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.

Fig. 1.

(A) CMCase activity of Paenibacillus ICGEB2008 on a CMC-containing agar plate after 48 h of growth. (B) Zymogram analysis of Paenibacillus ICGEB2008 culture supernatant. The arrows indicate the endocellulase and xylanase protein bands with maximum hydrolyzing activity.

Fig. 2.

Purification and activity profiles of recombinant and native Endo5A. (A) Purification profile of recombinant Endo5A on an SDS-PAGE gel. Recombinant Endo5A was expressed in E. coli and purified by immobilized metal affinity chromatography (IMAC). Lane 1, molecular mass marker (in kDa); lane 2, uninduced sample; lane 3, induced sample; lane 4, insoluble cell lysate fraction; lane 5, soluble cell lysate fraction; lane 6, flowthrough; lane 7, wash; lanes 8 to 10, chromatography eluates from IMAC columns. (B and C) Purification profiles of native Endo5A on an SDS-PAGE gel. Culture supernatant from Paenibacillus ICGEB2008 was used for purification by ultrafiltration (lane 1), hydrophobic interaction chromatography (lane 2), gel permeation chromatography (lane 3), and ion-exchange chromatography (lane 4) and then was separated by SDS-PAGE in a gel containing CMC. The gels were stained with either Coomassie blue (B) to visualize the proteins or Congo red (C) to visualize the CMCase clearance zone. A molecular mass marker (in kDa) was loaded in lane M. The arrow indicates the location of Endo5A. (D and E) Temperature (D) and pH (E) optima for native Endo5A, recombinant Endo5A, and the recombinant Endo5A-GS-Xyl11D fusion protein. All three enzymes were purified and tested for activity at various temperatures and pHs. The results indicate that CMCase activities for all three enzymes are similar at the different temperatures and pH conditions.

Fig. 3.

Purification and activity profiles of recombinant and native Xyl11D. (A) SDS-PAGE gel profile for purification of recombinant Xyl11D. Recombinant Xyl11D was expressed in E. coli and purified by IMAC. Lane 1, clarified culture lysate; lane 2, flowthrough; lanes 3 to 5, chromatography eluates of IMAC. Molecular mass markers (in kDa) are shown. (B and C) SDS-PAGE gel profiles for purification of native Xyl11D. Native Xyl11D from culture supernatant of Paenibacillus ICGEB2008 was purified by ultrafiltration (lane 1), hydrophobic interaction chromatography (lane 2), gel permeation chromatography (lane 3), and ion-exchange chromatography (lane 4) and then separated on an SDS-PAGE gel containing xylan. Gels were stained with either Coomassie blue (B) to visualize the proteins or Congo red (C) to visualize the xylanase clearance zone. A molecular mass marker (in kDa) was loaded in lane 5. (D and E) Temperature (D) and pH (E) optima for xylanase activities of native Xyl11D, recombinant Xyl11D, and the recombinant Endo5A-GS-Xyl11D fusion protein. The results indicate that xylanase activities are similar for all three enzymes over a range of temperatures and pH conditions.

Fig. 4.

CD spectroscopy of recombinant bifunctional chimeric proteins. (A) Recombinant proteins used for CD analysis. All four fusion proteins and their individual counterparts were expressed in E. coli, purified by metal affinity and gel permeation chromatography, and then used for CD analysis. Lane M, molecular mass markers; lane 1, Endo5A-Xyl11D; lane 2, Xyl11D-Endo5A; lane 3, Endo5A-GS-Xyl11D; lane 4, Xyl11D-GS-Endo5A; lane 5, Xyl11D; and lane 6, Endo5A. (B) CD spectra of Endo5A, Xyl11D, and an equimolar mixture of Endo5A and Xyl11D. (C) CD spectra of all four chimeric models.

Fig. 5.

Positive-mode matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of sugars (M + Na⁺) present in the hydrolyzed products of CMC after incubation with Endo5A (A), hydrolyzed products of xylan after incubation with Xyl11D (B), and hydrolyzed products of a xylan-CMC mixture following incubation with the Endo5A-GS-Xyl11D chimeric protein (C).