RESUMEN

We evaluated the effects of fermentation time and acid casein content on the microbial rennet obtained by solid-state fermentation using wheat bran as the carbon source. The experiments used two fermentation times (72 and 96 h), while acid casein content was 1.5, 2.0, 2.5, and 3.0 g. Rennet strength from eight enzymatic extracts was measured using pasteurized whole milk. Rennet strength of samples from 72 h of fermentation showed an increase when acid casein content increased. The rennet strength increased at 96 h of fermentation with increasing amount of casein (up to 2.5 g), and then decreased with the largest addition (3.0 g) of casein. Coagulation time for the sample with highest rennet strength was 420 s.

Asunto(s)

Bacterias/metabolismo , Caseínas/química , Caseínas/metabolismo , Quimosina/metabolismo , Nitrógeno/metabolismo , Fermentación

RESUMEN

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.

Asunto(s)

Calotropis/enzimología , Caseínas/metabolismo , Queso , Proteasas de Cisteína/metabolismo , Animales , Bovinos , Quimosina/metabolismo , Hidrólisis , Látex/metabolismo , Proteínas de Plantas/metabolismo

RESUMEN

Binary blends of S. marianum-flower extract and chymosin, as coagulant preparations, enabled the manufacture of miniature cheeses with distinctive characteristics compared to those of chymosin-renneted cheeses. The physicochemical parameters, sensory attributes of the cheeses, and in-vitro water-soluble antioxidant activity were analyzed and compared to those properties obtained from control chymosin-renneted cheeses. The preponderant proteolytic constituent in the flower extract was isolated in a two-step-purification protocol. The thus purified aspartic peptidase was maximally active at acidic pHs and exhibited a preference for peptide bonds between hydrophobic residues. Enzymologic characterization revealed differences in the kinetic parameters and specificity compared to other enzymes employed, such as rennet. S. marianum-flower extract, as a source of peptidase with distinctive characteristics, is a suitable substitute for chymosin in miniature-cheese production. The addition of vegetable rennet contributed to the development of an intense aroma and conferred antioxidant activity to the cheeses and wheys.

Asunto(s)

Queso/análisis , Quimosina/metabolismo , Manipulación de Alimentos/métodos , Silybum marianum/enzimología , Animales , Flores/enzimología , Leche

RESUMEN

High pressure processing (HPP) is able to promote changes in enzymes structure. This study evaluated the effect of HP on the structural changes in milk-clotting enzymes processed under activation conditions for recombinant camel chymosin (212MPa/5min/10°C), calf rennet (280MPa/20min/25°C), bovine rennet (222MPa/5min/23°C), and porcine pepsin (50MPa/5min/20°C) and under inactivation conditions for all enzymes (600MPa/10min/25°C) including the protease from Rhizomucor miehei. In general, it was found that the HPP at activation conditions was able to increase the intrinsic fluorescence of samples with high pepsin concentration (porcine pepsin and bovine rennet), increase significantly the surface hydrophobicity and induce changes in secondary structure of all enzymes. Under inactivation conditions, increases in surface hydrophobicity and a reduction of intrinsic fluorescence were observed, suggesting a higher exposure of hydrophobic sites followed by water quenching of Trp residues. Moreover, changes in secondary structure were observed (with minor changes seen in Rhizomucor miehei protease). In conclusion, HPP was able to unfold milk-clotting enzymes even under activation conditions, and the porcine pepsin and bovine rennet were more sensitive to HPP.

Asunto(s)

Quimosina , Manipulación de Alimentos/métodos , Presión , Animales , Camelus , Bovinos , Quimosina/química , Quimosina/metabolismo , Quimosina/efectos de la radiación , Estabilidad de Enzimas , Interacciones Hidrofóbicas e Hidrofílicas , Pepsina A/química , Pepsina A/metabolismo , Pepsina A/efectos de la radiación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Porcinos , Temperatura

RESUMEN

Codon optimization of the Bos taurus Chymosin gene (CYM) for its expression in Pichia pastoris was performed in this study. A synthetic CYM gene was designed in silico by replacing codons rarely used by P. pastoris with equivalent nucleotide combinations that codify for the same amino acid but that are more frequently encountered in the genome of P. pastoris. A total of 332 nucleotides were modified to optimize 289 codons. The synthetic CYM gene was cloned into the expression vector pPICZαA and transformed into P. pastoris. The transformed strains were grown in artificial media supplemented with glycerol as a carbon source to increase biomass and then cultured in a similar medium replacing glycerol with methanol as a carbon source to initiate gene induction. Raw extracts of the growth media exhibited milk-clotting activity of 146.11 SU/mL. Produced recombinant chymosin showed coagulant activity from 25 to 50 °C, and within a pH range of 5-6.9, having optimum activity at 35-40 °C, and pH 5.0. These results show that codon optimization is a viable strategy to improve CYM gene expression levels in P. pastoris for the production of recombinant chymosin.

Asunto(s)

Quimosina/genética , Quimosina/metabolismo , Pichia/genética , Análisis de Secuencia de ADN/métodos , Animales , Bovinos , Codón , Medios de Cultivo/química , Genes Sintéticos , Proteínas Recombinantes/metabolismo , Transformación Genética

RESUMEN

In this work, we studied the influence of the type of coagulant enzyme and the curd scalding temperature on the proteolysis and residual coagulant and plasmin activities of a cooked cheese, Reggianito, in the interest of reducing ripening time. A two-factor experimental design was applied in two levels: type of coagulant enzyme, bovine chymosin or camel chymosin, and curd scalding temperature, 50 or 56 °C. The experimental treatments were applied in Reggianito cheese making experiments, and the samples were ripened for 90 d at 12 °C. Scalding temperature influenced residual coagulant activity; the cheeses cooked at 50 °C had significantly higher activity than those treated at 56 °C. In contrast, scalding temperature did not modify plasmin activity. Proteolysis was primarily affected by curd cooking temperature because chymosin-mediated hydrolysis of αs1 casein was slower in cheeses treated at 56 °C. Additionally, the nitrogen content in the cheese soluble fractions was consistently lower in the cheeses scalded at 56 °C than those cooked at 50 °C. A significant influence of the type of coagulant enzyme was observed, especially in the nitrogen fractions and peptide profiles, which demonstrated that camel chymosin was slightly less proteolytic; however, these differences were lower than those caused by the scalding temperature.

Asunto(s)

Queso/análisis , Quimosina/metabolismo , Fibrinolisina/metabolismo , Manipulación de Alimentos/métodos , Calor , Proteolisis , Animales , Argentina , Camelus , Caseínas/metabolismo , Bovinos , Concentración de Iones de Hidrógeno , Nitrógeno/análisis , Péptidos/análisis

RESUMEN

Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.

Asunto(s)

Quimosina/metabolismo , Precursores Enzimáticos/metabolismo , Lactococcus lactis/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Bovinos , Quimosina/genética , Precursores Enzimáticos/genética , Proteínas Recombinantes/genética

RESUMEN

Proteases are some of the most important industrial enzymes, and one of their main applications is for the production of cheese in the dairy industry. Due to a shortage of animal rennet, microbial coagulant proteases are being sought. In this work, the production of microbial rennet from Thermomucor indicae-seudaticae N31 was studied in submerged fermentation. The best enzyme production was obtained in a fermentation medium containing 4 % wheat bran as the substrate in 0.3 % saline solution, incubated for 72 h at 45 °C and 150 rpm. The value of the milk clotting activity (MCA) was 60.5 U/mL, and the ratio to proteolytic activity (MCA/PA) was 510. The crude enzyme showed optimum pH at 5.5 and two peaks of optimum temperature (MCA at 65 °C and PA at 60 °C). The MCA was stable in the pH range 4.0-4.5 for 24 h and up to 55 °C for 1 h. It was stable during storage at different temperatures (-20 to 25 °C) for 10 weeks. Based on these results, we conclude that microbial rennet from T. indicae-seudaticae N31 produced by submerged fermentation showed good prospects of replacing traditional rennet.

Asunto(s)

Quimosina/metabolismo , Microbiología Industrial/métodos , Mucorales/metabolismo , Fibras de la Dieta , Fermentación , Péptido Hidrolasas/metabolismo

RESUMEN

The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.

Asunto(s)

Aldehído Oxidasa/genética , Quimosina/metabolismo , Pichia/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/metabolismo , Animales , Ácido Ascórbico/metabolismo , Reactores Biológicos , Bovinos , Quimosina/análisis , Quimosina/química , Quimosina/genética , Medios de Cultivo , Fermentación , Concentración de Iones de Hidrógeno , Pichia/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sorbitol/metabolismo , Temperatura

RESUMEN

The objective of this study was to evaluate the effect of inulin as a fat replacer on the rheological properties, coagulation kinetics, and syneresis of milk gels. A randomized factorial design, replicated 3 times, with 3 inulin concentrations (0, 3, and 6%), 2 levels of fat (<0.2 and 1.5%), and 3 coagulation temperatures (27, 32, and 37°C) was used. The coagulation process was monitored using near-infrared spectrometry, small amplitude oscillatory rheometry, and visual coagulation indexes. The syneresis was evaluated by volumetric methods. Inulin addition increased the rates of aggregation and curd firming reactions in the casein gels. The observed effect, which was more evident on the aggregation reaction, depended on the concentration of inulin and the coagulation temperature. Addition of 6% inulin reduced the clotting time by approximately 26% and the time at which the gel reached a storage modulus equal to 30 Pa by approximately 36%. The optical parameter R'max, defined as the maximum value of change in light backscatter profile/change in time (where R' = dR/dt), was used to calculate an approximation of the temperature coefficients (Q10) for milk coagulation. Increasing fat concentration induced a consistent increase in all the optical, rheological, and visual parameters studied, although the observed trend was not statistically significant. The addition of inulin at a level of 6% produced a reduction in syneresis and increased the curd yield by approximately 30%. It was concluded that the addition of inulin affects the kinetics of milk coagulation and the cutting time and, therefore, the use of inline sensors such as near-infrared spectrometry may be necessary for optimal process control.

Asunto(s)

Quimosina/metabolismo , Sustitutos de Grasa/farmacología , Inulina/farmacología , Leche/química , Animales , Caseínas/química , Caseínas/metabolismo , Fenómenos Químicos , Geles/química , Cinética , Reología

RESUMEN

Kappa-casein (kappa-CN) is a milk phospho-glycoprotein that plays an important role in the electrostatic and steric stabilization of casein micelles suspensions. kappa-CN characteristics such as its aminoacidic composition and its high denaturalization temperature make this protein an excellent nutrient. The enzymatic destabilization of kappa-CN is the basis of cheese and yoghurt manufacture. In this paper, modifications on the kappa-CN aggregation process by hydrolysis with chymosin derived from the initial protein state and the presence of sucrose and lactose were studied. Our study shows that, during its self-association, kappa-CN opens its conformation by raising the exposition of hydrophobic regions to the medium. Hence, the initial polymerization or association state of kappa-CN affects on the aggregation stage after the enzymatic action of chymosin. The inhibition of aggregation when the protein was previously associated suggests that the region which participates in the aggregation of p-kappa-CN particles, would be the one involved in the self-association during the heating of kappa-CN samples. Sucrose and lactose also affect the aggregation of proteolized particles of kappa-CN. Furthermore, the nature and the concentration of the added sugars had significant influence on the protein aggregation process.

Asunto(s)

Caseínas/química , Caseínas/metabolismo , Quimosina/metabolismo , Animales , Biocatálisis , Bovinos , Quimosina/química , Calor , Tamaño de la Partícula , Conformación Proteica , Desnaturalización Proteica , Propiedades de Superficie

RESUMEN

Strongly proteolytic starters seem to improve the growth of nonstarter lactobacilli during cheese ripening, but no information is available on the impact of the nonmicrobial proteases usually active in cheese on their development. In the current study, the influence of chymosin- and plasmin-mediated proteolysis on the growth and biochemical activities of lactobacilli during ripening of miniature Cheddar-type cheeses, manufactured under controlled microbiological conditions, was studied. Two experiments were performed; in the first, residual chymosin activity was inhibited by the addition of pepstatin, and in the second, plasmin activity was increased by adding more enzyme, obtained in vitro through the activation of plasminogen induced by urokinase. Cheeses with or without a Lactobacillus plantarum I91 adjunct culture and with or without added pepstatin or plasmin solution were manufactured and ripened for 60 d. The addition of the adjunct culture resulted in enhancement of secondary proteolysis, evidenced by an increase in the total content of free amino acids (FAA) and modifications of the individual FAA profiles. Reduction in residual chymosin activity caused a decrease in primary and secondary proteolysis, characterized by the absence of alpha(s1)-casein hydrolysis and reduced production of peptides and FAA, respectively. The increase in plasmin activity accelerated primary proteolysis but no enhancement of secondary proteolysis was observed. Chymosin- and plasmin-mediated proteolysis did not influence the growth and biochemical activities of adventitious or adjunct lactobacilli, indicating that it is not a limiting factor for the development and proteolytic-peptidolytic activities of lactobacilli in the cheese model studied.

Asunto(s)

Queso/microbiología , Quimosina/metabolismo , Fibrinolisina/metabolismo , Lactobacillus/crecimiento & desarrollo , Lactobacillus/metabolismo , Proteínas de la Leche/metabolismo , Aminoácidos/química , Animales , Queso/análisis , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Lactobacillus plantarum/crecimiento & desarrollo , Lactobacillus plantarum/metabolismo , Leche/química , Péptidos/química , Análisis de Componente Principal

RESUMEN

Refolding of proteins from inclusion bodies is a field of increasing interest for obtaining large amounts of active enzymes. Consequently, the development of inexpensive and scalable processes is required. This is particularly challenging in the case of eukaryotic proteins containing cysteines, which may form disulfide bonds in the native active protein. Previous studies have shown that the formation of disulfide bonds is essential for the refolding of prochymosin. In this work we demonstrate that air oxidation can be efficiently used for the refolding of prochymosin and that 48% of the unfolded protein can be recovered as active enzyme at a final protein concentration of 0.8 mg/ml. Refolding of the protein strictly correlates with the change in pH of the refolding solution. We were able to follow the degree of oxidative renaturation of the prochymosin by simply measuring pH. Thus, the scaling up of the refolding system under controlled conditions was easily achieved. Analyses of different substances as folding aids indicate that the use of L-arginine or neutral surfactants improves the recovery of active protein up to 67% of the initial protein. The overall results indicate that prochymosin can be efficiently and inexpensively refolded with high yields by controlled air oxidation.

Asunto(s)

Quimosina/aislamiento & purificación , Quimosina/metabolismo , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Oxígeno/farmacología , Aire , Concentración de Iones de Hidrógeno , Oxidación-Reducción/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Pliegue de Proteína , Renaturación de Proteína/efectos de los fármacos

RESUMEN

The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309].

Asunto(s)

Quimosina/química , Mucor/enzimología , Biotecnología , Rastreo Diferencial de Calorimetría , Quimosina/genética , Quimosina/metabolismo , Dicroismo Circular , Estabilidad de Enzimas , Expresión Génica , Genes Fúngicos , Glicosilación , Concentración de Iones de Hidrógeno , Peso Molecular , Mucor/genética , Pichia/genética , Conformación Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Temperatura

RESUMEN

A refined three-dimensional molecular model of kappa-casein has been produced using energy minimization techniques and a Kollman force field on a previously reported predicted three-dimensional structure. This initial model was constructed via molecular modeling techniques from sequence-based secondary structural prediction algorithms. Both the initial and refined structures agreed with global secondary structure analysis from vibration spectroscopy. The refined structure contained many of the features of the initial model, including two sets of antiparallel beta-sheet structures containing predominantly hydrophobic side chains, which could form interaction sites with alpha s1-casein. Two types of energy-minimized dimer and tetramer models are presented: 1) using Cys as potential intermolecular disulfide binding sites and 2) using the two sheets as possible hydrophobic self-association sites, without Cys interactions. All structures yielded good stabilization energies and are in agreement with chemical, biochemical, and physical chemical results obtained for kappa-casein.

Asunto(s)

Caseínas/química , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Bovinos , Fenómenos Químicos , Química Física , Quimosina/metabolismo , Simulación por Computador , Sustancias Macromoleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

Three-dimensional structures derived from X-ray crystallography are extremely important in elucidating relationships between structure and function for many proteins. However, not all proteins can be crystallized. The caseins of bovine milk are one class of noncrystallizable proteins. The complete primary and partial secondary structures of these proteins are known, but homologous proteins with known crystallographic structure are not available. In this report, sequence-based predictions of secondary structure were made and adjusted to conform with global secondary structures derived from Fourier transform infrared spectroscopy. With this information, a three-dimensional structure for kappa-casein was constructed using molecular modeling computer programs. The constructed model contains two unstranded beta-sheets; both are predominantly hydrophobic and capable of forming quaternary structural interaction sites with alpha s1-casein. This unrefined structure is in good agreement with much of the biochemical information available for kappa-casein.

Asunto(s)

Caseínas/química , Secuencia de Aminoácidos , Animales , Caseínas/metabolismo , Bovinos , Quimosina/metabolismo , Simulación por Computador , Disulfuros/metabolismo , Glicosilación , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Espectrofotometría Infrarroja , Compuestos de Sulfhidrilo/metabolismo , Difracción de Rayos X

Asunto(s)

Catecolaminas/metabolismo , Hipertensión/metabolismo , Quimosina/metabolismo , Humanos , Riñón/metabolismo