Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01(T).
Genome sequencing of Catenovulum agarivorans YM01T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K(m) of 3.78 mg mL⁻¹ for agarose and a Vmax of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. joyceyujian@163.com.
Fangyuan
F
Cui
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. dongsujie310@163.com.
Sujie
S
Dong
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. shixiaochong@ouc.edu.cn.
Xiaochong
X
Shi
School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China. zhaoxia@ouc.edu.cn.
Xia
X
Zhao
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. xhzhang@ouc.edu.cn.
Xiao-Hua
XH
Zhang
D000362:Agar / Q000737:chemistry; D034241:Alteromonadaceae / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D016680:Genome, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D011994:Recombinant Proteins; D000362:Agar; D006026:Glycoside Hydrolases; C019624:agarase