Evaluation of the milk clotting properties of an aspartic peptidase secreted by <i>Rhizopus microsporus</i>

Silva, Ronivaldo Rodrigues da; Souto, Tatiane Beltramini; Rosa, Nathalia Gonsales da; Oliveira, Lilian Caroline Gonçalves de; Juliano, María A.; Juliano, Luiz; Rosa, José César; Cabral, Hamilton

doi:10.1080/10826068.2019.1683861

Cited by 9 publications

(8 citation statements)

References 23 publications

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“…Their molecular masses are in the ranges of 30–45 kDa (Shankar et al., 2010). Many microorganisms are being studied in detail to investigate the production of aspartic proteases and different fungal and microbial strains are reported as excellent producers of milk‐clotting enzymes such as Talaromyces leycettanus (Guo et al., 2020), Rhizomucor miehei (Wang et al., 2021), R. pusillus , Endothia parasitica (Thakur et al., 1990), Mucor circinelloides (Fernández ‐Lahore, et al., 1997), Aspergillus oryzae (Vishwanatha et al., 2009), Amylomyces rouxii , (Hsu et al., 2005), Rhizopus microsporus (da Silva et al., 2020), Mucor mucedo (Yegin et al., 2010), Mucor pusillu (Arima et al., 1970), Mucor racemosus (Bernardinelli et al., 1983), and Iprex lactis (Bailey and Siika‐aho, 1988).…”

Section: Introductionmentioning

confidence: 99%

Production of a novel milk‐clotting enzyme from solid‐substrate Mucor spp. culture

Qasim

Diercks-Horn

Gerlach

et al. 2022

Journal of Food Science

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Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk-clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk-clotting activity under a solid-state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25 • C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45 • C. The investigated enzyme was pronounced as thermal-sensitive and lost activity completely after 10 min incubation at 55 • C. The remarkable qualities of the studied enzyme, such as cost-effective production, milk-clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese-making industry.

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Section: Introductionmentioning

confidence: 99%

Production of a novel milk‐clotting enzyme from solid‐substrate Mucor spp. culture

Qasim

Diercks-Horn

Gerlach

et al. 2022

Journal of Food Science

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“…Chymosin particularly targets the κ-casein peptide link between Phe105 and Met106 ( Figure 1 A), triggering the formation of destabilized casein micelles, milk coagulation, and separation of the milk into curds and whey [ 5 ]. Although plants [ 6 , 7 , 8 , 9 , 10 , 11 ] and microorganisms [ 12 , 13 , 14 , 15 , 16 , 17 ] can be used as different sources of milk-coagulating enzymes, plant and microbial rennet are characterized by nonspecific proteolytic activity against casein, and animal rennet is more frequently used in the dairy sector for cheese making. Because of the high specific milk-clotting activity, chymosin is considered the most efficient protease for the cheese-making industry.…”

Section: Introductionmentioning

confidence: 99%

Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris

Akishev

Kiribayeva

Mussakhmetov

et al. 2021

Heliyon

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Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in Pichia pastoris cells under glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. It was purified by sequential anion and cation exchange chromatography. SDS-PAGE analysis resulted in two bands, approximately 42 and 35 kDa. The 42 kDa band vanished when the sample was treated with endoglycosidase H, indicating that the recombinant protein is partially glycosylated. Optimal pH for the activity of the highest-purity recombinant chymosin was pH 4.5 for cow's milk and pH 4.0 for mare's milk. The range 45–50 °C and 70 °C for cow's and mare's milk types, respectively, was found to be the most appropriate for maximal relative milk-clotting activity. Concentration of CaCl 2 that ensured the stability of the chymosin milk-clotting activity was between 20 and 50 mM with an optimum at 30 mM. Milk-clotting activity of camel recombinant chymosin and ability to make curd was successfully tested on fresh mare's milk. Pichia pastoris strain with integrated camel chymosin gene showed high productivity of submerged fermentation in bioreactor with milk-clotting activity 1412 U/mL and 80 mg/L enzyme yield. These results suggest that the constitutive expression of the camel chymosin Camelus bactrianus in the yeast Pichia pastoris has good prospects for practical applications.

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“…In cheese production, proteases play the role of milk coagulation enzymes, destabilizing the milk casein micelles, and stimulating their coagulation; this coagulation step is the main step in cheese production 15 . All proteolytic enzymes can coagulate milk, but for the enzyme to be considered a good coagulant it must have a high coagulation activity and low proteolytic activity 16 .…”

Section: Introductionmentioning

confidence: 99%

“…[9][10][11][12][13][14] In cheese production, proteases play the role of milk coagulation enzymes, destabilizing the milk casein micelles, and stimulating their coagulation; this coagulation step is the main step in cheese production. 15 All proteolytic enzymes can coagulate milk, but for the enzyme to be considered a good coagulant it must have a high coagulation activity and low proteolytic activity. 16 Bovine chymosin (EC 3.4.23.4) is the main milk coagulation enzyme used to make cheese; it is called rennet because it comes from the fourth stomach of calves 17 and is considered to be the most suitable due to its high coagulant activity and low proteolytic activity.…”

Section: Introductionmentioning

confidence: 99%

“…16 Bovine chymosin (EC 3.4.23.4) is the main milk coagulation enzyme used to make cheese; it is called rennet because it comes from the fourth stomach of calves 17 and is considered to be the most suitable due to its high coagulant activity and low proteolytic activity. 15,18 However, the limited availability of calf rennet and the increase in cheese consumption has led to a search for alternative substitutes for milk coagulation, such as microbial and plant-derived proteolytic enzymes. 17,19 Proteases can be obtained from various sources, such as plants, animals, and microorganisms.…”

Section: Introductionmentioning

confidence: 99%

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Thermostable trypsin‐like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting

Nogueira

Tavares

Santana

et al. 2021

Biotech and App Biochem

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The increased demand for cheese and the limited availability of calf rennet justifies the search for milk-clotting enzymes from alternative sources. Trypsin-like protease by Penicillium roqueforti was produced by solid-state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert-type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10-12) and an optimum temperature of 80 • C, being stable at temperatures above 60 • C. Enzymatic activity was maximized in the presence of Na + (192%), Co 2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin-like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.

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Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus

Cited by 9 publications

References 23 publications

Production of a novel milk‐clotting enzyme from solid‐substrate Mucor spp. culture

Production of a novel milk‐clotting enzyme from solid‐substrate Mucor spp. culture

Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris

Thermostable trypsin‐like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting

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