Ruminants are capable of hydrolyzing lignocellulosic residues to absorbable sugars by virtue of the microbial communities residing in their rumen. However, large sections of such microbial communities are not yet culturable using conventional laboratory techniques. Therefore in the present study, the metagenomic DNA of swamp buffalo (Bubalus bubalis) rumen contents was explored using culture-independent techniques. The consensus regions of glycosyl hydrolase 5 (GH5) family of cellulases were used as primers for PCR amplification. A full-length metagenomic cellulase gene, Umcel5B29, with a complete open reading frame (ORF) of 1611 bp was identified. The similarity search analysis revealed that Umcel5B29 is closely related to the cellulases (73% to 98% similarity) of ruminal unculturable microorganisms, indicating its phylogenetic origin. Further analysis indicated that Umcel5B29 does not contain a carbohydrate binding module (CBM). Subsequently, Umcel5B29 was overexpressed in Escherichia coli. The recombinant enzyme worked optimally at pH 5.5 and 45°C, a condition similar to the buffalo's rumen. However, the enzyme retained more than 70% of its maximal activity after incubation at pH 4-7 and more than 50% maximal activity after incubation at 30-60°C for 30 min. These characteristics render Umcel5B29 as a potential candidate for the bio-stoning process of denim.
Textile wastewater is the major problem in the world as production of colorant reached 8 × 10 6 kg per year, of which 10-15 % goes into effluents during different textile processes 1,2 . These colorants are primarily designed to resist fading of colour in the presence of intense light and moisture conditions to make good quality fabric. Discharge of such dyes in wastewater is a great threat to the natural ecosystems because these are toxic as well as carcinogenic. Thus, there is a dire need to develop a cost effective strategy to deal with such dyes which are harmful to life 3,4 . In this scenario, biological processes to treat such dyes could be environment friendly and cost effective procedures 5 . For example, degradative enzymes can be utilized to degrade textile effluents. These enzymes have low energy requirement, minimum operation cost and eco-friendly nature which make them suitable for such treatments. Laccases are the enzymes which are used for biotreatment of dyestuffs. Information regarding effluent treatment by lacases is available in the literature but potential of other enzymes such as oxidases and reductases is yet to be explored 1 .
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