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Vavilovskii Zhurnal Genetiki I Selektsii May 2022Recombinant chymosins (rСhns) of the cow and the camel are currently considered as standard milk coagulants for cheese-making. The search for a new type of...
Recombinant chymosins (rСhns) of the cow and the camel are currently considered as standard milk coagulants for cheese-making. The search for a new type of milk-clotting enzymes that may exist in nature and can surpass the existing "cheese-making" standards is an urgent biotechnological task. Within this study, we for the first time constructed an expression vector allowing production of a recombinant analog of moose chymosin in the expression system of Escherichia coli (strain SHuffle express). We built a model of the spatial structure of moose chymosin and compared the topography of positive and negative surface charges with the correspondent structures of cow and camel chymosins. We found that the distribution of charges on the surface of moose chymosin has common features with that of cow and camel chymosins. However, the moose enzyme carries a unique positively charged patch, which is likely to affect its interaction with the substrate. Biochemical and technological properties of the moose rChn were studied. Commercial rСhns of cow and camel were used as comparison enzymes. In some technological parameters, the moose rChn proved to be superior to the reference enzymes. Сompared with the cow and camel rСhns, the moose chymosin specific activity is less dependent on the changes in CaCl2 concentration in the range of 1-5 mM and pH in the range of 6-7, which is an attractive technological property. The total proteolytic activity of the moose rСhn occupies an intermediate position between the rСhns of cow and camel. The combination of biochemical and technological properties of the moose rСhn argues for further study of this enzyme.
PubMed: 35774365
DOI: 10.18699/VJGB-22-31 -
Nanomaterials (Basel, Switzerland) Oct 2021The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of...
The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS-PAGE investigation showed that β-CN, κ-CN, α-CN, and a portion of β-lactoglobulin (β-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS-PAGE analysis suggested that chymosin hydrolyzed individual β-CN, κ-CN, and α-CN, but not β-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed β-CN, κ-CN, and α-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed β-CN and α-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of β-CN and α-CN.
PubMed: 34685035
DOI: 10.3390/nano11102594 -
Kidney International May 2006The existence of a tissue renin-angiotensin (RAS) system independent of the circulating RAS has prompted the search for cellular binding sites for angiotensinogen and... (Review)
Review
The existence of a tissue renin-angiotensin (RAS) system independent of the circulating RAS has prompted the search for cellular binding sites for angiotensinogen and for renin in order to explain their tissue uptake. Two receptors that bind with similar affinity mature renin and prorenin were identified, the mannose-6-phosphate receptor (M6P-R) and a specific receptor. The M6P-R is a clearance receptor that binds exclusively the glycosylated forms of renin and prorenin. Binding of renin and prorenin to the M6P-R is followed by internalization and degradation, and the intracellular proteolysis of prorenin in mature renin did not provoke any generation of intracellular angiotensins. In contrast to the M6P-R, (pro)renin bound to the specific receptor was not degraded. Instead, receptor-bound renin showed increased catalytic activity, and receptor-bound prorenin exhibited full catalytic activity. This 'gain of activity' was explained by a conformational change of the (pro)renin molecule upon binding. Furthermore, (pro)renin binding provoked a rapid activation of the mitogen-activated protein kinases p44/p42, indicating that the receptor has mediated specific, angiotensin II-independent effects of (pro)renin. This receptor represents an elegant concept to explain the existence of active prorenin in vivo, and it provides a pathological role for prorenin in situations with paradoxical low renin and high prorenin concentrations such as in diabetes. Experimental models of rats overexpressing the receptor either in vascular smooth muscle cells and developing high blood pressure or with ubiquitous expression associated with glomerulosclerosis and proteinuria confirm a role for the receptor in cardiovascular and renal diseases.
Topics: Animals; Cardiovascular Diseases; Chymosin; Enzyme Precursors; Humans; Kidney Diseases; Mice; Rats; Receptor, IGF Type 2; Receptors, Cell Surface; Renin; Prorenin Receptor
PubMed: 16672920
DOI: 10.1038/sj.ki.5000265 -
Scientific Reports Jun 2021Withania coagulans (W. coagulans) extract and camel chymosin have aspartic protease capable of coagulating milk for cheese production. This study investigated the...
Withania coagulans (W. coagulans) extract and camel chymosin have aspartic protease capable of coagulating milk for cheese production. This study investigated the quality of camel and bovine milk cheeses coagulated using Withania extracts, came chymosin, and their mixture in two experiments. In Experiment (1), a factorial design with four factors (W. coagulans, camel chymosin, incubation time, and incubation temperature) was performed. The effect of these factors on cheese's yield and hardness were assessed. An enzyme concentration corresponding to a 36 µg/L of milk of W. coagulans, 50 IMCU/L of camel chymosin, holding time of 4 h, and incubation temperature of 60 °C provided the optimal textural hardness for both camel and bovine milk cheeses. Seven treatments were analyzed in experiment (2) were analyzed for physicochemical properties, yield, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGEitation). The results showed that pure Withania extract exhibited the lower coagulating effect resulting in cheeses with low yield, hardness, fat, protein, and total solids. The SDS-PAGE electropherograms of camel cheese showed several low molecular weight bands as compared to bovine cheese. This phenomenon is due to excessive proteolysis in camel cheese, which we believed is caused by the presence of endogenous enzymes.
Topics: Animals; Camelus; Cattle; Cheese; Chymosin; Milk; Plant Extracts; Solanaceae
PubMed: 34193923
DOI: 10.1038/s41598-021-92797-6 -
Journal of Dairy Science Mar 2021Chymosin is a predominant enzyme in rennet and is used in cheese production because of its excellent milk-clotting activity. Herein, we proposed a facile and label-free...
Chymosin is a predominant enzyme in rennet and is used in cheese production because of its excellent milk-clotting activity. Herein, we proposed a facile and label-free electrochemical method for determining chymosin activity based on a peptide-based enzyme substrate. The synthesized substrate peptide for chymosin was assembled onto the surface of the Au-deposited grassy carbon electrode. The current was proportional to chymosin activity, and thus chymosin activity could be determined. The detection ranges of chymosin activity were 2.5 to 25 U mL. The detection limit of chymosin activity was 0.8 U mL. The sensing platform was used to quantify chymosin activity in commercial rennet with high selectivity, excellent stability, and satisfactory reproducibility. We developed a facile, fast, and effective electrochemical assay for detecting chymosin activity, which has potential applications in cheesemaking.
Topics: Animals; Cheese; Chymosin; Milk; Peptides; Reproducibility of Results
PubMed: 33455776
DOI: 10.3168/jds.2020-19282 -
Nutrients Feb 2022The use of casein glycomacropeptide (CGMP) as a protein substitute in phenylketonuria (PKU) has grown in popularity. CGMP is derived from κ casein and is a sialic-rich... (Review)
Review
The use of casein glycomacropeptide (CGMP) as a protein substitute in phenylketonuria (PKU) has grown in popularity. CGMP is derived from κ casein and is a sialic-rich glycophosphopeptide, formed by the action of chymosin during the production of cheese. It comprises 20-25% of total protein in whey products and has key biomodulatory properties. In PKU, the amino acid sequence of CGMP has been adapted by adding the amino acids histidine, leucine, methionine, tyrosine and tryptophan naturally low in CGMP. The use of CGMP compared to mono amino acids (L-AAs) as a protein substitute in the treatment of PKU promises several potential clinical benefits, although any advantage is supported only by evidence from non-PKU conditions or PKU animal models. This review examines if there is sufficient evidence to support the bioactive properties of CGMP leading to physiological benefits when compared to L-AAs in PKU, with a focus on blood phenylalanine control and stability, body composition, growth, bone density, breath odour and palatability.
Topics: Animals; Caseins; Peptide Fragments; Phenylalanine; Phenylketonurias
PubMed: 35215457
DOI: 10.3390/nu14040807 -
Acta Crystallographica. Section D,... May 2013Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein,...
Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N-terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin was redetermined at a slightly higher resolution (1.8 Å) than previously determined structures. Camel and bovine chymosin share the same overall fold, except for the antiparallel central β-sheet that connects the N-terminal and C-terminal domains. In bovine chymosin the N-terminus forms one of the strands which is lacking in camel chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids delineating the substrate-binding cleft suggest a greater flexibility in the ability to accommodate the substrate in camel chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel chymosin towards bovine milk.
Topics: Animals; Camelus; Caseins; Cattle; Cheese; Chymosin; Crystallography, X-Ray; Glycosylation; Models, Molecular; Protein Conformation; Static Electricity; Structure-Activity Relationship
PubMed: 23633601
DOI: 10.1107/S0907444913003260 -
Biology Oct 2022In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel () chymosin (BacChym) has a milk-clotting activity higher than...
In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel () chymosin (BacChym) has a milk-clotting activity higher than that of calf chymosin for cow's, goat's, ewes', mare's and camel's milk. A procedure for obtaining milk-clotting reagent based on recombinant camel chymosin is proposed here. Submerged fermentation by a recombinant yeast ( GS115/pGAPZαA/ProchymCB) was implemented in a 50 L bioreactor, and the recombinant camel chymosin was prepared successfully. The activity of BacChym in yeast culture was 174.5 U/mL. The chymosin was concentrated 5.6-fold by cross-flow ultrafiltration and was purified by ion exchange chromatography. The activity of the purified BacChym was 4700 U/mL. By sublimation-drying with casein peptone, the BacChym powder was obtained with an activity of 36,000 U/g. By means of this chymosin, cheese was prepared from cow's, goat's, ewes', camel's and mare's milk with a yield of 18%, 17.3%, 15.9%, 10.4% and 3%, respectively. Thus, the proposed procedure for obtaining a milk-clotting reagent based on BacChym via submerged fermentation by a recombinant yeast has some prospects for biotechnological applications. BacChym could be a prospective milk-clotting enzyme for different types of milk and their mixtures.
PubMed: 36358248
DOI: 10.3390/biology11111545 -
Heliyon May 2021Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals...
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 (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in 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 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. 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 in the yeast has good prospects for practical applications.
PubMed: 34113734
DOI: 10.1016/j.heliyon.2021.e07137 -
Asian-Australasian Journal of Animal... Sep 2016Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene...
Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.
PubMed: 27004812
DOI: 10.5713/ajas.16.0038