Cloning, Expression, And Enzymatic Characterization Analysis Of CBH Gene From Rhizopus Stolonifer

Cellulase is a multi-enzyme consists of three different enzymes: cellobiohydrolase (CBH), endoglucanase (EG) and β-glucosidase (BGL). It is the three enzymes that rupture β-1,4 glycosidic bond formation of oligosaccharides. In the enzymatic hydrolysis, cellobiohydrolase mainly acts on the cellulose crystalline regions, endoglucanase mainly acts on the non-crystalline region, and cellobiose and cellooligosaccharide are degraded by P-glucosidase. Taking Rhizopus stolonifer TP-02 as template, research of cellobiohydrolase gene cbh1 and cbh2 structure and analysis of the substrate binding site and the key catalytic process. The two novel cellobiohydrolase genes(cbh1 and cbh1) were cloned from it. cbh1 and cbh2 were connected to pET22b vector and transformed into E.coli BL21. Purification and characterization of the analysis was aiso done. Main research results are as follows:(1) Successfully extracted Rhizopus stolonifer genomic DNA and synthesized cDNA first line, and two novel exoglucanases genes (cbh1 and cbh1) were cloned from it. Sequence analysis showed that the full-length of cbh1 was 1578 bp, encoding 536 amino acids and belonging to Glycoside Hydrolase family 7, however, cbh1 was 1647 bp with three introns, encoding 440 amino acids and belonging to Glycoside Hydrolase family 6. Discover Studio 2.5 was used to build homology modeling. The result showed that the active site of CBHI was covered by loops, forming a ’barrel’ of the tunnel structure. Glu213, Glu218 and Trp41 residues in the tunnel played an important role in the process of substrate hydrolyses, however, the key amino acid residues were Arg179, Trp371, Lys399 and His418 in CBHII.(2) cbhl and cbh2 without signal peptide were connected to pET22b vector and transformed into E.coli BL21(DE3), using congored screening medium to screen positive clones, E.coli BL21(DE3)/pET22b-cbh1 and E.coli BL21(DE3)/pET22b-cbh2. After optimized culture conditions, the recombinant bacteria fermentation experiments showed with 0.5 mmol/L IPTG induction, the activity of E.coli BL21 (DE3)/pET22b-cbh1 reached a maximum value of 0.61 IU/mL after 14 h of fermentation in 20℃, however, the activity of E.coli BL21(DE3)/pET22b-cbh2 reached a maximum value of 0.78 IU/mL after 14 h of fermentation in 25℃ with 0.2 mmol/L IPTG induction.(3) Purification of CBHI and CBHII by ermentation and characterization of the analysis was done. Using affinity chromatography and ion exchange chromatography to purify CBHI, yielding 56 kDa protein with 3.57 IU/mg specific activity. The optimum temperature and pH studies showed the optimum temperature and pH of CBHI was 55℃ and 5.5, maintaining mostly enzyme activity between pH 4.5 to 6.5. Km value was 6.1 mg·mL-1 when taking microcrystalline cellulose as substrate. Using affinity chromatography, ion exchange chromatography and G75 chromatography to purify CBHII, yielding 46 kDa protein with 4.67 IU/mg specific activity. The optimum temperature and pH studies showed the optimum temperature and pH of CBHI was 50℃ and 5.0, maintaining mostly enzyme activity between pH 4.0 to 7.0. Km value was 7.257 mg·mL-1. Fe3+ and Co2+ had promotion on CBHI, however, Fe2+ and Co2+ had promotion on CBHI.This study successful cloned cbhl and cbh2 gene from Rhizopus stolonifer. Homologous simulation analysis found that the three-dimensional conformation of CBHI and CBHII was a tunnel contains key amino acid residues similar to Trichoderma harzianum and Trichoderma reesei. It was worth noting that CBHI contained a Trp41 hydrogen bonding with fiber six sugar, however, in CBHII the residues were Arg179, Trp371, Lys399 and His418. Cloning, expression and protein separation and purification techniques are used to study the characteristics of the enzyme. The results showed that CBHⅠ had a wide pH stability, while CBHⅡ having better heat resistance compared Trichoderma reesei, providing a theoretical basis for further study of Rhizopus stolonifer cellulase.