Photoinduced Decarboxylation-dependent Transamination of Pyridoxal 5′-Phosphate-α-Amino Acid Schiff Bases

Bilirubin oxidase was purified from the culture filtrate of Myrothecium verrucaria MT-1 by a procedure involving ammonium sulfate precipitation, charcoal treatment, and QAE-Sephadex A-50 and Sephadex G-100 column chromatographies. The purified enzyme was homogeneous on disc gel electrophoresis.

Copper and carbohydrate were contained in the enzyme. The enzyme was inhibited by Fe²⁺ and compounds that complex with copper. Bilirubin, biliverdin, hemin and chlorophyllin which consist of tetrapyrrole, and substrates of laccase were oxidized by the enzyme. Bilirubin was oxidized more rapidly than other substances. Bilirubin oxidase differed from laccase in reactivity with substances consisting of tetrapyrrole. Substances consisting of tetrapyrrole were oxidized only a little by laccase but rapidly oxidized by bilirubin oxidase. The apparent Km value for bilirubin was calculated to be 190 μm.     

DOPA Production with Enterobacter cloacae NB 320 by Transamination Reaction

Taxonomical investigation was performed on the bacterium, strain NB 320 isolated from soil, and it was identified as Enterobacter cloacae. This bacterium produced the enzyme which catalyzed the transamination reaction between 3,4-dihydroxyphenyl pyruvate and an amino acid to form l-Dopa.

The optimum culture conditions for the enzyme production were studied along with the characteristics of the enzyme. The enzyme of the strain was different in some properties from that of Alcaligenes faecalis IAM 1015 which had been already studied. The former utilized glutamate as an amino donor best among the amino acids tested for transamination and was induced by the addition of glutamine and asparagine. Intact cells of the strain did not catalyze the reaction unless they were treated with sonication or with a detergent.     

Properties of the recombinant α-glucosidase from Sulfolobus solfataricus in relation to starch processing

An α-glucosidase activity (EC 3.2.1.20) isolated from Sulfolobus solfataricus strain MT-4 was characterised and found of interest at industrial level in the saccharification step of hydrolysis process of starch. The gene encoding for the enzyme was expressed in Escherichia coli BL21 (DE3) with a yield of 87.5 U/g of wet biomass. The recombinant cytosolic enzyme was purified to homogeneity with a rapid purification procedure employing only steps of selective and progressive thermal precipitations with a final yield of 75.4% and a purification of 14.5-fold. The properties of this thermophilic α-glucosidase were compared with those of the α-glucosidase of a commercial preparation from Aspergillus niger used in the starch processing.     

Identification of Potent Bacterium for Production of Chillproofing Enzyme

A bacterium was isolated which produced an extracellular proteolytic enzyme preventing chill haze of beer. This strain was named B–103.

Taxonomical studies on strain B–103 indicated that it was Gram-negative, aerobic, motile, and catalase-positive. Cells were single rods (0.2~0.6×1.0~1.8 μ) with a peritrichous flagella. The bacterium produced nitrite from nitrate, extracellular DNase and 5′-nucleotide from nucleoside. It was capable of nitrate respiration and of oxidative or fermentative cleavage of carbohydrates.

From these characteristics, it was identified as a strain of Serratia marcescens.     

Isolation and Identification of Microorganism,Producing Microbial Alkaline Proteinase Inhibitor (MAPI)

In the screening of actinomycetes’ culture filtrate for inhibitor of subtilisin and various microbial alkaline proteinases, a novel inhibitor was found in a cultured broth of strain WT-27. This inhibitor was named as MAPI, abbreviation of microbial alkaline proteinase inhibitor.Judging from the morphological and physiological properties of the actinomycetes which produced MAPI, this strain was identified as Streptomyces nigrescens.

For the production of MAPI, this strain was aerobically cultured at 25 ~ 27%C in a jar fermentor which contained an optimum medium consisting of polypepton 3 %, meat extract 1%, glucose 1%, NaCl 0.1%, K₂HPO₄ 0.1% and MnSO₄·nH₂O 0.0001%, pH 7.0. The production of MAPI reached its maximum after 21 ~ 24 hr cultivation.

MAPI had an inhibitory activity against various microbial alkaline proteinases, α-chymotrypsin and papain but not against trypsin, kallikrein, thermolysin, or pepsin.     

An α-1, 3-Glucanase from Streptomyces sp. KI-8 Production and Purification

An α-l,3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-l,3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-l,3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied.

The enzyme was purified by (NH₄)₂SO₄ precipitation, column chromatography on DEAE-cellulose and P(phospho)-cellulose. To eliminate the concomitant β-l,3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-l,3-glucanase was eluted with a solution of α-1,3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78,000 by SDS-polyacrylamide gel electrophoresis.     

Extremely Barophilic Bacteria Isolated from the Mariana Trench, Challenger Deep, at a Depth of 11,000 Meters

Two strains of obligately barophilic bacteria were isolated from a sample of the world’s deepest sediment, which was obtained by the unmanned deep-sea submersible Kaiko in the Mariana Trench, Challenger Deep, at a depth of 10,898 m. From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to Shewanella benthica and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus Moritella. The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain. This is the first evidence of the existence of an extreme-barophile bacterium of the genus Moritella isolated from the deep-sea environment.     

Studies on Transglycosidation to Vitamin B6 by Microorganisms

The enzyme activity to synthesize pyridoxine glucoside was demonstrated in intact cells and cell extracts of genera, Sarcina and Micrococcus. The isolated and identified strain, Micrococcus sp. No. 431 was found to have high activity of this enzyme in its cell extract.

The enzyme activity reached to a maximum after 20 hr of cultivation.

The enzyme which synthesized pyridoxine glucoside via transglucosidation from sucrose to pyridoxine was purified from Micrococcus sp. No. 431 by means of ammonium sulfate fractionation, DEAE-Sephadex, hydroxylapatite and Sephadex G–100 column chromatographies. The enzyme was purified about 354–fold and confirmed to be homogenous in polyacrylamide-gel electrophoresis and ultracentrifugation.     

Serratia Protease

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease production of this strain was over 3 times as much as that of Serratia marcescens which was known as a protease-producing organism. The highly purified enzyme was prepared from the culture supernatant through ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75.

The purified enzyme moved homogeneously with a sedimentation constant, s_20,w of 3.8 S in ultracentrifugation and the molecular weight was determined to be 6.0 × 10₄ by the Archibald method. Determination of the ultraviolet absorption spectrum indicated the E^1%_{280 mμ,1 cm} was 13.0. Neither carbohydrate nor sulfur-containing amino acid was detected in the purified enzyme preparation. The enzyme showed maximal activity at pH 9.0 and at 40°C, and was stable under lower temperatures over the pH range from 5 to 10, whereas it was unstable at 37°C in alkaline conditions. The enzyme was completely inactivated by heating at 55°C for 15 min.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

C3d and Epstein-Barr Virus (CR2/CD21 Ligands) Stimulate Cells of an HTLV-I Line,MT-2

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with ³²P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Degradation of Barley Glucan and Lichenan by a Bacillus pumilus Enzyme

A bacterial strain was isolated from soil, which rapidly degraded purified barley β-glucan as well as lichenan. The strain belonged to Bacillus pumilus, and some authentic strains of this species were also shown to hydrolyze the gluean. An enzyme active on the above substrates but not on laminaran and on CM-cellulose was partially purified from the culture fluid. This enzyme, about 27,000 in molecular weight, was found to cleave a β-(1 → 4) linkage adjacent to a β-(1 → 3) in the polymers. It was suggested that only an enzyme of this type should be called a ‘lichenanase’ and discriminated from cellulases and laminaranases.     

Prospects for the use of new basidiomycete strains for the direct conversion of lignocellulose into ethanol

Sixty-six isolates of basidiomycete fungi were screened for the ability to synthesize cellulase. The effect of temperature on cellulase activity was studied for eight basidiomycete strains as perspective producers of ethanol. The temperature optima of enzyme activity ranged between 26 and 32°C. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and rye straw were studied for seven basidiomycetes strains: Fomitopsis pinicola MT-5.09, F. pinicola MT-5.21, Piptoporus betulinus MT-30.04, Fomes fomentarius MT-4.05, F. fomentarius MT-4.23, Trametes hirsuta MT-24.24, Flammulina velutipes MT-3.03 Maximum ethanol production from Na-carboxymethyl cellulose (1.3 g/dm³) was achieved by strain F. velutipes MT-3.03. Strain F. fomentarius MT-4.05 more effectively converted rye straw to ethanol with yield of 1.1 g/dm³.     

Screening Test of Microbes

Potent bacteria for production of chillproofing enzyme were isolated during screening tests on 1670 strains of microorganisms.

All but one of these bacteria were classified as Serratia marcescens and the exceptional strain was tentatively designated as B–103. These bacteria produced an extracellular proteolytic enzyme which prevented chill haze of beer.     

Cloning and characterization of indolepyruvate decarboxylase from <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> AM1

For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k _cat value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k _cat/K _m). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown.     

A denitrifying bacterium from the deep sea at 11 000-m depth

The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30°C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4°C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies. Received: June 2, 1997 / Accepted: August 9, 1997     

Agarolytic Pathway in the Newly Isolated Aquimarina sp. Bacterial Strain ERC-38 and Characterization of a Putative β-agarase

Marine microbes, particularly Bacteroidetes, are a rich source of enzymes that can degrade diverse marine polysaccharides. Aquimarina sp. ERC-38, which belongs to the Bacteroidetes phylum, was isolated from seawater in South Korea. It showed agar-degrading activity and required an additional carbon source for growth on marine broth 2216. Here, the genome of the strain was sequenced to understand its agar degradation mechanism, and 3615 protein-coding sequences were predicted, which were assigned putative functions according to their annotated functional feature categories. In silico genome analysis revealed that the ERC-38 strain has several carrageenan-degrading enzymes but could not degrade carrageenan because it lacked genes encoding κ-carrageenanase and S1_19A type sulfatase. Moreover, the strain possesses multiple genes predicted to encode enzymes involved in agarose degradation, which are located in a polysaccharide utilization locus. Among the enzymes, Aq1840, which is closest to ZgAgaC within the glycoside hydrolase 16 family, was characterized using a recombinant enzyme expressed in Escherichia coli BL21 (DE3) cells. An enzyme assay revealed that recombinant Aq1840 mainly converts agarose to NA4. Moreover, recombinant Aq1840 could weakly hydrolyze A5 into A3 and NA2. These results showed that Aq1840 is involved in at least the initial agar degradation step prior to the metabolic pathway that uses agarose as a carbon source for growth of the strain. Thus, this enzyme can be applied to development and manufacturing industry for prebiotic and antioxidant food additive. Furthermore, our genome sequence analysis revealed that the strain is a potential resource for research on marine polysaccharide degradation mechanisms and carbon cycling.