The xylanase gene (xynA) of Bacillus licheniformis 9945A was cloned and expressed in Escherichia coli BL21(DE3) using pET-22b(+) as an expression vector. The recombinant xylanase enzyme was purified by ammonium sulfate precipitation, followed by single-step immobilized metal ion affinity chromatography with a 57.58-fold purification having 138.2 U/mg specific activity and recovery of 70.08 %. Molecular weight of the purified xylanase, 23 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable for up to 70 °C with a broad pH range of 4-9 pH units. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA, indicating that the xylanase was a metalloenzyme. However, an addition of 1-4 % Tween 80, β-mercaptoethanol, and DTT resulted in the increase of enzyme activity by 51, 52, and 5 %, respectively. Organic solvents with a concentration of 10-40 % slightly decreased the enzyme activity. The xylanase enzyme possesses the ability of bioconversion of plant biomasses like wheat straw, rice straw, and sugarcane bagasse. Among the different tested biomasses, the highest saccharification percentage was observed with 1 % sugarcane bagasse after 72 h of incubation at 50 °C with 20 units of enzyme. The results suggest that recombinant xylanase can be used in the bioconversion of natural biomasses into simple sugars which could be further used for the production of biofuel.

Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose.

The production of an extracellular β-glucosidase by Aspergillus niger NRRL 599 was optimized using submerged fermentation technique. Effect of different media, different carbon sources, initial pH of the fermentation medium, temperature, incubation period and inoculum size on the production of β-glucosidase enzyme was investigated. A. niger NRRL 599 produced maximum extracellular β-glucosidase (4.48 U/mg) in Eggins and Pugh medium with 1% wheat bran (w/v) at pH 5.5 inoculated with 4% conidial suspension after 96 h of incubation at 30°C. Purified β-glucosidase gave K m and V max values of 3.11 mM and 20.83 U/mg respectively for pNPG hydrolysis. The enzyme was optimally active at pH 4.8 and at temperature of 60°C. Thermodynamic parameters, E a , ∆H and ∆S were found to be 52.17 KJ/mol, 49.90 J/mol.K and -71.69 KJ/mol, respectively. The pKa 1 and pKa 2 of ionizable groups of active site residues involved in V max were calculated to be 4.1 and 6.0 respectively.

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The present study is concerned with biosynthesis of Endo-1, 4-␤-glucanase by locally isolated strain of Trichoderma viride in submerged fermentation using shake flask. Cultural conditions were optimized for enhanced production of endoglucanase. Different fermentation media were examined and it was found that Mandel and Reese (1960) medium composed of 1.4 g (NH 4 ) 2 SO 4 , 2.0 g KH 2 PO 4 , 0.3 g urea, 0.3 g MgSO 4 ·7H 2 O, 0.0014 g ZnSO 4 ·7H 2 O, 0.005 g FeSO 4 ·7H 2 O, 0.0016 g MnSO 4 , 0.002 g CoCl 2 , 0.002 g CaCl 2 , 2.0 ml Tween-80 and 1.0 g polypeptone with wheat bran (1%) as carbon source gave better production of enzyme. Effect of incubation temp, pH, inoculum and fermentation time on endoglucanase production was also carried out. Optimal endoglucanase activity (2.25 U/ml/min) was achieved with 4% spore inoculum after 72 h of inoculation at 30 • C and initial pH 5.5. Enzyme produced was further characterized in terms of kinetic and thermodynamic parameters. K m and V max were determined using various transformations of Michaelis-Menten equation, i.e., Lineweaver-Burk plot, Eadie-Hofstee plot and Hanes-Wolff plot. It was found that Lineweaver-Burk plot gave more accurate K m and V max values of 0.46% and 2.20 U/ml/min respectively. The energy of activation Ea, enthalpy ( H) and entropy ( S) of activation were calculated using Arrhenius equation and found to be Ea = 53.56 kJ/mol, H = 51.29 kJ/mol, S = −9.68 J/mol/K respectively. Endo-1, 4-␤-glucanase produced was most active at 50 • C and pH 5.0 after reaction time of 30 min.