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Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40.

ABSTRACT: Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha-neoagarobiose hydrolase to release galactose from agarose.

SUBMITTER: Ekborg NA

PROVIDER: S-EPMC1472325 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40.

Ekborg Nathan A NA Taylor Larry E LE Longmire Atkinson G AG Henrissat Bernard B Weiner Ronald M RM Hutcheson Steven W SW

Applied and environmental microbiology 20060501 5

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans ...[more]

PMID: 16672483

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Project description:The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-D-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB) N-acetyl-beta-D-glucosaminidases and one cytoplasmic (HexC) N-acetyl-beta-D-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E. coli K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.

Project description:BackgroundLaminarin is a potential biomass feedstock for the production of glucose, which is the most preferable fermentable sugar in many microorganisms by which it can be converted to biofuels and bio-based chemicals. Also, laminarin is a good resource as functional materials because it consists of β-1,3-glucosidic linkages in its backbone and β-1,6-glucosidic linkages in its branches so that its oligosaccharides driven from laminarin have a variety of biological activities. It is industrially important to be able to produce laminarioligosaccharides as well as glucose from laminarin by a single enzyme because the enzyme cost accounts for a large part of bio-based products. In this study, we investigated the industrial applicability of Bgl1B, a unique β-glucosidase from Saccharophagus degradans 2-40T, belonging to the glycoside hydrolase family 1 (GH1) by characterizing its activity of hydrolyzing laminarin under various conditions.ResultsBgl1B was cloned and overexpressed in Escherichia coli from S. degradans 2-40T, and its enzymatic activity was characterized. Similar to most of β-glucosidases in GH1, Bgl1B was able to hydrolyze a variety of disaccharides having different β-linkages, such as laminaribiose, cellobiose, gentiobiose, lactose, and agarobiose, by cleaving β-1,3-, β-1,4-, and β-1,6-glycosidic linkages. However, Bgl1B showed the highest specific activity toward laminaribiose with a β-1,3-glycosidic linkage. In addition, it was able to hydrolyze laminarin, one of the major polysaccharides in brown macroalgae, into glucose with a conversion yield of 75% of theoretical maximum. Bgl1B also showed transglycosylation activity by producing oligosaccharides from laminarin and laminaribiose under a high mass ratio of substrate to enzyme. Furthermore, Bgl1B was found to be psychrophilic, exhibiting relative activity of 59-85% in the low-temperature range of 2-20 °C.ConclusionsBgl1B can directly hydrolyze laminarin into glucose with a high conversion yield without leaving any oligosaccharides. Bgl1B can exhibit high enzymatic activity in a broad range of low temperatures (2-20 °C), which is advantageous for establishing energy-efficient bioprocesses. In addition, under high substrate to enzyme ratios, Bgl1B can produce high-value laminarioligosaccharides via its transglycosylation activity. These results show that Bgl1B can be an industrially important enzyme for the production of biofuels and bio-based chemicals from brown macroalgae.

Dataset Information

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40.

Publications

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40.

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