Dataset Information

Purification and cDNA cloning of NADPH-dependent aldoketoreductase, involved in asymmetric reduction of methyl 4-bromo-3-oxobutyrate, from Penicillium citrinum IFO4631.

ABSTRACT: Penicillium citrinum was found to catalyze the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate [(S)-BHBM] with high optical purity. From the strain, a cDNA clone encoding a novel NADPH-dependent alkyl 4-halo-3-oxobutyrate reductase (KER) was isolated. Escherichia coli cells overexpressing KER produced (S)-BHBM in the presence of an NADPH-regeneration system.

SUBMITTER: Asako H

PROVIDER: S-EPMC546806 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Purification and cDNA cloning of NADPH-dependent aldoketoreductase, involved in asymmetric reduction of methyl 4-bromo-3-oxobutyrate, from Penicillium citrinum IFO4631.

Asako Hiroyuki H Wakita Ryuhei R Matsumura Kenji K Shimizu Masatoshi M Sakai Jun J Itoh Nobuya N

Applied and environmental microbiology 20050201 2

Penicillium citrinum was found to catalyze the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate [(S)-BHBM] with high optical purity. From the strain, a cDNA clone encoding a novel NADPH-dependent alkyl 4-halo-3-oxobutyrate reductase (KER) was isolated. Escherichia coli cells overexpressing KER produced (S)-BHBM in the presence of an NADPH-regeneration system. ...[more]

PMID: 15691974

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Project description:BackgroundEndophytic fungi are known plant symbionts. They produce a variety of beneficial metabolites for plant growth and survival, as well as defend their hosts from attack of certain pathogens. Coastal dunes are nutrient deficient and offer harsh, saline environment for the existing flora and fauna. Endophytic fungi may play an important role in plant survival by enhancing nutrient uptake and producing growth-promoting metabolites such as gibberellins and auxins. We screened roots of Ixeris repenes (L.) A. Gray, a common dune plant, for the isolation of gibberellin secreting endophytic fungi.ResultsWe isolated 15 endophytic fungi from the roots of Ixeris repenes and screened them for growth promoting secondary metabolites. The fungal isolate IR-3-3 gave maximum plant growth when applied to waito-c rice and Atriplex gemelinii seedlings. Analysis of the culture filtrate of IR-3-3 showed the presence of physiologically active gibberellins, GA1, GA3, GA4 and GA7 (1.95 ng/ml, 3.83 ng/ml, 6.03 ng/ml and 2.35 ng/ml, respectively) along with other physiologically inactive GA5, GA9, GA12, GA15, GA19, GA20 and, GA24. The plant growth promotion and gibberellin producing capacity of IR-3-3 was much higher than the wild type Gibberella fujikuroi, which was taken as control during present study. GA5, a precursor of bioactive GA3 was reported for the first time in fungi. The fungal isolate IR-3-3 was identified as a new strain of Penicillium citrinum (named as P. citrinum KACC43900) through phylogenetic analysis of 18S rDNA sequence.ConclusionIsolation of new strain of Penicillium citrinum from the sand dune flora is interesting as information on the presence of Pencillium species in coastal sand dunes is limited. The plant growth promoting ability of this fungal strain may help in conservation and revegetation of the rapidly eroding sand dune flora. Penicillium citrinum is already known for producing mycotoxin citrinin and cellulose digesting enzymes like cellulase and endoglucanase, as well as xylulase. Gibberellins producing ability of this fungus and the discovery about the presence of GA5 will open new aspects of research and investigations.

Project description:Backgroundβ-Glucosidases are components of the cellulase system, a family of enzymes that hydrolyze the β-1,4 linkages of cellulose. These proteins have been extensively studied due to the possibility of their use in various biotechnological processes. They have different affinities for substrates (depending on their source) and their activities can be used for saccharification of different types of biomass. In this context, the properties and the synergistic capacity of β-glucosidases from different organisms, to supplement the available commercial cellulase cocktails, need a comprehensive evaluation.ResultsTwo β-glucosidases belonging to GH3 family were secreted by Penicillium citrinum UFV. PcβGlu1 (241 kDa) and PcβGlu2 (95 kDa) presented acidic and thermo-tolerant characteristics. PcβGlu1 showed Michaelis-Menten kinetics for all substrates tested with Km values ranging from 0.09 ± 0.01 (laminarin) to 1.7 ± 0.1 mM (cellobiose, C2) and kcat values ranging from 0.143 ± 0.005 (laminarin) to 8.0 ± 0.2 s-1 (laminaribiose, Lb). PcβGlu2 showed substrate inhibition for 4-methylumbelliferyl-β-d-glucopyranoside (MUβGlu), p-nitrophenyl-β-d-glucopyranoside (pNPβGlu), cellodextrins (C3, C4, and C5), N-octil-β-d-glucopyranoside, and laminaribiose, with Km values ranging from 0.014 ± 0.001 (MUβGlu) to 0.64 ± 0.06 mM (C2) and kcat values ranging from 0.49 ± 0.01 (gentiobiose) to 1.5 ± 0.2 s-1 (C4). Inhibition constants (Ki) for PcβGlu2 substrate inhibition ranged from 0.69 ± 0.07 (MUβGlu) to 10 ± 1 mM (Lb). Glucose and cellobiose are competitive inhibitors of PcβGlu1 and PcβGlu2 when pNPβGlu is used as a substrate. For PcβGlu1 inhibition, Ki = 1.89 ± 0.08 mM (glucose) and Ki = 3.8 ± 0.1 mM (cellobiose); for PcβGlu2, Ki = 0.83 ± 0.05 mM (glucose) and Ki = 0.95 ± 0.07 mM (cellobiose). The enzymes were tested for saccharification of different biomasses, individually or supplementing a Trichoderma reesei commercial cellulose preparation. PcβGlu2 was able to hydrolyze banana pseudostem and coconut fiber with the same efficiency as the T. reesei cocktail, showing significant synergistic properties with T. reesei enzymes in the hydrolysis of these alternative biomasses.ConclusionsThe β-glucosidases from P. citrinum UFV1 present different enzymatic properties from each other and might have potential application in several biotechnological processes, such as hydrolysis of different types of biomass.

Dataset Information

Purification and cDNA cloning of NADPH-dependent aldoketoreductase, involved in asymmetric reduction of methyl 4-bromo-3-oxobutyrate, from Penicillium citrinum IFO4631.

Publications

Purification and cDNA cloning of NADPH-dependent aldoketoreductase, involved in asymmetric reduction of methyl 4-bromo-3-oxobutyrate, from Penicillium citrinum IFO4631.

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