Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus

Anna, Li; Xie, Chen; Zhang, Jie; Zhang, Jia; Li, Duochuan

doi:10.1007/s12275-011-9355-6

Cited by 17 publications

(8 citation statements)

References 50 publications

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“…The analysis of three conserved domains from different organisms shows that these domains perform function through identical mechanism. Serine proteases from these organisms are being used in many important applications such as biocontrol agents and in industries that use high temperature, especially detergent industry . It is well established that out of many enzymes produced by Bacillus species, proteases are important for diverse industrial applications specifically detergent industry .…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus

Iqbal

Aftab

Afzal

et al. 2014

J. Basic Microbiol.

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Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus

Iqbal

Aftab

Afzal

et al. 2014

J. Basic Microbiol.

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“…The accession numbers in NCBI (http://www.ncbi.nlm.nih.gov/) are gi: 367020944, gi: 367022650 and gi: 367031002, respectively. Various glucoside hydrolases belonging to GH family 16,17,25,47,55,72 and 125 were identified in bands no. 1-7, but no cellulase was identified, which was consistent with the enzyme activity results.…”

Section: Mass Spectrometry Analysis Of Protein Differential Expressiomentioning

confidence: 99%

“…Their extracellular enzymes might be more active and stable under extreme conditions and may provide economic advantages in the production of chemicals and biomass-based fuels [17,23]. Myceliophthora thermophila is a mild thermophilic fungus which can grow in temperatures between 25 and 55 °C.…”

Section: Introductionmentioning

confidence: 99%

Enhancing xylanase production in the thermophilic fungus Myceliophthora thermophila by homologous overexpression of Mtxyr1

Wang

Gong

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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The xylanase regulator 1 protein in Myceliophthora thermophila ATCC42464 (MtXyr1) is 60 % homologous with that of Trichoderma reesei. However, MtXyr1's regulatory role on cellulolytic and xylanolytic genes in M. thermophila is unknown. Herein, MtXyr1 was overexpressed under the control of the MtPpdc (pyruvate decarboxylase) promoter. Compared with the wild type, the extracellular xylanase activities of the transformant cultured in non-inducing and inducing media for 120 h were 25.19- and 9.04-fold higher, respectively. The Mtxyr1 mRNA level was 300-fold higher than in the wild type in corncob-containing medium. However, the filter paper activity and endoglucanase activities were unchanged in corncob-containing medium and glucose-containing medium. The different zymograms between the transformant and the wild type were analyzed and identified by mass spectrometry as three xylanases of the glycoside hydrolase (GH) family 11. Thus, overexpression of xyr1 resulted in enhanced xylanase activity in M. thermophila. Xylanase production could be improved by overexpressing Mtxyr1 in M. thermophila.

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“…A novel aspartic protease gene (RmproA) from Rhizomucor miehei CAU432 cloned into P. pastoris was also successfully expressed in P. pastoris (Sun et al 2018). In another study, an alkaline protease gene from A. oryzae and a serine protease gene from Thermoascus aurantiacus var.levisporus were cloned in P. pastoris GS115 were fruitfully expressed in P. pastoris (Guo & Ma, 2008;Li et al, 2011).…”

Section: Cloning and Expression Of Aspartic Proteinase Gene In Pastorismentioning

confidence: 99%

Cloning and Expression of an Active Aspartic Proteinase Gene From Aspergillus Oryzae DRDFS13 in Pichia Pastoris

Mamo¹,

Kostadinovska²,

Kangwa³

et al. 2020

Preprint

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BackgroundPichia pastoris is a yeast widely used in expressing recombinant proteins from eukaryotic organisms. In the present study, the total RNA was extracted from a eukaryotic fungus; Aspergillus oryzae DRDFS13 and reverse transcribed into cDNA using specific primers. The gene for aspartic protease was amplified and sequenced and then cloned into pGAPZαA for further expression in P. pastoris. The recombinant yeast (P. patoris X-33Ap) was cultivated in YPD media at pH 5 and 7 for 6 days and the production of recombinant proteins was checked by total protein determination, milk-clotting activity assay, and SDS-PAGE analysis. ResultsThe gene sequence results showed 98% similarity with aspartic protease gene from A. oryzae RIB40. The aspartic protease gene cloned into pGAPZαA (later pMKAP) was successfully expressed in P. pastoris as an active extracellular protease with the highest MCA (190.47 MCU/mL) of secreted enzyme from the recombinant yeast was obtained at pH 5 and 6 days of incubation time. The major protein expressed by the recombinant P. Pastoris X-33 AP has a molecular mass between 32 and 46 kDa. When analyzed for clotting activity, the protein was able to clot skim-milk in 2 min. The clotting activity was found to be 190.47 U/mL.ConclusionThus, the milk-clotting protease extracted form the recombinant yeast in the present study could be a suitable candidate for cheese production. However, further study of the recombinant proteins need to be carried out and its application in cheese production by analyzing the organoleptic and chemical properties of the cheese produced.

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Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus

Cited by 17 publications

References 50 publications

Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus

Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus

Enhancing xylanase production in the thermophilic fungus Myceliophthora thermophila by homologous overexpression of Mtxyr1

Cloning and Expression of an Active Aspartic Proteinase Gene From Aspergillus Oryzae DRDFS13 in Pichia Pastoris

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