It’s known that actual problem of enzyme technology is keeping and increasing activity and stability of enzymes after immobilization process. The reason of the issue is limitation of enzyme activity by blocking functional active groups of catalytic site. It was reported [1] that high substrate concentration inhibits functional active groups of catalytic site.
The effectiveness of enzyme immobilization depends on the amount of enzyme that is precoupled into sorbent. It was reported [2] that activated carbon does not possess functional active groups on the surface to occur chemical reaction with others. Having familiar with this, it was required to reactivate activated carbon.
It was presented some enzyme immobilization approaches for the industrial purposes but some of them are quite applicable in water-organic media. The main reason of above mentioned problem is destruction of selected sorbent by chemical and biological factors, their high cost for installation or not representing stability and catalytic activity in scale up processes. The usefulness of the present method is possessing insoluble and non-destructive sorbent therefore keeping stability in watery and alcoholic media.
In the present work isolated and purified beta-fructofuranosidase from Saccharomyces vini used as an object of immobilization.
Materials and methods
Enzymes, chemicals and instruments. -fructofuranosidase obtained [3] from Saccharomyces vini Rkaciteli-6 strain. Glutaraldehyde (MERCK, Darmstadt, Germany), Silufol UV, glucooxydase, sucrose, D-glucose, dimethylformamide, urea, ammonium sulfate, n-butanol, acetic acid, isoamylol [Reachim, Russia] and other chemicals purchased from local sources [Chemreactivecomplect, Uzbekistan] were analytical pure. Centrifuge SLR-1 UCh-2, magnetic circulator MM5, Ultrathermostate MTAKUTESZ TYPE-57 and Autoclave “Bergius-1L” used for the reactivation of activated carbon.
Isolation and purification. -fructofuranosidase was isolated by twice freezing and thaw method under liquid nitrogen and mechanical destroying yeast cell membrane with quartz sand. Enzyme extracted in 1:1 ratio with 0,1M acetate buffer (pH 5.0) for 1h at 40C. The supernatant used as an initial material for the preparation of -fructofuranosidase. Protein (enzyme) was purified 3,92 times with ammonium sulfate (70 %), gel-filtration and dialysis against distilled water. After dialysis enzyme preparation was dried by freeze drying method.
Reactivation of activated carbon. Reactivation of the sorbent performed by J. Bimer [2]. According to this approach urea was used for the amination of activated carbon. Reaction mixture contained 10 g activated carbon, 20 g urea in 100 ml dimethylformamide condition. Amination process occurred at 3000C for 1h in Bergius-1L autoclave to destruct C-C bonds of the sorbent.
Chemical modifying of activated carbon. After completing reactivation procedure 5 ml 0,1 M borate buffer (pH 8,0) added into activated carbon and modification reaction was conducted by adding 0,25 % vol. glutaraldehyde (80 mkl). The reaction mixture was mixed actively at room temperature for 2 h. Excess glutaraldehyde removed by washing with distilled water.
Immobilization of yeast -fructofuranosidase. Chemical modified activated carbon was replaced in 0,1M borate buffer (pH 8.0) in 50 % sucrose solution and mixture incubated for 24h at 40C in active mixing condition. Obtained immobilized invertase preparation washed with distilled water (3 times) and NaCl and MgCl2 solution respectively. In the end of process preparation washed with 1 % NaCl and MgCl2 in 20 % alcoholic solution and dried at 40C.
Results. After successfully cultivation obtained yeast biomass used as a source of enzyme and the enzyme showed 331 (unitmg) specific activity. In the result of purification process, obtained enzyme preparation dialyzed and dried in vacuum condition.
As above mentioned, activated carbon doesn’t possess surface functional active groups therefore it was required to reactivate the selected sorbent.
Reactivation reaction occurred in the presence of activated carbon (40g) and urea (40g) in dimethylformamide solution at 3000C for 2h under high atmospheric pressure (step 1). In the result of the reaction it was obtained aminated activated carbon. Chemical modification of the sorbent performed using 2 % glutaraldehyde (step 2).
Covalent immobilization of -fructofuranosidase carried out by coupling of amino groups of -fructofuranosidase and active group of glutaraldehyde (step 3). Covalent immobilization of the beta-fructofuranosidase carried out following optimal conditions: pH optimum 7.6, optimal concentration of bifunctional agent (glutaraldehyde) 0,25 % vol. and optimal enzyme concentration 1200 mkg enzyme for 1 gr sorbent. The immobilization reaction can be imagined by following reaction.
C-NH-CO-N=СН-(СН2)3-CНО + H2N-E C-NH-CO-N=СН-(СН2)3-СН=N-E
Obtained immobilized -fructofuranosidase showed its transferase activity in water-organic media and the preparation can be applied in food technology for the fusel oil biotransformation into alkylfructosides.
References:
1. Mirzarakhmetova D. T., Abdurazakova S. H. Obtaining immobilized preparation of β-fructofuranosidase in the presence of high concentration of substrate. Chemistry of Natural Compounds. 1998. #3. P. 343–345
2. Bimer J., Salbut -P.D., Berlozecki S. Modified active carbons from precursors enriched with nitrogen functions; sulfur removal capabilities // Fuel. 1998. -V.77. — #6. -P.519–525.
3. Dekhkonov D. B., Mirzarakhmetova D. T., Rakhimov M. M. Obtaining high actively yeast β-fructofuranosidase. Acta NUUz. Tashkent. 2004. Vol. 4. -P.3–4
