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Thermostable enzymes are currently being investigated to improve industrial processes of starch saccharification. A novel glucoamylase was purified to electrophoretic homogeneity from the culture supernatant of Fusarium solani on a fast protein liquid chromatographic system (FPLC). The recovery of glucoamylase after gel filtration on FPLC was 31.8% with 26.2-fold increase in specific activity. The enzyme had a molecular mass of 40 kDa by SDS-PAGE and 41 kDa by gel filtration. The glucoamylase exhibited optimum activity at pH 4.5. The Kcat and Km were 441/min and 1.9 mg/ml, respectively, for soluble starch, specificity constant (Kcat/Km) was 232. The enzyme was thermally stable at 50 °C and retained 79% activity after 60 min at this temperature. The half-life of the enzyme was 26 min at 60°C. The enzyme was slightly stimulated by Cu2+ and Mg2+ and strongly inhibited by Hg2+, Pb2+, Zn2+, Ni2+ and Fe3+.

Two sensitive, high-resolution and exceedingly versatile methods for the detection of isoenzymes of polymer-degrading enzymes on high-resolution, alkaline, cold, in situ-native (HiRACIN)-PAGE and high-resolution in situ-inhibited, native (HiRISIN)-PAGE are described. Extracellular crude extracts containing xylanases and carboxymethylcellulases from Scopulariopsis sp. and glucoamylases from Aspergillus niger were subjected to non-denaturing PAGE containing substrates in the resolving gel. In case of HiRACIN-PAGE, the enzymes were prevented from degrading their respective substrates during run by carrying out electrophoresis at 4 °C and the pH of running and resolving gel buffer systems were increased from 8.5 to 10.6. In case of HiRISIN-PAGE, adding competitive inhibitor of the enzyme, cellobiose, in the resolving gel prevents the degradation of polymer during the run. These techniques were successfully applied, for the first time, to visualize four, three and four sharp and distinct bands of xylanases, glucoamylases and CMCases, respectively.

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.

The carboxyl groups of purified carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 were modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). The half-lives of GAM15 at different temperatures were significantly enhanced whereas those of GAM75 were reduced as compared with the native CMCase. The activation energies of denaturation of native, GAM15 and GAM75 were 40, 35 and 59kJ mol respectively. Native CMCase and GAM15 showed no compensation effect, whereas native and GAM75 gave temperature of compensation of 44iC. Gibb's free energy of activation for denaturation (DG*) of GAM15 was increased as compared with native CMCase. Surprisingly the entropies (DS*) of activation for denaturation were negative for native and GAM75 and decreased further for GAM15 between the temperature range of 45 to 65iC. A possible explanation for the thermal inactivation of native and increased thermal stability of GAM15 is also discussed.

Solid-state cultivation of Fusarium solani was carried out for enhanced production of glucoamylase (GA) using different substrates like wheat bran, rice bran, green gram bran, black gram bran and maize bran. The SSF medium containing wheat bran as a substrate yielded the highest enzyme activity. The physical and chemical parameters were optimized. Maximum enzyme activity (61.35±3.69) U/g of dry wheat bran was achieved under optimum growth conditions. The optimum conditions were fructose as carbon and energy additive 1 % (by mass), urea as nitrogen additive 1 % (by mass), initial moisture content of solid substrate 70 % (by mass per volume), incubation period 96 h, inoculum size 15 % (by mass per volume) having 10^6–10^7 spores/mL, incubation temperature (35±1) °C and pH=5.0. It was further observed that the addition of surfactants caused a decrease in enzyme biosynthesis by F. solani in SSF of wheat bran under optimum process conditions., Uzgojem gljive Fusarium solani fermentacijom na krutoj podlozi povećana je proizvodnja glukoamilaze (GA) primjenom različitih supstrata kao što su pšenične i rižine posije, te posije zelene soje, crne soje i kukuruza. Najveća aktivnost enzima dobivena je uporabom podloge od pšeničnih posija. Optimirani su fizikalni i kemijski parametri i pri optimalnim uvjetima rasta postignuta je maksimalna aktivnost enzima od (61,35±3,69) U/g suhih pšeničnih posija. Optimalni uvjeti bili su: 1 % (m/V) fruktoze kao izvora ugljika i energije, 1 % (m/m) uree kao izvora dušika, 70 % (m/V) početnog udjela vlage u krutoj podlozi, vrijeme inkubacije u trajanju od 96 h, 15 % inokuluma (m/V) s 10^6-10^7 spora/mL, temperatura inkubacije (35±1) °C i pH=5,0. Dodatkom površinski aktivnih tvari smanjila se biosinteza enzima proizvedenog fermentacijom pri optimalnim uvjetima na podlozi od pšeničnih posija s pomoću gljive F. solani.

Carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 was purified by a combination of ammonium sulphate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography on FPLC with 9-folds increase in specific activity. Native and subunit molecular weights were found to be 36 kDa each. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). Similarly, the enzyme was modified by EDC in the presence of ethylenediamine dihydrochloride plus cellobiose for 75 min (EDAM75). The neutralization (GAM15 and GAM75) and reversal (EDAM75) of negative charges of carboxyl groups of CMCase had profound effect on the specificity constant (k cat / K m ), pH optima, pK a ’s of the active-site residues and thermodynamic parameters of activation. The specificity constants of native, GAM15, GAM75, and EDAM75 were 143, 340, 804, and 48, respectively. The enthalpy of activation (ΔH # ) of Carboxymethylcellulose (CMC) hydrolysis of native (50 and 15 kJ mol −1 ) and GAM15 (41 and 16 kJ mol −1 ) were biphasic whereas those of GAM75 (43 kJ mol −1 ) and EDAM75 (41 k J mol −1 ) were monophasic. Similarly, the entropy of activation (ΔS # ) of CMC hydrolysis of native (−61 and −173 J mol −1 K −1 ) and GAM15 (−91 and −171 J mol −1 K −1 ) were biphasic whereas those of GAM75 (−82 J mol −1 K −1 ) and EDAM75 (−106 J mol −1 K −1 ) were monophasic. The pH optima/pK a ’s of both acidic and basic limbs of charge neutralized CMCases increased compared with those of native enzyme. The CMCase modification in the presence of glycinamide and absence of cellobiose at different pH’s periodically activated and inhibited the enzyme activity indicating conformational changes. We believe that the alteration of the surface charges resulted in gross movement of loops that surround the catalytic pocket, thereby inducing changes in the vicinity of active site residues with concomitant alteration in kinetic and thermodynamic properties of the modified CMCases.