National Institute for Interdisciplinary Science & Technology - CSIR, Cochin University of Science and Technology, Cochin 682022, INDIA.
Beta- Glucosidases (BGLs) are enzymes that hydrolyze aryl beta-glucosides and/or cellobiose and cello-oligosaccharides. In cellulolytic fungi, BGL completes the final step in cellulose hydrolysis by converting cellobiose to glucose. In several fungi, especially Trichoderma reesei the best known cellulase producer, the enzyme is produced in lower quantities compared to the other two classes of cellulases, and it is slow acting, making BGL the rate limiting component in cellulose hydrolysis. The less abundance of BGL even under conditions of cellulase induction and the product inhibition to which it is susceptible, limits the use of native cellulase preparations in lignocellulosic biomass hydrolysis for alcohol production.
Glucose tolerant BGL can circumvent the problem of feedback inhibition, and if available in an enzyme cocktail for biomass hydrolysis can improve the efficiency of hydrolysis by shifting the equilibrium towards a higher product concentration than otherwise achievable. Few species of Aspergilli are known to produce glucose tolerant β-glucosidases and some of these enzymes have been cloned and characterized. It is expected that more of such glucose tolerant BGLs may be prevalent in nature especially in filamentous fungi. Isolation of such enzymes and knowledge about their properties, sequences and expression patterns can help in design of better enzyme cocktails for biomass hydrolysis as well as in targeted approaches for modifying the glucose tolerance of existing BGLs.
An Aspergillus strain was isolated at NIIST from decaying wood which produced BGL that was active at 0.5M Glucose concentration. The fungus was identified as Aspergillus niger and was deposited in NII culture collection with accession number NII 08121. Multiple BGL isoforms could be detected in this fungus by Native PAGE followed by activity staining using a fluorescent substrate for BGL. The isoforms were differentially induced in response to carbon sources and four isomers were detected, two of which showed glucose tolerance at 250mM glucose. Degenerate primers designed for BGL gene belonging to families 1, 3 and 5 of glycosyl hydrolases could amplify stretches from the genomic DNA. The amplicons on sequencing confirmed homology to BGLs belonging to respective families indicating presence of BGLs belonging to all three families glycosyl hydrolases in the fungus. The major BGL which was the highly expressed isoform in native strain was purified and characterized. It was 120kDa in size and was most active at pH 5.0 and stable at 50°C till 72 hours. It retained more than 90% of the activity up to 48h which indicated the suitability for biomass hydrolysis.
Conditions for BGL production were optimized in Solid State Fermentation (SSF) employing A. niger which resulted in improved enzyme yield. The enzyme produced under SSF retained 19 % activity in presence of 0.25M glucose. The major beta glucosidase (BGL1) of this fungus showed interesting properties including an optimum temperature of 70 °C, optimum pH of 5.0 and extended stability at 50 °C for more than 48h. To test the efficacy of A. niger BGL in biomass saccharification and ethanol production, enzyme blends were created using T. reesei cellulase. Cellulase production from T. reesei under solid state fermentation was optimized and the enzyme was produced using this fungus under optimized conditions for saccharification trials. T. reesei cellulase and the BGL from A. niger were used to create blends for biomass hydrolysis. Optimized enzyme cocktails were derived for efficient hydrolysis of pretreated rice straw, and the hydrolysate was used to demonstrate ethanol production by alcohol fermentation employing Saccharomyces cerevisiae.
The study has helped to reveal the property of differential induction of BGL isoforms in Aspergillus niger which can be used to manipulate culture conditions to produce the desired isoform in excess. Also, a laboratory process for producing BGL enzyme in high titers was derived. Major BGL isoform was purified and characterized and partial gene sequences of BGLs belonging to families 1, 3 and 5 of glycosyl hydrolases were obtained. This information would be very useful in isolating the full length cDNAs of these genes for further studies and possible over expression in suitable vectors.