Stabilization of Raw Starch Digesting Amylase from Aspergillus Carbonarius and Effects of Metal Ions on Enzyme Activity and Stability

Abstract

Raw starch digesting amylase (RSDA) from Aspergillus carbonarius was purified by starch affinity chromatography and concentrated with (NH4)2SO4. RSDA was immobilized by adsorption on microbead (MB) silica gels; or on silica, chitosan, chitin, sepabeads, amberlite and agarose gels with glycidol (Gly), glutaraldehyde (G) or polyglutaraldehyde (PG) as activating/crosslinking agent. RSDA was covalently bound on Gly, PG or G activated beads or adsorbed on support prior to crosslinking (Seq X) or spontaneously crosslinked (Sp X). Out of 10 MB silica gels, MB 300A gave the highest immobilization yield (87.6%) and was used for further studies. To activate beads/gels 10-12% G was required while 1.5-3% was optimum for crosslinking. Duration of G or PG activation/crosslinking of beads/gel did not significantly influence immobilization yield (P>0.05). Immobilization efficiency as activity yield (U/g) was significantly influenced (P<0.05) by the concentration of RSDA offered and the duration of RSDA immobilization. pH of immobilization significantly influenced (P<0.05) activity yield on agarose and chitosan. Highest immobilization/activity yield during RSDA crosslinking/covalent binding was 100%/86.7% for PG-agarose, 85%/78.1% for PG-amberlite; 96.9%/86% for PG-sepabeads, 98.3%/89.4% for PG-silica, 95%/83.2% for G-chitosan and 62.5%/57.3% for Seq X-chitin. Optimum pH changed from 5 to 6-8 for PG-amberlite, 6-7 for G-sepabeads, 7 for Seq X-chitosan, chitin and G-amberlite. Optimum temperature changed from 30 oC to 40 oC for Gly-agarose, PG-agarose, seq X-silica, to 50 oC for Seq X and Sp X agarose, Sp X sepabeads and G-chitin and to 60 oC for PG and seq X-sepabeads, PG and seq X-chitin. Seq X-agarose only lost 3.5%, G-amberlite 8%, PG-sepa 1%, PG-silica 2%, PG-chitosan 6%, G-chitin 6.5% while free RSDA lost 50% activity after incubation at 60 oC for 180 min. Gly-agarose and Seq X-sepabeads retained 100% activity after 10 cycles. Immobilization led to alteration in the kinetic constants (Km and Vmax) of the RSDA. RSDA was also modified using 10, 15, 20 μl or mg acetic (AA), succinic (SA), citraconic (CA), maleic (MA) or phthalic (PA) anhydride and different polysaccharides (chitosan, carboxymethylcellulose [CMC], dextran, β-cyclodextrin). RSDA derivatives were named based on type and quantity of anhydride used. Highest number (17) of lysine residues was modified with 20 mg SA and lowest (3) with 10 μl CA. Optimum pH varied from 5 to 3 or 3-4 for all SA, AA, MA, CMC and dextran derivatives, and increased to 7-8 for CA and PA derivatives. Optimum pH for chitosan and β-cyclodextrin was same with native RSDA. The most pH stable derivatives were 10 SA, 15 AA, 15 CA, 20 MA, 10 PA and CMC. Optimum temperature varied based on type and concentration of the modifier and highest optimum temperature of 60 oC was obtained for 15 AA, chitosan and dextran modified RSDA. At 80 oC, most thermostable were 10 SA, 15 AA, 10 CA, 20 MA, 20 PA, β-cyclodextrin and dextran. Probing the environment of tryptophan by fluorescence spectra confirmed changes in the secondary structure of the RSDA. Inactivated RSDA was activated with 5 mM Co2+ stabilized with Mn2+ ions. Km of reactivated RSDA and native RSDA were 0.18 and 0.30 mg/ml, respectively.