Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

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.     

锰过氧化物酶的耦合固定化及其性质研究

分别采用海藻酸钠、明胶和壳聚糖为载体,并以戊二醛为交联剂,通过包埋-交联和吸附-交联两种耦合固定化方法制备固定化锰过氧化物酶。探讨了酶的不同固定化条件和固定化酶的部分性能。与游离酶相比,制备的3种固定化酶最适反应pH分别由7·0降低到5·0、5·0和3·0,最适反应温度分别由35℃升高到75℃、55℃和75℃。3种固定化酶的耐热性都显著提高,其中用壳聚糖制成的固定化酶在pH2·2~11的宽范围内表现出很好的酸碱耐受性。30℃连续测定6~9次酶活力,重复使用的3种固定化酶显示出良好的稳定性。将固定化酶应用在偶氮染料的脱色中,用明胶制成的固定化酶在静置和摇床条件下,以及用海藻酸钠制成的固定化酶在摇床条件下,均表现出与游离酶相近的脱色能力,并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。     

High-temperature alkaline alpha-amylase from Bacillus iicheniformis TCRDC-B13

A bacterial strain, Bacillus licheniformis, has been isolated and identified which produces high-temperature alkaline alpha-amylase. Cultural conditions, such as types of carbon and nitrogen sources, temperature, pH, and time of reaction, have been optimized for production of alpha-amylase in shake flask and fermenter. The enzyme produced was quite active even at 100 degrees C; however, it showed optimum activity at 90 degrees C. It exhibited optimum activity in the broad pH range 5.5-10. The effects of Na(+) and Ca(2+) ions on enzyme activity was also studied.     

Immobilization of chitosan-invertase neoglycoconjugate on carboxymethylcellulose-modified chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on a carboxymethylcellulose-coated chitin support via polyelectrolyte complex formation. The yield of immobilized protein was determined to be 72% and the enzyme retained 68% of the initial invertase activity. The optimum temperature for invertase was increased by 5 degrees C and its thermostability was enhanced by about 9 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 12.6-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The prepared biocatalyst retained 98% and 100% of the original catalytic activity after 10 cycles of reuse and 70 h of continuous operational regime in a packed bed reactor, respectively. The immobilized enzyme retained 95% of its activity after 50 days of storage at 37 degrees C.     

Isoglucose production from raw starchy materials based on a two-stage enzymatic system

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.     

Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor

Ethyl acetate was explored as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel from the crude oils of Jatropha curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The optimum reaction conditions for interesterification of the oils with ethyl acetate were 10% of Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, ethyl acetate to oil molar ratio of 11:1 and the reaction period of 12h at 50 degrees C. The maximum yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils, respectively under the above optimum conditions. Reusability of the lipase over repeated cycles in interesterification and ethanolysis was also investigated under standard reaction conditions. The relative activity of lipase could be well maintained over twelve repeated cycles with ethyl acetate while it reached to zero by 6th cycle when ethanol was used as an acyl acceptor.     

棒杆菌固定化细胞生产L（＋）—酒石酸

以卡拉胶为载体,固定化棒状杆菌（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｓｐ．）ＪＺ－１菌株细胞,再经活化处理,顺式环氧琥珀酸水解酶（ＥＳＨ）酶活力总回收率在１００％以上。摇瓶反应１０批,酶活力没有明显降低。１５００Ｌ酶柱中连续运行９０ｄ,酶稳定性很好,固定化后酶反应的最适温度（４５℃）和最适ｐＨ（９．０）没有改变,而热稳定性、ｐＨ稳定性增强。     

Immobilization and stabilization of a cyclodextrin glycosyltransferase by covalent attachment on highly activated glyoxyl-agarose supports

Covalent immobilization of cyclodextrin glycosyltransferase on glyoxyl-agarose beads promotes a very high stabilization of the enzyme against any distorting agent (temperature, pH, organic solvents). For example, the optimized immobilized preparation preserves 90% of initial activity when incubated for 22 h in 30% ethanol at pH 7 and 40 degrees C. Other immobilized preparations (obtained via other immobilization protocols) exhibit less than 10% of activity after incubation under similar conditions. Optimized glyoxyl-agarose immobilized preparation expressed a high percentage of catalytic activity (70%). Immobilization using any technique prevents enzyme inactivation by air bubbles during strong stirring of the enzyme. Stabilization of the enzyme immobilized on glyoxyl-agarose is higher when using the highest activation degree (75 micromol of glyoxyl per milliliter of support) as well as when performing long enzyme-support incubation times (4 h) at room temperature. Multipoint covalent immobilization seems to be responsible for this very high stabilization associated to the immobilization process on highly activated glyoxyl-agarose. The stabilization of the enzyme against the inactivation by ethanol seems to be interesting to improve cyclodextrin production: ethanol strongly inhibits the enzymatic degradation of cyclodextrin while hardly affecting the cyclodextrin production rate of the immobilized-stabilized preparation.     

Immobilization of a soluble chemically thermostabilized enzyme

Cellobiase was coupled to a dialdehyde dextran by reductive alkylation in the presence of sodium cyanoborohydride. The resulting conjugate, obtained without loss of enzymic activity, presents properties of thermoresistance largely superior to those of native enzyme: the rate of inactivation is reduced compared to that of native enzyme and its optimal temperature of activity is 70-75 degrees C instead of 65 degrees C. Finally the conjugate presents increased longevity when subjected to experiments of operational stability; its hydrolytic activity is maintained at 60 degrees C in a 10% (w/v) cellobiose solution for more than 100 h whereas the native enzyme is inactivated after 45 h. The cellobiase-dextran conjugate was immobilized by covalent coupling on aminated silica by reductive alkylation in the presence of NaBH(3)CN. The characteristics of thermoresistance of this stabilized and immobilized conjugate were studied and compared to those of a preparation of native cellobiase immobilized on a silica support activated with glutaraldehyde. Analysis of the thermoresistance of these two cellobiase preparations clearly shows that immobilization has maintained and even enhanced their properties. In particular, the operational stability, measured at 68 degrees C on 10% (w/v) cellobiose shows an increased longevity of the stabilized and immobilized enzyme for 120 h compared to 60 h for the native immobilized enzyme. Two successive incubations of these cellobiase derivatives show that it is possible to obtain 2.5 times more glucose with the stabilized-immobilized enzyme than with the immobilized preparation. The procedure described above enables us to prepare a thermostabilized immobilized cellobiase.     

具有谷氨酸脱羧酶的大肠杆菌固定化细胞

本文研究了用海藻酸钙包埋法制备含谷氨酸脱羧酶固定化细胞的方法以及研究了制备的条件和影响其制备的因素。该法具有包埋细胞活力回收高,方法简便等优点。比较研究了固定化细胞和自然细胞谷氨酸脱羧酶的一些生物化学性质。其中固定化细胞最适pH和pH稳定性增加,最适温度及热稳定性下降;表观米氏常数增大;二价金属离子Zn~(++)、Cu~(++)、Mg~(++)、Fe~(++),Sr~(++)程度不同的抑制酶活性,Ca~(++)激活固定化细胞酶活性,EDTA无抑制作用。对固定化细胞和自然细胞酶活力活化的研究中发现这两种细胞经蒸馏水保温处理后酶活性都上升,且自然细胞酶活的上升较固定化细胞大;而用底物溶液处理后,自然细胞无变化,固定化细胞酶活下降。     

Properties of a glucose oxidase covalently immobilized on amorphous AlPO4 support

Glucose oxidase (GOD) was covalently immobilized on amorphous AlPO₄ as well as on an AlPO₄/clay mineral Sepiolite system. Immobilization of the enzyme was carried out through the -amino group of lysine residues through an aromatic Schiff's-base. Activation of the support was obtained after reaction of appropriate molecules with support surface –OH groups. The enzymatic activities of native, and different immobilized GOD systems and filtrates, were followed by the amount of liberated -gluconic acid obtained in the enzymatic β- -glucose oxidation with the aid of an automatic titrator. The kinetic properties of native and immobilized GOD were obtained for glucose concentrations in the range of physiological conditions and at different working conditions such as reaction temperature, reaction pH, and enzyme concentration.

The binding percentage of enzymes was in the 50–80% range, with residual and specific activities in the 65–80% and 90–150% ranges, respectively. No change in the pH optimum and only slight changes in the V_max and K_M kinetic parameters with respect to native GOD were observed, so that not only was little deactivation of enzyme obtained throughout the immobilization process but also that the stability of the covalently bound enzyme in the two supports appeared to have increased with respect to the soluble enzyme. GOD immobilization also increased its efficiency and operational stability in repeated uses on increasing the amount of immobilized enzyme.     

Preparation and properties of gelatin-immobilized beta-glucosidase from Pyrococcus furiosus

Hyperthermostable beta-glucosidase from Pyrococcus furiosus was enclosed in gelatin gel by cross-linking with transglutaminase. Gelatin-immobilized beta-glucosidase was considerably more thermostable than the native enzyme. Lyophilized immobilisate was stored at 90 degrees C for 1 month without loss of activity. The immobilized beta-glucosidase catalyzed transglucosylation of 5-phenylpentanol with 10.0 equivalent of cellobiose at pH 5.0 and 70 degrees C for 12 h to afford 5-phenylpentyl beta-D-glucopyranoside in 41% yield. The immobilized enzyme was more effective than the native one in transglucosylation. The gelatin-immobilized Pfu-beta-glucosidase recovered from the first run of the reaction was reusable on successive runs.     

Biocatalysis using an organic-soluble enzyme for the preparation of poly(lactic acid) in organic solvents

Proleather from Bacillus sp. was chemically modified with decanoyl chloride for enhanced activity for the preparation of poly(lactic acid) in organic solvents. The modified enzyme was highly soluble (up to 44 mg-protein/ml) and active in various organic solvents including chloroform, tetrahydrofuran (THF), pyridine and acetone. The organic-soluble proleather efficiently catalyzed the polymerization of ethyl lactate. The reaction rate was 4-22 times that of native proleather, depending upon the solvent applied. The solubilized enzyme showed a highest activity at 50 degrees C, the same optimum temperature for both the native proleather and an immobilized enzyme, Novozyme-435. Denaturation of the enzymes' protein structures appeared to be the critical factor regulating the optimum activity temperature. Differential scanning calorimetry (DSC) analyses of the enzymes showed endothermic peaks around 55 degrees C, indicating the proteins' structures altered in that temperature range. Interestingly, the activity of the solubilized enzyme showed a more complicated water dependence as compared to native proleather.     

Immobilization of Streptomyces phaerochromogenes by radiation-induced polymerization of glass-forming monomers.

Immobilization of Streptomyces phaerochromogenes was studied by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures. Radiation damage of the enzyme could be avoided by choosing irradiation at low temperatures. The enzymatic activity of immobilized cells increased remarkably with a decrease in the irradiation temperature of about -24 degrees C. In constrast to the case of cell-free enzyme immobilization, the most characteristic case was than in these immobilized cells, the enzymatic activity did not decrease with repeated use even in the composite obtained at much lower monomer concentrations. Another characteristic of immobilized cells was the increase in enzymatic activity in the initial stage of repeated use, which could be attributed to the swelling effect of the polymer matrix, thereby increasing the enzymatic activity of whole cells.     

尼龙网固定化果胶酶的制备及其性质研究

用尼龙网作载体,经3-二甲氨基丙胺活化,用戊二醛将果胶酶固定化。所得固定化酶Km值与自然酶接近;对温度的稳定性有较大的提高,100℃保温30min才能使其失活。固定化酶在较宽的pH范围内能保持其正常活力,它对金属离子抑制剂的耐受性有较显著的提高,用0.5％果胶溶液作底物,重复使用10次后酶活力保留44％。固定化果胶酶与自然酶相比较,对不同果汁的澄清效果不同。固定化果胶酶在无保护剂存在的条件下,室温放置四个月活力不减少。     

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.     

Degradation of the framework of the Chlamydomonas cell wall by proteases present in a commercially available alpha-amylase preparation

A commercially available alpha-amylase derived from Bacillus licheniformis contained an enzymatic activity able to degrade the inner portion or framework of the cell wall of Chlamydomonas reinhardtii. Both the wall-degrading activity and the contaminating protease were destroyed by heating the alpha-amylase preparation at 90 degrees C for 30 min. Since the alpha-amylase activity was uneffected by heat treatment, we conclude that it was not the alpha-amylase but the contaminating protease in the preparation that was responsible for the cell wall-degrading activity.     

Degradation of the framework of the Chlamydomonas cell wall by proteases present in a commercially available alpha-amylase preparation.

A commercially available alpha-amylase derived from Bacillus licheniformis contained an enzymatic activity able to degrade the inner portion or framework of the cell wall of Chlamydomonas reinhardtii. Both the wall-degrading activity and the contaminating protease were destroyed by heating the alpha-amylase preparation at 90 degrees C for 30 min. Since the alpha-amylase activity was uneffected by heat treatment, we conclude that it was not the alpha-amylase but the contaminating protease in the preparation that was responsible for the cell wall-degrading activity.     

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.