Stability of immobilized alcohol dehydrogenase prepared using the thermostable enzyme from yeast mitochondria

Alcohol dehydrogenase from yeast was partially purified by heat treatment (70°C, 30 min) and immobilized on porous glass, Enzacryl-TI0 and hornblende. The stabilities of these preparations were studied at 30°C and in the case of Enzacryl-TI0 and hornblende at 50°C also. These stabilities were compared with those of immobilized alcohol dehydrogenase from yeast cytosol. In all cases the mitochondrial enzyme provided the more stable bound enzyme conjugates. However, at 50°C the soluble mitochondrial enzyme was more stable than any of the immobilized derivatives: half-life values were 40, 14 and 8 h for the soluble, Enzacryl-TI0 and hornblende samples, respectively.     

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.     

Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports

Glucoamylase from four different companies was studied: three had similar stability (half-life at 50°C about 140 hr); the fourth was less stable (half-life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl-TIO was less stable than enzyme bound to hornblende (metal-link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half-life.     

Invertase covalently coupled to porous glass: preparation and characterization

The enzyme invertase has been covalently coupled to porous glass particles. The product is extremely stable over a long period of time. Kinetic values for the immobilized enzyme are similar to the native enzyme. Excellent enzymatic activity for the immobilized enzyme was exhibited over a broad pH range. The immobilized enzyme when continuously operated for one month was found to have an operational half-life of over 40 days.     

Characteristics of yeast invertase immobilized on porous cellulose beads.

Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond. The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1. Both temperature profiles were fairly similar up to 55 degrees C. However, above this temperature the immobilized enzyme was more stable than the free enzyme. From the temperature data, the activation energies were found to be 7,322 and 4,052 cal/g mol for the free and the immobilized enzyme, respectively. Candida invertase shows characteristics of substrate inhibition. Both the Km and Ki for the free and the immobilized enzymes were determined. The apparent Ki for the immobilized invertase was much higher than the Ki of the free enzyme, suggesting a diffusion effect. Immobilized invertase molecules deep in the pores only see sucrose concentrations much less than the bulk concentrations. Immobilization, thus, offers certain processing advantages in this regard.     

Immobilization of yeasts on titanium activated inorganic supports

Summary A study of the immobilization of yeast cells with invertase activity by the metal link method was performed. Baker's yeast cells were immobilized on titanium activated porous silica support and on its alkylamine and aldehyde derivatives, their initial activities being 19.6, 39.9 and 10.6 U/ml of reactor respectively. When crosslinking of the immobilized cells was performed, an initial activity of 48.2 U/ml was achieved on the titanium activated support. Batch long-term stability tests were car ried out for 400 hours and the crosslinked preparations showed an unsta ble behaviour compared with the very stable preparations obtained with the simple metal-link method.A higher activity (56.2 U/ml) was obtained when a titanium activated macroporous support, pumice stone, was used as cell carrier, which compared favourably with calcium alginate entrapped cells (17.7 – 31.3 U/ml)     

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.     

Invertase covalent grafting onto corn stover

The covalent coupling of an invertase from baker's yeast onto an agricultural by-product, corn grits, has been developed. The optimal conditions for each step of the chemical modification of the support have been determined: oxidation with sodium metaperiodate, amination with ethylenediamine, reduction with sodium cyanoborohydride, and activation with glutaraldehyde. Activities up to 7.2 x 10(4) mumol reducing sugars produced/min g support could thus be achieved. Invertase coupling onto corn grits yields a derivative with a 25 times higher activity than when coupling this enzyme onto porous silica. The operational stability of invertase immobilized onto corn stover was found to be very high, with a half-life of up to 365 days at 40 degrees C when using a 2M sucrose solution as substrate. This immobilization method could be easily scaled up to the preparation of 10 kg of invertase derivative.     

Immobilized Aminoacylase on Porous Glass Beads

The preparation and properties of immobilized aminoacylase on porous glass by covalent binding [Porous glass-CVB-aminoacylase] and the continuous enzymatic reactions using such preparations are described.

Two types of porous glass-CVB-aminoacylase were prepared. One was aminoacylase covalently bound to alkylaminosilane derivative of porous glass with glutaraldehyde as a coupling agent [Alkylamino-porous glass-CVB-aminoacylase], and the other was aminoacylase covalently bound to arylaminosilane derivative of porous glass with nitrous acid as a coupling agent [Arylamino-porous glass-CVB-aminoacylase]. The enzyme activities of such immobilized aminoacylases were 3.2~13.0 units/ml glass for the former and 1.9~6.8 units/ml glass for the latter. Especially, alkylamino porous glass-CVB-aminoacylase showed excellent stability at pH 6~9 and temperature below 50°C, and was able to be stored for more than six months without appreciable loss of the activity.

The continuous enzyme reaction using the alkylamino porous glass-CVB-aminoacylase packed in a column was operated for 54 days at 37°C, and the half-life of the immobilized enzyme was calculated to be 78 days. From these results, it was recognized that such an immobilized aminoacylase on porous glass would be applicable in an industrial preparation of various l-amino acids from their dl-forms.     

Sucrose hydrolysis by thermostable immobilized inulinases from aspergillus ficuum

The possibility of using thermostable inulinases from Aspergillus ficuum in place of invertase for sucrose hydrolysis was explored. The commercial inulinases preparation was immobilized onto porous glass beads by covalent coupling using activation by a silane reagent and glutaraldehyde before adding the enzyme. The immobilization steps were optimized resulting in a support with 5,440 IU/g of support (sucrose hydrolysis) that is 77% of the activity of the free enzyme. Enzymatic properties of the immobilized inulinases were similar to those of the free enzymes with optimum pH near pH 5.0. However, temperature where the activity was maximal was shifted of 10 degrees C due to better thermal stability after immobilization with similar activation energies. The curve of the effect of sucrose concentration on activity was bi-phasic. The first part, for sucrose concentrations lower than 0.3 M, followed Michaelis-Menten kinetics with apparent K(M) and Vm only slightly affected by immobilization. Substrate inhibition was observed at values from 0.3 to 2 M sucrose. Complete sucrose hydrolysis was obtained for batch reactors with 0.3 and 1 M sucrose solutions. In continuous packed-bed reactor 100% (for 0.3 M sucrose), 90% (1 M sucrose) or 80% sucrose conversion were observed at space velocities of 0.06-0.25 h(-1). The operational half-life of the immobilized inulinases at 50 degrees C with 2 M sucrose was 350 days.     

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.     

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 10 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.     

Immobilization of chitosan-modified invertase on alginate-coated chitin support via polyelectrolyte complex formation

Saccharomyces cerevisiae invertase was chemically modified with chitosan and further immobilized on sodium alginate-coated chitin support. The yield of immobilized protein was determined as 85% and the enzyme retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 9 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was four-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 80% of the original catalytic activity after 50 h under continuous operational regime in a packed bed reactor.     

Kerase Immobilization by Covalent Attachment to Porous Glass

Kerase, a serine protease from Streptomyces fradiae, was immobilized on porous glass (SIKUG®) by covalent attachment, through amino groups on the enzyme. Modifications of four lysine residues (44·4% of the accessible or superficial amino groups) results in a loss of 6·5% of the enzymic activity. After immobilization, the optimal reaction pH changed from a range of 7·5-8·5 to 9-10. The immobilized protease was stable in a broad pH range, 6-12, while the soluble protease was irreversibly denaturated at alkaline pHs (pH>8). The optimal reaction temperature was displaced from 55 to 65°C, showing a higher thermal stability of the immobilized enzyme. Kerase immobilized onto porous glass was stable for at least 28 days, working in a repeated-batch process of three cycles per day, with an activity loss of 22·1 ± 3·1%.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

Characteristics of immobilized invertase

Five kinds of immobilized invertases (IMI)—covalently of porous glass and ion-exchange resins and ionically on ion-exchange resins—have been prepared and their kinetic characteristics for sucrose hydrolysis, such as K_m, K, pH profile, and thermal stability were studied. Comparing the values of K_m and activation energy and the entropy of IMI with those of native invertase, it was concluded that the immobilization influences not binding but kinetic specificity. The effects of the immobilization method on thermal stability were also discussed.     

Improvement in stability of an immobilized fungal laccase

Summary A laccase of the basidiomyceteTrametes versicolor was immobilized on porous glass beads that were activated with 3-aminopropyltriethoxysilane and glutaraldehyde. The support immobilized 100% of the enzyme, whereupon 90% of the original activity was retained. After immobilization, the enzyme was active in a wider pH and temperature range, and its heat stability and reuse were greatly improved compared to those of the free laccase. The immobilized enzyme was found reusable in treating different substrates, either recycled alone or in a sequential order.     

Characterization and immobilization of E. coli (ATCC-26) Beta-galactosidase

The β-galactosidase from Escherichia coli ATTCC-26 was partially purified and characterized. It was found to be comparable to galactosidases from other E. coli strains in stability, pH and temperature maxima, and activity requirements, but it had a more favorable ratio of activity toward lactose versus synthetic substrates. The galactosidase was immobilized on porous glass beads by three covalent bonding methods. Kinetic data for the free and bound enzymes were determined using natural and synthetic substrates. Activity characteristics of the free and immobilized enzymes were comparable, however, the bound forms were less stable to heat.     

Immobilization of enzymes on magnetic particles

Trypsin (EC 3.4.4.4) was immobilized in low yield on aminoalkylsilylated magnetite (Fe₃O₄). Better results were obtained when trypsin was immobilized by crosslinking with glutaraldehyde on magnetite. The preparation contained 36 mg protein/g magnetite and the enzyme retained 46% and 11% of esterase and proteolytic activity. Immobilized trypsin was more heat stable than trypsin. Invertase (β-D -fructofuranoside fructohydrolase, EC 3.2.1.26) was cross-linked on magnetite with glutaraldehyde in low yield due to the inactivation of the enzyme. However in the presence of 1% sucrose, the total activity recovered was 79% of the initial activity and the preparation contained 4.4 mg/g of active invertase. Immobilized invertase was less active than invertase when acting on oligosaccharides of the raffinose family. The immobilized enzymes could be easily recovered, from solutions or suspensions, magnetically.