Tomohiro Hisano

Bacterial alginate lyase: Enzymology, genetics and application

Abstract Alginate is a heteropolysaccharide comprised of mannuronate and guluronate. Three different types of alginate-degrading enzymes, alginate lyases A1-I, A1-II and A1-III, were produced by a bacterium isolated from a ditch. A1-I (63 kDa) was active on both brown seaweed (non-acetylated) and bacterial (acetylated) alginates, whereas A1-II (23 kDa) and A1-III (40 kDa) were specific to brown seaweed and bacterial alginates, respectively. The gene for A1-I was cloned, analyzed and a possible molecular route for the generation of A1-II and A1-III through post-translational processing of A1-I was established. Novel physiological and food technological functions of alginate were derived by depolymerization of the polymer molecule. A pyrogen-free A1-III was massively produced by genetically engineered Gram-positive microbes and is considered as a possible therapeutic agent for the treatment of patients with cystic fibrosis.

Bacterial alginate lyase gene: Nucleotide sequence and molecular route for generation of alginate lyase species

Abstract A bacterium (strain A1) isolated from a ditch synthesized three types of intracellular alginate lyases: A1-I (molecular weight [M.W.] 60,000), A1-II-2 (M.W. 25,000) and A1-III (M.W. 38,000). The nucleotide sequence of the gene for A1-I lyase, which has been cloned in Escherichia coli DH1 was determined. The open reading frame of the gene encoded 622 amino acids with a calculated M.W. of 69,153. The N-terminal amino acid sequence of A1-I lyase purified from strain A1 or E. coli DH1 cells transformed with the A1-I lyase gene was consistent with the deduced sequence from 55 His to 74 Ala, indicating that the A1-I lyase was synthesized as a precursor with a M.W. of 69,153 and then processed to a mature form with a M.W. of 63,681. The N-terminal sequence of the first twenty amino acids of A1-III lyase was found to match that of A1-I lyase. The N-terminal sequence of the first twenty amino acids of A1-II-2 lyase was consistent with the deduced amino acid sequence from 414 Ala to 433 Val in the nucleotide sequence of the A1-I lyase gene. These results indicated that the A1-I lyase was further processed to generate A1-II-2 and A1-III lyase species.

Sphingomonas sp. A1 lyase active on both poly-β-D-mannuronate and heteropolymeric regions in alginate

Alginate lyase III of Sphingomonas sp. A1 cleaved the glycosidic linkage of polymannuronate and heteropolymeric region composed of mannuronate and guluronate, but was inert on polyguluronate. The enzyme was observed to act endolytically, interact with tetrasaccharide in alginate, and form di- and trisaccharides as final products. This result suggests that the enzyme recognizes the unit of tetrasaccharide in alginate and cleaves the middle linkage of the tetrasaccharide.

Direct uptake of alginate molecules through a pit on the bacterial cell surface: A novel mechanism for the uptake of macromolecules

A yellow-pigmented bacterium isolated from a ditch as a potent producer of aglinate lyase was a Gram negative rod with a G + C content of 63 mol%, and was classified in the genus Sphingomonas. Electron microscopy revealed that the bacterial cell surfaces were covered by many large plaits. When grown in a medium containing alginate, a pit of 0.02–0.2 μm in diameter was formed on the cell surface, and a thin section showed the presence of a region where the cell membrane sinks into the cytosol. The pit and its neighborhood on cells grown in the presence of alginate were specifically stained with ruthenium red. On the basis of these results, we propose, for the first time, the existence of a direct uptake mechanism for polysaccharides through a mouth-like pit on the bacterial cell surface. This finding may provide a new insight into the transport of macromolecules in microbial cell systems.

On the Self-Processing of Bacterial Alginate Lyase

Abstract All of the alginate lyase species (A1-I, A1-II and A1-III) in a bacterium (strain A1) isolated from a ditch are generated from a common precursor protein (A1-0) having a molecular size of 69 kDa (Murata et al. , J. Ferment. Bioeng., 76, 427–437, 1993). A1-I (63 kDa) generated after removal of an N-terminal peptide (6 kDa) from A1-0 was found to be converted into A1-II (23 kDa) and A1-III (40 kDa) by a self-processing reaction of the A1-I molecule itself.

Bacterial alginate lyase highly active on acetylated alginates

Abstract A bacterium (strain A1) isolated from a ditch synthesized three kinds of intracellular alginate lyases: A1-I (molecular weight [M.W.] 60,000), A1-II-1 (M.W. 60,000) and A1-II-2 (M.W. 25,000) in laboratory-scale cultures. However, when cells of strain A1 were grown on an industrial scale, another lyase (A1-III) was produced other than A1-I, A1-II-1 and A1-II-2. The A1-III lyase was a monomer with a M.W. of about 38,000, and its activity toward bacterial (acetylated) alginates was much higher (2-fold) than that toward seaweed (non-acetylated) alginates. The N-terminal amino acid sequence of A1-III lyase was consistent with that of A1-I lyase.

Isolation and properties of a collagenase with caseinolytic activity from a Pseudomonas sp

Abstract A bacterium having collagenolytic activity was isolated from soil and was identified as a Pseudomonas sp. The bacterium had two kinds of collagenolytic enzymes and one of them was purified to apparent homogeneity judging from polyacrylamide gel electrophoresis and ultracentrifugation. The purified enzyme had a molecular weight of about 45,000 and hydrolyzed insoluble collagen and synthetic oligopeptides at the site of Xaa-Gly most efficiently at pH 7.4 and 45°C. Besides collagenolytic activity, the purified enzyme showed caseinolytic activity. The collagenase had a serine residue in an active site and catalyzed the hydrolysis of both collagen and casein.

Bacterial Alginate Lyase Inactive on Alginate Biosynthesized by Pseudomonas aeruginosa

A bacterium (strain Al) isolated from a ditch produces three kinds of intracellular alginate lyases [Al-I (molecular weight: M.W. 60,000), Al-II-1 (M.W. 60,000) and Al-II-2]; the former two lyases have been purified and characterized (Yonemoto et al., J. Ferment. Bioeng., 72, 152–157, 1991). As part of a series of studies, Al-II-2 lyase was purified from cell-free extract of the bacterium. The lyase, with a M.W. of 25,000, depolymerized sodium-, potassium- and propyleneglycol alginates most efficiently at pH 8.0, 70°C, but it was inactive toward bacterial alginates with O-acetyl groups.

Microbial spermidine dehydrogenase: Purification and properties of the enzyme in Pseudomonas aeruginosa and Citrobacter freundii

Abstract Spermidine dehydrogenases were purified from Pseudomonas aeruginosa and Citrobacter freundii . P. aeruginosa cells contained only one constitutive spermidine dehydrogenase. C. freundii cells contained two kinds of spermidine dehydrogenases (I and II) and both were induced in the presence of substrate, spermidine. The enzymes purified from the two strains consisted of a single polypeptide chain with a molecular weight of about 63,000. The enzymes (I and II) in C. freundii had a sharp absorbance peak at 430 nm and showed the same N-terminal amino acid sequences (Ser-Gly-Lys-Gly-Asn-). The enzymes in P. aeruginosa and C. freundii specifically oxidized spermidine and spermine in the presence of electron acceptors such as potassium ferrycyanide and 2, 6-dichlorophenolindophenol.

Production of bacterial alginate-specific lyase by recombinant Bacillus subtilis

Abstract Conditions for the production of bacterial alginate-specific lyase (A1-III) by Bacillus subtilis transformed with an inducible secretion vector pISA412 harboring the A1-III gene from a bacterium ( Flavobacterium sp.) of strain A1 were studied. Galactose at around 3% was found as the most efficient carbon source in the pH region between 7.0∼8.5. A higher amount of potassium phosphate was required to repress degradation of A1-III produced in medium. Under the most preferable culture conditions determined, production of the lyase reached approximately 0.3 mg/ml. The properties of A1-III purified from the medium were comparable with those of A1-III present in strain A1 in terms of molecular size, substrate specificity and in N-terminal amino acid sequence.