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.

Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341

2009 ◽  
Vol 85 (6) ◽  
pp. 1849-1859 ◽  
Author(s):  
Kurutahalli S. Vishwanatha ◽  
A. G. Appu Rao ◽  
Sridevi Annapurna Singh
Keyword(s):  
Aspergillus Oryzae ◽  
Milk Clotting ◽  
Milk Clotting Enzyme

scholarly journals Study the influence of nitrogen on rennin production by fungi Rhizomucor miehei using solid-state fermentation

2016 ◽  
Vol 3 (5) ◽  
pp. 193
Author(s):  
Houthail Al-Ahmad Al-Jammas ◽  
Hassan Al-Fathi ◽  
Walid Al-Khalaf ◽  
Anton Taifor
Keyword(s):  
Enzyme Activity ◽  
Solid State ◽  
Protein Content ◽  
Yeast Extract ◽  
Wheat Bran ◽  
Solid State Fermentation ◽  
Rhizomucor Miehei ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Milk Clotting Activity

The effect of different nitrogen resources on the biosynthesis of milk clotting enzyme by Rhizmucor miehei was studied under solid state fermentation using wheat bran as base medium. Urea, peptone, albumin, casein, yeast extract were added with different concentrations (1%-10%). The response parameters were the ratio of milk clotting activity (MC) to proteolytic activity (PA) and protein content. The highest enzyme yield was achieved with casein at a rate of 2% w/w followed by 2% yeast extract, 1% albumin, 1% peptone, and 1% urea with values 5.6, 4.9, 4.2, 4, 3 mg/mL, respectively. Maximum enzyme activity (MCA/PA) was 50.4, 44.1, 37.8, 36, 27 SU for casein, yeast extract, albumin, peptone, and urea, respectively.

scholarly journals Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

2009 ◽  
Vol 52 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Ana Rodrigues Duarte ◽  
Débora Maria Rodrigues Duarte ◽  
Keila Aparecida Moreira ◽  
Maria Taciana Holanda Cavalcanti ◽  
José Luiz de Lima-Filho ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulphate ◽  
Iodoacetic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fractional Precipitation ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Milk Clotting Activity ◽  
Optimal Ph

The partial characterization and purification of milk clotting enzyme obtained from the (root latex) of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions (AS1- 0-20%; AS2 - 20-40%; AS3 - 40-60%; AS4 - 60-80%; AS5 - 80-100%) and among the fractions obtained, the 40-60% fraction (AS3) showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2), but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. The enzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.

scholarly journals An Evaluation of the Clotting Properties of Three Plant Rennets in the Milks of Different Animal Species

Foods ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 600 ◽  
Author(s):  
Katia Liburdi ◽  
Carlo Boselli ◽  
Gilberto Giangolini ◽  
Simonetta Amatiste ◽  
Marco Esti
Keyword(s):  
Carica Papaya ◽  
Animal Species ◽  
Promising Alternative ◽  
Milk Clotting ◽  
Sheep Milk ◽  
Milk Clotting Enzyme ◽  
Different Temperatures ◽  
Vegetable Coagulant ◽  
Proteolytic Activities ◽  
Milk Coagulation

Cynara cardunculus, Carica papaya and Ficus carica extracts are proposed as milk coagulants herein. Their coagulation efficiency was measured in bovine, buffalo, goat and sheep milk incubated at different temperatures. The milk-clotting and proteolytic activities as well as the lactodynamographic parameters were determined considering animal rennet as a reference coagulant. The vegetable coagulant, extracted from C. cardunculus pistils, proved to be the most suitable milk-clotting enzyme for cheesemaking, since it possesses similar milk clotting properties to conventional calf rennet. F. carica latex, but seemed to be a promising alternative coagulant at higher temperatures. The strong proteolytic activity of papain caused poor milk coagulation in all milk samples. To conclude, this result also supports the original hypothesis of this study that the excessive proteolytic nature of plant coagulants can negatively affect the cheesemaking process. The optimization of using a plant rennet in a dairy application can be done by selecting the appropriate plant rennet with a consistent clotting efficiency. These innovative manufacturing processes may also lead to the optimization and production of new cheese varieties.

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.

scholarly journals Production of Traditional Cheese Coagulant in Farmhouse and Coagulating Effect

2016 ◽  
Vol 4 (Special-Issue-October) ◽  
pp. 133-137
Author(s):  
Dilek Say ◽  
Nuray Guzeler
Keyword(s):  
Room Temperature ◽  
Cow’S Milk ◽  
Cow's Milk ◽  
Milk Clotting ◽  
Goat’S Milk ◽  
Traditional Cheese ◽  
Milk Clotting Enzyme ◽  
Ewe's Milk ◽  
Goat's Milk ◽  
Milk Clotting Activity

Milk-clotting enzymes are the primary active agents in the manufacture cheeses. Animal rennet, microbial coagulant and plant coagulant are used as milk coagulants in cheese making. However, alternative milk coagulants are investigated instead of animal enzymes due to slaughtering of young ruminant. Manufacturing of milk clotting enzyme in farmhouse have been employed successfully for many centuries in Mediterrenean region and Toros mountain villages of Turkey for the production of traditional Tulum cheese. Figs, raisins, white beans, chickpeas, carob, granulated sugar, salt, yoghurt and home-made rennet (sarkanak) are found in the content of this enzyme. This mixture is left at room temperature for 5-6 days. The enzyme is filtered from using cloth bag and added into milk for coagulation. In this research; chemical composition of cow’s milk, goat’s milk and ewe’s milk were determined and obtained enzymes from different manufacturers were investigated of clotting effects on cow’s milk, goat’s milk and ewe’s milk. Four different coagulants had a strong coagulating effect on raw and pasteurized ewe's milk. The highest milk clotting activity of all coagulant samples were seen in ewe's milk, followed by cow's milk and goat's milk.

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.

Milk-clotting and proteolytic activities of rennet, and of bovine pepsin and porcine pepsin

1969 ◽  
Vol 36 (3) ◽  
pp. 427-433 ◽  
Author(s):  
P. F. Fox
Keyword(s):  
Porcine Pepsin ◽  
Milk Clotting ◽  
Proteolytic Activities ◽  
Ph Dependent ◽  
Rapid Drop ◽  
Milk Clotting Activity

SummaryThe milk-clotting and proteolytic activities of rennet, bovine pepsin and porcine pepsin were compared. The milk-clotting activity of porcine pepsin was extremely pH-dependent around pH 6·6 and coagulation did not occur above pH 6·68. The clotting activity of bovine pepsin was slightly more dependent on pH than that of rennet but no rapid drop-off in activity occurred as with porcine pepsin. Temperature influenced the clotting activity of rennet and bovine pepsin similarly but the behaviour of porcine pepsin was markedly different.For equal milk-clotting activity, the proteolytic activities of rennet and bovine pepsin were approximately equal and substantially lower than that of porcine pepsin. Electrophoretic examination showed that the proteolysis products of rennet and bovine pepsin were similar and quite different from those produced by porcine pepsin.The suitability of bovine pepsin as a rennet substitute is discussed.

scholarly journals Technology of obtaining milk-clotting enzyme from fungal culture Funalia Sp. for application in cheese production

2019 ◽  
Author(s):  
Ekaterina Gannochka ◽  
◽  
Boris Kolesnikov ◽  
Alisa Salamahina ◽  
Mark Shamtsyan
Keyword(s):  
Fungal Culture ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Cheese Production
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