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

2020 ◽  
Author(s):  
Jermen Mamo ◽  
Konstantina Kostadinovska ◽  
Martin Kangwa ◽  
Marcelo Fernandez-Lahore ◽  
Fassil Assefa
Keyword(s):  
Pichia Pastoris ◽  
Aspergillus Oryzae ◽  
Recombinant Proteins ◽  
Aspartic Protease ◽  
Cloning And Expression ◽  
Major Protein ◽  
Protease Gene ◽  
Recombinant Yeast ◽  
Milk Clotting ◽  
Cheese Production

Abstract 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.

A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme

2022 ◽  
Author(s):  
Yoko Takyu ◽  
Taro Asamura ◽  
Ayako Okamoto ◽  
Hiroshi Maeda ◽  
Michio Takeuchi ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Proteolytic Activities ◽  
Homologous Enzyme ◽  
Traditional Fermentation ◽  
Enzyme Source ◽  
Cheese Production ◽  
Milk Clotting Activity ◽  
Aspartic Endopeptidase

Abstract Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, two enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The two enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris

2018 ◽  
Vol 268 ◽  
pp. 28-39 ◽  
Author(s):  
Lucía Feijoo-Siota ◽  
José Luis R. Rama ◽  
Angeles Sánchez-Pérez ◽  
Tomás G. Villa
Keyword(s):  
Pichia Pastoris ◽  
Aspartic Protease ◽  
Milk Clotting

Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris

2012 ◽  
Vol 34 (10) ◽  
pp. 1937-1943 ◽  
Author(s):  
Nassapat Boonvitthya ◽  
Phatrapan Tanapong ◽  
Patcharaporn Kanngan ◽  
Vorakan Burapatana ◽  
Warawut Chulalaksananukul
Keyword(s):  
Pichia Pastoris ◽  
Aspergillus Oryzae ◽  
Cloning And Expression ◽  
Beta Glucosidase

scholarly journals Characterization of a Novel Aspartic Protease from Rhizomucor miehei Expressed in Aspergillus niger and Its Application in Production of ACE-Inhibitory Peptides

Foods ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2949
Author(s):  
Shounan Wang ◽  
Peng Zhang ◽  
Yibin Xue ◽  
Qiaojuan Yan ◽  
Xue Li ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Biochemical Characterization ◽  
Rhizomucor Miehei ◽  
Aspartic Protease ◽  
Protease Gene ◽  
Milk Clotting ◽  
Inhibitory Peptides ◽  
Ace Inhibitory Peptides ◽  
Ace Inhibitory

Rhizomucor miehei is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (RmproB) was cloned from R. miehei CAU432 and expressed in Aspergillus niger. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from Saccharomyces cerevisiae. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had Km values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC50 of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides.

scholarly journals Application of Milk-Clotting Protease from Aspergillus oryzae DRDFS13 MN726447 and Bacillus subtilis SMDFS 2B MN715837 for Danbo Cheese Production

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Jermen Mamo ◽  
Paulos Getachew ◽  
Mbugua Samuel Kuria ◽  
Fassil Assefa
Keyword(s):  
Bacillus Subtilis ◽  
Mineral Composition ◽  
Aspergillus Oryzae ◽  
The Body ◽  
Milk Clotting ◽  
Fungal Enzyme ◽  
Organoleptic Characteristics ◽  
Bacterial Enzyme ◽  
Significant Difference ◽  
Cheese Production

This study aimed to investigate the efficiency, biochemical composition, and sensory quality of Danbo cheese produced using proteases derived from the fungus and bacterium compared to the commercial product. A fungal enzyme from Aspergillus oryzae DRDFS13MN726447 and a bacterial enzyme from Bacillus subtilis SMDFS 2B MN715837 were produced by solid-state and submerged fermentation, respectively. The crude enzyme from A. oryzae DRDFS13 and B. subtilis SMDFS 2B was partially purified by dialysis and used for Danbo cheese production using commercial rennet (CHY-MAX® Powder Extract NB, Christian Hansen, 2235 IMCU/g) as a control. The Danbo cheese produced using dialyzed fungal enzyme (E1) (267 U/mL), dialyzed bacterial enzyme (E2) (522 U/mL), and commercial rennet (C) were analyzed for body property, organoleptic characteristics, and proximate and mineral composition when fresh and after 2 months of ripening. There was no significant difference in the cheese yield (C = 9 kg, E1 = 8.6 kg, and E2 = 8.9 kg) among the three treatments. The body properties of Danbo cheese produced with the fungal enzyme (E1) were firm and acceptable as the control (C), whereas the Danbo cheese produced by bacterial enzymes has shown a watery body. The overall organoleptic characteristics of Danbo cheese produced by the fungal enzyme (5.3) were similar to control cheese produced by commercial rennet (5.5). Both cheese types were significantly different in organoleptic properties from Danbo cheese produced by the bacterial enzyme (4.9). There was no significant difference (p>0.05) in the proximate composition between the ripened Danbo cheese produced by fungal enzyme and the control cheese except for crude protein content. However, the ripened cheese products showed a significant difference in their mineral composition except for sodium. In conclusion, this study demonstrated that the fungal enzyme from Aspergillus oryzae DRDFS 13 is more appropriate for Danbo cheese production than the bacterial enzyme from Bacillus subtilis SMDFS 2B. However, it requires further application of the enzymes for the production of other cheese varieties.

scholarly journals Cloning and expression of a family 10 xylanase gene (Aoxyn10) from Aspergillus oryzae in Pichia pastoris

2013 ◽  
Vol 59 (6) ◽  
pp. 405-415 ◽  
Author(s):  
Xin Yin ◽  
Yan-Yan Gong ◽  
Jun-Qing Wang ◽  
Cun-Duo Tang ◽  
Min-Chen Wu
Keyword(s):  
Pichia Pastoris ◽  
Aspergillus Oryzae ◽  
Cloning And Expression ◽  
Xylanase Gene ◽  
Family 10 Xylanase

Cloning, Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus

2013 ◽  
Vol 172 (4) ◽  
pp. 2119-2131 ◽  
Author(s):  
Chaomin Yin ◽  
Liesheng Zheng ◽  
Liguo Chen ◽  
Qi Tan ◽  
Xiaodong Shang ◽  
...  
Keyword(s):  
Pleurotus Ostreatus ◽  
Aspartic Protease ◽  
Protease Gene ◽  
Milk Clotting

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

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