Tatsuo Morishita

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, 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.

Cloning, DNA sequencing, and expression of the β-1,4-mannanase gene from a marine bacterium, Vibrio sp. strain MA-138

Abstract The manA gene encoding an extracellular β-1,4-mannanase of a marine bacterium, Vibrio sp. strain MA-138, was cloned and sequenced. The manA gene consists of an open reading frame of 1185 nucleotides encoding a protein of 395 amino acids with a molecular weight of 43,097. A putative ribosome-binding site and a promoter region were identified in the DNA sequence. ManA of Vibrio sp. strain MA-138 was classified into family 5 of the glycosyl hydrolases and is highly homologous to the ManAs of Caldocellum saccharolyticum (sequence identity: 53%) and Streptomyces lividons (sequence identity: 51%). The N-terminal amino acid sequence of the recombinant enzyme was completely in agreement with that deduced from the nucleotide sequence and that of the enzyme purified from strain MA-138. Although themolecular weight of the recombinant ManA was smaller than that of the enzyme from strain MA-138 on SDS-polyacrylamide gel electrophoresis, this was attributed to linkage of the carbohydrate chain to the latter protein. The enzymatic properties of the recombinant ManA was similar to those of the enzyme from strain MA-138.

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.

Gustatory and olfactory sensitivity to extracts of jack mackerel muscle in young yellowtail Seriola quinqueradiata.

Abstract The stimulatory effects of extract fractions of jack mackerel muscle, obtained using anion exchange chromatography, and their components on the gustatory and olfactory receptors of young yellowtail were investigated by recording electrical responses from the palatal taste nerve and the olfactory bulb. Ultrafiltrate (mol. wt. cut-off, 10,000 Da) of the muscle extract (FE) stimulated both chemosensory systems. FE was treated with a DEAE-Sephadex A-25 column (pH 5.5). The olfactory receptors showed a high sensitivity to both the adsorbate and non-adsorbate from the DEAE-Sephadex column. Their thresholds in the olfactory bulb response were around 10−5 and 10−8 of the original concentration, respectively. Those in the gustatory nerve response were 10−4 and 10−2. Amino acids were highly effective for the olfactory receptors. The threshold of l -glutamine was around 10−8 M. 5′-Inosinic acid and l -lactic acid were more effective for the gustatory receptors than for the olfactory receptors; their thresholds for the former were 10−4 M and 10−6 M, while those for the latter were 10−3 M and above 10−3 M. These findings suggest that the gustatory receptors are involved in discriminating food items during feeding and that the olfactory receptors may detect foods at some distance.