title: Molecular cloning, purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223.
abstract: A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4 kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0-10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4 min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4 min at 60, 62, and 65°C, respectively. The Kmand Vmaxvalues for ChA were 0.153 mM and 559.6 µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca2+, Cu2+, and Fe2+significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.
authors.affiliation: a Department of Food Science and Human Nutrition, and Fermented Food Research Center , Chonbuk National University , Jeonju , Korea.|b Korea Food Research Institute , Sungnam , Gyeonggi-do , Korea.
authors.forename: Young-Ran
authors.initials: YR
authors.lastname: Song
authors.affiliation: a Department of Food Science and Human Nutrition, and Fermented Food Research Center , Chonbuk National University , Jeonju , Korea.
authors.forename: Sang-Ho
authors.initials: SH
authors.lastname: Baik
mesh_terms: D000595:Amino Acid Sequence; D002109:Caffeic Acids / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002934:Cinnamates / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003373:Coumaric Acids / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D052198:Lactobacillus helveticus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002109:Caffeic Acids; D002934:Cinnamates; D003373:Coumaric Acids; D011994:Recombinant Proteins; C478851:methyl-p-coumarate; C042405:methyl caffeate; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; C530806:methyl ferulate
keywords: Caffeic acid; Lactobacillus helveticus; chlorogenic acid; cinnamoyl esterase; thermal stability