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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0393

PubMed

20686828, Mar Biotechnol (NY). 2011 Jun;13(3):411-22. doi: 10.1007/s10126-010-9312-0. Epub 2010 Aug 5.

Characterization method

enzyme activity assay,analysis of reaction products

Genomic accession number

AB516430

Nucelotide position range

932-22419

Substrate

galactan

Loci

BAK08901.1-BAK08910.1

Species

Microbulbifer thermotolerans/252514

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- - BAK08901.1 932 - 1529 (-) AB516430.1:1864-2461 -
- - BAK08902.1 1899 - 3741 (+) AB516430.1:2831-4673 -
agaO - BAK08903.1 4305 - 7833 (+) AB516430.1:5237-8765 -
cgiA - BAK08904.1 8846 - 10556 (+) AB516430.1:9778-11488 -
- - BAK08905.1 10633 - 14143 (-) AB516430.1:11565-15075 -
- - BAK08906.1 14580 - 15552 (+) AB516430.1:15512-16484 -
- - BAK08907.1 15691 - 16411 (-) AB516430.1:16623-17343 -
- - BAK08908.1 16428 - 17982 (-) AB516430.1:17360-18914 -
- - BAK08909.1 18071 - 20876 (-) AB516430.1:19003-21808 -
agaA - BAK08910.1 21117 - 22419 (+) AB516430.1:22049-23351 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 933 - 1529 (-) CDS No
- 1900 - 3741 (+) CDS No
- 4306 - 7833 (+) CAZyme: GH86|CBM6 Yes
- 8847 - 10556 (+) TC: gnl|TC-DB|Q9A608|1.B.14.12.1 Yes
- 10634 - 14143 (-) other Yes
- 14581 - 15552 (+) other Yes
- 15692 - 16411 (-) other Yes
- 16429 - 17982 (-) other Yes
- 18072 - 20876 (-) other Yes
- 21118 - 22419 (+) CAZyme: GH16|CBM6 Yes

PUL ID

PUL0393

PubMed

20686828, Mar Biotechnol (NY). 2011 Jun;13(3):411-22. doi: 10.1007/s10126-010-9312-0. Epub 2010 Aug 5.

Title

Hyper-production and characterization of the iota-carrageenase useful for iota-carrageenan oligosaccharide production from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94T, and insight into the unusual catalytic mechanism.

Author

Hatada Y, Mizuno M, Li Z, Ohta Y

Abstract

A gene of unknown function from the genome of the agar-degrading deep-sea bacterium Microbulbifer thermotolerans JAMB-A94(T) was functionally identified as a iota-carrageenase gene. This gene, designated as cgiA, is located together with two beta-agarase genes, agaA and agaO in a cluster. The cgiA gene product is 569 amino acids and shares 29% identity over 185 amino acids with the iota-carrageenase from Zobellia galactanivorans Dsij DSM 12802. Recombinant, cgiA-encoded iota-carrageenase (55 kDa) was hyper-produced in Bacillus subtilis. The recombinant enzyme shows maximal activity at 50 degrees C, the highest reported optimal temperature for a carrageenase. It cleaved beta-1,4 linkages in iota-carrageenan to produce a high ratio of iota-carrageenan tetramer, more than 75% of the total product, and did not cleave the beta-1,4 linkages in kappa- or lambda-carrageenan. Therefore, this enzyme may be useful for industrial production of iota-carrageenan oligosaccharides, which have demonstrated antiviral potential against diverse viruses. Furthermore, we performed site-directed mutagenesis on the gene to identify the catalytic amino acid residues. We demonstrated that a conserved Glu351 was essential for catalysis; however, this enzyme lacked a catalytic Asp residue, which is generally critical for the catalytic activity of most glycoside hydrolases.