Calf Chymosin Research Articles

Rennets differing in chymosin-to-pepsin ratio shape the metabolomic and sensory profile of Grana Padano PDO cheese during ripening

Utilizing different chymosin and pepsin ratios in cheesemaking may represent a potential strategy to shape the sensory profile of hard cheeses. This study investigated the impact of rennet with varying chymosin and pepsin ratios on the chemical profile and sensory attributes of Grana Padano PDO cheese at different ripening times (10 to 20 months). The research involved the analysis of hard cheese manufactured with distinct calf chymosin percentages (99 %, 95 %, and 83 %), exploiting sensory analyses and untargeted metabolomics to identify marker compounds correlating with specific sensory traits. The results demonstrated that varying the rennet composition significantly affected sensory profile; in particular, the rennet made by 83 % chymosin and 17 % pepsin generated a more complex sensory profile starting from 12 months. AMOPLS and ASCA analysis on untargeted metabolomics signatures revealed that ripening time was the only significant factor when compared with rennet type and the interaction ripening x rennet. Finally, at more advanced ripening times, 3-methylbutanoic acid and homoethone were significantly up-accumulated in cheese samples manufactured with higher pepsin percentages, likely explaining sensory outcomes. This study provides valuable insights into using rennet to tailor the sensory qualities of hard cheeses, underscoring the importance of enzyme selection in cheese manufacturing to drive innovation in the dairy industry.

Functionality of process cheese made from Cheddar cheese with various rennet levels and high-pressure processing treatments

Due to its versatility and shelf stability, process cheese is gaining interest in many developing countries. The main structural component (base) of most processed cheese formulations is young Cheddar cheese that has high levels of intact casein. Exporting natural Cheddar cheese base from the United States to distant overseas markets would require the aging process to be slowed or reduced. As Cheddar cheese ripens, the original structure is broken down by proteolysis and solubilization of insoluble calcium phosphate. We explored the effect of varying rennet levels (we also used a less proteolytic rennet) and application of high-pressure processing (HPP) to Cheddar cheese, as we hoped these treatments might limit proteolysis and concomitant loss of intact casein. To try to retain high levels of insoluble Ca, all experimental cheeses were made with a high-draining pH and from concentrated milk. To compare our intact casein results with current practices, we manufactured a Cheddar cheese that was prepared according to typical industry methods (i.e., use of unconcentrated milk, calf chymosin [higher levels], and low draining pH value [∼6.2]). All experimental cheeses were made from ultrafiltered milk with protein and casein contents of ∼5.15% and 4.30%, respectively. Three (low) rennet levels were used: control (38 international milk clotting units/mL of rennet per 250 kg of milk), and 25% and 50% reduced from this level. All experimental cheeses had similar moisture contents (∼37%) and total Ca levels. Four days after cheese was made, half of the experimental samples from each vat underwent HPP at 600 MPa for 3 min. Cheddar cheese functionality was monitored during aging for 240 d at 4°C. Cheddar cheese base was used to prepare process cheese after aging for 14, 60, 120, 180, and 240 d. Loss tangent (LT) values of cheese during heating were measured by small strain oscillatory rheology. Intact casein levels were measured using the Kjeldahl method. Acid or base titrations were used to determine the buffering capacity and insoluble Ca levels as a percentage of total Ca. The LTmax values (an index of meltability) in process cheese increased with aging for all the cheese bases; the HPP treatment significantly decreased LTmax values of both base (natural) and process cheeses. All experimental cheeses had much higher levels of intact casein compared with typical industry-make samples. Process cheese made from the experimental treatments had visually higher stretching properties than process cheese made from Cheddar with the typical industry-make procedure. Residual rennet activity was not affected by rennet level, but the rate of proteolysis was slightly slower with lower rennet levels. The HPP treatment of Cheddar cheese reduced residual rennet activity and decreased the reduction of intact casein levels. The HPP treatment of Cheddar cheese resulted in process cheeses that had slightly higher hardness values, lower LTmax values, and retained higher storage modulus values at 70°C. We also observed that the other make procedures we used in all experimental treatments (i.e., using a less proteolytic chymosin, using a concentrated cheese milk, and maintaining a high draining pH value) had a major effect on retaining high levels of intact casein.

THE OBTAINING OF THE RECOMBINANT CAMEL CHYMOSIN BY SUBMERGE FERMENTATION IN THE PILOT BIOREACTOR

In the cheese making industry, chymosin is used as a milk-clotting enzyme. With its high specific activity against κ-casein, chymosin better than other proteolytic enzymes. Bactrian camel chymosin has a milk-clotting activity higher than calf chymosin. A scheme for obtaining a milk-clotting preparation based on recombinant camel chymosin is proposed. Submerge fermentation of recombinant yeast Pichia pastoris was carried out in a 50-liter bioreactor and recombinant camel chymosin was obtained. The activity of chymosin in the yeast culture was 174.5 U/mL. Chymosin was concentrated 5.6-fold by cross-flow ultrafiltration with 10 kDa cut-off membrane, and chymosin was purified by ion exchange chromatography. The activity of purified chymosin was 4700 U/mL. By sublimation drying with casein peptone, the powder chymosin was obtained with an activity of 36,000 U/g. The proposed scheme for obtaining a milk-clotting drug based on recombinant camel chymosin using submerge fermentation of recombinant yeast has the prospect of being used at biotechnological enterprises.

Obtaining of Recombinant Camel Chymosin and Testing Its Milk-Clotting Activity on Cow's, Goat's, Ewes', Camel's and Mare's Milk.

In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel (Camelus bactrianus) 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 (Pichia pastoris 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.

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

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. PRACTICAL APPLICATION: The milk-clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.

Structure and shelf stability of milk protein gels created by pressure-assisted enzymatic gelation

In this work, pressure-assisted enzymatic gelation was applied to milk proteins, with the goal of enhancing the structure and stability of pressure-created milk protein gels. High-pressure processing (HPP) at 600 MPa, 3 min, and 5°C was applied to milk protein concentrate (MPC) samples of 12.5% protein concentration, both in the absence and in the presence of calf chymosin [up to 60 IMCU (international milk-clotting units)/kg of milk] or camel chymosin (up to 45 IMCU/kg of milk). Gel hardness, water-holding capacity, and degree of proteolysis were used to assess network strength and shelf stability. The processing trials and all measurements were conducted in triplicate. Statistical analyses of the data were performed by ANOVA, at a 95% confidence level. After HPP treatment, we observed significant structural changes for all samples. Pressurization of MPC, with or without chymosin addition, led to extensive protein aggregation and network formation. The strength of HPP-created milk protein gels without chymosin addition, as measured by the elastic modulus (G'), had a value of 2,242 Pa. The value of G' increased with increasing chymosin concentration, reaching as high as 4,800 Pa for samples with 45 IMCU/kg of camel chymosin. During 4 wk of refrigerated storage, the HPP and chymosin MPC gels maintained higher gel hardness and better structural stability compared with HPP only (no chymosin) MPC gels. The water-holding capacity of the gels without chymosin remained at 100% during 28 d of refrigerated storage. The HPP and chymosin MPC gels had a lower water-holding capacity (91-94%) than the HPP-only counterparts, but their water-holding capacity did not decrease during storage. Overall, these findings demonstrate that controlled, fast structural modification of high-concentration protein systems can be obtained by HPP-assisted enzymatic treatment, and the created gels have a strong, stable network. This study provides insights into the possibility of using HPP for the development of milk-protein-based products with novel structures and textures and long refrigerated shelf life, along with the built-in safety imparted by the HPP treatment.

Novel Bacillus Milk-Clotting Enzyme Produces Diverse Functional Peptides in Semihard Cheese.

Although rennet is one of the best choices for cheese manufacturing, its production cannot meet the growing demands of the cheese industry. Thus, new milk-clotting enzymes (MCEs) with similar or better properties as/than those of calf chymosin are needed urgently. Here, three MCEs, BY-2, BY-3, and BY-4, were mined by bioinformatic analysis and then expressed in and isolated from Escherichia coli. BY-4 had the highest milk-clotting activity/proteolytic activity (238.76) with enzyme properties similar to those of calf chymosin. BY-4 cheese had a composition, appearance, consistency/texture, and overall acceptability proximate to calf chymosin cheese. The EC50 values of peptides extracted from BY-4 cheese for 2,2-diphenyl-1-picrylhydrazyl inhibition (antioxidant property), angiotensin-converting enzyme inhibition (antihypertensivity), and growth inhibition of liver cancer cells (antitumor property) were found to be 81, 49, and 238 μg/mL, respectively, which were 2.35, 2.59, and 2.12 folds higher than those of calf chymosin cheese. These results indicated the potential of BY-4 as a supplement to calf chymosin in cheese manufacturing, especially for functional and health care purposes.

Effects of blends of camel and calf chymosin on proteolysis, residual coagulant activity, microstructure, and sensory characteristics of Beyaz peynir

Beyaz peynir, a white brined cheese, was manufactured using different blends of camel chymosin (100, 75, 50, 25, and 0%) with calf chymosin and ripened for 90 d. The purpose of this study was to determine the best mixture of coagulant for Beyaz peynir, in terms of proteolysis, texture, and melting characteristics. The cheeses were evaluated in terms of chemical composition, levels of proteolysis, total free amino acids, texture, meltability, residual coagulant activity, microstructure, and sensory properties during 90 d of ripening. Differences in the gross chemical composition were statistically significant for all types of cheeses. Levels of proteolysis were highly dependent on the blends of the coagulants. Higher proteolysis was observed in cheeses that used a higher ratio of calf chymosin. Differences in urea-PAGE and peptide profiles of each cheese were observed as well. Meltability values proportionally increased with the higher increasing levels of calf chymosin in the blend formula. These coagulants had a slight effect on the microstructure of cheeses. The cheese made with camel chymosin had a harder texture than calf chymosin cheese, and hardness values of all cheese samples decreased during ripening. The cheeses with a high ratio of calf chymosin had higher residual enzyme activity than those made with camel chymosin. No significant difference in sensory properties was observed among the cheeses. In conclusion, cheeses made with a high level of calf chymosin had a higher level of proteolysis, residual coagulant activity, and meltability. The cheeses also had a softer texture than cheeses made with a high content of camel chymosin. Camel chymosin may be used as a coagulant alone if low or limited levels of proteolysis are desired in cheese.

Effect of Oryctolagus cuniculus (rabbit) rennet on the texture, rheology, and sensory properties of white cheese.

Calf rennet has long been used in cheese‐making. Because of calf rennet shortage and high cost, novel proteases were needed to meet industry's increasing enzyme demand. Recombinant chymosins and camel chymosin were started to be used in the industry. There is no study in the literature subjecting use of rabbit rennet in cheese production. Chemical, rheological, and sensorial characteristics of white cheese made with rabbit rennet were investigated in this study. Quality characteristics of rabbit rennet cheese (RC) were compared to cheeses produced with commercial calf (CC) and camel chymosins (CLC). RC and CLC exhibited higher hardness and dynamic moduli values throughout the storage as compared to CC. Although moisture levels of cheese samples were similar at day 60, CC had much lower hardness and dynamic moduli values than CLC and RC. While the appearance and structure were better for CLC, the highest odor and taste scores were obtained by RC during 60 days of storage. The results of this investigation proposed that rabbit rennet could be a suitable milk coagulant for white cheese production. Our results showed that rabbit rennet has comparable cheese‐making performance with camel chymosin and could be a good alternative for calf chymosin.

Production of Mozzarella Cheese Using Rennin Enzyme from Mucor miehei Grown at Rice Bran Molasses Medium

The research aimed to study the characteristic and yield of Mozzarella cheese produced by using rennin enzyme from Mucor miehei which is grown at rice bran and molasses medium. The popularity of Mozzarella cheese in Indonesia is increased caused by the spreading of western foods in Indonesia such as pizza and spaghetti that use Mozzarella cheese for ingredient. In Italy, Mozzarella and pizza cheeses are dominating 78% of the total Italian Cheese products. In producing Mozzarella cheese, rennin enzyme is always used as milk coagulant. Even now, Indonesia has not produced the rennin enzyme yet. The rennin enzyme from Mucor miehei growing at rice bran and molases medium which have the availability can be managed purposively within short period of time. The completly randomized design methode used to get the best crude extracts of Mucor miehei rennin enzyme, then is employed to produce mozzarella cheese. The result of Mozzarella cheese has various characteristics such as the yield’s weight is 9.1%, which consists of 50% moisture content, 36.64% peotein levels, 0.1 melting ability and 82.72% stretch ability or 0.79/N. With that characteristic it is concluded that rennin enzyme from Mucor miehei grown at rice bran molasses medium has the potential to alternatively subtitute calf rennin to produce Mozzarella cheese, and the characteristics fulfill the standart.

Effect of camel chymosin on the texture, functionality, and sensory properties of low-moisture, part-skim Mozzarella cheese

The objective of this study was to compare the effect of coagulant (bovine calf chymosin, BCC, or camel chymosin, CC), on the functional and sensory properties and performance shelf-life of low-moisture, part-skim (LMPS) Mozzarella. Both chymosins were used at 2 levels [0.05 and 0.037 international milk clotting units (IMCU)/mL], and clotting temperature was varied to achieve similar gelation times for each treatment (as this also affects cheese properties). Functionality was assessed at various cheese ages using dynamic low-amplitude oscillatory rheology and performance of baked cheese on pizza. Cheese composition was not significantly different between treatments. The level of total calcium or insoluble (INSOL) calcium did not differ significantly among the cheeses initially or during ripening. Proteolysis in cheese made with BCC was higher than in cheeses made with CC. At 84 d of ripening, maximum loss tangent values were not significantly different in the cheeses, suggesting that these cheeses had similar melt characteristics. After 14 d of cheese ripening, the crossover temperature (loss tangent = 1 or melting temperature) was higher when CC was used as coagulant. This was due to lower proteolysis in the CC cheeses compared with those made with BCC because the pH and INSOL calcium levels were similar in all cheeses. Cheeses made with CC maintained higher hardness values over 84 d of ripening compared with BCC and maintained higher sensory firmness values and adhesiveness of mass scores during ripening. When melted on pizzas, cheese made with CC had lower blister quantity and the cheeses were firmer and chewier. Because the 2 types of cheeses had similar moisture contents, pH values, and INSOL Ca levels, differences in proteolysis were responsible for the firmer and chewier texture of CC cheeses. When cheese performance on baked pizza was analyzed, properties such as blister quantity, strand thickness, hardness, and chewiness were maintained for a longer ripening time than cheeses made with BCC, indicating that use of CC could help to extend the performance shelf-life of LMPS Mozzarella.

Proteomic Comparison of Equine and Bovine Milks on Renneting

Rennet-induced coagulation of bovine milk is a complex mechanism in which chymosin specifically hydrolyzes κ-casein, the protein responsible for the stability of the casein micelle. In equine milk, this mechanism is still unclear, and the protein targets of chymosin are unknown. To reveal the proteins involved, the rennetability of equine milk by calf chymosin was examined using gel-free and gel-based proteomic analysis and compared to bovine milk. RP-HPLC analysis of bovine and equine milks showed the release of several peptides following chymosin incubation. The hydrolyses of equine and bovine casein by chymosin were different, and the major peptides produced from equine milk were identified by mass spectrometry as fragments of β-casein. Using two-dimensional electrophoresis, equine β-casein was confirmed as the main target of calf chymosin over 24 h at 30 °C and pH 6.5. The gel-based analysis of equine milk discriminated between the different individual proteins and provided information on the range of isoforms of each protein as a result of post-translational modifications, as well as positively identified for the first time several isoforms of κ-casein. In comparison to bovine milk, κ-casein isoforms in equine milk were not involved in chymosin-induced coagulation. The intensity of equine β-casein spots decreased following chymosin addition, but at a slower rate than bovine κ-casein.

Optimization of Medium Composition for Production of Recombinant Calf Chymosin from Kluyveromyces lactis in Submerged Fermentation

Response surface methodology was applied to optimize medium components for production of recombinant calf chymosin by Kluyveromyces lactis GG799. The previous data indicated that the most suitable carbon source, nitrogen source, salt and vitamin were glucose, yeast extract, KH2PO4 and Ca D-Pantothenate, respectively. The concentration of four media components were optimized by using central composite design of response surface methodology. The optimum medium composition for recombinant calf chymosin production was found to contain glucose 29.84 g · L−1, yeast extract 19.85 g · L−1, KH2PO4 0.1 g · L−1 and Ca D-Pantothenate 4.49 mg · L. The enzyme activity of recombinant calf chymosin was 722 U · mL−1, which was in an excellent agreement with the predicted value (723 U · mL−1). The production of recombinant calf chymosin from Kluyveromyces lactis GG799 was effectively increased by response surface methodology.

Disruption of PMR1 in Kluyveromyces lactis improves secretion of calf prochymosin

Chymosin is an important industrial enzyme widely used in cheese manufacturing. Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, only low yields of chymosin (80 U mL(-1) in shake flask culture) have been obtained using K. lactis GG799. The aim of this study was to increase the amount of recombinant calf chymosin secreted by K. lactis GG799 by disrupting the PMR1 gene. Kluyveromyces lactis GG799 harbouring the disrupted PMR1 gene showed reduced growth in ethylene glycol tetraacetic acid-containing and Ca(2+) -deficient medium, but Ca(2+) supplementation eliminated the growth problem. The calf chymosin gene was ligated into the K. lactis GG799 expression vector, generating the plasmid pKLAC1-N-prochymosin. The linearised plasmid was homologously integrated into the genome of K. lactis GG799. In shake flask culture, chymosin activity was 496 U mL(-1) in the K. lactis PMR1-deficient mutant, sixfold higher than that in wild-type K. lactis GG799. Disrupting the PMR1 gene improved chymosin production in K. lactis GG799 sixfold. This knowledge could be applied to industrial chymosin production.

Inhibition of rennets by blood serum

Incubation of calf chymosin with the serum from blood of various mammalian species before addition to milk reduced its milk‐clotting activity at 30°C; the inhibitory potency of the sera was in the order horse > goat > cattle > donkey = human. For all species, inhibition of calf chymosin increased on increasing both the incubation time and the level of blood serum. Horse blood serum inhibited other milk coagulants in the order porcine pepsin > Cryphonectria parasitica proteinase = calf chymosin > Rhizomucor miehei proteinase = bovine pepsin. Methylamine‐treated blood serum did not inhibit calf chymosin, suggesting that α2‐macroglobulin may be the principal inhibitor of rennet in blood serum.

Characterization of wine rennet and its kinetics by gel electrophoresis

The rennet of glutinous rice wine (wine rennet) is an exclusive clotting agent for Chinese Royal cheese production. Some characterizations are reported herein in an attempt to provide evidence about the use of the protease as either a rennet substitute or an accelerator in cheese making and ripening. The results showed that wine rennet was a monomeric and unglycosylated protease. The N-sequencing indicated a high degree of similarity to other fungal rennets. The cleavage sites of wine rennet on oxidized insulin B chain identified by HPLC-mass spectrometry included Gln4-His5, Ala14-Leu15, Leu15-Tyr16, Tyr16-Leu17, and Phe24-Phe25 at pH 6.5, which were similar to those observed for Mucor rennet, but different from calf chymosin except for Leu15-Tyr16. A comparison study of the kinetic properties of wine rennet on bovine caseins with that of rennets from calf and Mucor miehei by gel electrophoresis showed that these rennets had similar coagulation efficiency but different reaction rates. Wine rennet exhibited a higher degree of degradation than the calf and Mucor enzymes at pH 6.5 and 40°C. Therefore, wine rennet would be an adjunct for calf rennet or an accelerator in cheese making.

Codon optimization of the calf prochymosin gene and its expression in Kluyveromyces lactis

Chymosin as an important industrial enzyme widely used in cheese manufacture. The yeast Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, low yields (80 U/ml in shake flask cultures) were obtained when the K. lactis strain GG799 was used to express chymosin. We hypothesized that the codon-usage bias of the host may have resulted in inefficient translation and chymosin production. To improve expression efficiency of recombinant calf chymosin in K. lactis strain GG799, we designed and synthesized a DNA sequence encoding calf prochymosin using optimized codons, while keeping the G + C content relatively low. We altered 333 nucleotides to optimize codons encoding 315 amino acids. In shaking flask culture, chymosin activity was 575 U/ml in the strain expressing the optimized gene, a sevenfold higher expression level compared with the non-optimized control. SDS–PAGE analysis revealed that the purified recombinant calf chymosin had a molecular mass of 35.6 kDa, the same as the molecular weight of native calf chymosin. Alpha-casein, beta-casein, and kappa-casein were incubated with the recombinant calf chymosin from K. lactis strain GG799 or chymosin from calf stomach and the breakdown products were analyzed by SDS–PAGE. Both the recombinant calf chymosin and the native calf chymosin specifically hydrolyzed kappa-casein. Our results show that codon optimization of the calf chymosin gene improves expression in K. lactis strain GG799. Genetic manipulation to optimize codon usage has important applications for industrial chymosin production.

Suitability of recombinant camel ( Camelus dromedarius) chymosin as a coagulant for Cheddar cheese

Cheddar-type cheeses were manufactured using fermentation-produced camel or calf chymosin. There were no significant differences in the composition and pH between the cheeses made with either coagulant. The extent of primary proteolysis was significantly lower in cheeses made with camel chymosin than in cheeses made with calf chymosin. There were large quantitative differences between the peptide profiles of cheeses; however, the levels of amino acids were similar except for isoleucine, histidine and lysine. The cheeses made with camel chymosin were characterized by lower intensities of sulphur and brothy flavours and showed less bitter taste; however, the cheeses made with calf chymosin had greater breakdown of texture, higher smoothness and mouthcoating and were more cohesive and adhesive. The results of this study suggest that camel chymosin appears to be suitable for making Cheddar cheese with lower levels of proteolysis but with good flavour.

A new approach to the use of fluorogenic dinitrophenyl-containing substrates for determining the proteolytic activity of aspartyl proteinases

A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.

A comparative study of functional properties of calf chymosin and its recombinant forms

The action of calf chymosin obtained from transgenic sheep milk and the recombinant protein expressed in yeast Kluyveromyces lactis (Maxiren) on fluorogenic peptide substrates, namely Abz-A-A-F-F-A-A-Ded, Abz-A-A-F-F-A-A-pNA, Abz-A-F-F-A-A-Ded, Abz-A-A-F-F-A-Ded, Abz-A-A-F-F-Ded, Abz-A-A-F-F-pNA, and heptapeptide L-S-F-M-A-I-P-NH2, a fragment of kappa-casein (the native chymosin substrate), was investigated. It has been established that transgenic chymosin and recombinant chymosin (Maxiren) differ from the native enzyme in their action on low molecular weight substrates, whereas there was no difference in enzymatic action on protein substrates. Pepstatin, a specific inhibitor of aspartic proteinases, inhibits the recombinant chymosin forms less efficiently than the native enzyme. Perhaps this is associated with local conformational changes in the substrate binding site of recombinant chymosin occurring during the formation of the protein globule.