Yoshiharu Maruyama

Amylase-pullulanase enzyme produced by B. circulans F-2

Abstract A novel amylase, which hydrolyzes both pullulan and soluble starch at the same rate, was found in the culture filtrate of Bacillus circulans F-2. The enzyme was purified by ammonium sulfate fractionation, starch adsorption, and ion-exchange and hydrophobic chromatographies. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis both in denatured and non-denatured conditions and activity bands toward soluble starch and pullulan comigrated on the gel. The molecular weight of the enzyme was determined to be 220 000 by gel filtration. Its isoelectric point was found to be 4.13. The optimum pH of amylase activity were determined to be around 7.0–8.5, while that of the pullulanase activity was around 7.0. The optimum temperature of both enzymes was about 50°C. The enzyme produced a series of maltooligosaccharides from amylase whereas it produced only maltotriose (G3) from pullulan. It was found that this enzyme possessed two active sites to hydrolyze α-1,4-glucosidic and α-1,6-glucosidic linkages with the same rate.

Distribution of Nitrogen-fixing Bacteria in the Central Pacific Ocean*

The plate culture method using the two formulae for non-nitrogenous media was adopted in this investigation for the purpose of counting and isolating nitrogen-fixing bacteria distributed in the open sea.Sea water samples were collected at eighteen different stations in the region of Lat. 50°N–15°S along Long. 155°W and two other stations in the Pacific Ocean. In order to compare with those samples from the open sea, water samples were also obtained at four stations in Suruga and Sagami Bays.Nitrogen-fixing bacteria appear to be widely but very unevenly distributed at all depths in sea water, in numbers approximately ranging from nil to 104 per 100 ml of sea water, and denser vertical populations have been found in the area of Lat. 40°N and 5°N along Long. 155°W, even at depths from 2,000 to 3,000m. A conparatively denser population of bacteria was found in sea water from Suruga Bay and Sagami Bay.The bacteria associated with plankton were abundantly demonstrated, in numbers ranging from 106 to 108 per 1 ml settling volume of plankton, in many plankton samples collected at four stations in the southern parts of the Pacific Ocean. Almost all the bacteria isolated from the samples of blue green algal colonies,Trichodesmium, sp., were able to grow on nonnitrogeneous media.

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×102 to 4.6×102/ml for seawater, 4.0×104 to 4.3×105/g wet wt for eelgrass-bed sediment, and 2.1 × 105 to 1.2 × 107/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.

Nitrogen Fixation by Marine Agar-degrading Bacteria

Summary: Several strains of agar-degrading bacteria capable of fixing N2 were isolated from seawater and eelgrass-bed sediment in Aburatsubo Inlet, Kanagawa, Japan, during the summer of 1986. All strains were Gram-negative, facultatively anaerobic, and required NaCl for growth. They were straight or slightly curved rods and were motile in liquid medium by means of a single polar flagellum. These characteristics as well as the G+C contents of their DNA (44.7-46.1 mol%) placed them in the family Vibrionaceae. These strains produced extracellular agarase on agar medium, yielding reducing sugars and acids as the end products. They expressed significant nitrogenase (acetylene reduction) activities after a few hours of incubation under anaerobic conditions. They utilized combined nitrogen sources both aerobically and anaerobically, but fixed N2 only under anaerobic conditions. Neither yeast extract nor vitamins were required for N2 fixation. These strains were demonstrated to fix N2 anaerobically using agar as the sole carbon source.

Cloning and expression of raw-starch-digesting α-amylase gene from Bacillus circulans F-2 in Escherichia coli

The raw potato-starch-digesting alpha-amylase gene of Bacillus circulans F-2 was cloned for the first time in Escherichia coli C600, using plasmid pYEJ001. The recombinant plasmid, named pYKA3, has a 5.4 kb insert from a chromosome of the donor bacterium. Subcloning of this amylase gene gave plasmid pHA300 which carried 3.15 kb of the inserted DNA. The transformed bacterium, E. coli C600 (pYKA3), produced the amylase in the periplasmic space, whereas it is secreted outside the cell in the donor bacterium. The cloned raw-starch-digesting alpha-amylase has a molecular weight of 93,000 on SDS-PAGE, and its action pattern was absolutely the same as that of the potent raw-starch-digestible amylase produced by B. circulans F-2. The periplasmic amylase produced by the transformed E. coli (pHA300) could digest raw starch granules such as potato, corn and barley raw starch granules, indicating that the raw-starch-digesting amylase is active in E. coli. Furthermore, this amylase crossreacted with the rabbit antiserum raised against the raw potato-digesting alpha-amylase of B. circulans F-2. From these results it was concluded that the cloned amylase is the same amylase protein as B. circulans F-2 amylase, which has a potent raw-starch digestibility. Thus, this paper is to our knowledge the first describing the molecular cloning of raw-starch-digesting alpha-amylase from Bacillus species and its successful expression in E. coli.

Purification of a amylase—pullulanase bifunctional enzyme by high-performance size-exclusion and hydrophobic-interaction chromatography

Abstract A novel bifunctional enzyme, amylase—pullulanase enzyme (APE), which is produced by Bacillus circulans F-2, was separated and purified in only two steps by high-performance size-exclusion (HPSEC) and high-performance hydrophobic-interaction chromatography (HPHIC) with 50 mM phosphate buffer (pH 7.3) containing 5 mM Co2+. About a 90% recovery of the total enzyme activity was achieved, together with a 1821-fold increase in specific activity. APE activity recovered from the column decreased rapidly in the absence of Co2+, which acts as an activator and stabilizer. However, most of the activity was restored on the addition of Co2+. When a descending salt gradient (1 to 0 M ammonium sulphate in 60 min) and a mobile phase containing Co2+ were used in HPHIC, the APE characteristics were altered, resulting in earlier elution of the enzyme. The results indicate that the hydrophobic properties of APE can be altered by the addition of Co2+, and that the application of HPSEC amd HPHIC with cations such as Co2+ results in the effective purification of a high-molecular-weight enzyme.

A Photobacterium-like bacterium able to fix nitrogen

A Photobacterium-like bacterium isolated from the roots of eelgrass (Zostera marina) was shown to fix nitrogen under anaerobic conditions. Nitrogen fixation by Photobacterium spp. has not been reported previous to this.

Characterization and symbiotic nitrogen fixation of Rhizobium that nodulates Chinese milk vetch (Astragalus sinicus, L.

Abstract Strains of Rhizobia were isolated from nodules of Chinese milk vetch (Astragalus sinicus, L.) collected from paddy fileds in Japan. Taxonomical investigation revealed that they belonged to the genus Rhizobium, a fast-growing bacterium on yeast extract-mannitol agar plate. As the nodules formed on the plants inoculated with these strains gave values of specific C2H2-reducing activity (10-20 nmol C2H4 production/(h·mg nodule fr. wt.)) similar to those of soybean nodules, the effectiveness of Chinese milk vetch as green manure was suggested. Since the addition of nitrate to the nutrient solution reduced the total C2H2-reducing activity of the plant, but not the specific activity of the nodules, it was considered that the addition of nitrate inhibited the formation and development of the nodules. The leg-haemoglobin content was not affected by the nitrate addition. The leghaemoglobin of the milk vetch nodules was separated into five components by PAGE (polyacrylamide gel electrophoresis) and their re...

Analysis of Nitrogenase Reaction Using Monoclonal Antibodies Against α-Subunit of Component I of A.Vinelandii

Conversion of atmospheric nitrogen to ammonia by nitrogen-fixing microorganisms is catalysed by the enzyme nitrogenase. Nitrogenase consists of two components, component I and component II. Component I is a Fe-S protein containing Mo and is a tetramer(α2s2)- Component I catalysed the reduction of nitrogen with component II. Reduced component II gives electrons to component I with the consumption of ATP. The reaction mechanism fo nitrogenase is complex and has been studied by kinetic analysis. We have generated monoclonal antibodies against the nitrogenase of A.vinelandii which show inhibitory effects agaist either acetylene reduction or hydrigen evolution. We have identified the peptide fragments to which each monoclonal antibody binds.