title: Molecular cloning, overexpression, and enzymatic characterization of glycosyl hydrolase family 16 β-Agarase from marine bacterium Saccharophagus sp. AG21 in Escherichia coli.
abstract: An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea.
authors.forename: Youngdeuk
authors.initials: Y
authors.lastname: Lee
authors.forename: Chulhong
authors.initials: C
authors.lastname: Oh
authors.forename: Mahanama
authors.initials: M
authors.lastname: De Zoysa
authors.forename: Hyowon
authors.initials: H
authors.lastname: Kim
authors.forename: Wickramaarachchige Don Niroshana
authors.initials: WD
authors.lastname: Wickramaarachchi
authors.forename: Ilson
authors.initials: I
authors.lastname: Whang
authors.forename: Do-Hyung
authors.initials: DH
authors.lastname: Kang
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000362:Agar / Q000378:metabolism; D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D059001:Aquatic Organisms / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000461:Rhodophyta / Q000382:microbiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D020536:Enzyme Activators; D008670:Metals; D011994:Recombinant Proteins; D000362:Agar; D006026:Glycoside Hydrolases; C019624:agarase