A novel aspartic protease from Rhizomucor miehei expressed in Pichia pastoris and its application on meat tenderization and preparation of turtle peptides

Sun, Qian; Chen, Fusheng; Geng, Fang; Luo, Yongkang; Gong, Siyi; Jiang, Zhengqiang

doi:10.1016/j.foodchem.2017.10.113

Cited by 75 publications

(45 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This posttranslational modification of the aspartic protease enzyme increased its thermal stability and resistant to proteolysis (Goettig 2016;Wang et al 2008). Similar to this study, two potential N-glycosylation sites were identified for the amino acid sequence of purified recombinant aspartic protease from Pichia pastoris (Sun et al 2018). The purified enzyme produced in this study was better in thermal stability and resistance to proteolysis than the aspartyl proteinase from M.…”

Section: Discussionsupporting

confidence: 63%

Purification and Characterization of Aspartic Protease Produced fromAspergillus oryzaeDRDFS13 under Solid-State Fermentation

Mamo

Orellana

Yelemane

et al. 2020

Preprint

View full text Add to dashboard Cite

Aspartic proteases (E.C.3.4.23.) are endopeptidases with molecular masses ranging between 30-45 kDa. They depend on aspartic acid residues for their catalytic activity and show maximal activity at low pH. Thus the main objective of the present study was to purify and characterize aspartic protease from locally identified fungi by solid-state fermentation. The aspartic protease in the current study was obtained from A. oryzae DRDFS13 under SSF. The crude enzyme extract was purified by size-exclusion (SEC) and ion-exchange (IEC) chromatography. The protein contents of crude enzyme and IEC fractions were determined by BCA methods while the presence of N-glycosylation was checked using Endo-H. Inhibition studies were conducted using protease inhibitors. The milk-clotting activity (MCA), protease activity (PA); molecular weight and enzyme kinetics were determined using standard methods. Optimum temperature and stability, optimum pH and stability, and the effect of cations on MCA were assessed using standard methods. The maximum MCA (477.11 U/mL) was recorded from IEC fraction A8. The highest specific activity (183.50 U/mg), purification fold (6.20) and yield (9.2%) were also obtained from the same fraction (IEC A8). The molecular weight of 40 kDa was assigned for the purified enzyme (IEC A8). However, its molecular weight was decreased to 30 KDa upon deglycosylation assay which infers that the protein is glycosylated. Incubation of the pure enzyme (IEC A8) with pepstatin A caused a 94 % inhibition on MCA. The dialyzed enzyme showed a Km and Vmax values of 17.50 mM and 1369 U, respectively. The enzyme showed maximum MCA at 60 oC and pH 5.0 with stability at pH 4.5-6.5 and temperature 35-45 oC. Most cat-ions stimulate the activity of the enzyme; moreover, the highest MCA was detected at 50 mM of MnSO4. Furthermore, the results obtained in the present study confirmed that the aspartic protease enzyme produced from A. oryzae DRDFS13 and purified in ion-exchange chromatography could be used as a substitute source of rennet enzyme for cheese production.

show abstract

Section: Discussionsupporting

confidence: 63%

Purification and Characterization of Aspartic Protease Produced fromAspergillus oryzaeDRDFS13 under Solid-State Fermentation

Mamo

Orellana

Yelemane

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…At pH 5.0, 7.0, and 8.0, the enzyme exhibited 96.5%, 86.8% and 82.2% residual activity. These results showed that the enzyme has the characteristic of alkali-resistance and acid-resistance, which means the strain, was suitable for animal intestinal environment (44). Therefore, it is one possible application of β-glucanase from B. velezensis Y1 in animal breeding as an exogenous.…”

Section: Enzyme Properties Of β-Glucanasementioning

confidence: 87%

Simultaneous Production of Β-glucanase and Protease From Bacillus Velezensis Strain Identified in the Manure of Piglets.

Khalid¹,

Miao²,

Wei³

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: β-glucanase and protease, are the representative of most worldwide enzyme market. In this study, a strain producing β-glucanase and protease, identified as one type of Bacillus velezensis and named Y1, was isolated from the manure of piglet.Results:Its optimal β-glucanase-producing conditions were as: fermentation temperature of 37 °C, fermentation time of 60 h, initial pH of 6.0, outfit fluid amount of 50 mL/250 mL and carbon to nitrogen ratio was 1:1. The properties of the β-glucanasewere that the most suitable reaction temperature was 65°C, the optimum pH was pH 6.0. The optimal protease-producing conditions were fermentation temperature of 37 °C, fermentation time of 60 h, initial pH of 7.0, outfit fluid amount of 75/250 mL and carbon to nitrogen ratio of 2:3.The properties of the protease showed that the most suitable reaction temperature was 50°C, the optimum pH was 6.0. And the enzyme had the best stability at 45°Cand pH 7.0. The effects of each factor on producing β-glucanase and protease by B. velezensis Y1 were as follows: temperature > time > pH > loaded liquid volume. The amplified PCR fragments of β-glucanase and protease were 1434 bp containing an open reading frame of 1413 bp encoding a protein with 444 amino acids and 1752 bp containing an open reading frame of 1521 bp encoding a protein with 506 amino acids, respectively.Conclusion: This study identified the enzymes production, optimized the fermentation conditions of β-glucanase and protease by B. velezensis. As well as the enzyme properties were investigated and genes were cloned.

show abstract

“…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%

“…Aspartic protease genes from fungi may be expressed in yeast for large scale fermentation as it is reported that yeast is good expression hosts for genes of fungal origin (Sun et al, 2018;Yegin and Fernandez-Lahore, 2013). The authors reported that the methylotrophic yeasts such as Pichia pastoris are widely used as expression platforms for recombinant proteins for basic research and industrial applications.…”

Section: Introductionmentioning

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

View full text Add to dashboard Cite

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.

show abstract

A novel aspartic protease from Rhizomucor miehei expressed in Pichia pastoris and its application on meat tenderization and preparation of turtle peptides

Cited by 75 publications

References 37 publications

Purification and Characterization of Aspartic Protease Produced fromAspergillus oryzaeDRDFS13 under Solid-State Fermentation

Purification and Characterization of Aspartic Protease Produced fromAspergillus oryzaeDRDFS13 under Solid-State Fermentation

Simultaneous Production of Β-glucanase and Protease From Bacillus Velezensis Strain Identified in the Manure of Piglets.

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

Contact Info

Product

Resources

About