CLONING OF α-AMYLASE AND PULLULANASE GENES OF BACILLUS LICHENIFORMIS-FAO.CP7 FROM COCOA (THEOBROMA CACAO L.) PODS AND BIOCHEMICAL CHARACTERIZATION OF THE EXPRESSED ENZYMES

Abstract

Alpha-amylase and pullulanase are important starch-utilizing enzymes gaining attention is diverse biotechnological industries. The production of these enzymes by wild-type microorganisms is yet to meet industrial needs thereby necessitating the use of Recombinant DNA Technology for their overexpression. In this study, α-Amylase and pullulanase genes from Bacillus licheniformis-FAO.CP7 were cloned into DH10B strain of Escherichia coli. Positive clones were screened by a combination of modified colony PCR, restriction digestion and Sanger DNA sequencing analysis. Plasmids containing the positive recombinant DNA construct were transformed and efficiently overexpressed in BL21(DE3) strain of Escherichia coli. Expressed proteins were purified to homogeneity by one-step nickel affinity chromatography using Hispur Ni (2+) -NTA resin. Molecular weight of the purified enzymes was determined by SDS-PAGE on 12% gel. Physicochemical properties such as effects of pH, temperature, organic solvents and metal ions on the activities and stabilities of α-amylase and pullulanase were determined. α-Amylase and pullulanase genes have an open reading frame of 1551 and 2247bp encoding 516 and 748 amino acids respectively. The molecular mass of α-amylase and pullulanase were 57.5 kDa and 79.4 kDa respectively. The specific activities of the α-amylase and pullulanase were 410.54 U/mg and 180.13 U/mg respectively. α-Amylase has an optimum pH and optimum temperature of 5.0 and 65 o C respectively while pullulanase has optimum pH and optimum temperature of 5.5 and 70 o C respectively. The enzymes were stable within the range of 40 to 80 o C showing residual activity of over 65% at 70 o C. The enzymes were also active over a wide range of pH 4.0-9.0 with a residual activity of over 50% between pH 4.0-9.0. While the Km of α-amylase for soluble starch was 0.28 mg/mL, the Km of pullulanase for pullulan was 0.18 mg/mL. Aminoacid sequence analysis of the proteins revealed that α-Amylase from Bacillus licheniformis- FAO.CP7 have three amino acid residues at the Na/Ca 2+ binding sites, and 17 amino acid residues at the active site. Pullulanase have 15 amino acid residues at the active sites and no calcium/sodium binding site and a consensus Tyr-Asn-Trp-Gly-Tyr-Asn-Pro motif which is only found in Type I pullulanases. The purified amylolytic enzymes have three similar amino acid residues at their catalytic site (Asp-Glu-Asp) forming their catalytic triad. The purified amylolytic enzymes at concentration of 5 mM were activated by Ca 2+ , and Mg 2+ and at a concentration of 20 mM, different levels of inhibition were observed Mg 2+ , K + , and Ca 2+ and other metallic ions such as Zn 2+ , Co 2+ , Fe 2+ , Mn 2+ , Pb 2+ , Ni 2+ , Hg 2+ . β-Mercaptoethanol, EDTA and selected detergents all had various levels of inhibition for both enzymes. Organic solvents such as ethanol, isoamylalcohol and acetone at 10% (v/v) are all activators of the enzymes while ethyl ether, benzene chloroform at the same concentration had various levels of inhibition on the enzymes. The thermostable properties and the stability of these amylolytic enzymes within the pH range of their activities implicate them as potential enzymes for industrial applications especially in starch/sugar utilizing industries. With the aid of recombinant DNA technology, these veritable enzymes can be produced in quantity that meets industrial need.