Thermostable Acid Protease Produced by Penicillium duponti K1014, a True Thermophilic Fungus Newly Isolated from Compost

Hashimoto, Hikotaka; Iwaasa, Takashi; Yokotsuka, Tamotsu

doi:10.1128/aem.24.6.986-992.1972

Cited by 18 publications

(9 citation statements)

References 6 publications

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“…25, 1973 have been isolated thus far. These data agree with the thermal properties obtained with a crude enzyme preparation (4).…”

Section: Discussionsupporting

confidence: 89%

“…Assay of protease activity. Protease activity was assayed as described in detail previously (4). When necessary, the casein-275-nm method (3) was used.…”

Section: Methodsmentioning

confidence: 99%

“…Milk-clotting acid proteases from thermophilic species of Mucor, Mucor pusillus (1, 10) and Mucor miehei (16,17), have been purified and characterized. However, the properties of acid protease from other thermophilic fungi have not been described in detail; only a few reports (4,5) have been published at this time.…”

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confidence: 99%

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Some Properties of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

Hashimoto

Iwaasa

Yokotsuka

1973

Applied Microbiology

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A purified acid protease from a true thermophilic fungus, Penicillium duponti K1014, was most active at pH 2.5 for milk casein and at pH 3.0 for hemoglobin. The enzyme was stable at a pH range of 2.5 to 6.0 at 30 C for 20 h. The acid protease retained full activity after 1 h at 60 C at a pH range between 3.5 and 5.5. At the most stable pH of 4.5, more than 65% of its activity remained after heat treatment for 1 h at 70 C. These thermal properties show the enzyme as a thermophilic protein. The enzyme activity was strongly inhibited by sodium lauryl sulfate and oxidizing reagents such as potassium permanganate and N-bromosuccinimide. No inhibition was caused by chelating reagents, potato inhibitor, and those reagents which convert sulfhydryl groups to mercaptides. Reducing reagents showed an activating effect. The enzyme showed the trypsinogen-activating property at an acidic pH range; optimal trypsinogen activation was obtained at a pH of approximately 3.0. The isoelectric point of the enzyme was estimated to be pH 3.89 by disk electrofocusing. By using gel filtration, an approximate value of 41,000 was estimated for the molecular weight.

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“…25, 1973 have been isolated thus far. These data agree with the thermal properties obtained with a crude enzyme preparation (4).…”

Section: Discussionsupporting

confidence: 89%

“…Assay of protease activity. Protease activity was assayed as described in detail previously (4). When necessary, the casein-275-nm method (3) was used.…”

Section: Methodsmentioning

confidence: 99%

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Some Properties of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

Hashimoto

Iwaasa

Yokotsuka

1973

Applied Microbiology

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“…As it is difficult to obtain sufficient amounts from the native strain of C. thermophilum, overexpression of the C. thermophilum gene pro in yeast provided an efficient method to produce the protease. The recombinant protease expressed in P. pastoris exhibited remarkable thermal stability and a high activity and it is one of the most thermostable proteases isolated from fungi (Hashimoto et al 1972;Stevenson and Gaucher 1975;Shenolikar and Stevenson 1982;Gaur et al 1989;Morton et al 2003;Bazarzhapov et al 2006;Wang et al 2006;Kotlova et al 2007;Liu and Yang 2007;Yang et al 2007b).…”

Section: Characterization Of Recombinant Protease Expressed In Pichiamentioning

confidence: 99%

Cloning, expression and characterization of the serine protease gene fromChaetomium thermophilum

2009

Journal of Applied Microbiology

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Aims: Microbial proteases play an essential role in scientific research and commercial applications. This study is to clone, sequence, and express a thermostable protease gene from the thermophilic fungi Chaetomium thermophilum and to generate yeast strains expressing C. thermophilum protease suitable for industrial applications. Methods and Results: Degenerate primers were designed based on the conserved domain of other identified serine proteases and cDNA fragment of C. thermophilum gene pro was obtained through reverse transcriptase‐polymerase chain reaction (RT‐PCR). The full‐length cDNA of 2007 bp was generated using RACE amplification. The cDNA contains an open reading frame of 1596 bp encoding 532 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the subtilisin serine proteases. The C. thermophilum gene pro was expressed in Escherichia coli BL21 (DE3) and Pichia pastoris, respectively and soluble protein was obtained in P. pastoris. The expressed protease was secreted into the culture medium by the yeast in a functional active form and the purified recombinant protease exhibits optimum catalytic activity at pH 8·0 and 60°C. The enzyme is stable at 60°C. The integration of gene pro into P. pastoris genome is stable after 10 generations and the yeast transformants showed a consistent protease expression. Conclusions: Gene pro encoding a serine protease from C. thermophilum was cloned, sequenced, and overexpressed successfully in P. pastoris. The expressed protease was purified and the properties of the recombinant protease are characterized. Significance and Impact of the Study: Chaetomium thermophilum is a soil‐borne thermophilic fungus and the protease cloned from it is stable in a high temperature and a wide rage of pH. The overexpression of the enzyme in a mesophilic micro‐organism offers a potential value for scientific research and commercial applications.

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“…Penicillium duponti K1014, which was isolated from the compost in Japan, was found to produce a large amount of extracellular acid protease and other hydrolytic enzymes such as cellulases, amylases, and hemicellulases (10).…”

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confidence: 99%

Production and Purification of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

Hashimoto¹,

Kaneko²,

Iwaasa³

et al. 1973

Applied Microbiology

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A thermophilic fungus, Penicillium duponti K1014, produced an acid protease in a submerged culture. Maximum enzyme production, in a 30-liter fermentor at a preselected optimum growth temperature of 50 C, occurred after 3 days. The productivity of the enzyme was associated with changes of viscosity and pH of culture broth during the growth. The acid protease was isolated from the culture filtrates and was purified ninefold by alcohol precipitation, column chromatography on O-(diethylaminoethyl)cellulose, batchwise treatment with O-(carboxymethyl)cellulose, and by gel filtration. The purified enzyme was homogeneous on disk electrophoresis and sedimentation analysis. The sedimentation constant S20.w, calculated at a concentration of 0.64%, was 3.98S.

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Thermostable Acid Protease Produced by Penicillium duponti K1014, a True Thermophilic Fungus Newly Isolated from Compost

Cited by 18 publications

References 6 publications

Some Properties of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

Some Properties of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

Cloning, expression and characterization of the serine protease gene fromChaetomium thermophilum

Production and Purification of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

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