Bovine Chymosin Research Articles

Construction of a Kluyveromyces lactis strain with multi-copy integration for enhanced bovine chymosin production by CRISPR/Cas9 and UV mutagenesis

Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcymD, and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcymD-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.

Tobacco Plant: A Novel and Promising Heterologous Bioreactor for the Production of Recombinant Bovine Chymosin.

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD600) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform.

Comprehensive bioinformatics-based annotation and functional characterization of bovine chymosin protein revealed novel biological insights

Chymosin, an aspartic protease present in the stomachs of young ruminants like cows (bovine), causes milk coagulation and cheese production through the breakdown of κ-casein peptide bonds at the Met105-Phe106 site. Bovine chymosin is first synthesized as a pre-prochymosin that is cleaved to produce the mature chymosin protein. Despite significant strides in research, our understanding of this crucial enzyme remains incomplete. The purpose of this work was to perform in silico evolutionary and functional analysis and to gain unique insights into the structure of this protein. For this, the sequence of Bos taurus chymosin from UniProt database was subjected to various bioinformatics analyses. We found that bovine chymosin is a low molecular weight and hydrophilic protein that has homologs in other Bovidae species. Two active sites of aspartic peptidases, along with a functional domain, were identified. Gene Ontology analysis further confirmed chymosin's involvement in proteolysis and aspartic endopeptidase activity. Potential disordered residues and post-translational modification sites were also uncovered. It was revealed that the secondary structure of bovine chymosin is comprised of beta strands (44.27%), coils (43.65%), and alpha helices (12.07%). A highly optimized 3D structure was also obtained. Moreover, crucial protein–protein interactions were unveiled. Altogether, these findings provide valuable insights that could guide future research on bovine chymosin and its biological roles.

Proteolytic activity of three variants of fermentation-produced chymosin on sodium caseinate and during Cheddar cheese ripening

Proteolytic activities and specificities of three types of fermentation-produced chymosin were investigated in cheese and in sodium caseinate (NaCN) digests made using these coagulants. The highest level of enzyme required to hydrolyse all intact αS1-CN in NaCN solution at pH 5.2 over 24 h was for a modified camel chymosin (mCC; 13.34 IMCU mL−1), followed by camel chymosin (4 IMCU mL−1) and bovine chymosin (0.4 IMCU mL−1). Many peptides were identified using liquid chromatography-mass spectrometry from both Cheddar cheese and NaCN digests produced using each chymosin. Besides previously reported casein-derived peptides produced by bovine and camel chymosins and corresponding cleavage sites, several new cleavage sites were identified. The proteolytic specificity of mCC on casein was determined. Most of the peptides observed in Cheddar cheese samples were also identified in NaCN digests. The overall proteolytic activity of mCC was the lowest, which may have implications for ripening and functionality of cheese.

The effect of thioredoxin and prochymosin coexpression on the refolding of recombinant alpaca chymosin.

The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pET-TrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage.

Milk-clotting activity of recombinant bovine and camel chymosin for cow’s, goat’s and ewes’ milk

Goats and sheep were domesticated during the Neolithic Revolution. Since then, various breeds of goats and sheep have been bred, including dairy ones. In this work, we tested the milk-clotting activity of recombinant bovine (Bos taurus) and camel (Camelus bactrianus) chymosins in cow, goat, and ewe milk. The milk-clotting activity of recombinant bovine chymosin for cow’s, goat’s and ewes’ milk was 12 854 ± 0,61, 5385 ± 0,25 and 14 811 ± 0,72 U/mg. The activity of recombinant camel chymosin was higher by 29%-46% and amounted to 16 590 ± 0,82, 7850 ± 0,34 and 20 700 ± 0,85 U/mg. Proteolytic activity was 1679.97 ± 9.54 and 10 767 ± 54.56 U/mg for recombinant camel and bovine chymosins, respectively. With the recombinant camel chymosin, cheeses were obtained from cow’s, goat’s and ewes’ milk. The yield of cheese from cow’s, goat’s and ewes’ milk was 18.0%, 17.3% and 15.3%, respectively. The results indicate the prospects for the use of recombinant camel chymosin as the coagulation enzyme in the processing of cow’s, goat’s and ewes’ milk to cheeses.
 

Biosynthesis of the acid protease produced by Lacticaseibacillus casei LBC 237 and Limosilactobacillus fermentum LBF 433 and their potential application in the bovine milk clotting

This study aimed to produce and evaluate proteases from Lacticaseibacillus casei (LBC 237) and Limosilactobacillus fermentum (LBF 433) for milk coagulation. Proteases were assessed for acid protease activity (PA) and stability within temperature range of 30–75 °C and pH levels of 5.0, 5.5, and 6.8. Substrate concentration (0.025 %–2.5 %) and storage conditions (up to 120 days) were also investigated. Milk-clotting activity (MCA) and the effect of NaCl (1–4 %) on coagulation were evaluated, with subsequent characterization of resulting clots in terms of molecular size, structure, and flavor profile. Chemical changes in fermentation, proteases, and clots were determined. The proteases showed better PA at 60 °C and a pH of 5.5, with no change over 120 days. LBC-P demonstrated a higher affinity for the substrate. The proteases generated bands at ∼35 kDa, similar to bovine chymosin bands, and LBC-P exhibited MCA (480 SU/mL). Clots formed using LBC-P and bovine chymosin displayed similar bands (50 kDa, 35 kDa, and 15 kDa), while the LBC-P clot exhibited a more porous structure. LBC-P was inactivated by NaCl concentrations (≥1 %), and the clots showed changes in amides I. The bitterness intensity was lower for the clot obtained with LBC-P. While both LBC-P and LBF-P showed potential for protein hydrolysis, only LBC-P demonstrated potential for milk coagulation for cheese production. This study suggests acid proteases with high hydrolytic activity. In conclusion, L. casei (LBC 237) derived proteases show potential for milk coagulation, offering a promising alternative to bovine chymosin in yellow cheese production.

Effect of the recombinant chymosins of different origins on the quality and shelf life of soft cheeses

The effect of the type (bovine chymosin (Chy-max Extra), camel chymosin (Chy-max M), and modified camel chymosin (Chy-max Supreme)) and applied dose (1500, 2500, or 3500 IMCU/100 kg of milk) of milk-clotting enzyme (MCE) on the proteolysis degree, microstructure, rheological and sensory properties of Crescenza soft cheese was studied. The proteolysis degree of cheeses was directly proportional to the general proteolytic activity (PA) and dose of the MCEs added into milk during the cheese production. With an equal dose of the MCEs added into milk, the highest level of proteolysis was noted in the cheese options produced with recombinant bovine chymosin (Chy-max Extra). There were no statistically significant differences (p > 0.05) in the proteolysis degree between the cheese options made with the same doses of the camel chymosin (Chy-max M) and modified chymosin (Chymax Supreme). After 21 days of storage, the cheeses with Chy-max Extra MCE had the most plastic consistency, while the cheeses with Chy-max Supreme MCE had the hardest and most elastic one. Cheeses produced with Chy-max M MCE occupied an intermediate position in terms of consistency density. Recombinant MCEs based on camel chymosin (Chy-max M) and modified chymosin (Chy-max Supreme) with low level of PA, may be recommended for use in the production of soft cheeses. This will extend the shelf life of cheeses by slowing-down the plasticization rate of their consistency.

Influence of different types of fermentation-produced chymosin on quality of soft cheeses

The disadvantage of soft cheeses is their short shelf life. In soft cheeses with a high moisture content, proteolysis occurs at a high rate, as a result of which the cheeses quickly overripe. The main proteolytic agent in soft cheeses is the milk-clotting enzyme (MCE). Increasing the shelf life of cheeses can be achieved by using MCE types having low proteolytic activity (PA). We have studied the effect of MCE based on different types of fermentation-produced chymosin: Chy-max® Extra (bovine chymosin), Chy-max® M (camel chymosin), Chy-max® Supreme (“modified” chymosin) on the dynamics of proteolysis in soft cheeses and related changes in the structure of cheeses during their storage. All 3 types of studied MCEs have different levels of nonspecific PA. The higher the level of nonspecific PA of the used MCE, the higher the rate of the proteolysis process in the resulting cheeses. Increasing the dose of MCE also increases the rate of proteolysis in cheeses. To increase the shelf life of soft cheeses, which depends on the period of preservation of a dense consistency, it is promising to use MCE with low PA based on camel chymosin (Chy-max® M) and “modified” chymosin (Chy-max® Supreme).

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

Cheddar-type cheese was manufactured using fermentation-produced bovine chymosin (BC), fermentation-produced camel chymosin (CC) and a modified fermentation-produced camel chymosin (mCC) and ripened for 180 days. Only minor differences were found in cheese composition and pH between the cheeses made with any of the chymosins studied. Proteolysis in cheese made with mCC was reduced compared with cheese made with BC or CC. Significantly higher instrumental and sensory hardness and significantly lower meltability were found in cheeses made using CC or mCC compared with cheese BC after 180 days of ripening. Descriptive sensory analysis results showed that cheese made with CC or mCC had less sulphur and barny flavour; the brothy flavour and bitter taste of cheese made with mCC were also lowest. In conclusion, the modified camel chymosin appears to be suitable for the manufacture of Cheddar cheese with modified functionalities.

The impact of chymosin and plant‐derived proteases on the acid‐induced gelation of milk

This work aimed to study milk gel formation during a slow fermentation process and the simultaneous action of different proteases like bovine chymosin, recombinant camel chymosin, ficin and bromelain. The acid‐induced gels with bovine chymosin and recombinant camel chymosin were firmer than the control gels without proteases or the gels added with plant proteases. However, the acid‐induced gels with chymosins had a considerable water release and shrunk in volume as the milk fermentation progressed. In contrast, the acid‐induced gels added with bromelain and ficin were softer and showed less syneresis, even at acidic pHs.

Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties.

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichia coli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis-Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1µM, 2.68 ± 0.08s-1 and 2.27± 0.10µmM-1·s-1, respectively.

INFLUENCE OF POINT AMINO ACID SUBSTITUTIONS IN THE RECOMBINANT COW CHYMOSIN MOLECULE ON THE DEPENDENCE OF ITS COAGULATION ACTIVITY ON THE CONCENTRATION OF CALCIUM CHLORIDE IN MILK

It is shown that point amino acid substitutions Asp198→Lys, Asp156→Val and their combination lead to a decrease in the sensitivity of coagulation activity of recombinant bovine chymosin to changes in the concentration of calcium chloride in milk.

Biotechnological potential of a cysteine protease (CpCP3) from Calotropis procera latex for cheesemaking

This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos’ morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin.

Microstructure and fracture properties of semi-hard cheese: Differentiating the effects of primary proteolysis and calcium solubilization.

The individual roles of hydrolysis of αS1- and β-caseins, and calcium solubilization on the fracture properties of semi-hard cheeses, such as Maasdam and other eye-type cheeses, remain unclear. In this study, the hydrolysis patterns of casein were selectively altered by adding a chymosin inhibitor to the curd/whey mixture during cheese manufacture, by substituting fermentation-produced bovine chymosin (FPBC) with fermentation-produced camel chymosin (FPCC), or by modulating ripening temperature. Moreover, the level of insoluble calcium during ripening was quantified in all cheeses. Addition of a chymosin inhibitor, substitution of FPBC with FPCC, or ripening of cheeses at a consistent low temperature (8 °C) decreased the hydrolysis of αS1-casein by ~95%, ~45%, or ~30%, respectively, after 90 d of ripening, whereas ~35% of β-casein was hydrolysed in that time for all cheeses, except for those ripened at a lower temperature (~17%). The proportion of insoluble calcium as a percentage of total calcium decreased significantly from ~75% to ~60% between 1 and 90 d. The rigidity or strength of the cheese matrix was found to be higher (as indicated by higher fracture stress) in cheeses with lower levels of proteolysis or higher levels of intact caseins, primarily αS1-casein. However, contrary to the expectation that shortness of cheese texture is associated with αS1-casein hydrolysis, fracture strain was significantly positively correlated with the level of intact β-casein and insoluble calcium content, indicating that the cheeses with low levels of intact β-casein or insoluble calcium content were more likely to be shorter in texture (i.e., lower fracture strain). Overall, this study suggests that the fracture properties of cheese can be modified by selective hydrolysis of caseins, altering the level of insoluble calcium or both. Such approaches could be applied to design cheese with specific properties.

Large-scale production of yak (Bos grunniens) chymosin A in Pichia pastoris

Milk-clotting enzymes used in the dairy industry can be obtained from different sources such as plants, animals, and microorganisms. Recombinant chymosin is the best alternative for the dairy industry due to the differences in physicochemical properties of coagulating enzymes and scarcity of chymosin from animal sources.In this study, glycosylated and non-glycosylated forms of yak chymosin were extracellularly produced in a methylotrophic yeast, Komagataella phaffii (Pichia pastoris). Synthetic yak prochymosin genes were cloned into the pPICZαA vector, expressed in P. pastoris GS115 (PDI) strain. Active chymosin expression was achieved into supernatant with Saccharomyces cerevisiae α-mating factor under the control of methanol-inducible AOXI promoter. The glycosylation of yak chymosin did not have a significant effect on yield and activity at shake flask level. In a 5L fermentor, production of native yak-chymosin was achieved and the enzyme activity was found as 214 IMCU/ml. pH of 6–7 and temperature of 40 °C values were optimum for the enzyme. The laboratory scale white cheese production yield with recombinant yak chymosin was very similar to a commercial bovine chymosin. These results indicate that P. pastoris expression system is very suitable for recombinant yak chymosin production to meet the needs of the cheese industry.

Effect of salt bridges rupture on the activity and thermostability of bovine chymosin

Effect of salt bridges rupture on the activity and thermostability of bovine chymosin

Production of transgenic sheep: Microinjection of the bovine P77 gene construct in Chios sheep breed zygotes

The feasibility of integration of the bovine P77 construct into zygotes and two cell embryos of Chios sheep breed was examined. The P77 gene construct comprised bovine as l casein promoter regulatory sequence fused to the fragment between exons 2 and 9 of the bovine chymosin gene. One of the two pronuclei of the zygote or a nucleus of a blastomere of a two cell embryo was microinjected with 1 pi of DNA solution containing approximately 1000 copies of the gene construct. In total 193 zygotes and 87 two cell embryos collected from 49 donor ewes were used. After 1 - 5 h culture microinjected zygotes (n=175) and two cell embryos (n=76) were transferred by a laparoscopic technique to the oviducts of 68 recipients. 22 recipients (32.4%) gave birth to 29 lambs which correspond to 11.6% of the total number of zygotes - embryos transferred. PCR analysis performed in skin samples of these lambs revealed that in one ewe lamb the P77 construct has been integrated in its genome. The growth and puberty of this lamb was physiological and is currently pregnant. This is the first transgenic farm animal born in Greece.

On the effect of mutations in bovine or camel chymosin on the thermodynamics of binding κ-caseins.

Bovine and camel chymosins are aspartic proteases that are used in dairy food manufacturing. Both enzymes catalyze proteolysis of a milk protein, κ-casein, which helps to initiate milk coagulation. Surprisingly, camel chymosin shows a 70% higher clotting activity than bovine chymosin for bovine milk, while exhibiting only 20% of the unspecific proteolytic activity. By contrast, bovine chymosin is a poor coagulant for camel milk. Although both enzymes are marketed commercially, the disparity in their catalytic activity is not yet well understood at a molecular level, due in part to a lack of atomistic resolution data about the chymosin-κ-casein complexes. Here, we report computational alanine scanning calculations of all four chymosin-κ-casein complexes, allowing us to elucidate the influence that individual residues have on binding thermodynamics. Of the 12 sequence differences in the binding sites of bovine and camel chymosin, eight are shown to be particularly important for understanding differences in the binding thermodynamics (Asp112Glu, Lys221Val, Gln242Arg, Gln278Lys. Glu290Asp, His292Asn, Gln294Glu, and Lys295Leu. Residue in bovine chymosin written first). The relative binding free energies of single-point mutants of chymosin are calculated using the molecular mechanics three dimensional reference interaction site model (MM-3DRISM). Visualization of the solvent density functions calculated by 3DRISM reveals the difference in solvation of the binding sites of chymosin mutants.

Effect of coagulant type and level on the properties of half-salt, half-fat Cheddar cheese made with or without adjunct starter: Improving texture and functionality

The potential of increasing proteolysis as a means of enhancing the texture and heat-induced flow of half-fat, half-salt Cheddar cheese made with control culture (CL, Lactococcus lactis subsp. cremoris/lactis) or adjunct culture (AC, CL + Lactobacillus helveticus) was investigated. Proteolysis was altered by substituting bovine chymosin (BC) with camel chymosin (CC), or by a 2.5-fold increase in level of BC. In cheese with CL-culture, increasing BC led to a large increase in pH and more rapid degradation of αS1-casein during maturation, and cheese that was less firm after 180 d. In contrast, substitution of BC with CC in cheeses made with CL-culture had an opposite effect. While chymosin type and level had a similar influence on αS1-casein hydrolysis in the AC-culture cheeses, it did not affect texture or flowability. Grading indicated that cheese made with AC-culture and with a higher level of BC was the most appealing.