Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the common sea hare Aplysia kurodai

Rahman, Mohammad Matiur; Inoue, Akira; Tanaka, Hiroyuki; Ojima, Takao

doi:10.1016/j.cbpb.2010.07.006

Cited by 43 publications

(29 citation statements)

References 49 publications

(58 reference statements)

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“…To date, the authors’ group has been studying on alginate lyases from herbivorous marine mollusks, such as abalone, sea hare, and small snails [24,25,26,27,28,29]. These molluscan enzymes were the prominent members of polysaccharide PL-14 according to the searches for CAZy data base and available for the investigation of catalytic mechanism of PL-14 enzyme [27,29].…”

Section: Introductionmentioning

confidence: 99%

Characterization of an Alginate Lyase, FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

et al. 2014

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A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.

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Section: Introductionmentioning

confidence: 99%

Characterization of an Alginate Lyase, FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

et al. 2014

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“…The common sea hare A. kurodai is a good source for polysaccharide-degrading enzymes since it harbor much digestive fluid in gastric lumen (Kumagai and Ojima, 2009; Rahman et al, 2010; Zahura et al, 2010; Tsuji et al, 2013; Kumagai et al, 2014). Recently, four cellulase isozymes, i.e., 21, 45, 66, and 95 K cellulases, were isolated from the digestive fluid of A. kurodai (Tsuji et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai

2014

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The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95, 66, 45, and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40°C, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55°C for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic β-1,4-glucanase (EC 3.2.1.4). A cDNA of 1013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47–62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.

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“…On the other hand, the extracts from bivalves, such as Mizuhopecten yessoensis and Spisula sachalinensis, did not show appreciable activity. The above gastropods are known to possess alginate lyase (37)(38)(39), whereas the bivalves have not been shown to possess alginate lyase. These facts imply that the alginate lyase and DEH reductase are intimately related enzymes and take part in the assimilation of alginate in gastropods.…”

Section: Familymentioning

confidence: 99%

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate

Mochizuki

Nishiyama

Inoue

et al. 2015

Journal of Biological Chemistry

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Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ϳ40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50°C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, ␣-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18 -60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family.Alginate is a structural polysaccharide of brown seaweeds and certain bacteria, comprising ␤-D-mannuronate (M) 2 and ␣-L-guluronate (G), which form poly(M), poly(G), and random(MG) blocks in alginate polymer (1-3). Alginate from brown seaweeds has been widely used as a viscosifier and gelling agent in the food and pharmaceutical industries because sodium alginate solution exhibits high viscosity and forms an elastic gel upon forming calcium salt (1, 4). Alginate oligosaccharides are also recognized as functional materials that exhibit various biological functions, such as promotion of root growth of higher plants (5, 6), acceleration of growth rate of Bifidobacterium sp. (7), and stimulation of proliferation of endothelial cells (8). Further, 4-deoxy-5-erythro-hexoseulose uronic acid (DEH), the end product of alginate lyases (EC 4.2.2.3 and EC 4.2.2.11) (see Fig. 1), was recently proven to be available for ethanol fermentation with genetically modified microorganisms (9 -11). These findings have increased the practical potentiality of both alginate and alginate-producing brown seaweeds. Besides such practical aspects, alginate is an important food source for herbivorous gastropods like abalone (12-16). Namely, abalone feeds brown seaweeds as a daily diet and digests poly(M) block of ...

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Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the common sea hare Aplysia kurodai

Cited by 43 publications

References 49 publications

Characterization of an Alginate Lyase, FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

Characterization of an Alginate Lyase, FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate

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