title: Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10.
abstract: The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.
authors.affiliation: Jiangnan University, Jiangsu, People's Republic of China. wmmu@jiangnan.edu.cn
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
authors.forename: Feifei
authors.initials: F
authors.lastname: Chu
authors.forename: Qingchao
authors.initials: Q
authors.lastname: Xing
authors.forename: Shuhuai
authors.initials: S
authors.lastname: Yu
authors.forename: Leon
authors.initials: L
authors.lastname: Zhou
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D046973:Ruminiclostridium cellulolyticum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C003243:psicose; D005632:Fructose; D019998:Racemases and Epimerases