Microbial Proteinases and Biotechnology

Outtrup, Helle; Boyce, Charlotte

doi:10.1007/978-94-009-0765-2_6

Cited by 48 publications

(35 citation statements)

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“…R. miehei is well known from biotechnological applications in consequence of its extracellular enzyme production (e.g. proteases, lipases; Outtrup and Boyce 1990). At the same time, both species have been reported as agents of opportunistic fungal infections in men and animals (Ribes et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides

Lukács

Papp

Somogyvári

et al. 2008

Antonie van Leeuwenhoek

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In this study, the gene hmgR encoding the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) was cloned and characterized in the zygomycete fungus Rhizomucor miehei. The hmgR gene comprises a total of 3,585 bp including the coding sequence of a 1,058 amino acids length putative protein and five introns (137, 83, 59, 60 and 69 bp in length) dispersed in the whole coding region. Southern hybridization analysis revealed that the gene is present only in one copy in the R. miehei genome. The isolated Rhizomucor gene was expressed in the related fungus, Mucor circinelloides. Transformants harbouring the Rhizomucor hmgR gene in an autoreplicative plasmid proved to be more tolerant to statins (e.g. lovastatin, simvastatin, and fluvastatin), the competitive inhibitors of the HMG-CoA reductase, than the original M. circinelloides strain. At the same time, heterologous expression of the Rhizomucor hmgR did not affect the carotenoid production of M. circinelloides.

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Section: Introductionmentioning

confidence: 99%

Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides

Lukács

Papp

Somogyvári

et al. 2008

Antonie van Leeuwenhoek

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“…Extracellular proteases are important for the hydrolysis of proteins in cell-free environments and enable the cell to absorb and utilize hydrolytic products 4 . At the same time, these extracellular proteases have also been commercially exploited to assist protein degradation in various industrial processes 5,6 . It is important to screen the media components and fermentation conditions for cost-efficient enzyme production; proteases are generally produced by submerged fermentation.…”

Section: Introductionmentioning

confidence: 99%

Screening of Nutritional Parameters for the Production of Protease from Aspergillus Oryzae

Gedela

Lokeswari

Jayaraju

2006

Journal of Chemistry

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Production of protease enzyme by fungus Aspergillus oryzae was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing glucose, malt extract, yeast extract, peptone, K 2 HPO 4 , MgSO 4 and FeSO 4 . The present paper describes the screening of media components and fermentation conditions in shake flask. The organism utilized carbon sources glucose, fructose, sucrose, lactose, dextrin and starch among them glucose was found to be the best carbon source, for nitrogen sources various inorganic and organic media components were investigated among them peptone is found to be the best nitrogen source. 1% cottonseed followed by 2% Soya bean meal was found to be the best inducer. With optimized media two-fold increase in the protease production. The fungus growth depends on the concentration of carbon, nitrogen and salt solution, where as the enzyme production was also influenced by the culture time, pH and interaction between these two variables.

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“…A preliminary report of the amino acid sequence of Rt41A proteinase, deduced from the nucleotide sequence of the gene 1151, shows it is 70% similar to aqualysin I [16] and has high similarity to the industrially important subtilisin-like proteinases [17]. The differences in the properties of Thermus proteinases [I 81 potentially make them a commercially interesting group of enzymes, the diversity of which is not yet fully understood.…”

Section: Members Of the Extremely Thermophilic Eubacterial Genusmentioning

confidence: 99%

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A

Peek

Daniel

Monk

et al. 1992

European Journal of Biochemistry

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Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25 -10.5) which can be exploited in purification by using cationexchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90°C with 5 mM CaCI, present. No loss of activity was observed after 24 h at 70°C and the half-lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 "C and the half-life at 70 "C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents.Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.Generally there is no firm evidence to suggest that an enzyme isolated from an extremely thermophilic organism will have a higher specific activity than its mesophilic counterpart (when assayed at a temperature appropriate to the growth temperature of the producing organism). However, mesophilic proteins tend to denature and become more susceptible to proteolysis at the temperatures at which thermostable enzymes operate optimally. Thus, proteinases from extreme thermophiles have higher specific activities against mesophilic proteins than do most microbial proteinases [l]. The inherent stability of thermophilic proteinases to high temperatures as well as to detergents, organic solvents and chaotropic agents, makes these enzymes potentially useful in a range of biotechnological applications [2, 31.

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Microbial Proteinases and Biotechnology

Cited by 48 publications

References 50 publications

Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides

Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides

Screening of Nutritional Parameters for the Production of Protease from Aspergillus Oryzae

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A

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