Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1.
A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734bp and encodes 578 amino acids with a molecular mass of 61,757Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.
Kasuya
Ken-ichi
K
Material Science Laboratory, Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, 1-5-1 Tenjin, Kiryu-shi, Gunma 376-8515, Japan. kkasuya@bce.gunma-u.ac.jp
Takano
Tsutomu
T
Tezuka
Yoko
Y
Hsieh
W-C
WC
Mitomo
Hiroshi
H
Doi
Yoshiharu
Y
D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002087:Butyrates / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D006885:Hydroxybutyrates / Q000378:metabolism; D008969:Molecular Sequence Data; D011091:Polyesters / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D000094122:Polyhydroxybutyrates
D002087:Butyrates; D015820:Cadherins; D002265:Carboxylic Ester Hydrolases; D005982:Glutathione Transferase; D006885:Hydroxybutyrates; D011091:Polyesters; D011993:Recombinant Fusion Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C033592:4-nitrophenyl butyrate; C022417:poly-beta-hydroxybutyrate depolymerase