High-Level Expression and Substrate-Binding Region Modification of a Novel BL312 Milk-Clotting Enzyme To Enhance the Ratio of Milk-Clotting Activity to Proteolytic Activity

2019 ◽  
Vol 67 (49) ◽  
pp. 13684-13693
Author(s):  
Yao Zhang ◽  
Yongjun Xia ◽  
Xiaofeng Liu ◽  
Zhiqiang Xiong ◽  
Shijie Wang ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Substrate Binding ◽  
Binding Region ◽  
High Level Expression ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
High Level ◽  
Milk Clotting Activity

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.

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 CHEMICAL CHANGES OF THE PAPAYA PLANT DURING DEVELOPMENT, WITH SPECIAL REFERENCE TO ITS PROTEOLYTIC ACTIVITY

1969 ◽  
Vol 27 (1) ◽  
pp. 1-15
Author(s):  
C. F. Asenjo ◽  
D. H. Cook ◽  
M. del C. De Fernández ◽  
L. A. Alvarez
Keyword(s):  
Special Reference ◽  
Proteolytic Activity ◽  
Maximum Activity ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Fruit Pulp ◽  
Hydrogen Ion Concentration ◽  
Milk Clotting ◽  
Green Fruit ◽  
Milk Clotting Activity

1. All parts of the papaya plant, with the exception of the seed, showed varying degrees of proteolytic activity. 2. Milk clotting activity appeared first in the leaf and later, in the stem and root. 3. The largest amount of proteolytic activity was concentrated in the green fruit rind and, in decreasing quantities, in the leaf, fruit pulp, stem, and root. 4. In the leaf and, to a lesser extent, in the stem and root, a definite variation took place in proteolytic activity during the thirteen initial months of growth. Maximum activity was reached between the fourth and ninth months. 5. Data have been collected regarding the moisture, hydrogen ion concentration, and nitrogen content of the various parts of the papaya plant during its initial thirteen months of growth.

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 Demonstration of chymosin (EC 3.4.23.4) in the stomach of newborn pig

1978 ◽  
Vol 169 (2) ◽  
pp. 425-427 ◽  
Author(s):  
B Foltmann ◽  
P Lønblad ◽  
N H Axelsen
Keyword(s):  
Proteolytic Activity ◽  
Milk Clotting ◽  
Milk Clotting Activity

The stomach of newborn pig contains a proteinase that is immunologically closely related to calf chymosin (rennin) (EC 3.4.23.4.). None of the pepsins from the stomach of adult pig is present in the newborn pig. Pig chymosin has optimal general proteolytic activity around pH 3.5. The ratio of milk-clotting activity to general proteolytic activity is about 30–70 times higher than that of pyloric and fundic pepsins.

scholarly journals Optimization of Crude Papaya (Carica papaya) Protease in Soft-Unripened Cheese Preparation

2020 ◽  
Vol 12 (12) ◽  
pp. 1-8
Author(s):  
Bunty Maskey ◽  
Nabindra Kumar Shrestha
Keyword(s):  
Numerical Optimization ◽  
Carica Papaya ◽  
Enzyme Concentration ◽  
The Other ◽  
Milk Clotting ◽  
Optimization Study ◽  
Plant Protease ◽  
Milk Clotting Enzyme ◽  
Papaya Latex ◽  
Milk Clotting Activity

The use of plant protease instead of chymosin for producing cheese has become a trend which is aimed at lacto-vegetarian consumers and religion based ecological markets. In this context, the present investigation was carried out in order to utilize milk clotting enzyme from Papaya (Carica papaya). Numerical optimization study revealed that maximum milk clotting activity was achieved at pH 6.5, temperature 70℃ and enzyme concentration 1 g/1000 ml milk using papaya protease as coagulant. Protein, ash and calcium showed no significant (p>0.05) difference among the cheeses made using different coagulants. However, significantly (p<0.05) higher levels of moisture and ash, and lower levels of fat were observed in the cheese produced by papaya protease compared to that made using rennet. Papaya protease significantly enhanced the spreadability of cheese while the other sensory properties were similar to the control except aftertaste. The results revealed that the papaya latex as crude papaya protease may have potential application for the manufacture of soft-unripened cheese and further could be utilized as a milk coagulant in cheese making.

scholarly journals Characteristics of Curds With Milk Clotting Enzyme from Indonesian Local Isolate of Lactic Acid Bacteria

2020 ◽  
Vol 1 (2) ◽  
pp. 79-86
Author(s):  
Wendry Putranto ◽  
Apon Mustopa ◽  
Jendri Mamangkey ◽  
Netty Aritonang
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Observational Research ◽  
Ammonium Sulfate Precipitation ◽  
Fermentation Time ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Qualitative Observation ◽  
Milk Clotting Activity

To get the potential of lalcat acid bacteria isolate to produce Milk Clotting Enzyme (MCE), it is necessary to screen milk clotting activity both quantitatively and qualitatively. Through qualitative observation, the characteristics of the curd resulting from enzyme activity can be obtained. MCE is a protease that has the characteristics of milking. Based on the results of this observational research, the curd characteristic produced can be used as a benchmark to determine the length of time of fermentation and optimization of the determination of ammonium sulfate precipitation concentration. Isolate BAL shows the results of a compact curd at a fermentation time of 25 hours at 37 ℃ and the optimization results of the deposition of ammonium sulfate which shows the characteristics of a compact curd by 45% ammonium sulfate.

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