Noshi Minamiura

Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature

A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp. from Thailand soil. The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies. The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa. This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability. The optimum temperature and pH were 60 degrees C and 6.0, respectively. The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+). The optimum temperature for levan production was 50 degrees C. Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C.

Characterization of levan produced by Serratia sp.

Abstract Levan was produced by a newly isolated bacterium from soil, taxonomically identified as a Serratia sp. This is the first report of levan production by Serratia sp. The levan was digested by levanase, which cannot hydrolyze β-2,1 linkages and the remaining substrate was analyzed by NMR. It was found that this levan had less β-2,1 linkage than other microbial levans, and that the structure was quite different from the levan produced by other bacteria such as genus Bacillus .

A novel method of isolation and some characteristic properties of human pancreatic elastases

One component of elastases of human pancreatic juice and pancreatic extract was obtained in a highly purified state by chromatography on a column of sawdust. The elastase obtained after repeated adsorption chromatography with NaCl-containing buffers was almost homogeneous by gel filtration and polyacrylamide gel electrophoresis. This elastase showed relatively high elastolytic activity, but relatively low hydrolytic activity towards succinyl trialanine p-nitroanilide, as compared with another component of pancreatic juice elastase (which was not absorbed onto sawdust). Both elastases isolated were alkaline earth metal-dependent enzymes.

Three Different Types of α-Amylases from Aspergillus awamori KT-11: Their Purifications, Properties, and Specificities

Three forms of α-amylases, designated Amyl I, Amyl II, and Amyl III were purified to a homogenous state by several column chromatographies from a koji culture in wheat bran of a strain of black mold, which was isolated in Indonesia and identified as Aspergillus awamori. They have molecular weights of 49,000, 63,000, and 97,000 by SDS-PAGE, respectively, and the optimum pHs were 4.0 for Amyl I and 5.5 for both Amyl II and Amyl III on soluble starch. Amyl I hydrolyzed malto-tetraose, -pentaose, -hexaose, -heptaose, and β- and γ-cyclodextrin to produce maltose and maltotriose as major products but not or hardly hydrolyzed maltose, isomaltose, maltotriose, isomaltotriose, α-cyclodextrin, or raw corn starch. On the other hand, both Amyl II and Amyl III hydrolyzed maltotriose as well as all the maltooligosaccharides described above and α-, β-, and γ-cyclodextrin, and even raw corn starch as well as heat-gelatinized corn starch to produce maltose as a major product and glucose and maltotriose as minor products, but they did not hydrolyze maltose, isomaltose, or isomaltotriose. The limit hydrolyses of soluble starch with three kinds of enzymes were 33% for Amyl I, 35% for Amyl II, and 38% for Amyl III, the reaction products had α-anomeric forms by NMR analysis, and the blue color reaction with I2 disappeared completely at about 18% of hydrolysis of the starch for all enzymes.

Isolation of a Bacterium Assimilating (R)-3-Chloro-1,2-Propanediol and Production of (S)-3-Chloro-1,2-Propanediol Using Microbial Resolution

A bacterium capable of assimilating 3-chloro-1,2-propanediol was isolated from soil by enrichment culture. The strain was identified as Alcaligenes sp. by taxonomic studies. The crude extracts of the cells had dehalogenating activities and converted various halohydrins to the corresponding epoxides. 3-Chloro-1,2-propanediol was degraded stereospecifically by the strain, liberating chloride ion. The residual isomer was found to be the (S)-form (99.4% enantiomeric excess). (S)-3-Chloro-1,2-propanediol was obtained from the racemate by use of this strain in 38% yield, and (S)-glycidol (99.4% enantiomeric excess) was subsequently synthesized from the obtained (S)-3-chloro-1,2-propanediol by alkaline treatment.

Some properties of levansucrase of Bacillus natto stabilized with periodate oxidized yeast glucomannan

Abstract It was observed that levansucrase from Bacillus natto became unstable and was easily inactivated when the salts were removed from the enzyme solution, while the enzyme was stable for long time in a buffered saline. After modification with periodate oxidized yeast glucomannan, the enzyme increased thermal stability up to 45°C, in which it conserved more than 90% of its activity after 15 min treatement. The optimum temperature was also shifted from 40°C in the case of original enzyme to 50°C for the modified enzyme after 10 min reaction time. The half-life time increased significantly from 9 min to 55 min at 50°C, however it increased from 30 min and 22 min respectively at 40°C and 45°C to more than 1 h at the same temperature. The content of carbohydrates of modified enzyme was 25% that increases the molecular weight from 57 KDa to 80 KDa. The products from sucrose by the modified enzyme were the same as the case using original enzyme. Namely, the products confirmed were levan and 3 kestoses (6-, 1-, and neo-kestose).

A novel generation of optically active 1,2-diols from the racemates by using halohydrin dehydro-dehalogenase

Abstract A novel enzyme dehalogenating halohydrins, designated as halohydrin dehydo-dehalogenase (HDDase), was purified from Alcaligenes sp. DS-S-7G. The enzyme catalyzed oxidative dehalogenation of ( R )-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] to acetic acid and formaldehyde via hydroxyacetone stereoselectively by the addition of artificial electron acceptors. The dehalogenating activity was much higher in the presence of 2,6-dichlorophenolindophenol (DCIP) and phenazine methosulfate (PMS). The resulting stereoselective dehydro-dehalogenation was applicable to preparation of various optically active halohydrins and 1,2-diols so that the respective residual isomers had excellent enantiomeric excesses (ee) (60–99% ee).

Development of a radioimmunoassay for human deoxyribonuclease I

A reliable radioimmunoassay (RIA) for human deoxyribonuclease I (DNase I) is described. Using delayed addition of tracer antigen, the method is sensitive (9.5 reproducible and specific. A good parallel relationship was observed between the standard curve and dilution curves for human urine and human pancreatic juice. G-actin, a naturally occurring DNase I inhibitor, caused no change in the immunoreactivity of DNase I. In healthy individuals, aged 11-90 yr, the mean serum DNase I was 18.4 ng/ml (SD 6.7 ng/ml). Increased serum DNase I occurred in patients with acute pancreatitis, renal failure, and in about one-third of patients with various malignant tumors.

Detection and serological relationships of cymbidium mosaic potexvirus isolates.

Twenty-two isolates of Cymbidium mosaic virus (CyMV) were isolated from 35 orchid plants suspected of being infected with CyMV. Among the three methods used for detecting CyMV, immunoelectron microscopy (IEM-1) was shown to be the most sensitive method, being able to detect the virus in 71.43% of suspected CyMV-infected plants while the electron microscopic method and the indexing plant method could detect 51.43 and 42.86%, respectively. Out of 12 symptomless plants investigated, 25% were found by IEM-1 method to be infected with the virus. Purified CyMV were flexuous rods having lengths between 470-490 nm. A few end-to-end aggregates were also observed and the 280 260 absorbance ratios were from 0.884 to 0.929. The yield of CyMV was 31.07 to 44.09 mg per kg of Datura leaves. Antibodies against purified CyMV D2 were produced in rabbits and hens. The antibody titers in the yolk and sera of hens indicated that 0.5 mg of virus per immunization efficiently generated an abundant supply of IgY in the yolk, however 1 mg of virus per immunization gave a stronger immune response in both sera and yolk. The average yields of IgY were 6.5 +/- 0.6 and 9.4 +/- 0.9 mg/ml of yolk in the group that received 0.5 mg and the group that received 1.0 mg of the virus, respectively. Positive ELISA reactions were observed in 18 and 20 of 22 CyMV isolates when detected with rabbit IgG and IgY, respectively, demonstrating that those isolates were serologically related and the ELISA reactions were shown to be stronger with IgY than those with rabbit IgG in most isolates. The degree of reaction between the CyMV isolates, O(2) and O(4), and IgY was less than that of the other isolates. The two isolates, D(6) and Cat(6), gave negative reactions to rabbit IgG. The results of ELISA assays showed that the homologous serological reaction was not consistently stronger than the heterologous one. Twelve isolates out of twenty-two gave stronger reactions than the homologous antigen (CyMV D(2)) when IgY was used as the detecting antibody while nine isolates gave stronger reactions when using rabbit IgG. No reactions were observed with other plant viruses and plant proteins from healthy Datura.

Evaluation of enzyme-linked immunosorbent assays for the detection of cymbidium mosaic virus in orchids

Abstract Indirect enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich (DAS) ELISA using polyclonal antibodies (PAb), egg yolk immunoglobulin G (IgY), rabbit antisera (IgG) or monoclonal antibodies (MAb) alone, or any two in combination, were compared for the detection of Cymbidium mosaic virus (CyMV) in orchids. An indirect ELISA using IgG, IgY or MAb was able to detect 50–100 ng of purified virus and 2–8-fold dilution of infected leaf extract. Detection of purified CyMV by DAS-ELISA using IgY as the detecting antibody gave 4–16 times higher sensitivity than that using IgG on either MAb or PAb coated microplates. The DAS-ELISA using MAb as the coating antibody and IgY as the detecting antibody was shown to be the most sensitive, being able to detect 6.25 ng of purified virus and 1024-fold dilution of an infected leaf extract.