Sibtain Ahmed

Molecular Cloning of Cellulase Genes from Trichoderma harzianum

Cellulases are attractive source for utilization of the agro-industrial waste materials. Exoglucanase (EC; 3.2.1.91), Endoglucanase (EC. 3.2.1.4) and βglucosidase (EC. 3.2.1.21) were isolated from Trichoderma harzianum (E-58 strain). The fungus was grown on Vogels medium with different carbon sources. Maximal production of the enzymes was achieved at 28 C, pH 5.5 under continuous shaking at 120 rpm for 5 days. Glucose repressed the synthesis of the enzymes whereas carboxymethylcellulose (CMC) produced the enzymes in substantial amounts. Maximum activity of cellulases (exoglucanase, endoglucanase and β-glucosidase) were found to be 2.764, 14.4 and 0.629IU mL, respectively. Corresponding genes for cellulases were isolated with the help of RTPCR. RNA was isolated from mycelia of T. harzianum grown on CMC and xylan. First strand of cDNA was synthesized using oligo dT (18) primer and subjected to PCR with specific primers. The amplified products were purified through agarose gel electrophoresis and ligated into SmaI site of pUC18. The plasmids containing exg, egl and bgl genes were transformed into E. coli for further characterization. INTRODUCTION Despite the fact that the world community is no longer pre-occupied with fossil fuel shortage, there is still a considerable research and development directed towards understanding and commercializing enzymatic hydrolysis of cellulose [1]. Cellulose is the most abundant organic polymer in this planet and is an important renewable energy source along with sugars and starches [2]. Energy production from cellulosic raw material involves its hydrolysis into glucose. Highly ordered cellulose substrates are converted into soluble sugars only when exoglucanase, endoglucanase and β-glucosidase are present in solution simultaneously in right proportion. A number of fungal species are known for the production of cellulases such as Aspergillus niger, Sporotrichum spp, Chaetomium thermophile, Trichoderma species etc [3]. Gene cloning is recently being employed for studying the structure and function of a number of enzymes and proteins and their over expression. This strategy has been found very efficient as compared to other traditional methods. Present study was designed to layout strategy for enhanced production of cellulases. In this paper we have reported the isolation and cloning of cellulase genes (exg ,egl and bgl) from T.harzianum (E-58 strain). MATERIALS AND METHODS Growth Conditions and Enzyme Assay Trichoderma harzianum (E58) was grown at 28C with shaking at 120 rpm in Vogels medium (0.5% Trisodium citrate, 0.5% KH2PO4.0.2% NH4NO3, 0.4% (NH4)2SO4, 0.02%

Optimization of antioxidant exopolysaccharidess production by Bacillus licheniformis in solid state fermentation

Response surface methodology was applied to optimize physical and nutritional variables for the production of antioxidant exopolysaccharidess (EPSs) by Bacillus licheniformis UD061 in solid state fermentation with squid processing byproduct and maize cob meal used as a carbon and nitrogen source and solid matrix. The factors noted with Plackett-Burman design for optimization of EPSs production were NaCl, MgSO4·7H2O, and moisture level. These factors were further optimized using Box-Behnken design and response surface methodology. Using this methodology, the quadratic regression model of EPSs production was built. Maximum EPSs production was obtained under the optimal conditions of 4.08 g L(-1) NaCl, 0.71 g L(-1) MgSO4·7H2O, and 60.49% moisture level. A production of 14.68 mg gds(-1), which was well in agreement with the predicted value, was achieved by this optimized procedure.

Corn stover-enhanced cellulase production by Aspergillus niger NRRL 567

High cost involved in micro propagation is a major constraint to its popular use in sugarcane (Saccharum spp. hybrid). This study describes two technologies for cost reduction in sugarcane micropropagation, that is, direct regeneration of complete plantlets on the same medium and substituting in vitro rooting by ex vitro rooting in conventional micro propagation. A protocol for one step regeneration of complete plantlets was developed for sugarcane cultivar CoS96268. Complete plantlets were regenerated in 42 days on regeneration medium using leaf disc explants, pretreated on MS medium supplemented with 3 mg/l 2,4-D for eight days. More than 95% explants exhibited regeneration with an average of 23 shoots per explants. After 42 days on regeneration medium, each explants produced 6 to 8 healthy plantlets which could be successfully hardened. A protocol for ex vitro rooting of micro shoots, raised from axillary bud culture was also standardized. In vitro shoots, 5 to 6 cm long, treated overnight with 20 mg/l NAA, led to formation of complete plantlets with more than 90% root induction. These plantlets possessed more than 6 roots of 4 cm average length per plantlet and exhibited 95% survival when transferred to polybags containing soil. Thus, direct adventitious regeneration and ex vitro rooting can be applied to sugarcane micro propagation to reduce cost of plant production.

Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing

Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings.