Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

Substrate complexation and aggregation influence the cyclodextrin glycosyltransferase (CGTase) catalyzed synthesis of alkyl glycosides

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) was used to convert dodecyl-β-maltoside (DDM) to dodecyl-β-maltooctaoside (DDMO) using α-cyclodextrin (α-CD) or starch as glycosyl donors. At 300 mM α-CD, varied DDM concentration and 60 °C, the reaction obeyed Michaelis-Menten kinetics with a K_m value of 18 mM and a V_max value of 100 U/mg enzyme. However, at 25 mM α-CD the reaction rate decreased with increasing DDM concentration (5-50 mM), and when the α-CD concentration was varied at fixed DDM concentration an S shaped curve was obtained. The deviations from Michaelis-Menten kinetics were interpreted as being caused by formation of inclusion complexes between α-CD and DDM and by micellation of DDM. To achieve a high reaction rate, a high concentration of free α-CD is necessary, since α-CD in the form of a complex has low reactivity. When starch is used as glycosyl donor in the CGTase catalyzed alkyl glycoside elongation reaction, it is thus important to choose reaction conditions under which the cyclization of starch to α-CD is efficient.     

Covalent immobilization of triacylglycerol lipase onto functionalized novel mesoporous silica supports

A novel mesoporous silica material was synthesized via a silicate salt route in the presence of polyvinyl alcohol as the structure-directing agent under acidic conditions. The material was functionalized and employed as the supports (LPS-1 and LPS-2) for immobilizing triacylglycerol lipase from porcine pancreas (PPL). Not only they had a good thermal stability and reusability but also the activity recovery of LPS-1 and LPS-2 reached to 69% and 76%, respectively. The optimal pH and temperature region of the LPS supports immobilized PPL for hydrolysis of olive oil were at 8.0 and 55-60 degrees C. Kinetic parameters such as maximum velocity (V(max)) and the Michaelis constant (K(m)) were determined for the free and the immobilized lipase and LPS-2 immobilized PPL had the highest catalytic efficiency in the three. Meanwhile, the LPS supports exhibited many advantages than small porous materials for immobilizing PPL.     

Efficient synthesis of a long carbohydrate chain alkyl glycoside catalyzed by cyclodextrin glycosyltransferase (CGTase)

Alkyl glycosides with long carbohydrate groups are surfactants with attractive properties but they are very difficult to synthesize. Here, a method for extension of the carbohydrate group of commercially available dodecyl‐β‐d ‐maltoside (DDM) is presented. DDM was converted to dodecyl‐β‐d ‐maltooctaoside (DDMO) in a single step by using a CGTase as catalyst and α‐cyclodextrin (α‐CD) as glycosyl donor. The coupling reaction is under kinetic control and the maximum yield depends on the selectivity of the enzyme. The Bacillus macerans CGTase favored the coupling reaction while the Thermoanaerobacter enzyme also catalyzed disproportionation reactions leading to a broader product range. A high ratio α‐CD/DDM favored a high yield of DDMO and yields up to 80% were obtained using the B. macerans enzyme as catalyst. Biotechnol. Bioeng. 2009; 104: 854–861. © 2009 Wiley Periodicals, Inc.     

Covalent immobilization of laccase on activated carbon for phenolic effluent treatment

Summary Laccase was covalently immobilised to activated carbon using four derivatisation methods. The highest bound activity was obtained using diimide coupling of laccase to carboxyl groups on the carbon. The maximum bound activity was reached at 11.5 mg laccase/g carbon. The carbon-immobilised laccase (CIL) was stable at pH values from 4.0 to 9.0. CIL stored at 4°C lost 38± 5% activity in the first 4 days, then a further 22±5% in 126 days. CIL showed increased stability to low pH although the pH optimum was unchanged. The activation energy of CIL was lower than soluble laccase. Oxidation of 2,6-dimethoxyphenol (DMP) by CIL in a packed-bed system was only 30±10% of that in a fluidised bed system. Of the initial activity 10–30% was retained after oxidation of seven batches of DMP. CIL removed colour from two industrial effluents. Colour was removed from pulp mill bleach plant effluent at 115 colour units per enzyme unit per hour and the removal rate increased with increasing effluent concentration. Correspondence to: R. G. Burns     

Improvement of cyclodextrin glycosyltransferase (CGTase) production by recombinant Escherichia coli pAD26 immobilized on the cotton

The cyclodextrin glycosyltransferase (CGTase) of the recombinants Escherichia coli pAD26 cells immobilized on cotton was optimally produced by statistical methodology. Primarily, carbon and nitrogen sources were selected by one-factor-at-a-time method. Wheat starch, Casamino acid, Edamin and Hy-soy were identified as the best nutrients. These sources were secondly confirmed by Plackett-Burman design (fifteen variables were studied with sixteen experiments), as the most significant components with respect to CGTase production. In the third step, concentration of most significant factors and their interaction were optimized with a Box-Behnken experimental design. Under the optimized conditions (agitation 200 rpm, yeast extract concentration 20 g/L, wheat starch concentration 10 g/L and Hy-soy concentration 2.5 g/L), CGTase yield 145.11 U/mL was 3.6 and 23 folds higher than those obtained by the use of the initial conditions (39.77 U/mL) and free cells (6.37 U/mL), respectively.     

Covalent coupling method for lipase immobilization on controlled pore silica in the presence of nonenzymatic proteins

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica previously activated with glutaraldehyde in the presence of nonenzymatic proteins. This strategy is suggested to protect the enzyme from aggregation effects or denaturation that occurs as a result of the presence of silane precursors used in the formation of the silica matrix. The immobilization yield was evaluated as a function of the lipase loading and the additive type (albumin and lecithin) using statistical concepts. In agreement with the mathematical model, the maximum coupling yield (32.2%) can be achieved working at high lipase loading (450 units x g(-1) support) using albumin as an additive. In these conditions, the resulting immobilized lipase exhibits high hydrolytic (153.2 U x mg(-1)) and esterification (337.6 mmol x g(-1) x min) activities. The enhanced activity of the final lipase derivative is the sum of the benefits of the immobilization (that prevents enzyme aggregation) and the lipase coating by additives that increases the accessibility of active sites to the substrate.     

The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-A resolution

The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-A structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and +1, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195. On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.     

Covalent immobilization of lipase in organic solvents

Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.     

Formation of cyclodextrins with cyclodextrin glucosyltransferase stimulated with polarized light

After illumination with white, linearly polarized light (WLPL), cyclodextrin glycosyltransferase produced mixture of α‐, β‐, and γ‐cyclodextrins (CD) with higher overall yield than did that enzyme when nonilluminated. The illumination also influenced the ratio of those CD and that effect depended on concentration of enzyme and illumination time. At a high enzyme concentration (0.64 U/cm³), regardless the illumination time, formation of β‐CD predominated. The highest yield of β‐CD was afforded after 1 h illumination and 2 h illumination led to a significant increase in the yield of γ‐CD. Three‐month storage of enzyme illuminated with WLPL did not reduce its enhanced activity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Trypsin immobilization in silica gel

Trypsin immobilization in an inorganic polymer, i.e. silica gel, was investigated. Properties of the immobilized enzyme were examined. It was found that the enzyme retained its activity, depending on the gel genesis and dehydration of the carrier. The effect of stabilizing supplements on the enzyme activity was studied. The enzyme immobilization was accompanied by an increase in its thermostability.     

Acetylcholinesterase with mesoporous silica: Covalent immobilization,physiochemical characterization,and its application in food for pesticide detection

Toxic contamination of commonly consumed food products and water due to food chain vulnerability via agricultural products and commodities is a serious health hazard. This study reports on Santa Barbara Amorphous (SBA-15), a type of mesoporous silica nanoparticles, for efficient and stable acetylcholinesterase (AChE) adhesion toward detection of toxic pesticides. AChE was immobilized to the inert framework of mesoporous materials viz. SBA-15 with a proficient hydrolytic response toward acetylthiocholine. The immobilized system acts as a biosensor for the detection of pesticides, which are organophosphorus compounds in food. Both the SBA-15 and immobilized SBA-15 were characterized to give an insight on the physiochemical and morphological modification properties. The enzyme activity was accessed by Ellman’s spectrophotometric bioassay for bare and enzyme-immobilized SBA-15 that resulted in promising enzymatic activity with the counterpart. Enzyme stability was also studied, which exhibited that immobilized AChE retained its catalytic activity up to 60 days and retained 80% of the hydrolytic activity even at 37°C. On the basis of the success of immobilized enzyme (covalent) being inhibited by acetylthiocholine, the sensor was administered for the inhibition by monocrotophos and dimethoate that are used widely as pesticides in agricultural. The inhibitory concentration (IC₅₀) value was found to be 2.5 ppb for monocrotophos and 1.5 ppb for dimethoate inhibiting immobilized AChE. This was verified using cyclic voltammetry, an electrochemical analysis thus proving that the SBA-15@AChE complex could be used as a sensitive and highly stable sensor for detecting the concentration of hazardous pesticide compounds.     

Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.     

环糊精葡萄糖基转移酶182位点定点改造催化合成糖基化染料木素

染料木素及其单糖苷衍物在食品和医药领域具有重要作用,但难溶于水的特性极大地限制了其应用范围,研究表明糖基化反应可有效提高其水溶性.文中针对来源于软化芽孢杆菌的环糊精葡萄糖基转移酶,研究其对染料木素单糖苷衍生物槐角苷的糖基化反应.通过对D182位点的定点饱和突变,突变酶D182C较WT转化率提高了13.42％,主要糖基化...     

Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Covalent immobilization of chitosan and heparin on PLGA surface

Chitosan and heparin were covalently immobilized onto a poly(lactic acid-co-glycolic acid) (PLGA) surface using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) in a 2-morpholinoethane sulfonic acid (MES) buffer system. The properties of the modified PLGA surface and the control were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The water contact angle of the modified film was greatly decreased and the element content on the surface of the films changed correspondingly. Platelet adhesion assay showed that blood compatibility of the chitosan/heparin modified film was improved while hepatocyte culture indicated that the cell compatibility of the modified film was enhanced.     

Covalent immobilization of penicillin acylase from Streptomyces lavendulae

Penicillin acylase from Streptomyces lavendulae has been covalently immobilized to epoxy-activated acrylic beads (Eupergit C). Consecutive modification of the matrix with bovine serum albumin leads to a new biocatalyst (ECPVA) with enhanced activity (1.5 fold) in the hydrolysis of penicillin V respect to its soluble counterpart. This biocatalyst had a K _m value of 7.6 mM, slightly higher than K _m for native acylase (3 mM). In addition, ECPVA can be recycled for at least 50 consecutive batch reactions without loss of catalytic activity.     

Covalent immobilization of enzymes within micro-aqueous organic media

Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes.This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly.     

葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。     

Optimized immobilization of activated sludge in poly(ethylene glycol) gels by UV technology

Conventional methods for immobilization of microorganisms in hydrogel are not very efficient and cost-effective. Ultraviolet (UV) technology, a rapid method for hydrogel preparation in ambient environment, was applied to immobilize activated sludge in poly(ethylene glycol) (PEG) gel carriers synthesized from PEG pre-polymer. Conditions for cell immobilization were optimized by selecting suitable UV photoinitiators, crosslinker concentrations, and protection gas. The optimum conditions are the following: 12% PEG pre-polymer with 1% MBA and 0.1% DEAP. More than 90% of ammonia was reduced, and 75% of the total organic compound was removed after treating 40 and 10 mg/L synthetic ammonia wastewater with different C/N ratios in 5 h under aeration conditions. Ammonia degradation increased with increasing C/N ratios, and the scanning electron microscopy images revealed that the immobilized beads had porous structures dominantly filled with nitrifiers. The bioactivity of the immobilized activated sludge was maintained during immobilization by UV technology.