Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

Improved Transformation Method for Acetobacter with Plasmid DNA

An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4,000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca²⁺ . The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 105 transformants per γg plasmid DNA was achieved.     

Isolation of Allo-isoleucic Acid (α-Hydroxy-β-methylvaleric Acid) and β-Hydroxy-β-methylvaleric Acid from Turkish Tobacco Leaves

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity.

The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added.

After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol.

The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation. $u=114\times \frac{E_{t1}?E_{t2}}{t_2?t_1}$

Where E_t1 and E_t2 mean the optical density of the ethanol solution at 310 mμ prepared after t₁ and t₂ minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute.

The substrate tannic acid used in this determining method was purified. It was composed of one mole of glucose and nine moles of gallic acid, and eight moles of which formed four moles of m-digallic acid.     

Characterization of a Potato Starch-digesting Bacterium and Its Production of Amylase

A bacterium which can utilize potato starch granules as sole carbon source was isolated and identified as Bacillus circulans from its physiological and biochemical properties. Scanning electron microscopic observation of potato starch granules recovered from the culture broth revealed that granules were degraded gradually from their surface resulting in elongated granules with layered structures on their surface. This bacterium produced extracellular amylase which can digest potato starch granules in vitro. The amylase has a unique property in that it produces only maltohexaose from gelatinized starch in the early stage of the reaction. For the production of this amylase potato starch was found to be most effective while soluble sugars including gelatinized starch and maltose had little effect.     

Formation of Threonine Aldolase by Bacteria and Yeasts

Threonine aldolase was found to be formed in various strains of bacteria and yeasts when they were grown in media containing l-threonine as a sole source of carbon. As the other sources of carbon, d, l-allothreonine, l-serine and glycine were effective but glucose and sucrose were inert for the formation of the enzyme.

The maximal formation of the enzyme was observed in the initial of stationary phase of growth and, thereafter, the enzyme disappeared with the consumption of l-threonine. It seems that the enzyme is adaptive in nature and that it is responsible for the growth in threonine as the carbon source.     

A Growth Factor for Malo-lactic Fermentation Bacteria

A growth factor (TJF) for a malo-lactic fermentation bacterium has been isolated from tomato juice, and found to be a β-glucoside. The NMR spectra of TJF and its acetate revealed that the glucosyl residue linked to the hydroxyl group at C-2′ or C-4′ of d- or l-pantothenic acid moiety. Then, 2′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (I), 4′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (II) and 4′-O-(β-d-glucopyranosyl)-d(R)-pantothenic acid (II-a) were synthesized, and Il-a and 4′-O-(β-d-glucopyranosyl)-l-pantothenic acid (II-b) were obtained by the optical resolution of the acetate of II. Among the above compounds, II-a was identical with natural TJF regarding to the biological activity, NMR and ORD spectra, and thin-layer chromatography.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

It has been found that although Brevibacterium lactofermentum No. 2256 is incapable of accumulating l-glutamic acid in a biotin sufficient medium, it produces a large quantity of the acid in the presence of sucrose fatty acid ester. In a biotin deficient medium, however, the ester brought the unfavorable diminution of l-glutamic acid accumulation caused by the decrease of glucose consumption in an incubation period. The undesirable effects were practically lost when the ester was added to the culture medium after more than eight hours in the course of incubation. This fact suggests that the ester is concerned with the growth of microorganism. It is very interesting to elucidate the interrelation between sucrose fatty acid ester and biotin. For the maximum accumulation of l-glutamic acid corresponding increase in amount of the ester to the increasing concentration of biotin was necessary. The proportional relation did not extend to excedingly high levels of the two implicating factors. The further observations concerning the effects of the individual fatty acid esters such as sucrose stearate remain unsatisfactory.     

Synthesis and Some Properties of UDP-(1)fructose

UDP-(1)fructose was synthesized essentially by the method of Michelson or Roseman et al. The product obtained was much more stable to acid than UDP-fructose isolated from Jerusalem artichoke tubers by Umemura et al.⁷⁾ and UDP-glucose. Hydrolysis time curves of UDP-(1)fructose and fructose-1-phosphate in 0.01N HGl and 0.1N HCl both at 100°C are presented. It was concluded from these curves that UDP-(1)fructose was first hydrolyzed into UMP and fructose-1-phosphate, and then fructose-1-phosphate was hydrolyzed more slowly into free fructose and inorganic phosphate.