Characterizaton and Immobilization of Dextranase from Bacillus Species Using Different Synthetic and Non-Synthetic Polymers

Hydrothermal springs are renowned as ideal ecological niche for wide species of thermophilic microbes. Thermozymes of thermophiles have enticed commercial application owing to their stability against inimical industrial approaches. Current study is designed to explore hot springs of Gilgit, Pakistan for the isolation of thermophile capable of degrading complex polymer dextran. Dextranase not only plays a vital role in sugar processing industry but the enzymatic fractions of polymer are also of significant interest in cosmetics, pharmaceutical and food industries. Besides their vast commercial application, thermostable dextranase from non-toxin producing microbe is still a dilemma in food processing industry in order to develop an efficient and cost effective process. Therefore, efforts are being made to explore new source of thermostable dextranase.Bacillus megaterium KIBGE-IB31 [GenBank accession: KF241867] was identified as thermophile accountable for enhanced production of valuable extracellular dextranase under specific fermentation conditions. Strain characterization was based on phenotypic and genotypic analysis. The distinctive feature of this strain classified it as a GRAS and aerobic thermophile. Maximum production of 464.68 U ml-1 with a specific activity of 160.2 U mg-1 was achieved when 10.0 kDa dextran (15.0 g L-1) was amalgamated with various trace elements and nitrogen sources at 60°C up to 24 hours of fermentation time. Inoculum size and agitation speed also had a positive impact on maximum enzyme yield. Gradient precipitation resulted in 61% recovery of partially purified dextranase from crude sample. Whilst, steady state kinetics of dextranase exhibited high catalytic activity within 5.0 minutes at 50°C in 50 mM sodium phosphate buffer of pH: 07 with Vmaxand Km value of 5191 U ml-1 min-1 and 4.38 mg ml-1 respectively. The behavior of different metal ions revealed activating as well as inhibitory action on the catalytic performance of dextranase however, none of the metal ion was found to be essential for enzymatic reaction. Similar findings was observed with surfactant and solvents. SDS (10 mM) was found to be strong inhibitor while triton X-100 (10 mM) decreased 77% activity of dextranase. Data of stability studies proved thermophilic origin of dextranase as it showed stability against wide range of temperature and pH. Hydrolytic action of dextranase was observed by studying surface topology of dextran through scanning electron microscopy. Thin layer chromatography suggested that the hydrolytic response resulted in the formation of various isomalto-oligosaccharides and these oligosaccharides could be utilized as commercially important prebiotics. Immobilization of dextranase using different interactions with different matrices, bestowed covalent cross linking as influential contact of dextranase with matrix in contrast, to adsorption and entrapment methods. However, cross linking without any matrix that is CLEAs was proven to be an efficient immobilization protocol in terms of stability, reusability as well as reduced the cost due to absence of any matrix. Although the reaction time after immobilization remains same except in entrapment but the temperature and pH optima was shifted after covalent cross linking in both protocols. Being a thermostable biocatalyst the stability of dextranase was high at extreme temperature and pH, but was further improved after immobilization. The recycling efficiency of dextranase in the forms of CLEAs was highest (10 cycles with 67% residual activity) than that of other three immobilized form.