الفهرس 
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Abstract
The recent concerns regarding global climate change and the need for renewable and sustainable source of liquid fuel has become one of the most challenging issues in the 21st century. Therefore, the interest has been focused recently on the production ofibiofuel frominon-food plantibiomass. Lignocellulosic materials are the most abundant biomass and energy source on earth that can be converted to cellulosic ethanol known as second-generation biofuel. Cellulose and hemicelluloses are the major component of plant biomass and are the most abundant polysaccharides on earth. Lignocellulolytic microorganisms are widely distributed in diverse habitats. Bacteria play a key role during composting and degradation of lignocellulose due to their ability to produce potent lignocellulolytic enzymes.
In this study, bacterial isolates were isolated from agricultural residues compost located at the Faculty of Agriculture, Minia University. Nine (CX1-CX9) cellulose and hemicellulose-degrading bacteria were isolated on basal salt culture medium supplied with filter paper as the sole carbon source. The cellulolytic and xylanolytic activities of the nine isolates were determined using Congo red clearing zone assay. Five ofithe nine isolatesiwere ableito degradeiboth CMC and xylan and they were subjected to further investigation. The production oficellulolytic andixylanolytic enzymesiby the five isolates grownion basal salt culture medium supplemented with agricultural waste materials was quantified. CX2 and CX9 isolates exhibited the highest cellulolytic and xylanolytic activities and were able to depolymerize both rice straw and sugarcane bagasse. Higher enzyme activities were obtained on rice straw.
The five isolates were identified on molecular basis by amplifying and sequencing their 16S rRNA genes. The similarity betweenithe 16SirRNA gene sequencesiof the isolatesiand the known sequences in the bacterial and archaeal ribosomal RNA database was determined through BLAST tool at theiNational Centreifor BiotechnologyiInformation (NCBI). A phylogenetic tree was constructed using Neighbor-Joining algorithm based on the 16SirRNA gene sequences to determine the revolutionary relationship between the five lignocellulolytic bacterial isolates and the closely related bacterial species. The five isolates were identified as: Paracoccus kondratievae strain GB (CX2 and CX4), Paracoccus communis (CX5), Bacillus australimaris (CX7) and Bacillus pumilus (CX9). The sequences of the 16SirRNA genesiof the five isolatesiwere submitted to the Genbank and they were assigned accession numbers.
The production and activity of both cellulase and xylanase from Paracoccus kondratievae strain GB (CX2) were determined. The highest cellulase production was obtained at incubation temperatureiofi35°C, pH 8, agitationispeed ofi150irpm, incubation period of 5-7 days, CMC as a carbon source and ammonium sulphate as a nitrogen source. Xylanase was produced maximally at 35°C, pH 8, agitation speed of 150 rpm, incubation period of 4 days, xylan as a carbon source and ammonium nitrate as a nitrogen source. On the other hand, the highest cellulase activity was obtained at 45-55°C and pH 6 after 60-70 minutes of incubation and the highest xylanase activity was obtained at 45°C and pH 6 after 50 minutes of incubation.
Similarly, the production and activity of both cellulase and xylanase from Bacillus pumilus (CX9) were determined. The highest cellulase production was obtained at an incubation temperatureiofi35°C, pH 8, agitationispeed ofi150irpm, incubation period of 3 days, CMC as a carbon source and ammonium nitrate as a nitrogen source. Xylanase was produced maximally at 35°C, pH 8, agitation speed of 150 rpm, incubation period of 4 days, xylan as a carbon source and ammonium nitrate as a nitrogen source. On the other hand, the highest cellulase and xylanase activities were obtained at 55°C and pH 6 after 60 minutes of incubation.
A GH10-endoxylanase encoding gene was identified in the genome of Bacillus pumilus strain ATCC 7061. The 1230 bp-long gene was amplified by PCR and cloned into the expression vector pET28-a (+) using NcoI and XhoI restriction sites. The gene was cloned in frame with the codons for the six histidine in the expression vector to produce a protein with a his-tag attached to its C-terminus.
The recombinant plasmid (pET28-GH10-endoxylanase) was introduced into the Escherichia coli expression host strain BL 21-codon plus (DE3)-RIPL. The gene expression was induced, and the recombinant protein (about 50 kDa) was produced. Affinity purification of the his-tagged GH10-endoxylanase protein was conducted using Ni-NTA resin under native conditions.
The optimumiconditions forithe activity of the pure recombinant Bacillusipumilus (CX9) GH10-endoxyalase were determined. The maximal endoxylanase activity was obtained at 50°C and pH 6 after 40 minutes of incubation. TheiVmax and Kmiof theirecombinant GH10-endoxyalase were determined on breach wood xylan using Lineweaver–Burk plot and they were 1666.7 nmol s-1 mg protein-1 and of 4.7 mg ml-1, respectively. The recombinant GH10-endoxylanase exhibited a high ability to depolymerize the different agricultural lignocellulosic materials. However, the highest activity was noticed on wheat bran.
The cellulolytic and xylanolytic bacterial strains isolated in this study together with the recombinant endoxylanase could prove useful in the conversion of lignocellulosic residues abundant in Egypt to clean renewable fuel.