title: Isolation and characterization of a proteinaceous α-amylase inhibitor AAI-CC5 from Streptomyces sp. CC5, and its gene cloning and expression.
abstract: An α-amylase inhibitor producing Streptomyces sp. strain CC5 was isolated from soil. A proteinaceous α-amylase inhibitor AAI-CC5 was purified from strain CC5. AAI-CC5 specifically inhibited mammalian α-amylases. The molecular weight of the inhibitor was determined to be 8,212 Da by MALDI-TOF Mass Spectrum. The N-terminal 15 amino acid residues of the purified AAI-CC5 were DTGSPAPECVEYFQS, which is dissimilar to other reported proteinaceous α-amylase inhibitors. AAI-CC5 is a pH insensitive and heat-stable protein, and cannot be hydrolysed by trypsin. AAI-CC5 was cloned and expressed in Escherichia coli BL21 (DE3) with a hexa-histidine tag on the C terminal. AAI-CC5 shared 82 % identity with Parvulustat. The recombinant α-amylase inhibitor was purified to homogeneity by one-step affinity chromatography using Ni(2+)-NTA resin with molecular mass of 9,404 Da. Steady state kinetics studies of α-amylase and the inhibitor revealed an irreversible, non-competitive inhibition mechanism with IC50 and Ki value of 6.43 ×1 10(-11) and 4.45 × 10(-11) M respectively. These results suggest this novel α-amylase inhibitor possessed powerful inhibitory activity for α-amylase, and it may be a candidate in research of diabetes therapy and obesity treatment.
authors.affiliation: Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Sun
authors.forename: Weihao
authors.initials: W
authors.lastname: Lu
authors.forename: Pingping
authors.initials: P
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yi
authors.initials: Y
authors.lastname: Kong
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055550:Protein Stability; D059748:Proteolysis; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D000516:alpha-Amylases