyme. It is suggested that the greater mobility of the enzyme molecules is responsible for the enhanced activity. It has also been shown that the stability of the immobilized enzyme systems was good; they retained their activity during several washing/assay cycles. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 1783–1796, 2000     

Study on the activity and stability of urease immobilized onto nanoporous alumina membranes

Nanoporous alumina membranes were employed as substrate materials for urease immobilization. Anodic porous alumina was prepared by the two-step anodization of high purity aluminum. By controlling anodization conditions, the nanoporous structure with desired dimension was obtained. Urease immobilization onto nanoporous alumina membranes was performed by four different protocols. Effect of pore diameter, pore length and immobilization methods on the activity and stability of immobilized enzyme was discussed in detail. The results show that the enzymes immobilized onto porous alumina with big pore diameter possess high activity and poor stability as compared to small pore diameter. The effect of pore length is complicated, the activity of enzyme increases with the increasing pore length for big pore size; while for correspondingly small pore size, enzymatic activity slightly depends on pore length. The immobilization methods have a slight effect on enzymatic activity, whereas enzyme immobilization by chitosan coating and reticulation with glutaraldehyde exhibits a good long-term stability as compared to that only via physical adsorption.     

壳聚糖载体柔性固定化木瓜蛋白酶

用酶柔性固定化模型,以壳聚糖为载体,双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化. 通过对固定化条件的优化,得出选用壳聚糖、双醛淀粉制得的柔性载体(Chitosan-DAS50)在酶用量为14.4 mg/g(酶/干球)、pH 8的条件下,固定木瓜蛋白酶18 h,所得的固定化酶活力回收率达72%,相当于采用壳聚糖-戊二醛(Chitosan-GA)手臂载体的3倍. 结果表明,酶的柔性固定化模型可以改善传统共价结合法固定化及手臂固定化酶活力回收率不高的缺陷.     

Immobilization of enzymes to porous-bead polymers and silica gels activated by graft polymerization of 2,3-epoxypropyl methacrylate

Three types of organic polymers and bead-shape silica gels were activated by graft polymerization of 2,3-epoxypropyl methacrylate; in some cases, epoxide groups on the support surface were modified to NH₂ groups. Eight active matrices so obtained were assessed as supports for immobilized enzymes using peroxidase, glucoamylase and urease. The immobilization yield of protein and specific activities of enzymes were better with supports containing NH₂ groups than with those containing epoxide spacer arms. Maximum enzyme immobilization and storage stabilities were obtained with silica-gel beads activated by graft polymerization of 2,3-epoxypropyl methacrylate. With all eight matrices tested, the immobilized enzymes showed good stability with not less than 82% of the original activity persisting after 28 days. The developed matrices have potential for use in process-scale biotechnological operations.     

Invertase immobilized on spacer‐arm attached poly(hydroxyethyl methacrylate) membrane: Preparation and properties

Microporous poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane was prepared by UV‐initiated photopolymerization. The spacer arm (i.e., hexamethylene diamine) was attached covalently and then invertase was immobilized by the condensation reaction of the amino groups of the spacer arm with carboxyl groups of the enzyme in the presence of carbodiimides. The values of the Michael's constant K_m of invertase were significantly larger (ca. 2.5 times) upon immobilization, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the immobilized invertase. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 70°C the half times for the activity decay were 12 min for the free enzyme and 41 min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1685–1692, 2000     

Optimization of covalently coupling enzymes to polymeric membranes: EPR studies of papain

In an effort to better understand bioreactor systems, papain (EC 3.4.22.2) was covalently immobilized onto vinyl alcohol/vinyl butyral copolymer (PMB) membrane by means of glutaraldehyde (GA), 1,1'-carbonyldiimidazole (CDI), or 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP). Various kinetic and performance characteristics of the immobilized papain were evaluated. It was found that the characteristics of the membranebound papain depended on the immobilization methods. The CDI- and FMP-immobilized papain bioreactors showed better storage and thermal stability than did the GA-immobilized papain bioreactor, although the apparent Michaelis constant, K_m, of the GA-immobilized papain was closer to the free enzyme than to the corresponding CDI- and FMP-immobilized enzymes. In separate experiments, a 6-carbon spacer was inserted between the membrane surface and the covalently bound enzyme. It was found that the insertion of a spacer reduced the disturbance of the enzyme systems, resulting in K_m values intermediate between the free and directly bound enzymes for all three immobilization methods. Electron paramagnetic resonance spectroscopy was also used to investigate the conformational change and the active site structure of papain. It was found that the active site SH group of papain immobilized with a 6-carbon spacer had faster motion than that of directly bound enzyme, but slower motion than that of the free enzyme. With both direct-coupling and with a spacer, the SH group motion at the active site of papain by CDI and FMP immobilizations was similar, but slower than the corresponding GA immobilization. The conformational changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme. There was a good correlation between the motion of spin-labeled cysteine in the active site of papain and kinetic properties of this protease: As motion slowed, K_m increased and V_max decreased. Of the immobilization procedures used, GA immobilization with a spacer yielded kinetic and structural characteristics most similar to the free enzyme while providing increased stability and reusability relative to the latter. © 1993 John Wiley & Sons, Inc.     

Improvement in the Cleaning Performance Towards Protein Soils in Laundry Detergents by Protease Immobilization on the Silica Nanoparticles

This work aimed to understand the effect of protease immobilization on silica nanoparticles and how such immobilization affects protease performance as catalysis for enhancing the removal of protein soils. Detergent products contain many components that may affect the free enzyme activity and stability. Various factors such as temperature, pH and humidity are know to affect enzyme activity and cleaning efficiency. Therefore, the effect of enzyme immobilization on the removal of protein based soil was investigated on cotton fabrics as the model soil. The effect of temperature and humidity on the stability of free and immobilized enzyme was compared. It was found that the immobilized enzyme increased cleaning efficiency toward protein soil removal on cotton fabrics, whereas the free enzyme imposed a small effect on the enzymatic activity towards the same soil substrates. In addition, the stability of the immobilized enzyme against temperature and humidity was much higher than its corresponding value by free enzyme.     

Immobilization of DNA on poly(glycidyl methacrylate-co-ethylene dimethacrylate), bead cellulose and sepharose

Summary The copolymer of glycidylmethacrylate with ethylene-dimethacrylate, its derivatives, bead cellulose and Sepharose were used for immobilization of DNA via new coupling methods: reaction with oxirane, acylhydrazide hydrazide, and diazonium salt. The susceptibility of immobilized DNA for DNAse was tested. The most efficient immobilization was achieved on carriers with acylhydrazide groups. The binding capacity of the carrier and the susceptibility of immobilized DNA for the DNase depends on the length of the spacer. The immobilized DNA is stable in solution for several weeks, and in the lyophilized state for several months.     

Enzymic synthesis of fatty esters by hydrophobic lipase derivatives immobilized on organic polymer beads

Lipase fromCandida rugosa was modified with several hydrophobic modifiers before being adsorbed onto organic polymer beads. The effects of different enzyme modifiers, supports, solvents, reaction temperatures, fatty acids, and alcohols on the activity of the immobilized enzyme were investigated. The immobilized lipases were good biocatalysts for esterification reactions in organic solvents. They exhibited high activities in all solvents tested, including polar solvents. The activity seemed to depend on the type of support rather than on the modifier of the enzyme. The medium polar support, XAD7, appeared to be the best for the modified lipases. The immobilized lipase favored the medium-chain fatty acids rather than the long-chain fatty acids as acyl donors. The alcohol selectivity of the enzyme was unchanged upon immobilization. The native and immobilized lipases favored the short-chain and terpene alcohols as nucleophiles.     

Cinnamic ester of D‐sorbitol for immobilization of mushroom tyrosinase

Mushroom tyrosinase was immobilized by adsorption onto the totally cinnamoylated derivative of D ‐sorbitol. The polymerization and cross‐linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of tyrosinase on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. The pH value, enzyme concentration and immobilization time were all important parameters affecting immobilization efficiency; also, enzyme immobilization efficiency correlated well with the tyrosinase isoelectric point. The immobilized enzyme showed an optimum measuring pH of 3.5 and greater activity at acid and neutral pH values than the soluble enzyme. The optimal reaction temperature was 35 °C and the temperature profile was broader than that of the free enzyme or of the enzyme immobilized on other supports. The apparent Michaelis constant of mushroom tyrosinase immobilized on the SOTCN derivative acting on 4‐tert‐butylcatechol (TBC) was 0.40 ± 0.02 mmol dm^?3, which was lower than for the soluble enzyme, suggesting that the affinity of this enzyme for this substrate was greater when immobilized than when in solution. Immobilization stabilized the enzyme and made it less susceptible to activity loss during storage at pH values in the range 4–5.5, and the suicide inactivation of the immobilized tyrosinase was null or negligible in a reaction medium with 4‐tert‐butylcatechol at a concentration of 0.4 mmol dm^?3. The results show that cinnamic carbohydrate esters of D ‐sorbitol are an appropriate support for tyrosinase immobilization and could be of use for several tyrosinase applications. Copyright © 2005 Society of Chemical Industry     

Acid activated montmorillonite: an efficient immobilization support for improving reusability,storage stability and operational stability of enzymes

Three enzymes, α-amylase, glucoamylase and invertase, were immobilized on acid activated montmorillonite K 10 via two independent techniques, adsorption and covalent binding. The immobilized enzymes were characterized by XRD, N₂ adsorption measurements and ²⁷Al MAS-NMR spectroscopy. The XRD patterns showed that all enzymes were intercalated into the clay inter-layer space. The entire protein backbone was situated at the periphery of the clay matrix. Intercalation occurred through the side chains of the amino acid residues. A decrease in surface area and pore volume upon immobilization supported this observation. The extent of intercalation was greater for the covalently bound systems. NMR data showed that tetrahedral Al species were involved during enzyme adsorption whereas octahedral Al was involved during covalent binding. The immobilized enzymes demonstrated enhanced storage stability. While the free enzymes lost all activity within a period of 10 days, the immobilized forms retained appreciable activity even after 30 days of storage. Reusability also improved upon immobilization. Here again, covalently bound enzymes exhibited better characteristics than their adsorbed counterparts. The immobilized enzymes could be successfully used continuously in the packed bed reactor for about 96 hours without much loss in activity. Immobilized glucoamylase demonstrated the best results.     

触角状环氧链结构对固定化脂肪酶(YCJ01)酶活的影响

为了改善酶的刚性固定化对酶蛋白构象的负面影响,以原子转移自由基聚合法（ATRP）合成了亲水性、柔性、触角状环氧化酶固定化载体PS-acyl-P（AM-co-GMA）,通过改变单体总量与引发剂量比例得到不同链长触角状环氧化酶固定化载体,并将其用于耐有机溶剂脂肪酶（YCJ01）的共价柔性固定化,重点考察了链长对固定化酶酶活的影响。结果表明,链长（增重率不超过3200%）越长,酶活越高。     

Activity of immobilized lipoxygenase used for the formation of perhydroxyacids

Lipoxygenase-produced oxidized fatty acids may serve as intermediates in chemical and pharmaceutical syntheses. For practical applications the enzyme should be immobilized to prevent its loss at product retrieval and to enable a continuous process. In this study the immobilization of soybean lipoxygenase on various neutral and charged supports was investigated. The best results were obtained using cellulose-based anion exchangers. The activity of the immobilized lipoxygenase was lower than the activity of the free enzyme, which was possibly caused by diffusion limitation of the fatty acids to or from the support beads.     

Modulation of the Selectivity of Immobilized Lipases by Chemical and Physical Modifications: Release of Omega-3 Fatty Acids from Fish Oil

Three different lipases (from Candida antarctica fraction B (CALB), Thermomyces lanuginose (TLL), and Rhizomucor miehei (RML)) were immobilized by two different methods, immobilization on CNBr-activated Sepharose via a mild covalent immobilization or adsorption onto hydrophobic supports (Octyl-Sepharose). These immobilized preparations were chemically and physically modified on the protein surface (enzyme carboxylic groups with ethylenediamine, amino groups with succinic anhydride, or coating with polyethyleneimine).The activity and selectivity in the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by enzymatic hydrolysis of sardine oil were evaluated. Activity and selectivity were dependent on the different lipases, the immobilization protocols, the modification methods, and the pH of the reaction media. The selectivity (EPA/DHA ratio) of RML immobilized on CNBr-activated Sepharose was increased after succinylation from 7.5 to 34 at pH 6.0. The selectivity of octyl-RML improved from 1.5 to 8.5 when pH was increased from 6 to 8. The selectivity and activity of octyl-TLL increased twofold after PEI coating at pH 6. The properties of CAL-B derivatives were slightly altered after modification.     

Evaluation of nanogels as supports for enzyme immobilization

Nanogels are hydrophilic polymers made up of crosslinked nano‐sized particles. These nanogels have large surface area that offers several functional groups as reserves for binding drugs, generating biosensors and as supports for enzyme immobilization. This mini‐review is an attempt to evaluate the recent developments in the use of nanogels as supports in enzyme immobilization. Emphasis is laid on the effect of nanogel structure and immobilization protocol on the property profile of the immobilized enzymes as compared with their free counterparts. The prospective applications of the nanogel‐immobilized enzymes are also evaluated. © 2014 Society of Chemical Industry