Yeast alcohol dehydrogenase was immobilized on weakly
basic macroporous anion exchange resin beads Lewatit MP-64.
After the adsorption the enzyme was crosslinked by glutaraldehyde:
The activity of the immobilized enzyme was investigated
in the pH 8.9 recirculation reactor system at 303 K. It was found
that the immobilized enzyme was destabilized upon addition of
semicarbazide hydrochloride to the buffer solution.
A greater amount of protein was attached to the support when
ethanol was present in the enzyme solution, but the activity of
the bound enzyme was lower than in the absence of ethanol

Đurđa Vasić-Rački

English

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CROATICA CHEMICA ACTA CCACAA 57 (2) 305-311 (1984) CCA-1445 YU ISSN 0011-1643 UDC 547/577.15 Original Scientific Paper Immobilization of Yeast Alcohol Dehydrogenase on Weakly Basic Anion Exchange Resin Beads fJuraa V asic-Racki Faculty of Technology, The University of Zagreb, Zagreb, Croatia, Yugoslavia Received October 13, 1983 Yeast alcohol dehydrogenase was immobilized on weakly basic macroporous anion exchange resin beads Lewatit MP-64. After the adsorption the enzyme was crosslinked by glutaraldehyde: The activity of the immobilized enzyme was investigated in the pH 8.9 recirculation reactor system at 303 K. It was found that the immobilized enzyme was destabilized upon addition of semicarbazide hydrochloride to the buffer solution. A greater amount of protein was attached to the support when ethanol was present in the enzyme solution, but the activity of the bound enzyme was lower than in the absence of ethanol. INTRODUCTION Alcohol dehydrogenase from yeast (YADH) has been previously immobili-zed on supports such as s-triazinyl DEAE cellulose,1 silanized aluminia,2 s-tri-azinyl resin,3 derivatives of agarose,4 Enzyacryl T-10, amberlite IR-resin, porous glass5 and polymethylmethacrylate.6 In order to immobilize the enzyme on amberlite IR-45 resin the glutaraldehyde-linkage and azo-linkage methods were employed.5 In this work, in distincUon from the previously mentioned methods, the immobilization of YADH on macroporous weakly basic anion exchange resin Lewatit MP-64 was performed by adsorption after which the enzyme was crosslinked with glutaraldehyde. This procedure was suggested by Haynes and Walsh7 for the immobilization of bovine trypsin onto colloidal silica particles. The aim of this research was to compare the storage stability of the soluble and bound enzyme as well as to study the factors affecting the stability of the bound enzyme including the effect of semicarbazide hydrochloride in the buffer solution. EXPERIMENTAL Materials and Methods Yeast alcohol dehydrogenase (YADH)-lyophilized, specific activity of ca. 400 U/mg enzyme protein (25 °C, ethanol as subsfrate), and ~-nicotin-amide-dinucleotide (NAD+) free acid, grade II, 98°/o, were purchased from Boehringer Mannheim, Ger-many. Lewatit MP-64 macroporous weakly basic anion exchange resin in OH-form was obtained from Bayer, Germany. It is a polystyrene crosslinked with the divinyl--benzene, having the polar groups of type -- N(R3)'. Glut araldehyde 25°/o solution, abs. ethanol and sodium pyrophosphate (N4P20 7 • 10 H 20) were purchased from Merck, Germany, and glycine from Carlo Erba, Italy. 306 D. V ASIC-RACKI Preparation of the Immobilized Enzyme For the immobilization of Y ADH the static procedure8 was used as a repre-sentative process. A measured quantity (dry weight at 333 K) of anion exchange resin beads was loaded into a column of 0.5 cm internal diameter. They were then soaked overnight in the standard buffer solution pH 8.9 (0.05 mol dm-3 sodium pyrophosphate and 0.05 mol dm-3 glycine). The enzyme solution (a measured weight of lyophilized enzyme in standard buffer solution) was added to the anion exchanger and it was slowly discharged frorr1 the column after half an hpur. The resin beads were then washed with the standard buffer until A2sonm of the effluent reached 0. After that the exchange resin beads with immobilized YADH were treated with glutaraldehyde for 5 minutes. Excess glutaraldehyde was then removed by washing the column with buffer. The protein content was estimated by spectral absorption at 280 nm of YADH with a molar absorption coefficient0 of 1.89 x 105 cm2 mo1-1 and molecular weight of 150 000. The amount of the protein bound on the anion exchange resin beads was calculated as the difference between the amount allded to the column and that found in solution after completion of the loading process and washing procedures. Kinetic Assays The kinetics of ethanol oxidation were determined by recording the rate of formation of NADH at 340 nm in a recirculation reactor system which was similar to that described by J . R. Ford.10 The initial rate of ethanol oxidation was calculated from the linear change of Aa4on m during the first two minutes of the reaction, by using the molar extinction coefficient for NADH11 6.22 cm2 mo1-1. Each reaction was repeated three times under the same conditions and the mean value was calculated. The experimental error was estimated to be 10°/o. For a typical assay the final volume was 21 cm3• The activity of free YADH was determined in the same reactor system. RESULTS AND DISCUSSION The activity of the immobilized YADH was determined when YADH was supported on the beads of Lewatit MP-64 with particle diameter of 0.2 mm. This diameter was chosen for the reason that internal diffusional resistance is negligible12 under these conditions. The activity of the immobilized YADH was 17-280/o of that of the soluble enzyme. J . R. Wykes' reported it was as 12.5-150/o and W . Schopp13 as 10.0-15.00/o. The Quantities of the Attached Proteins The quantity of the attached proteins on the 0.1 g (dry weight) anion exchanger with particle diameter of 0.8 mm depends on the enzyme concen-tration in the standard buffer solution (Table I). The support is saturated at an enzyme concentration of = 0.7 mg enzyme cm-3 standard buffer solution. The quantity of attached proteins decreases with the increasing particle size dP (Table II) . This can be expected since the enzyme adsorbs mainly onto the external surface of the anion exchange resin beads. Lewatit MP-64 has a higher capacity for YADH than previously used supports. 1,5 ,14 This higher capacity can be ascribed to strong polar ion exchange groups present in Lewatit MP-64. IMMOBILIZATION OF YEAST ALCOHOL DEHYDROGENASE 307 TABLE I The Effect of Enzyme Concentration on the Adsorption of Y ADH on Lewatit MP-64 YADH concentration mg/cm3 0.132 0.395 0.591 0.745 0.903 1.098 Amount of attached proteins* mg/g dry support 0.62 ± 0.033 0.80 ± 0.035 2.24 ± 0.042 2.82 ± 0.092 2.95 ± 0.049 2.87 ± 0.057 * The values given for the amounts of the attached proteins correspond to the arithmetic mean ± standard deviation of all measurements performed in five experiments. TABLE II The Effect of Support Particle Size on the Adsorption of Y ADH. The Initial Enzyme Concentration in Buffer was 0.6 mg/cm3 Support particle size mm 0.2 0.2-0.3 0.3-0.4 0.4-0.5 0.5--0.6 0.6-0.8 Amounts of attached proteins* mgl g dry support 12.57 ± 1.032 6.68 ± 0.077 5.59 ± 0.141 4.94 ± 0.021 4.68 ± 0.156 3.03 ± 0.146 * The values given correspond to the arithmetic mean ± standard deviation of all measurements performed in five experiments. The Storage Stability Stability of the adsorbed enzyme was checked by storing both soluble YADH without any stabilizing agent and immobilized enzyme at +4 °c. As shown in Figure 1. the immobilized enzyme loses activity about twice as slowly as soluble YADH. The Effect of Glutaraldehyde Treatment on the Activity and Stability of Immobilized Y ADH The effect of treatment of adsorbed YADH with various concentrations of glutaraldehyde for 5 minutes at 303 K is shown in Table III. Since higher concentrations of glutaraldehyde lower the enzyme activity, a 0.20/o solution was routinely used to cross-link the enzyme to Lewatit MP-64. This treatment results in a marked increase in the stability of adsorbed YADH in repeated runs of the same column (Figure 2). 308 D. VASIC-RACKI 100 'c!2. >: > -0 ct! 4> > - 50 ct! 4> .... 1 2 3 4 5 6 7 storage time~ Figure 1. Storage stability of yeast alcohol dehydrogenase. Soluble enzyme (0 ) solution 2.5 X 10·• mo! m -• in standard buffer pH 8.9 and immobilized enzyme <•> having 2.78 mg protein/g dry support were stored at + 4 •C and assayed at 303 K . The initial coenzyme and ethanol con-centrations were 1.26 X 10-• mo! dm-• and 0.5 mo! dm·', respectively. TABLE III The Effect of Glutaraldehyde Treatment on the Observed Activity of Immobilized Y ADH* Glutaraldehyde solution wt 0/o 0.1 0.2 0.3 0.6 0.8 LO Specific immobilized enzyme* activity U/mg attached protein 179.1 ± 1.82 181.7 ± 2.67 160.6 ± 3.92 116.7 ± 4.10 77 .8 ± 2.75 29.3 ± 1.04 • The particle size of the support was 0.8 mm. The bound enzyme contained 3.04 mg protein/g dry support. The activity of the immobilized enzyme was assayed at pH 8.9 in standard buffer and temperature 303 K. The initial concentrations of coenzyme and ethanol were 2.5 X· l0-4 mol dm·• and 0.5 mo! dm·• respectively. The values given correspond to the arithmetic mean ± standard deviation of all measurements performed in three runs. The Effect of Semicarbazide Hydrochloride on the Stability of the Immobilized Y ADH According K. Watanabe et al.3 the conversion of ethanol with immobilized YADH increases when semicarbazide hydrochloride is present in the buffer solution. To investigate this effect on the Lewatit bound YADH, the kinetics of ethanol oxidation were measured in standard buffer solution containing 0.09 mol dm-3 semicarbazide hydrochloride. The initial concentration of NAD+ IMMOBILIZATION OF YEAST ALCOHOL DEHYDROGENASE 309 was 1.26 X 10-4 mol dm-3 and of ethanol 0.5 mol dm-3• As shown in Figure 2., the presence of semicarbazide has no influence on the stability of YADH bound to Lewatit MP-64 in the absence of g1utaraldehyde. On the other hand the activity of the bound enzyme cross-linked by glutaraldehyde decreases markedly. Hence, contrary to the findings of K. Watanabe et al.3, semicarbazidt-cannot be recommended in the case of Lewatit MP-64 - bound enzyme. 100 ~ >--> -" Cll G> > -50 Cll G> .. 3 4 5 6 number of runs Figure 2. The '.effect of glutaraldehyde and semicarbazide hydrochloride on the stability of immobilized Y ADH. (0 ) - enzyme adsorbed on Lewatit MP-64 {2.87 mg protein/g dry support) and assayed in standard buffer pH 8.9 without and containing 0.09 mol dm-• semicarbazide hydrochloride, respectively; identical values were obtained in •both experiments. c•> - adsorbed enzyme (3 .18 mg proteing/g dry support) was treated with 0.2°/o glutaraldehyde for 5 min and assayed in standard buffer pH 8.9. (()) - adsorbed enzyme (3.07 mg protein/g dry support) was treated with 0.2' /o glutaraldehyde and assayed in buffer containing 0.09 mol dm-• semicarbazide hydrochloride. All assays were performed at 303 K with initial coenzyme and ethanol concentrations 1.26 X 10-• mol dm-s and 0.5 mol dm-•, respectively. The Effect of Ethanol on the Immobilization of Y ADH When the immobilization procedure was carried out in the presence of ethanol (0.5 cm3 of abs. ethanol/2.0 mg enzyme dissulved in the standard buffer as usual), 12.5 mg proteins/g dry support were attached to the anion, exchange resin with particle diameter 0.5 mm. The specific activity of the bound enzyme was 88.5 ± 3.13 U/mg attached proteins. Without ethanol, only 4.94 mg proteins/g dry support were attached. In this case the specific activity · of the bound enzyme was 166.1 ± 8.32 U/mg attached proteins. In both cases the enzyme was assayed in the standard buffer at pH 8.9 and 303 K. The initial ethanol and coenzyme concentrations were 0.5 mol dm-3 and 1.26 X 10-4 mol dm-3, respectively. Lower specific activity of the enzyme bound to the resin in the presence of ethanol can be due to enzyme-enzyme interactions, which are more frequent 310 D. V ASIC-RACKI at a higher number of enzyme molecules bound to the same surface of the resin beads. Lineweaver-Burk Plot A Lineweaver-Burk plot of reciprocal initial velocity versus reciprocal NAD+ concentrations for immobilized YADH on macroporous exchange resin departs from linearity showing a slightly concave form (Figure 3.). A similar departure from linearity for soluble YADH and ethanol as a substrate was observed by A. P. Nygaard15 and explained by the change of the dielectric constant of the reaction medium. However, the effect observed with the immobilized enzyme and NAD+ requires another explanation. Cll C'? 60 E 50 ... I -~ 40 ~ l 30 > 20 0 10 20 Figure 3. Lineweaver-Burk plot for immobilized YADH. The initial rates v', were measured in pyrophosphate glycine buffer, pH 8.9 at temperature 303 K. The initial ethanol concentration was 0.5 mol dm·•. The coenzyme concentrations ranged from 2.5 x 10-• mol dm-• to 1.26 x 10-•. mol dm·•. The bound enzyme contained 3.05 mg proteing/g dry support. CONCLUSIONS 1. The macroporous weakly basic anion exchange resin has a high capacity for yeast alcohol degydrogenase. 2. Cross-linking of YADH by glutaraldehyde increases the stability of the immobilized enzyme. 3. Semicarbazide hydrochloride acts as a destabilizing agent for immobi-lized YADH. 4. The presence of ethanol in the enzyme solution increases the quantity of attached proteins, but the activity of the immobilized enzyme does not increase proportionally with the amount of attached protein. IMMOBILIZATION OF YEAST ALCOHOL DEHYDROGENASE 311 REFERENCES 1. J. R. Wykes, P . Dunn i 11, and M. D. Li 11 y, Biotech. Bioeng. 17 (1975) 51. 2. R. W. Cough 1 in, M. Ai z aw a, B. F . A 1 ex and er, and M. Ch a r 1 es, Biotech. Bioeng. 17 (1975) 515. 3. K . W a t a n a b e, K. K i mu r a, H. M a r u m o, and H. S a m e j i m a , Agric. Biol. Chem. 41 (1977) 5467. 4. S . B arr y, T. Griffin, and D. B. Johns on, Int. J. Biochem. 9 (1978) 289. 5. M. J. Brougham and D. B. Johnson, Int. J . Biochem. 9 (1978) 283. 6. J . R. Mi 11 is and L. B. Wingard, Biotech. Bioeng. 23 (1981) 965. 7. R. Haynes and K . A. W a 1 sh, Biochim. Biophys. Res. Comm. 36 (1969) 235. 8. R. A. Mes s i rig, Adv. Biochem. Eng. 10 (1978) 52. 9. J . E. Hayes and S. F. Ve 1 i ck, J. Biol. Chem. 207 (1954) 225. 10. J. R. Ford, A.H. Lambert, W. Cohen, and R. P . Chambers, Biotech. Bioeng. Symp. No. 3, (1972) 267. 11. B. L. Hore ck er and A. Korber g, J . Biol. Chem. 175 (1948) 385. 12. D. Vasi c - Racki, E. Pa j c, and J. Nov a k, Proc. A. I. Y. Chem. Eng. Conf., Vol. II, Graz, 1982, 683. 13. W. Schopp, J. Thyfronitou, and H. Aurich, Acta Biol. Med. Germ. 35 (1976) 1443. 14. M. Ai z aw a, Biotech. Bioeng. 17 (1975) 1369. 15. A. P . Nygaard and H. Theo re 11, Acta Chem. Scanq. 9 (1955) 1551. SAZETAK Imobilizacija alkoholne dehidrogenaze kvasca na slabobaziean anionski izmjenjivac f>. Vasic-Racki Alkoholna dehidrogenaza kvasca je imobilizirana na slabo baziean makropo-rozan anionski izmjenjivac Lewatit MP-64. Nakon adsorpcije na izmjenjivac, enzim je na njegovoj povrsini umrezen s glutaraldehidom. Aktivnost imobiliziranog enzima je ispitivana u reaktoru s recirkulacijom kod pH 8.9 i na temperaturi 303 K. Pronadeno je da se imobilizirani enzim destabilizira u prisustvu semikarbazid-hidroklorida u otopini pufera. Uz etanol u enzimskoj otopini veea kolicina proteina se veze na nosilac, ali je aktivnost tako imobiliziranog enzima manja. 

Immobilization of Yeast Alcohol Dehydrogenase on Weakly Basic Anion Exchange Resin Beads

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