title: Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium.
abstract: Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a Paenibacillus strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of Paenibacillus. Endo5A and Xyl11D were overexpressed in Escherichia coli, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.
authors.affiliation: Synthetic Biology and Biofuel Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Adlakha
authors.forename: Raman
authors.initials: R
authors.lastname: Rajagopal
authors.forename: Saravanan
authors.initials: S
authors.lastname: Kumar
authors.forename: Vanga Siva
authors.initials: VS
authors.lastname: Reddy
authors.forename: Syed Shams
authors.initials: SS
authors.lastname: Yazdani
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D041981:Gastrointestinal Tract / Q000382:microbiology*; D007915:Lepidoptera / Q000382:microbiology*; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases