Some properties of free and immobilized alpha-amylase from Penicillium griseofulvum by solid state fermentation

Alpha-amylase was produced from Penicillium griseofulvum by an SSF technique. Alpha-amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5 degrees C and 40 degrees C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver-Burk plots for both enzymes. Km values were 9.1 mg mL(-1) for free enzyme, and 7.1 mg mL(-1) for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the a-amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.     

Evaluation of the performance of immobilized penicillin G acylase using active-site titration

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.     

Properties of a reversible soluble-insoluble cellulase and its application to repeated hydrolysis of crystalline cellulose

Cellulase was covalently immobilized on an enteric coating polymer, Eudragit L, that is reversibly soluble and insoluble depending on the pH of the medium. The hydrolysis of solid cellulose with the immobilized enzyme can take advantage of the soluble property of the immobilized enzyme itself at the most reactive pH value; on the other hand, recovery of the enzyme can take advantage of the insoluble property of the enzyme at other pH values. It was experimentally confirmed that 100% of immobilized enzyme activity in solution can be recovered by precipitation and by dissolving it again by alternative change of pH. After a period of hydrolysis, immobilized enzyme and unreacted cellulose were precipitated together to remove the product-the soluble sugar solution-by changing pH. Following this, a new buffer solution was added to the precipitate to dissolve it and resume the reaction. This was repeated several times. The hydrolysis rate of this process increased significantly compared with that of a batch process. Utilization of the reversible soluble-insoluble carrier for immobilizing enzyme is promising, not only for cellulose-cellulase systems, but also for other heterogeneous reaction systems.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

Effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase in a capillary bed reactor

The effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase were studied in a continuous flow capillary bed reactor. Temperature affects the rates of enzymatic reactions in two ways. Higher temperatures increase the rate of the hydrolysis reaction, but also increase the rate of thermal deactivation of the enzyme. The effect of temperature on the kinetic parameters was studied by performing lactose hydrolysis experiments at 15, 20, 25, 30, and 40 degrees C. The kinetic parameters were observed to follow an Arrhenius-type temperature dependence. Galactose mutarotation has a significant impact on the overall rate of lactose hydrolysis. The temperature dependence of the mutarotation of galactose was effectively modelled by first-order reversible kinetics. The thermal deactivation characteristics of the immobilized enzyme reactor were investigated by performing lactose hydrolysis experiments at 52, 56, 60, and 64 degrees C. The thermal deactivation was modelled effectively as a first order decay process. Based on the estimated thermal deactivation rate constants, at an operating temperature of 40 degrees C, 10% of the enzyme activity would be lost in one year.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.     

Kinetic studies of sepharose-and CH-sepharose-immobilized dihydrofolate reductase

Dihydrofolate reductase, purified to homogeneity from amethopterin-resistant Lactobacillus casei, was immobilized by coupling to cyanogen bromide-activated Sepharose or carbodiimide-activated CH-Sepharose. Coupling yields were determined by amino acid analysis following the hydrolysis of the gel. Enzyme activity was measured by the conventional spectrophotometric procedure, thus permitting the facile characterization of the immobilized enzyme. The pH optimum of the immobilized enzyme was shifted to 5.8 compared with pH 5.5 for the soluble enzyme. The immobilized enzyme retained greater than 90%of the initial activity over a six-month period and could be reused as many as ten times without loss of activity. As observed with the soluble enzyme, the activity of immobilized enzyme, which was lost on denaturation with 4M guanidine hydrochloride, was recovered rapidly and completely by washing the gel with buffer. The K(m) (app) values for dihydrofolate and NADPH for the immobilized enzyme were increased 15-164-fold over the K(m) values measured for soluble dihydrofolate reductase. Scatchard analysis of the interaction of amethopterin with the immobilized enzyme yielded linear plots and a K(d) (app) value of 0.56 x10(-8)M, and revealed that all of the immobilized enzyme molecules were capable of binding the ligand.     

Properties and applications of Penicillium funiculosum cellulase immobilized on a soluble polymer

Summary The cellobiase and xylanase activities of Penicillium funiculosum were immobilized on a soluble polymer poly(vinyl alcohol) (PVA). The kinetic parameters and the adsorption characteristics of the bound and free enzymes were compared. The Km value of the immobilized preparation was the same as the free enzyme. The hydrolysis of different cellulosic substrates by the bound enzyme is investigated.     

Production of omega-3 polyunsaturated fatty acids through hydrolysis of fish oil by Candida rugosa lipase immobilized and stabilized on different supports

This paper describes the fish oil hydrolysis performed to obtain Omega-3 fatty acids using Candida rugosa lipase (CRL) immobilized and stabilized on different supports. The enzyme was successfully immobilized, presenting higher thermal stability than the free enzyme. Besides, the cationic derivatives were more stable than the others derivatives and free enzyme in methanol, propanol and cyclohexane. Reactions of fish oil hydrolysis were carried out in organic aqueous medium using 10?U of biocatalyst per gram of oil, at 37?°C. After 96?h, the CRL immobilized on cyanogen bromide agarose rendered the lower fish oil hydrolysis, producing 218?μM of Omega-3, which was 1.1-fold more than the hydrolysis catalyzed by free enzyme, while the ionic derivatives rendered the highest fish oil hydrolysis producing 582 and 577?μM of Omega-3 using the carboxymethyl and sulfopropyl derivatives, respectively. The carboxymethyl and the sulfopropyl derivatives resulted in a 2.9-fold increase in the hydrolysis of fish oil, making these derivatives attractive for industrial applications.     

葡聚糖磁性毫微粒固定化L－天冬酰胺酶的研究

葡聚糖磁性毫微粒固定化Ｌ－天冬酰胺酶的研究徐慧显,李民勤,潘再群,马建标,何炳林（南开大学高分子化学研究所,天津３０００７１）大肠杆菌天冬酰胺酶对急性淋巴白血病有明显疗效［１］,注射入体内以后,可迅速清除血清中的天冬酰胺──敏感性肿瘤细胞的必需营养成...     

The effect of methylacrylate on the activity of glucomylase immobilized on granular polyacrylonitrile

Glucoamylase was immobilized on granular polyacrylonitrile (PAN) and the optimum condition in its immobilization reaction was determined. The effect of the ratio of the imidoester and methylester to the total cyanogen on the activity of the immobilized enzyme was studied. The activity of the immobilized enzyme increased in proportion to the molar number of imidoester and decreased with that of methylester. The K(m) and V(m) values of immobilized glucoamylase which were prepared at various conditions of immobilization were determined. There were opposite trends in K(m)S between glucoamylase immobilized on imidoester-rich support and immobilized on methylester in the support, evidenced as functions of temperature. This suggests that opposite charges in the support, produced by heat deformation of PAN by hydrolysis of methylester, were an influence on the apparent K(m) of immobilized glucoamylase, besides the diffusional limitation.     

Biocatalytic properties of immobilized inulinase

A heterogeneous biocatalyst based on inulinase immobilized on a nonionic sorbent of Stirosorb series was proposed. Thermal and acid inactivation of free and immobilized inulinase was examined and the corresponding inactivation constants were calculated. An increase in the thermal stability of the immobilized enzyme in comparison to the free one was found. The possibility of using the immobilized enzyme in the hydrolysis of inulin for ten cycles was determined.     

Immobilization of glucoamylase by adsorption on carbon supports and its application for heterogeneous hydrolysis of dextrin

Glucoamylase (GA) was immobilized by adsorption on carbon support: on Sibunit, on bulk catalytic filamentous carbon (bulk CFC) and on activated carbon (AC). This was used to prepare heterogeneous biocatalysts for the hydrolysis of starch dextrin. The effect of the texture characteristics and chemical properties of the support surface on the enhancement of the thermal stability of the immobilized enzyme was studied, and the rates of the biocatalyst's thermal inactivation at 65-80 degrees C were determined. The thermal stability of glucoamylase immobilized on different carbon supports was found to increase by 2-3 orders of magnitude in comparison with the soluble enzyme, and decrease in the following order: GA on Sibunit>GA on bulk CFC>GA on AC. The presence of the substrate (dextrin) was found to have a significant stabilizing effect. The thermal stability of the immobilized enzyme was found to increase linearly when the concentration of dextrin was increased from 10 wt/vol % to 50 wt/vol %. The total stabilization effect for glucoamylase immobilized on Sibunit in concentrated dextrin solutions was about 10(5) in comparison with the enzyme in a buffer solution. The developed biocatalyst, 'Glucoamylase on Sibunit' was found to have high operational stability during the continuous hydrolysis of 30-35 wt/vol % dextrin at 60 degrees C, its inactivation half-time (t1/2) exceeding 350 h. To improve the starch saccharification productivity, an immersed vortex reactor (IVR) was designed and tested in the heterogeneous process with the biocatalyst 'Glucoamylase on Sibunit'. The dextrin hydrolysis rate, as well as the process productivity in the vortex reactor, was found to increase by a factor of 1.2-1.5 in comparison with the packed-bed reactor.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Immobilization of alpha-glucosidase in chitosan coated polygalacturonic acid

Crude alpha-glucosidase from Baker's yeast was immobilized in polygalacturonic acid beads and coated with chitosan. Chemical and physical characterization were performed by using p-nitrophenyl-alpha-D-glucopyranoside (pNPG) as an artificial substrate. Operation, thermal, pH, and strorage stabilities of the free and immobilized enzyme were also examined. The stabilities of immobilized enzyme were found to be better than that of the free enzyme. Furthermore, the hydrolysis rate of the chitosan coated alpha-glucosidase polygalacturonic acid beads were studied. In conclusion, the enzyme beads appear to have good characteristics and offer the prospect that this system may find application in enzyme immobilization, in addition to controlled drug release studies.     

Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application

A simple way of fabricating enzymatic membrane reactor with high enzyme loading and activity retention from the conjugation between nanofibrous membrane and lipase was devised. Poly(acrylonitrile-co-2-hydroxyethyl methacrylate) (PANCHEMA) was electrospun into fibrous membrane and used as support for enzyme immobilization. The hydroxyl groups on the fibrous membrane surface were activated with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes. The resulted bioactive fibrous membranes were examined in catalytic efficiency and activity for hydrolysis. The observed enzyme loading on the fibrous membrane with fiber diameter of 80–150 nm was up to 1.6% (wt/wt), which was as thrice as that on the fibrous membrane with fiber diameter of 800–1000 nm. Activity retention for the immobilized lipase varied between 32.5% and 40.6% with the activation methods of hydroxyl groups. Stabilities of the immobilized lipase were obviously improved. In addition, continuous hydrolysis was carried out with an enzyme-immobilized fibrous membrane bioreactor and a steady hydrolysis conversion (3.6%) was obtained at a 0.23 mL/min flow rate under optimum condition.     

Hydrolysis of concentrated sucrose syrups by invertase immobilized on anion exchanger waste cotton thread.

Yeast invertase was immobilized on polyethyleneimine-coated cotton thread by adsorption followed by crosslinking with glutaraldehyde. The thread-bound invertase was used as an easily retrievable system for the hydrolysis of 80% w/v commercial sucrose syrups. The immobilized enzyme was stable for over 90 days to a temperature of 50 degrees C, only when stored in 80% sucrose solution. Above this temperature, inactivation of enzyme was observed. The cotton threads were used in a batch reactor for hydrolysis of sucrose in about 30 batches carried out over a period of 50 days without loss in activity. The threads could also be used in a packed bed reactor (1.51) for 97% hydrolysis of 80% sucrose syrups at 50 degrees C at a rate of about 360 kg per month for a period of 3 months.     

A simple kinetic model for the hydrolysis of α-d-glucans using glucoamylase immobilized on titanium (IV) activated porous silica

A simple kinetic model which describes the hydrolysis of α-d-glucans by immobilized glucoamylase (exo-1,4-d-glucosidase, EC 3.2.1.3) is reported. The hydrolysis of starch, amylose, amylopectin, maltose and 40DE starch hydrolysates using glucoamylase immobilized on alkylamine derivatives of titanium(IV) activated porous silica are described by a kinetic model based on Langmuir-Hinshelwood kinetics. This model involves enzyme kinetics with or without product inhibition and reverse reactions as well as mass transfer and diffusion effects in immobilized enzyme reactors. The results of other authors are also interpreted by the model developed in this article.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

无花果蛋白酶在阴离子交换树脂上的固定化

无花果蛋白酶通过８％戊二醛活化载体,共价结合到聚苯乙烯阴离子交换树脂ＧＭ２０１上,固定化作用在ｐＨ７．７,酶浓度０．８ｍｇ／ｇ树脂,４℃下进行６ｈ。得到的固定化酶表观Ｋ_ｍ值（酪蛋白,１．１１×１０~（－４）ｍｏｌ／Ｌ）小于溶液酶Ｋ_ｍ值（１．９６×１０~（－４）ｍｏｌ／Ｌ）;固定化酶活性在ｐＨ６～８保持稳定,溶液酶最适ｐＨ为７．２;固定化酶最适温度由溶液酶的５０～６０℃移至３７℃;固定化酶２５℃保持７ｄ,重复水解酪蛋白７次后,保留８３．３％活性。固定化酶对酪蛋白水解度达４７．５％,对大豆球蛋白达１１．６％。