Engineering Of Microbial (fungal) Glucoamylases By Chemical Modification Techneaques For Industrial Application

Glucoamylase (exo-1,4-α-D-glucan glucanohydrolase: EC 3.2.1.3) yields s-Dglucose from the non-reducing chain ends of a wide range of polysaccharide substrates and play a tremendous role in the improvement of starch based food products. Non-traditional use of glucoamylases in detergent industry has a great scope of expansion the world over in the coming days.Industrial employment of glucoamylase demands its hardiness to harsh operational environment.Thermo-stable enzymes can withstand extreme environmental conditions and can resist against detergents, organic solvents, temperature & pH.The operational stability of enzymes is of paramount importance for any bioprocess, which can be improved through various protein engineering techniques.With the development of novel procedures that exploit selective and efficient protein chemistry, chemical modification, either alone or combination with other mutagenesis techniques, could make a significant contribution to the development of enzymes that cope with the industrial demands.Various fungal strains (A. niger, A.oryzae, A.tamarii, A.fumigatus, A.flavus and Humicola sp) were grown in submerged cultivation at different temperatures, pH and substrate concentrations in order to optimize GA production.A.niger and Humicola sp were selected for their higher GA production ability as compared to other fungi.The apparent molecular mass of Humicola sp and A.niger GAs determined through SDS-PAGE were 72.8 and 93 kDa.A.niger GA was proved to be much better as compared to Humicola sp GA on the basis of, wider pH range, higher optimum temperature, more turn over (kcat) and higher specificity constant.It also showed 10 fold higher resistance against thermal unfolding and an activation trend against α- chymotrypsin A. niger was decided to be improved through γ-rays treatment with the main objective to enhance the production, stability and function of the enzyme. The 1 kGy γ-rays exposed mutant (M-7) of A.niger was selected on the basis of deoxy-Dglucose resistance.The mutant M-7 was about two fold efficient for extracellular GA production in submerged cultivation on Vogel’s medium containing 4% (w/v) wheat bran, pH 5.0 at 30 oC. The mutant GA had same molecular mass on SDS-PAGE, however on MALDI-TOF a difference of about 814 Da was observed.The mutant GA proved to be two fold efficient for hydrolysis of soluble starch and was thermodynamically more stable at higher temperatures (60-70 oC). The mutant GA was further engineered with pyrrole and sulfanilamide nucleophiles in the presence of EDC.The additional aromatic interactions due to pyrrole and sulfanilamide coupling resulted into decrease in the activation energy and increase in the specificity towards soluble starch hydrolysis of A.niger GA.Sulfanilamide broadened the pH range and increased optimum temperature from 60 to 65 oC, while pyrrole improved the thermostability of GA towards higher temperatures (60-66 oC). Pyrrole and sulfanilamide also improved the resistance of the GA towards α-chymotrypsin, while urea had detrimental effect. Mn+2 increased the optimum temperature of apo GA by 5 oC, while Ca+2 remained unaffected.Higher concentrations of Ca+2 and Mn+2 decreased the activation energy of GA resulting efficient formation of ES*-complex as compared to apo-GA. Both Ca+2 and Mn+2 increased the catalytic properties of GA by increasing kcat.The resistance of Ca+2- and Mn+2- bound GAs against unfolding at temp 50-55 oC was improved.The activation mechanism of A.niger GA by Ca+2 and Mn+2 was of partially competitive and both of the metals liked to bind with the ES-complex rather than free enzyme.