Laboratory of
Applied Microbiology
Course of Biofunction
Department of
Agro-bioscience
Faculty of
Agriculture
Iwate University (1st
Apr, 2001)
Link
to [Staff/Students] [Teaching Subjects] [Present Researches] [Published Researches]
Staff and Students
of Laboratory
Administrative Staff
Ø Norio
Wakao
Professor//Doctor of Agriculture (Tohoku University)
Tel (Fax) : 019-621-6156 E-mail: wakao@iwate-u.ac.jp
Ø Kimiyasu
Isobe Associate Professor//Doctor of Agriculture (Kyoto University)
Tel
(Fax) : 019-621-6155 E-mail: kiso@iwate-u.ac.jp
Students
Ø
Graduate School of Agricultural Sciences
( Master Program )
1st year ( Takuya Ippaimori, Keego Tokuta )
2nd
year ( Rie Onodera, Yoshiaki Takeda )
Ø Undergraduate
4th year ( Yasutaka Sasaki, Yuuki Takamatsu, Masanao
Minamino, Seishi Chiba )
Cooperative Staff
Ø Yoshikazu
Inaba ( Dai-ichi Kagaku Yakuhin Co.)
Ø
Naoko Mori ( Researcher of
Food Science Laboratory, Taishi Food Inc.)
Teaching subjects (Lectures
and Laboratories)
For
Undergraduate
Ø
Principle of Microbiology（Wakao） 2nd year/Second Semester
Ø
Physiology/Function of
Microorganisms（Wakao） 3rd year/First Semester
Ø
Environmental Microbiology（Wakao） 3rd year/Second Semester
Ø
Microbial Genetics（Wakao） 3rd year/Second Semester
Ø
Industrial Microbiology（Isobe） 4th year/First Semester
Ø
Laboratory Microbiology（Isobe） 3rd year/Second Semester
For Graduate School
for Mater’s Program
Ø
Advanced Environmental
Microbiology（Wakao） First Semester
Ø
Advanced Applied
Microbiology（Isobe） Second Semester
Present Researches of Laboratory
N.Wakao
Key words : Microbial ecology, Acid natural environments,
Acidophilic bacteria, Zn-BChla, Photosynthesis.
Microorganisms can inhabit many extreme natural
environments, such as temperature, pH, oxidation/reduction potential, salts,
nutrients, pressure, light, water activity and so on. Among these extreme environments, we have been studying the
world of acidophilic aerobic bacteria living under strongly acid conditions:
isolation, classification and physiology of the acidophiles. Especially, we
focused on the acidophilic aerobic photosynthetic bacteria containing the new
pigment, Zn-BChla, which was firstly discovered in our laboratory.
The first report on the new pigment was [Discovery of
natural photosynthesis using Zn-containing bacteriochlorophyll in an aerobic
bacterium Acidiphilium rubrum] published in the journal of Plant Cell
Physiol., 37:889-893. The work was awarded with a prize of the Japanese Society
of Plant Physiologists. http://www.nacos.com/jspp/gakkaisho.htm
Screening of acidophilic aerobic heterotrophic
bacteria.
ØIsolation of many acidophilic aerobic bacteria from various acid natural
environments, such as acid mine drainage waters (metal sulfide mines), acid hot
springs and acid sulfate soils.
ØClassification and identification of the isolates: Analysis of
morphological and physiological/biochemical properties, characteristics of
chemical classification, and phylogeny based on 16S rRNA base sequence.
Analysis
of photosynthetic pigments.
ØAnalysis of characteristics of photosynthetic
pigments (Zn-BChla and/or Mg-BChla) of the isolates by HPLC to find the
acidophilic aerobic photosynthetic bacteria.
Analysis of cultural
conditions influencing biosynthesis of the pigments.
ØThe
effects on growth of various cultural conditions, such as nutrients (carbon and
nitrogen sources) medium pH, dissolved oxygen, and light irradiation.
ØInvestigation
of photosynthesis, that is, production of ATP and reducing power and fixation
of carbon dioxide by light irradiation.
K.Isobe
【Screening, analysis of function, and
application of new enzymes produced by microorganisms】
Key words : Bioconversion, Diagnostic enzymes, Lipolytic enzymes.
Microorganisms are very important and essential for our living,
e.g., production of such fermented foods as sake, wine, soy, natto, yogurt and
cheese, production of such seasoning as amino acids and nucleic acids,
production of such law materials of medical supplies as antibiotics and
medicals, bioremediation of natural environments and so on. The aim of this
study is isolation of many microorganisms inhabiting under the specific
conditions of natural ecosystem, such as soils and waters, and then screening
of the enzymes of specific functions and activities among the isolates. In
addition, we study the characteristics of the enzymes newly found, structures
and functions of the enzymes and further their applications.
Methodology of Research
1. Screening of microorganisms which produce new enzymes from natural samples
(soils and waters).
2. Purification of the enzymes from the isolates and elucidation of their
properties.
3. Development of usefulness and applications of the enzymes.
4. Analysis of structures and functions of the enzymes and also relationship
between them.
Enzymes for analyses of living constituents (utilization
in medical field)
Ø
On screening,
elucidation and application of microbial enzymes useful for analyses of living
constituents, such as blood constituents. For example: Cholesterol oxidase,
cholesterol dehydrogenase, creatinine-metabolizing enzymes.
Enzymes for bioconversion (utilization in chemical
industrial field)
Ø On
screening, elucidation and
application of microbial enzymes useful for conversion of substances. For
example: Production of useful aldehydes from ethyleneglycol and synthesis of
glycolipids by esterases.
Enzymes available at acid condition (utilization in both
food and environmental fields)
Ø On
screening of thermo- and acido-tolerat microorganisms and the enzymes produced by them. For example:
Catalase (for the treatment of drainage waters), lipase (quality improvement of
oils and fats, application to organic synthesis reactions), esterase
(application to organic synthesis reactions), protease (innovation and
modification of biofunction by the enzymes).
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Main Researches Published
N.Wakao
1.
Wakao, N., Shiba, T., Hiraishi, A.,
Ito, M., Sakurai, Y. (1993)
Distribution of bacteriochlorophyll a in species of the genus Acidiphilium. Curr. Microbiol. 27:277-279.
2.
Wakao, N., Nagasawa, N., Matsuura, T., Matsukura,
H., Matsumoto, T., Hiraishi, A., Sakurai, Y., Shiota, H. (1994) Acidiphilium multivorum sp. nov., an acidophilic chemoorganotrophic bacterium from pyritic acid
mine drainage. J. Gen. Appl.
Microbiol. 40:143-159.
3.
Wakao, N., Yokoi,
N., Isoyama, N., Hiraishi, A., Shimada, K., Kobayashi, M., Kise, H., Iwaki, M.,
Itoh, S., Takaichi, S., Sakurai, Y. (1996) Discovery of natural photosynthesis using Zn-containing
bacteriochlorophyll in an aerobic bacterium Acidiphilium rubrum. Plant Cell Physiol., 37:889-893.
4.
Suzuki, K., Wakao,
N., Sakurai, Y., Kimura, T., Sakka, K., Ohmiya, K. (1997) Transformation of Escherichia coli
with a large plasmid of Acidiphilium multivorum AIU 301 encoding arsenic
resistance. Appl. Environ.
Microbiol., 63:2089-2091.
5.
Wakao, N., Hiraishi,
A., Shimada, K., Kobayashi, M., Takaichi, S., Iwaki, M., Itoh, S. (1997) Zinc-containing bacteriochlorophyll in
aerobic acidophilic bacteria of the genus Acidiphilium. J. Inorganic Biochemistry,
67:217. 8th International
Conference on Bioinorganic Chemistry (ICBIC-8) (Yokohama)
6.
Takaichi, S., Wakao,
N., Hiraishi, A., Shimada, K., Nagashima, V.P., Matsuura, K., Kobayashi, M.,
Iwaki, M., Itoh, S. (1997)
Discovery of natural photosynthesis using Zn-containing
bacteriochlorophyll in an aerobic bacterium Acidiphilium rubrum. Tetrapyrrole Photoreceptors in
Photosynthetic Organisms. Blarney,
Cork, Ireland: R.J. Cogdell (Glasgow, Berlin) .
7.
Nagashima, K.V.P. ,
Matsuura, K., Wakao, N., Hiraishi, A., Shimada, K. (1997) Nucleotide sequences of genes coding
for photosynthetic reaction centers and light-harvesting proteins of Acidiphilium
rubrum and related aerobic acidophilic bacteria. Plant Cell Physiol., 38:
1249-1258.
8.
Suzuki, K., Wakao,
N., Y., Kimura, T., Sakka, K., Ohmiya, K. (1998) Expression and regulation of the arsenic resistance
operon of Acidiphilium multivorum AIU 301 plasmid pKW301 in Escherichia
coli. Appl. Environ.
Microbiol., 64: 411-418.
9.
Shimada, K., Itoh,
S., Iwaki, M., Nagashima, K., Matsuura, K., Kobayashi, M., Wakao, N.
(1998) Reaction center complex
based on Zn-bacteriochlorophyll from Acidiphilium rubrum. 11th International Photosynthesis
Congress (Budapest).
10.
Wakao, N., Hiraishi,
A., Shimada, K., Kobayashi, M., Takaichi, S., Iwaki, M., Itoh, S. (1998) Discovery, characteristics and
distribution of zinc-BChl in aerobic acidophilic bacteria including Acidiphilium
species and other related acidophilic bacteria. The Phototrophic Prokaryotes ed.by G.A.Peschek, Plenum
Press, London. p.745-750.
11.
Hiraishi, A.,
Nagashima, K.V.P., Matsuura, K., Shimada, K., Takaichi, S., Wakao, N.,
Katayama, Y. (1998) Phylogeny and
photosynthetic features of Thiobacillus acidophilus and related
acidophilic bacteria: its transfer to the genus Acidiphilium as Acidiphilium
acidophilum comb. nov. Int. J.
Syst. Bacteriol. 48:1389-1398.
12.
Itoh, S., Iwaki, M.,
Wakao, N., Yoshizu, K., Aoki, A., Tazaki K. (1998) Accumulation of Fe, Cr and Ni metals inside cells of
acidophilic bacterium Acidiphilium rubrum that produces Zn-containing
bacteriochlorophyll a. Plant Cell
Physiol., 39:740-744.
13.
Suzuki, K., Wakao,
N., Kimura, T., Sakka, K., Ohmiya,K. (1998) Metalloregulatory properties of the ArsR and ArsD repressors
of Acidiphilium multivorum AIU 301. J. Ferment. Bioengineer., 85: 623-626.
14.
Kobayashi, M.,
Yamamura, M., Akiyama, M., Kise, H., Inoue, K., Hara, M., Wakao, N., Yahara,
K., Watanabe, T. (1998) Acid
resistance of zinc-bacteriochlorophyll a from an acidophilic bacterium Acidiphilium
rubrum. na. Sci.,
14:1149-1152.
15.
Akiyama, M.,
Kobayashi, M., Kise, H., Hara, M., Wakao, N., Shimada, K. (1998) Pigment composition of the
reaction center complex isolated from an acidophilic bacterium Acidiphilium
rubrum grown at pH 3.5.
Photomed. Photobiol., 20:85-87.
16.
Kobayashi, M.,
Akiyama, M., Yamamura, M., Kise, H., Takaichi, S., Watanabe, T., Shimada, K.,
Iwaki, M., Itoh, S., Ishida, N., Koizumi, M., Kano, H., Wakao, N., Hiraishi, A.
(1998) Structural determination of
the novel Zn-containing bacteriochlorophyll in Acidiphilium rubrum, otomed. Photobiol., 20:75-80.
17.
Wakao, N., Hiraishi,
A., Shimada, K. Kobayashi, M. Takaichi, S.,Iwaki, M., Itoh, S. (1999) Discovery, characteristics, and
distribution of Zinc-BChl in aerobic acidophilic bacteria including Acidiphilium
species and other related acidophilic bacteria. The Phototrophic Prokaryotes, ed by Peschek et al. Kluwer
Academic/Plenum Publishers, p.745-750.
18.
Sakka, K., Suzuki,
K., Wakao, N., Kimura, T., Ohmiya,K. (1999) Characterization of the arsenic resistance operon of an
acidophilic bacterium Acidiphilium multivorum. Recent Res. Devel. Agric. Biol. Chem., 3:1-922.
19.
Takaichi, S., Wakao,
N., Hiraishi, A., Itoh, S., Shimada, K. (1999) Nomenclature of metal-substituted
(bacterio)chlorophylls in natural photosynthesis: Metal-(bacterio)chlorophyll
and M-(B)Chl. Photosyn. Res.,
59:255-256.
20.
Akiyama, M., Miyake,
M., Kise, H., Narato, M., Wakao, N., Inoue, K., Kobayashi, M. (1999) Accumulation of poly-β-hydroxybutyrate in an acidophilic photosynthetic purple
bacterium Acidiphilium rubrum.
Photomed. Photobiol., 21:101-104.
21.
Akiyama, M.,
Nagashima, K.V.P., Hara, M., Wakao, N., Tominaga, K., Kise, H., Kobayashi,
M.(1999) Stoichiometries of LH1/RC determined by the molar ratio of BChl/BPhe analyzed
by HPLC in seven species of purple bacteria containing LH1 only. Photomed. Photobiol., 21:105-110.
22.
Kobayashi, M.,
Akiyama, M., Yamamura, M., Kise, H., Wakao, N., Ishida, N., Koizumi, M., Kano,
H., Watanabe, T. (1999)
Comparison of physicochemical properties of Metallo-bacteriochlorophylls
and metallochlorophylls.
Zeitshrift fur Physikalische Chemie, Bd.213, S.202-214.
23.
Wakao, N., Kudo, S.,
Isoyama, N., Isobe, K. (2000)
Analysis by colony-forming curves of growth characteristics of Acidiphilium
rubrum, an acidophilic aerobic photosynthetic bacrterium with
zinc-bacteriochlorophyll. 10th
International Symposium on Phototrophic Prokaryotes (Abstracts). p.131.
(Barcelona)
24.
Wakao, N., Tamo, R.,
Isobe, K., Chortip Lorrungruang, Napavarn Noparatnaraporn (2000) Screening of acidophilic aerobic
mesophilic and thermotolerant bacteria containing Zn-BChl and/or Mg-BChl in
natural environments in both Japan and Thailand: Preliminary report. 2nd Joint
Seminar on Development of Thermotolerant Microbial Resources and their
applications. p.123. (JSPS-NRCT) (Yamaguchi University).
25.
Hiraishi, A., Matsuzawa, Y., Kanbe, T.,
Wakao, N. (2000) Acidisphaera
rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing
bacterium isolated from acidic environments. Int. J. Syst. Evol. Microbiol. 50:1539-1546.
26. Akiyama,
M., Nagashima, K.V.P., Wakao, N., Kise, H., Hara, M., Kobayashi, M. (2001) Models of the RC/LH1 complexs of
photosynthetic purple bacteria.
Photomedicine and Photobiology, 23:27-34.
27. Wakao,
N., Yasuda, T., Jojima, Y., Yamanaka, S. and Hiraishi, A.(2002) Enhanced growth of Acidocella
facilis and related acidophilic bacteria at high concentrations of
aluminum. Microbes and Environments, 17:98-104.
K.Isobe
1.
Yamada, H., Isobe,
K., Tani, Y., Hiromi, K. (1979)
A differential determination procedure for spermine and spermidine with
beef plasma amine Oxidase. Agric.
Biol. Chem., 43:2487-2491.
2.
Sakurai, Y.,
Isobe, K., Shiota, H. (1980)
Partial purification and some properties of an alkaline arylsulfatase
produced by Aspergillus Fungi. Agric. Biol. Chem., 44:1-7.
3.
Isobe, K.,
Tani, Y, Yamada, H., Hiromi, K. (1980)
Determination of polyamines with immobilized beef plasma amine
oxidase. Agric. Biol. Chem.,
44:615-619.
4.
Yamada, H.,
Isobe, K., Tani, Y. (1980)
Oxidation of polyamines by fungal enzymes. Agric. Biol. Chem., 44:2469-2476.
5.
Isobe, K.,
Tani, Y., Yamada, H. (1980)
Crystallization and characterization of polyamine oxidase from Penicillium chrysogenum. Agric. Biol. Chem., 44:2651-2658.
6.
Isobe, K.,
Tani, Y., Yamada, H. (1980)
Crystallization and characterization of polyamine oxidase from Aspergillus terreus. Agric. Biol. Chem., 44:2749-2751.
7.
Isobe, K.,
Tani, Y., Yamada, H. (1980)
Kinetic properties of fungal polyamine oxidases and their application to
differential determination of spermine and spermidine. Agric. Biol. Chem., 44:2955-2960.
8.
Isobe, K.,
Tani, Y., Yamada, H. (1981)
Differential determination procedure for putrescine, spermidine and
spermine with polyamine oxidase from fungi and putrescine oxidase. Agric. Biol. Chem., 45:727-733.
9.
Isobe, K.,
Tani, Y., Yamada, H. (1982)
A new enzyme, agmatine oxidase, from fungi. Agric. Biol. Chem., 46:1345-1351.
10.
Isobe, K.,
Tani, Y., Yamada, H. (1982)
Crystallization and characterization of agmatine oxidase from Penicillium chrysogenum. Agric. Biol. Chem., 46:1353-1359.
11.
Otsuji, S.,
Soejima, Y., Isobe, K., Yamada, H., Takao, S., Nishi, M. (1985) An enzymatic differential assay for
urinary diamines, spermidine, and spermine. J. Cancer Res. Clin. Oncol., 109:115-121.
12.
Isobe, K.,
Yamada, H., Soejima, Y., Otsuji, S. (1987) A simple enzymatic differential assay for diamines,
spermidine, and spermine in urine and Blood. Biochem. Med., 37:110-120.
13.
Isobe, K.,
Matsuzawa, T., Soda, K., (1987)
Crystallization of 1-pyrroline-5-carboxylate dehydrogenase from Bacillus sphaericus. Agric. Biol. Chem., 51:1947-1953.
14.
Isobe, K.,
Yamada, H., Soejima , Y., Otsuji, S. (1987) A rapid enzymatic assay for total blood polyamines. Clin. Biochem, 20:157-161.
15.
Isobe, K.,
Akiba, T., Yamaguchi, S., Naka, Y., (1988) Crystallization and characterization of lipase
from Penicillium cyclopium. Agric. Biol. Chem., 52:41-47.
16.
Matsuzaki,
S., Hamana, K., Isobe, K. (1990)
Occurrence of N6-methylagmatine in seeds of leguminous plants. Phytochemistry, 29:1313-1315.
17.
Nokihara,
K., Isobe, K. (1990) Amino acid
sequence and glycosylation site of lipase from Penicillium camembertii.
J. Protein Chem., 9:298.
18.
Isobe, K.,
Nokihara, K., Yamaguchi, S., Mase, T., Schmid, R. D. (1992) Crystallization and
characterization of monoacylglycerol and diacylglycerol lipase from PenicilliumCamembertii. Eur.J.Biochem., 203:233-237.
19.
Matsuzaki,
S., Hamana, K., Okada, M., Isobe, K. (1992) Identification of unusual polyamines by the combined
use of high performance liquid chromatography and polyamine-metabolizing
enzymes. Dokkyo J. Med. Sciences, 19:117-124.
20.
Chemnitius,
G.C., Suzuki, M., Isobe, K., Kimura, J., Karube, I., Schmid, R.D.(1992) Thin-film polyamine biosensor :
Substrate specificity and application to fish freshness Determination. Anal. Chim. Acta, 263:93-100.
21.
Isobe, K.,
Matsuzawa,T., Y. Nagamura (1993) A
new enzymatic determination of ornithine carbamoyltransferase activity. Anal.
Lett., 26:475-486.
22.
Isobe, K.,
Nokihara, K. (1993) Primary
structure determination of mono- and diacylglycerol lipase from Penicillium camembertii. FEBS Lett., 320:101-106.
23.
Hedrich,
H.C., Isobe, K., Stahl, B., Nokihara, K., Kordel, M., Schmid, R.D., Karas, M.,
Hillenkamp, F., Spener, F. (1993)
Matrix-assisted ultraviolet laser desorption/ionization mass
spectrometry applied to multiple forms of lipases. Anal. Biochem., 211:288-292.
24.
Isobe, K.,
Aumann, K.-D. , Schmid, R. D. (1994) A structural model of mono- and diacylblycerol lipase
from Penicillium camembertii. J. Biotechnol., 32:83-88.
25.
Isobe, K.,
Nishise, H. (1994) Enzymatic
production of glyoxal from ethylene glycol using alcohol oxidase from methanol
Yeast. Biosci. Biotech. Biochem.,
58:170-173.
26.
Isobe, K.
(1995) Oxidation of ethylene
glycol and glycolic acid by glycerol oxidase. Biosci. Biotech. Biochem., 59:576-581.
27.
Isobe, K.,
Nishise, H. (1995) A new
enzymatic method for glycolaldehyde production from ethylene glycol. J. Mol. Catal. B : Enzymatic,
1:37-43.
28.
Isobe, K.,
Wakao, N. (1996)
Tween-hydrolyzing esterase produced by Acidiphilium sp. J.
Gen. Appl. Microbiol. 42:285-295.
29.
Isobe, K.,
Wakao, N. (1997) Purification and
some properties of thermostable acid esterase from Acidiphilium sp. AIU 409.
J. Gen. Appl. Microbiol. 43:151-156.
30.
Inagaki, K., Tanaka,
N., Isobe, K., Wakao, N., Tanaka, B. (1997) Cloning of the extracellular esterase gene from Acidocella
facillis. International
Biohydrometallurgy Symposium and Biomine Conference (Abstract) (Sydney).
31.
Isobe, K., Wakao, N.
(1998) Purification and
characterization of a thermostable acid esterase from Acidiphilium
sp.AIU409. Abstracts of 1st
JSPS-NRCT Joint Seminoar (Development of Thermotolerant Microbial Resourcess and
Their Applications in Thailand and Japan, Kasetsart University, Bangkok), p.104.
32.
Isobe, K., Wakao, N.
(1998) Screening of thermostable
acid esterase from acidophilic bacteria. Abstracts of 1st ISPS-NRCT Joint
Seminoar (Development of Thermotolerant Microbial Resourcess and Their
Applications in Thailand and Japan. Kasetsart University, Bangkok), p.140.
33.
Ukeda, H., Ishii,
T., Sawamura, M., Isobe, K. (1998)
Glycoaldehyde production from ethylene glycol with immobilized alcohol
oxidase and catalase. Biosci.
Biotechnol. Biochem. 62:1589-1591.
34.
Isobe, K., Nishise,
H. (1999) A method for glyoxylic
acid production using cells of Alcaligenes sp.GOX 373. J. Biotechnol.,75:265-271.
35.
Isobe, K., Inoue,
N., Kamata, K., Aran H.-Kittikum, Wakao, N. (2000) Screening of thermostable lipolytic enzyme from acid-
and thermotolerant fungi. 2nd
Joint Seminar on Development of Thermotolerant Microbial Resources and their
Applications. p.150 . (Yamaguchi
University).
Review
1. Matsuzaki, S., Hamana, K., Isobe, K., Niitsu, M., Samejima, K.
(1988) Novel Polyamines and
guanidinoamines in higher plants. The Biology and Chemistry of Polyamines (Ed.
S. H. Goldemberg and I. S. Algranati, ICSU Press, Oxford) pp. 159-167.
2. Matsuzaki, S., Hamana, K., Isobe, K. (1989) g -Guanidinooxypropylamine and
NG-methylagmatine : Novel guanidinoamines found in seeds of leguminous plants,
GUANIDINE 2 (Ed. A. Mori, et al,
Plenum Publishing Co.) pp. 15-21.
3. Sztajer, H., Erdmann, H., Isobe, K., Morelle, G., Schmid, R. D.
(1991) Production and some
properties of partially purified lipase from Penicillium simplicissimum, Lipases: Structure, Mechanism and
Genetic Engineering (Ed. L.Alberghina, et al., VCH Verlagsgesellschaft, F. R.
Germany) pp. 339-344.
4. Isobe, K., Nokihara, K. (1991) Physicochemical properties of mono- and diacylglycerol
lipase from Penicillium camembertii,
Lipases: Structure, Mechanism and Genetic Engineering (Ed. L. Alberghina, et
al., VCH Verlagsgesellschaft, R. Germany)
pp.345-348.
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