Characterization of Matrix Bound (Immobilized) Thermostable Amyloglucosidase from Aspergillus fumigatus

Abstract

Exploration of new microbial organisms from diverse environment and different geographical locations has turned pivotal for the production of industrially important extracellular enzymes. Amyloglucosidase is an important hydrolase that is used in various industries for the bioconversion of starch. Current study is an attempt to produce higher titers of amyloglucosidase from indigenously isolated filamentous fungi. Preliminary screening showed that maximum amyloglucosidase production was achieved after optimization of conditions for Aspergillus fumigatus KIBGE-IB33 which was identified on the basis of molecular phylogeny and taxonomy. The cultivation of isolate and production of amyloglucosidase was enhanced by manipulating different parameters and maximum yield was attained at 30°C in starch containing medium (pH-7.0) after 04 days of fermentation. In case of chemical parameters, potato starch (10.0gL-1), yeast extract (10.0gL-1) and peptone (5.0gL-1) were found to be suitable carbon and nitrogen sources. Gradient precipitation method was performed for partial purification of amyloglucosidase which resulted in approximately 6.3 times purification. The kinetic properties of amyloglucosidase suggested that maximum catalysis of starch was observed in 50 mM citrate buffer of pH-5.0 at 60°C after 05 minutes with Vmax and Km values of 947 kU mg-1 and 1.417 mg ml-1, respectively. Amyloglucosidase retained approximately 50% of its activity when exposed at 60°C after 04 hours suggesting its thermostable nature. Storage of amyloglucosidase at 37°C and 4°C showed 30% and 62% of residual activity after 40 days. Na+, K+ and Ca+2 enhanced the activity of amyloglucosidase while, Cu2+, Fe2+, Hg2+, Ni2+, Zn2+ and Al3+ were found to be the inhibitors of this enzyme. Isopropanol (10 mM) was observed as an activator while, 100 mM concentration of DMSO, chloroform and formaldehyde acted as inhibitors of amyloglucosidase after 02 hours. Nonionic detergents (tween-80 and triton-X100) showed no effect on catalytic activity of enzyme however, anionic detergents (EDTA and SDS) exhibited negative effect on its activity. Native-PAGE and In situ electrophoresis revealed that the apparent molecular weight of amyloglucosidase was approximately 175 kDa. End product of amyloglucosidase was analyzed using thin layer chromatography. Amyloglucosidase was further immobilized using different strategies. Among them, the carrier free cross linking proved to be the most suitable condition for immobilization of amyloglucosidase with percent recovery of 94% followed by chitosan (85%) > agar-agar (80%) > alginate (66%). Catalytic efficiency of amyloglucosidase was slightly changed after immobilization as pH of amyloglucosidase shifts from pH-5.0 to 6.0 in case of alginate and CLEAs while, temperature optima increased 5 degrees from 60°C to 65°C in all except in case of alginate. Activation energy decreased after immobilization due to which stability of amyloglucosidase increased at higher temperatures for longer time period as compared to soluble enzyme. Kinetic behavior (Km and Vmax) of enzyme also changed in carrier bound strategies due to mass transfer limitations whereas, no effect was observed in CLEAs. Results of recycling studies showed that covalently bounded amyloglucosidase retained more enzymatic activities even after 15 cycles as compared to the entrapped enzyme that lost their activities within 10 cycles. In a nut shell, current research demonstrates successful improvement in the kinetic behavior as well as stability of immobilized amyloglucosidase as compared to soluble enzyme. Thus it can be anticipated that immobilized amyloglucosidase can be used in different industrial sectors.