Laccase immobilization on the tailored cellulose-based Granocel carriers

Extracellular laccase produced by Cerrena unicolor was immobilized by adsorption or covalent bonds formation on the cellulose-based carrier Granocel. Immobilization was optimized by changing the anchor groups and the methods of activation/immobilization. On the base of measured activity and stability of immobilized preparations, the covalent method was selected. It was shown that coupling of the enzyme to the carrier via divinyl sulfone or glutaraldehyde yielded an enzyme-carrier preparation of high activity and storage stability. Further optimization of the carrier's superstructure consisted in changing pore diameters and amount of functional groups on the carriers surface. Three-fold higher activity was noted when the enzyme was immobilized on NH2-modified Granocel with the highest size exclusion limit and amino group content. Relatively low products sorption was observed on the carrier surface. The effects of protein concentration and pH-value of the coupling mixture on immobilization efficiency were evaluated also.     

Immobilization of enzymes on porous silica supports

Four silica supports differing in pore dimensions were activated by treatment with SiCl₄ and then with ethylenediamine to obtain alkylamine groups on the silica surface. Three enzymes, peroxidase from cabbage, glucoamylase from Aspergillus niger C and urease from soybean were immobilized on these supports using glutaraldehyde as coupling agent. It was found that the protein content, the retained enzymatic activity and the storage stability of the silica supported enzymes were considerably affected by support pore size and enzyme molecular weight, the factors which are supposed to alter protein distribution inside the support pores. The highest activity was found for peroxidase and glucoamylase attached to the silica with the widest pores, but their loss in activity during storage was considerable. The urease retained less activity after immobilization, but its storage stability was excellent.     

Immobilization of glucoamylase on polyamide nets

The formation of reactive groups on polyamide nets (nylon 6) and the subsequent immobilization of glucoamylase were investigated. Different mesh sizes of the nets and two chemical methods of enzyme coupling - i( partial hydrolysis of the polyamide with subsequent glutaraldehyde binding and ii) O-alkylation of the carrier using a treatment with a benzene-methyl sulphate mixture – were used. The reactivity of immobilized glucoamylase (GA) was tested by hydrolysis reactions using 1% starch solutions. The highest reactivity (140 μg glc/)min × cm² was obtained for methylated nylon samples attached to a glass rod and by coupling glucoamylase on the nylon surface which had been treated with lysine and glutaraldehyde. This method resulted in a more reactive and more stable preparation of immobilized glucoamylase as compared to a simpler method of coupling glutaraldehyde to partially hydrolyzed nylon.     

铜金属螯合载体固定糖化酶

[目的]制备出含Cu2+的琼脂糖-IDA螯合载体及对其固定糖化酶工艺条件进行优化.[方法]利用金属螯合配体(IDA-Cu2+)与蛋白质表面供电子氨基酸相互作用的原理制备载体,采用紫外分光光度法测定不同影响因素下固定化糖化酶的酶活.[结果]Cu2+的加入量和固定化过程的酸度比给酶量对固定化糖化酶的活性影响还要大,在给酶量80 mg/g载体、1.0× 10-2 mol Cu2+/g载体、pH 4.6和固定化4h的固定化条件下,固定化酶活为252.1 U/g,重复使用5次后酶活为首次固定化酶活的65.1％.[结论]该Cu2+-IDA-金属螯合琼脂糖可用于淀粉水解糖化酶的优良固定化载体材料.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Application of bacterial cellulose pellets in enzyme immobilization

Over recent years, there has been a growing interest in the use of cellulose materials in bioprocessing technologies. Bacterial cellulose which is the pure cellulose has unique physical properties which differ from those of plant cellulose and has therefore attracted attention as a new functional material. The applications of bacterial cellulose rarely use the pellet type but it has potential in enzyme immobilization since pellet form is usually used in this field. In this research, Glucoamylase which is widely used in the food industry was immobilized on bacterial cellulose beads after testing using various activation procedures. The results showed that the epoxy method with glutaraldehyde coupling was the best method. After comparison of the different types of bacterial cellulose beads for glucoamylase immobilization, the wet bacterial cellulose beads of the smallest size (0.5–1.5 mm) were the best support. The immobilization of enzyme enhances its stability against changes in the pH value and temperature especially in the lower temperature region. The relative activity of the immobilized glucoamylase was still above 77% at pH 2.0 and it was the highest value in the literature. The relative activities were more than 68% in the lower temperature region even at 20 °C. Thus, bacterial cellulose beads are a practical potential support for the preparation of immobilized enzymes in industrial applications.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Activity and stability of glucoamylase preparations in different methods of immobilization

Catalytic activity and stability of glucoamylases immobilized by different methods (adsorption, covalent binding) are studied comparatively. The highest stability is shown to be obtained under covalent binding. The binding efficiency and immobilized glucoamylase properties depend on the nature of insoluble carrier and a purification degree of the enzyme preparations. The choice of the cross-linking agent promoting a binding between the enzyme and the carrier is very significant. The activity and stability of immobilized glucoamylases obtained when using different cross-linking agents rise in such a sequence: 2,4-toluylenediisocyanate, cyanurochloride, glutaric dialdehyde, gossypol. Catalytic properties and stability are determined for soluble and immobilized glucoamylase forms from different sources.     

L-DOPA production from tyrosinase immobilized on nylon 6,6

The production of L-DOPA immobilized on chemically modified nylon 6,6 membranes was studied in a batch reactor. Tyrosinase was immobilized on nylon using glutaraldehyde as a crosslinking agent. The effects of membrane pore size and glutaraldehyde concentration upon enzyme uptake and L-DOPA production were investigated. Enzyme uptake was unaffected by glutaraldehyde concentration; approximately 70% uptake was observed when 25% w/v (group 1), 5% (group 2), and 3% (group 3) glutaraldehyde were used, indicating that glutaraldehyde was in excess. Similarly, uptake was the same for membranes with 0.20 and 10 mum pore sizes.Membranes produced using different levels of glutaraldehyde exhibited dramatically different capacities for L-DOPA production, despite the fact that enzyme uptake was equivalent. Membranes from groups 2 and 3 (5% and 3% glutaraldehyde) produced L-DOPA at a rate of 1.70 mg L(-1) h(-1) over 170 h in a 500-mL batch reactor. However, no free L-DOPA was detected when group 1 membranes were used. Experimental evidence suggests that L-DOPA was produced, but remained bound to these membranes via excess glutaraldehyde left over from the immobilization process. Membrane pore size also effected L-DOPA production; less production was observed when 10-mum membranes were used, despite equivalent enzyme uptake. The observed difference in production may be due to differences in the pore density on the two types of membranes which could affect the access of the substrate to the immobilized enzyme.The results of these studies indicate that tyrosinase can be effectively immobilized on nylon 6,6. L-DOPA production was optimal when 0.20-mum-pore-size membranes were activated with 3-5% glutaraldehyde. Stability studies indicated a 20% reduction in activity over 14 days when the immobilized enzyme was used under turnover conditions. (c) 1996 John Wiley & Sons, Inc.     

Hydrolysis of proteins by immobilized-stabilized alcalase-glyoxyl agarose

This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.     

Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol

Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS) modification) in order to check the effect of these modifications on the catalytic features of the immobilized enzymes (including stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobiled enzyme is even negative. Most of the modifications presented a positive effect on some enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymes at pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, while it reduced the stability of the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent preparation at pH 9 (by a 10-fold factor).     

Immobilization of glucoamylase on porous silicas

The influence of the pore structure of silica carriers (macroporous silica gels, silochromes and porous glasses) on the catalytic activity of immobilized glucoamylase (exo 1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase EC 3.2.1.3) has been studied. The dependence of the immobilized glucoamylase activity, in units g^?1, on the carrier pore diameter was found to pass through a maximum within a range 70–100 nm. Macroporous silica gels can be used with success as carriers for glucoamylase immobilization instead of porous glasses and silochromes.     

Immobilization of glucose oxidase on polymer membranes treated by low-temperature plasma

Low-temperature plasma was employed for activation of polymer membranes as a carrier for enzyme immobilization. Glucose oxidase was immobilized on polypropylene (PP), polyvinylidene fluoride (PVDF), or polytetrafluoroethylene (PTFE) membrane surfaces treated by nitrogen or ammonia gas plasma using glutaraldehyde as a linking agent. Enzyme activity was evaluated by the response of glucose sensor composed of the immobilized enzyme membrane and a dissolved oxygen electrode. The sensor response was found to depend on the kind of carrier membrane and to become maximum at suitable conditions of plasma treatment.     

固定化酶的微环境效应Ⅰ——最适pH的移动

以葡聚糖为载体,在葡聚糖上引入了不同量的羧甲基,采用CNBr法活化葡聚糖上剩余的羟基,使之与葡萄糖淀粉酶(GA)共价结合,从而观察载体羧甲基定量变化对固定化葡萄糖淀粉酶pH——活性曲线的影响。分别使用了两种不同孔度的葡聚糖(SephadxG100、G200)作为载体,首次发现在载体带有离子基因的情况下,载体的孔度变化对固定化酶的pH——活性曲线影响极大,甚至可以完全消除载体静电荷对此带来的影响。这些结果表明:固定化酶的微环境不仅与载体的静电势有关,也与载体分子空间结构的疏密程度有关。本文对固定化葡萄糖淀粉酶(IGA)的热稳定性也作了评价。     

Immobilization of lipase from Candida rugosa on Sepabeads(®): the effect of lipase oxidation by periodates

The objective of this paper was the investigation of a suitable Sepabeads^? support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads^? EC-EA and Sepabeads^? EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads^? EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.     

氨基树脂固定化L-苏氨酸醛缩酶及其应用*

L-苏氨酸醛缩酶(L-Threonine aldolase,L-TA)可以催化甘氨酸和醛合成β-羟基-α-氨基酸。β-羟基-α-氨基酸具有两个手性中心,是多种手性药物的中间体。但是,游离的L-TA难以重复利用,分离纯化困难,严重阻碍了工业化应用。固定化技术可以有效解决这些问题。利用氨基树脂NAA固定化来源于Bacillus nealsonii的L-苏氨酸醛缩酶,采用戊二醛作为交联剂,经过条件优化确定最佳固定化条件为:加酶量13 U、载体量0.6 g、0.4%(V/V)戊二醛、活化时间2 h、pH 8.5、35℃、固定化5 h。在此条件下,固定化酶酶活回收率为85.7%。在30℃下半衰期可达59天,为游离酶的6.5倍。将其应用于合成L-syn-对甲砜基苯丝氨酸,使用460 h后,残余酶活为79.4%。进一步开发了载体再利用策略,将失活固定化酶表面的氨基用戊二醛活化后,再与新的游离酶进行固定化,实现载体的再利用。利用该方法载体可重复利用两次,制备的固定化酶仍能使用460 h。该方法大大降低了固定化成本,为固定化L-TA的工业化应用打下坚实的基础。     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

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.