Toshiyoshi Araki

ISOLATION AND REGENERATION OF HAPLOID PROTOPLASTS FROM BANGIA ATROPURPUREA (RHODOPHYTA) WITH MARINE BACTERIAL ENZYMES1

Three kinds of enzymes, agarase, β‐1,4‐mannanase, and β‐1,3‐xylanase, required for isolation of protoplasts from the red alga Bangia atropurpurea (Roth) C. Ag. were prepared from bacterial culture fluids of Vibrio sp. PO‐303, Vibrio sp. MA‐138, and Alcaligenes sp. XY‐234, respectively, isolated from the sea environment. The optimal pH of all enzymes was around 7.5. Suitable conditions for protoplast isolation from B. atropurpurea were examined. The pretreatment of the fronds with pa‐pain solution (20 mM Mes buffer, pH 7.5, containing 2% papain and 0.5 M mannitol) contributed to successful protoplast isolation. When razor‐cut fragments of the fronds (about 200 mg in fresh weight) immersed in 20 mM Mes buffer, 7.5, containing 0.5 M mannitol and one unit each of agarase, β‐1,4‐mannanase, and β‐1,3‐xylanase were incubated at 22°C for 90 min with gentle agitation, 5.7 × 106 protoplasts were released from them. Many protoplasts regenerated into fronds of regular or irregular shape.

Cloning of the Novel Gene Encoding β-Agarase C from a Marine Bacterium, Vibrio sp. Strain PO-303, and Characterization of the Gene Product

ABSTRACT The β-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. β-Agarase C was identified as the first β-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.

Cloning, sequencing, and expression in Escherichia coli of the new gene encoding β-1,3-xylanase from a marine bacterium, Vibrio sp. strain XY-214.

ABSTRACT The Vibrio sp. strain XY-214 β-1,3-xylanase gene cloned in Escherichia coli DH5α consisted of an open reading frame of 1,383 nucleotides encoding a protein of 460 amino acids with a molecular mass of 51,323 Da and had a signal peptide of 22 amino acids. The transformant enzyme hydrolyzed β-1,3-xylan to produce several xylooligosaccharides.

Molecular Cloning, Expression, and Characterization of a β-Agarase Gene, agaD, from a Marine Bacterium, Vibrio sp. Strain PO-303

The β-agarase-d gene (agaD) from a marine bacterium, Vibrio sp. strain PO-303, was cloned and expressed in Escherichia coli. The gene consists of 1,362 bp and encodes a protein of 453 amino acids with a predicted molecular weight of 50,824. The full length of agarase-d consists of a signal peptide, a glycoside hydrolase family 16 catalytic module (CM), and a carbohydrate binding module (CBM). The full length of agarase-d without the signal peptide (rAgaDΔfull), the catalytic module (rAgaDCM), or the CBM (rAgaDCBM) was expressed in E. coli as recombinant proteins. rAgaDCM exhibited higher enzyme activity (63.6 units/mg) than rAgaDΔfull (1.20 units/mg) against agarose. rAgaDCM hydrolyzed agar and porphyran to several oligosaccharides and acted on neoagarohexaose to produce neoagarotetraose and neoagarobiose, but did not act on neoagarotetraose. rAgaDCBM bound to agarose.

Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

A beta-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the beta-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed beta-1,3-xylan but not other polysaccharides such as beta-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or beta-1,4-mannan. TxyA was capable of binding specifically to beta-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the K(d) and the maximum amount of protein bound to beta-1,3-xylan were 4.2 microM and 18.2 micromol/g of beta-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of beta-1,3-xylan.

Cloning and Characterization of a β-1,4-Mannanase 5C Possessing a Family 27 Carbohydrate-Binding Module from a Marine Bacterium, Vibrio sp. Strain MA-138

The β-1,4-mannanase 5C gene (man5C) of Vibrio sp. strain MA-138 was cloned and expressed in Escherichia coli. The man5C gene consisted of 2,010 bp nucleotides encoding a protein of 669 amino acids with a predicted molecular weight of 76,309. β-1,4-Mannanase (Man5C) is a modular enzyme composed of a catalytic module belonging to glycoside hydrolase family 5, a linker region, and a putative carbohydrate-binding module (CBM) belonging to family 27. Recombinant Man5C exhibited maximal activity at 50 °C at pH 7.0, and it had a K m of 0.6 mg ml−1 and a V max of 556.2 μmol min−1 μmol−1 for glucomannan. Binding studies revealed that the C-terminal putative CBM27 had the ability to bind soluble β-mannans and contributed to increasing the rate of depolymerization by binding to the polymeric substrate. Man5C of Vibrio sp. MA-138 is the first non-extremophile enzyme to be identified as a β-mannanase possessing CBM27.

Use of Porphyra spheroplasts as feed additive for red sea bream

Two test diets with and without 5% Porphyra spheroplasts (PS) were formulated using white fishmeal as the main protein source. Red sea bream Pagrus major (mean body weight 15.4±0.1 g) were maintained in a flow-through system (100 L) of thermo-controlled sea water (salinity 32–34, 25°C, 8 L/min) with ordinary aeration (400–600 mL/min) under laboratory light conditions (light-dark 12 h:12 h). Fish were fed three times a day at 10:00, 14:00 and 18:00 hours by hand for 42 days at 6% body weight on each experimental diet. Studies revealed that growth performance, survival and nutrient retention were significantly (P<0.05) higher in the groups fed a diet containing spheroplasts (PS diet). Further, the fish fed the PS diet showed significantly (P<0.05) lower feed conversion rates. Both groups of the PS and control diets had similar levels of body nutritional profile in terms of proximate compositions and fatty acids without compromising blood serum related parameters. From these experimental results, thus, it is comprehensible that a supplementary diet containing Porphyra spheroplasts can be used for maximizing not only growth of P. major but also for utilization of the feed ingredients.

Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

We cloned a novel beta-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the beta-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed beta-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse beta-1,4-xylan, beta-1,4-mannan, beta-1,4-glucan, beta-1,3-xylobiose or p-nitrophenyl-beta-xyloside. When beta-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of beta-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble beta-1,3-xylan, but not that of soluble glycol-beta-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

Supplemental effect of Porphyra spheroplasts on the growth and feed utilization of black sea bream

A 56-day feeding trial was conducted to evaluate the effects of Porphyra spheroplasts (PS) as a feed additive on growth, carcass composition and feed utilization of black sea bream. Four experimental diets with or without PS (0, 1, 3, 5% inclusion level) were formulated to determine the proper incorporation level for best growth and utilization using white fishmeal as the principal protein source. Three replicate groups of fish averaging 1.28±0.05 g (mean ± SD) were maintained in flow-through thermo-controlled sea water (32–34 psu, 25°C) aquaria (100 L) under the laboratory conditions (L12: D12) and fed three times a day at 09:00, 13:00 and 17:00 hours by hand at approximately 3% body weight on each diet. The rearing trial revealed that growth performance (in terms of weight gain, specific growth rate) are almost the same level in all the dietary groups, nevertheless the survival, nutrient utilizations and retentions (fee efficiency, protein efficiency ratio, protein retention rate, lipid retention rate) were significantly higher (P<0.05) in a group fed on the diet containing 3% spheroplasts. Further, the fish fed on the diet containing PS had significantly higher (P<0.05) crude lipid level in their dorsal muscles and viscera. The fish fed a PS-based diet showed higher level of eicosapentaenoic acid and docosahexaenoic acid (DHA) in viscera and DHA in dorsal muscles. The results obtained in this feeding trial suggest that the optimum dietary PS supplementation level up to 3% for black sea bream can be considered as improved consequence on growth performance, nutrient utilization or body composition were noticed.

Identification of feeding stimulants from a jack mackerel muscle extract for young yellowtail Seriola quinqueradiata

The feeding-stimulatory components of an aqueous extract of jack mackerel white muscle for young yellowtail were identified. The extract was fractionated using anion-exchange chromatography, and the feeding-stimulatory effectiveness of fractionated components examined by adding them to starch pellets and feeding to yellowtail. The filtrate (FE) of the muscle extract filtered through a membrane having a mol. wt. cut-off of 10,000 Da was found to have a high feeding-stimulatory effectiveness on the yellowtail. FE was fractionated using DEAE-Sephadex A-25 at pH 5.5. The DEAE-Sephadex adsorbate (AA) showed an effectiveness close to but slightly weaker than that of FE. The DEAE-Sephadex non-adsorbate (NA) had no appreciable effect. Subdividing AA components by stepwise elutions with NaCl solutions suggested that inosine-5′-monophosphate and lactic acid were largely responsible for the stimulatory effectiveness of AA. Some components in NA might also be synergistic with AA components to elicit the full effectiveness of FE.