Milk-clotting Protease Research Articles

Proteomic analysis, purification and characterization of a new milk-clotting protease from Tenebrio molitor larvae

• A new milk clotting protease from Tenebrio molitor larvae was isolated and characterized. • The protease was confirmed its Gene Ontology term, serine-type endopeptidase, with MW of 29.68 kDa. • The protease had a Michaelis constant (K m ) of 1.39 g/L with casein and a good C/P value of 104.7. • The main cleavage site in κ-casein (κ-CN) was only Lys116-Thr117 bond for the protease. • This study could provide a theoretical basis for the development of insect chymosin. Tenebrio molitor larvae is an edible insect with high protein content; researchers have extracted several proteases from gut of T. molitor larvae. However, information on the milk-clotting protease of T. molitor larvae remains unknown. We found that while most of the milk-clotting activity (MCA) was concentrated in the midgut (H) of T. molitor larvae, some activity was also seen in the hindgut (T). To evaluate the possible functional activity of the protease, we used TMT labelling-coupled with LC-MS/MS for proteomics analysis of T and H. The results showed that a total of 223 differentially expressed proteins (DEPs) between T and H. The upregulated proteins were enrich the cysteine-type endopeptidase and serine-type peptidase. A serine endopeptidase with a molecular mass of 29.681 kDa and a C/P value of 104.7 with milk-clotting activity was obtained from midgut of T. molitor larvae by ultrafiltration and multistage chromatography, and characterized by Edman degradation. The main cleavage site in κ-casein (κ-CN) was Lys116-Thr117 bond for the protease, as determined by mass spectrometry analysis. The protease had a Michaelis constant (K m ) of 1.39 g/L, an optimal pH of 5.5, and an optimal temperature of 65 °C, indicating it could serve as a new milk coagulant and provide a theoretical basis for the development of insect chymosin.

Three phase purification of milk clotting protease from Wrightia tinctoria fruit and studies on its casein subunit specificity

Crude aqueous extract of Wrightia tinctoria fruit was subjected to three phase partitioning (TPP) with three different ammonium sulphate concentrations (40, 60, 80%) and their effect on concentrating milk clotting proteases were examined with 1:1 ratio of crude extract to t-Butanol. Proteases that concentrated in the interphase (IP) fraction of 60 and 80% salt concentration exhibited high milk clotting activity. TPP contributed to reduction in Caseinolytic Activity (CA) and enhancement of Milk Clotting Index (MCI) in these IP fractions compared to the crude enzyme. Casein (whole and kappa) hydrolytic pattern was analyzed by tricine SDS PAGE. Electrophoretic pattern of hydrolysate after 1 hr incubation revealed hydrolysis of κ-CN by CE, IP 60 and IP 80 fractions. Appearance of two low molecular weight bands approximately around 15 kDa indicated the specific affinity and controlled hydrolytic behavior by the proteases. Study provides evidence towards the utility of TPP in concentrating these milk clotting proteases and in improving their MCI. Keywords : Milk clotting • Wrightia tinctoria • Proteases• cheese

PRODUCTION AND PARTIAL CHARACTERIZATION OF A MILK-CLOTTING PROTEINASE PRODUCED BY BACILLUS SUBTILIS SMDFS-2B MN715837 IN SUBMERGED CULTURES

This study focused on the production and partial characterization of a milk-clotting protease produced by Bacillus subtilis SMDFS 2B in submerged cultures, under partially optimized conditions. The crude enzyme was recovered in the culture supernatant and concentrate was produced after cell removal and subsequent dialysis. Inhibition studies were conducted employing four distinct protease inhibitors: Pepstatin-A, Phenylmethane-sulphonyl-fluoride (PMSF), Ethylenediaminetetraacetic acid (EDTA), and iodoacetamide (IA). The effect of temperature, pH, metal ions and substrate concentration on milk-clotting activity were also evaluated. The thermal stability of the enzyme was determined by incubating the crude enzyme at a temperature value ranging from 35 oC to 60 oC. Similarly, pH stability was determined at pH values ranging between 4.5 and 8.0. The highest milk-clotting activity was observed at a temperature of 55 oC and pH 5.5. The crude enzyme preparation remained stable on incubation at 35 oC and 40 oC for 15 min and at pH 5.5. The enzyme also showed the lowest residual milk-clotting activity in the presence of EDTA (7.94%) and Pepstatin-A (26.71%). The addition of Mg2+ and Mn2+ significantly increased milk-clotting activity. The enzyme also showed an elevation in its apparent milk-clotting activity upon increasing the substrate (skim-milk) concentration. Thus, the milk-clotting protease produced by B. subtilis SMDFS 2B by submerged fermentation revealed some interesting milk-clotting characteristics. This may open the way for applications in the food and dairy industries.

Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation.

This study reports the optimization of milk-clotting protease production from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) in both one-variable-at-a-time and response surface methodology (RSM). The production and optimization of milk-clotting protease obtained from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) using different agro-industrial wastes as solid substrates were studied. The agro-industrial wastes used included wheat bran, rice bran, pea bran, and grass pea bran. The chemical composition of the best solid substrate was tested using standard methods. Others cultivation parameters were studied, and the results showed that the optimum fermentation medium composed of wheat bran, casein (1% w/w), and glucose (0.5% w/w) and the conditions for maximum milk-clotting protease production were at the moisture content of 55.0%, inoculum of 0.5*106 spores/mL, incubation temperature of 30°C, pH of 6.0, and fermentation time of 5days. The highest milk-clotting activity was obtained from the crude enzyme extracted using 0.1M NaCl and partial purification of the crude enzyme using chilled acetone, and 80% (NH4)2SO4 increased the ratio of MCA/PA from 0.56 to 1.30 and 0.65, respectively. Moreover, the highest MCA (137.58U/mL) was obtained at a casein concentration of 0.5%, pH 4.0, and 25°C, using RSM. Thus, results from the present study showed that the optimization of milk-clotting protease production from A. oryzae DRDFS 13 under SSF by both one-variable-at-a-time and RSM significantly increased the milk-clotting activity. This is the first report from a fungus in the Ethiopian setting and a modest contribution to highlight the potential of harnessing microbial protease enzymes for industrial applications.

Production of Extracellular Rennin-Like Enzyme by a Newly Isolate Mucor circinelloides (von Tieghem) and its Application in Camembert Cheese Making

Introduction: Aspartic proteases produced by different non-pathogenic fungi belonging to mucorales, are commonly used as milk-coagulants. The present study aims to optimize conditions for milk-clotting protease production by a new strain of Mucor genus and to assess its ability in soft cheese making. Materials and Methods: About 20 fungal strains isolated from soil were investigated for their potential to produce milk-clotting proteases for further applications in cheese making. The hyper producer strain Mucor circinelloides 2095-2047 was selected for optimization of rennin-like enzyme production under solid-state fermentation (SSF) using the stepwise modifications of the selected parameters. The enzyme produced under the optimal conditions was partially purified and then applied in Camembert cheese making trials compared to the crude extract and commercial rennet. Results: The maximum milk-curdling activity achieved after optimization (571.43 SU/mL) was obtained using wheat bran (10 g) as the mainly source of carbon containing 1% galactose; moistened with the M-9 solution (pH 6.0) and incubated at 30°C for 96 hours. The enzyme of M. circinelloides was partially purified with a high recovery of 105% and 6.23-fold purity after (NH4)2SO4 fractionation and dialysis. The physicochemical properties of the three produced cheeses were very close. A low sensory quality of cheese was obtained with the crude extract which was getting better using the pre-purified enzyme. This extract was able to develop a very close or even a similar sensory quality to that obtained by the commercial rennet. Conclusions: According to findings, it is possible to propose the purified enzyme of M. circinelloides as a new alternative for rennet, but further optimization, purification and cheese production tests are required.

Label-free quantitative proteomic analysis of the biological functions of Moringa oleifera seed proteins provides insights regarding the milk-clotting proteases

In this study, label-free quantitative proteomics was used to investigate the biological functions of M. oleifera seed proteins, which resulted in the identification of milk-clotting proteases. In total, 921 proteins were identified, and proteins within the molecular weight range of 30–50 kDa were abundant. The identified proteins were mainly involved in catalytic activity and metabolic processes associated with carbohydrate and protein metabolism, among which, proteases in the observed molecular weight range could possibly be responsible for the previously reported milk-clotting activity. An aspartic-type endopeptidase with molecular mass of 45,517 Da was purified from M. oleifera seeds using ammonium sulfate precipitation, ultrafiltration, and preparative high performance liquid chromatography, and was characterized using liquid chromatography-mass spectrometry (LC-MS)/MS. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the purified protease exhibited hydrolase activity and was involved in several metabolic pathways, which further confirmed that proteomic analysis can assist in the purification of the milk-clotting protease. The optimal temperature and pH required for protease activity were 60 °C and 5.0, respectively. The high thermal stability and good pH stability of the protease indicated that it can be used in the dairy industry.

The non-edible parts of fennel (Fœniculum vulgare) as a new milk-clotting protease source

Although fennel stems and leaves represent an abundant biomass, they remain poorly exploited in terms of extraction of biomolecules. This work aimed at the valorization of the non-edible parts of fennel by the extraction and the characterization of a new milk-clotting protease. Both, enzymatic extraction and milk-clotting conditions were optimized in the context of developing new aspects of cheese. Fennel stems and leaves were sorted and coarsely grounded. The extraction consists in an aqueous maceration and extracts were used to coagulate raw cow's milk. Maximum milk-clotting activity was obtained under a temperature of 20°C, extraction time of 12h and solid-to-solvent ratio of 150:100gmL−1. Besides, the impact of extract concentration, milk pH, calcium content and temperature on clotting-time were studied. The statistical analysis of the results confirmed significant effects of the four studied parameters. The use of specific inhibitors suggested the presence of a serine protease in the crude enzymatic extracts. The findings of this research confirmed that the use of fennel protease could be a new alternative for the commercial proteases in dairy products.

English

Proteolytic enzymes are metabolites that can be produced by microbial sources and develop important functions in food industry as in cheese manufacturing. However, it is necessary to ensure the final product safety by testing toxins production by microorganisms. As a result of this, the aim of this study was to investigate the production of proteases by Aspergillus flavo furcatis DPUA 1608 in submerged and solid state fermentation and also certify the non-production of aflatoxin by this species. The aflatoxin test was carried out using the method of ammonia vapor. In this test, A. flavo furcatis DPUA 1608 was inoculated in seven different media cultures (COA, YES, CZ, CYA, GMS, PMS and PDA) and the production of toxins was confirmed by the color change of culture reverse after adding a 25% (w/v) ammonia solution. The protease production was conducted using four inoculums (SAB+GLI, SAB+SAC, BDA+GLI and BDA+SAC) in three media cultures of submerged fermentation obtained by a base mineral solution (MA01, MAGli and MASac) and in solid state fermentation using acai seeds and rice bran as substrate (SAFA). According to ammonia vapor test, A. flavo furcatis is not an aflatoxin producer. There was no color change in the colonies reverse of any culture media. All crude extracts obtained in both fermentations were tested for protease production. The best protease activity was observed in the medium MA01 (inoculums SAB+SAC, BDA+GLI and BDA+SAC). Milk clotting activity was determined in all crude extracts of submerged and solid fermentation. However, the clot formed was considered as strong milk coagulation only in the media MA01 of submerged fermentation (inoculums SAB+SAC, BDA+GLI and BDA+SAC) and in all inoculums of solid state fermentation (SAFA). A. flavo furcatis DPUA 1608 showed potential milk-clotting protease production in both fermentations media used. Key words: Ammonia vapor test, Aspergillus, milk-clotting enzymes, liquid fermentation, solid fermentation.

A new milk-clotting enzyme produced by Bacillus sp. P45 applied in cream cheese development

The growing demand for natural coagulants led to an increased necessity for rennet substitutes, promoting a search for new sources of proteases with coagulant properties. The aim of this study was to investigate the application of a bacterial enzyme as a novel milk-clotting protease in the development of cream cheese enriched with chia and quinoa flour. At the concentration of 30 mg/mL, the milk-clotting strength was similar to that observed for commercial chymosin, demonstrating the enzyme ability to catalyze the hydrolysis of milk casein. The cheese developed showed high water retention (≥99.0%) and consequently low syneresis process. The results indicate that the product made using the enzyme showed adequate sanitary conditions and technological characteristics indicated that the product is highly stable and viable.

OPTIMIZATION OF MILK-CLOTTING PROTEASE PRODUCTION BY A LOCAL ISOLATE OF ASPERGILLUS NIGER FFB1 IN SOLID-STATE FERMENTATION

The need to surmount the limitation of obtaining rennin, has been actively pushed researches to find new substitutes that present high milk-clotting activity which enables the production of high yields of cheese. In this study, the production of extracellular milk-clotting protease by locally isolated fungal specie, Aspergillus niger FFB1 under solid-state fermentation (SSF) using cheep agro-industrial byproduct (wheat bran) was optimized. The effects of several physicochemical and environmental factors were investigated to select the optimal conditions that ensure the best milk-clotting activity by application of "One-factor-at-a-time" method. A trial of cheese production using the crude extract was also carried out. The maximum enzyme activity (830 SU/g bran with a ratio MCA/PA of 4.25) was obtained under the optimum conditions of temperature (30°C), spores concentration (106 spores/mL), incubation time (72 hours), and moisture content of solid substrate (39.2%) adjusted suitably with mineral solution (Czapek-Dox) of pH 4.

Production and Characterization of a Milk-clotting Protease Produced in Submerged Fermentation by the Thermophilic Fungus Thermomucor indicae-seudaticae N31

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.

Use of a new milk-clotting protease from Thermomucor indicae-seudaticae N31 as coagulant and changes during ripening of Prato cheese

Prato cheeses were manufactured using coagulant from Thermomucor indicae-seudaticae N31 or a commercial coagulant. Cheeses were characterised using the following analysis: yield; fat; acidity; moisture; ash; salt; pH; total nitrogen; total protein; NS-pH 4.6/NT*100; NS-TCA 12%/NT*100; casein electrophoresis; and RP-HPLC. The results were statistically analysed and revealed that the proteolytic indices were not significantly different throughout the 60days of ripening of cheeses made with either coagulant. Even though there were some quantitative differences in the peptide profile of cheeses, the enzyme from T. indicae-seudaticae N31 was used in the production of good quality Prato cheese without having to change the established technological parameters of the process.

Production of milk clotting protease by a local isolate of Mucor circinelloides under SSF using agro-industrial wastes

Agro-industrial residues, a cheap source of energy have high potential in the area of fermentation for the production of enzymes. Twenty agro-industrial residues were evaluated to check the possibility of potential utilization of substrates in SSF for milk clotting enzyme protease production by Mucor circinelloides. In this study, dhal husk holds the greatest promise for cost effective production of the milk clotting enzyme. The dhal husk supported maximum milk clotting protease production, and yield was improved with the supplementation of sucrose and yeast extract as carbon and nitrogen source, respectively. Among all the physico-chemical parameters tested, the best results were obtained in a medium having moisture content of 20% at pH 7.0, when inoculated with 30% of spore suspension and incubated at 30°C for 5 days. The activity was increased further on addition of Ca2+, Cu2+, and Mg2+ ions. The purified milk-clotting protease obtained from M. circinelloides was successfully applied and compared with commercial rennet in the manufacture of a cheddar cheese.

Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31 : (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

Microbial rennet-like milk-clotting enzymes are aspartic proteinases that catalyze milk coagulation, substituting calf rennet. Crude enzymatic extract produced by the thermophilic fungus, Thermomucor indicae-seudaticae N31, on solid state fermentation (SSF) using wheat bran, exhibited high milk-clotting activity and low proteolytic activity after 24 h of fermentation. Highest milk-clotting activity (MCA) was at pH 5.7, at 70 °C and in 0.04 M CaCl 2; it was stable in the pH range 3.5–4.5 for 24 h and up to 45 °C for 1 h. MCA was highly inhibited by pepstatin A. Hydrolytic activity profile of the crude enzymatic extract on whole bovine casein, analyzed by gel electrophoresis (Urea–PAGE) and RP-HPLC revealed low proteolytic action towards casein fractions and a peptide profile similar to the one obtained with commercial Rhizomucor miehei protease (Hannilase).

Production of Milk-Clotting Protease from Bacillus subtilis

An indigenous Bacillus subtilis strain isolated from soil was found to be a potent milk-clotting protease (mcp) producer. Production optimized using response surface methodology (RSM) yielded 1,190 U/ml of enzyme in medium containing 6% fructose, 1% casein, 0.3% NH4NO3, 10 mM CaCl2, pH 6.0 and inoculated with 3% inoculum and incubated at 250 rpm for 72 h. Solid-state fermentation resulted in 1,080 and 952.3 U/gds of milk-clotting protease using soybean meal and rice bran, respectively, with higher proteolytic values of 18.97 and 9.1 IU/gds. Production in a biphasic system using an overlay of RSM-optimized medium on solid layer of 6% fructose and 1% casein with 1.5% agar resulted in significant enzyme production. Maximum mcp was obtained using a biphasic system where solid: liquid ratio of 3.0 resulted in a final yield of 1,276.65 U/ml with a yield index of 1.80 as compared to static liquid culture. However, significant increase or difference was noted as compared to yield obtained after RSM. This is the first report on the use of RSM for production of mcp from a bacterial species.

Role of casein on induction and enhancement of production of a bacterial milk clotting protease from an indigenously isolated Bacillus subtilis

To isolate and enhance the yield of a bacterial milk clotting protease (MCP) through process optimization and scale up. Bacillus subtilis was isolated as MCP producer with good milk clotting activity (MCA) per proteolytic activity (PA) index. The enzyme production was inducible with casein and enhanced with fructose and ammonium nitrate resulting in 571.43 U ml(-1) of enzyme. Medium containing 4% fructose, 0.75% casein, 0.3% NH(4)NO(3) and 10 mmol l(-1) CaCl(2), pH 6.0, inoculated with 4% (v/v) inoculum, incubated at 37 degrees C, 200 rev min(-1) for 72 h gave maximum production. A 6.67-fold increase in MCP yield with very high MCA per PA index was observed after final optimization indicating similarity to rennets. Mostly fungal MCPs have been reported. The MCA and MCA per PA index of this bacterium is comparable to that of many fungal reports and better than quite a few bacterial MCPs. Thus, this enzyme by B. subtilis has good probability of successful use in cheese production.

Purification and characterization of a milk-clotting protease from Bacillus licheniformis strain USC13

The study of a milk-clotting protease secreted by Bacillus licheniformis strain USC13. Growth of B. licheniformis USC13 in LB medium resulted in the production of a serine protease with a molecular weight of 62 kDa processed to its mature form of 34 kDa, both forms were found in the extracellular medium. The enzyme exhibited typical milk-clotting kinetics. The capacity of this protease to produce milk curds could make it useful as a new source of milk coagulants. Cheese-making industry seeks for novel enzyme sources, and microbial coagulants have several advantages over animal and plant counterparts. The protease from B. licheniformis has the ability to produce milk curds although more studies about quality of both the enzyme and the milk curds formed should be carried out in the future to confirm its usefulness in the dairy industry.

Isolation and Partial Characterisation of Milk-clotting Aspartic Protease from Streblus asper

A rennin-like milk-clotting protease from the twigs of Streblus asper was purified by a factor of 65 times with 36% recovery using ethanol precipitation, ion-exchange and size-exclusion chromatographic techniques. The enzyme was found to be monomeric in nature having a molecular mass of 55kDa. The enzyme acts optimally at 55°C and was stable in the temperature range of 30–40°C. Easy enzyme inactivation by moderate heating, makes this protease extract potentially useful for cheese production. The purified enzyme is an acid protease with an optimum pH of 5.5 and it retained 96% of its residual activity between pH 5.0 and 6.0. Pepstatin A inhibited the proteinase activity, whereas iodoacetamide, phenylmethyl sulphonyl fluoride, β-mercaptoethanol and ethylenediaminetetraacetic acid had no significant inhibitory effect suggesting the presence of aspartic acid residue at the active site. The milkclotting aspartic protease showed predominant α-helical conformation in phosphate buffer as evidenced from circular dichroic spectroscopy.

Milk-clotting protease production by Nocardiopsis sp. in an inexpensive medium

The milk-clotting and proteolytic activities of extracellular enzyme preparations from Nocardiopsis sp. were investigated under different culture conditions. A soybean flour medium was used, with concentrations of soybean flour and of glucose varying from 0.25 to 1% w/v and from 0 to 1% w/v, respectively. Growth was monitored with 2 ml samples withdrawn from the culture medium at 8-h intervals, for determination of total protein, proteolytic activity, milk-clotting activity and sugar reduction. The best milk-clotting protease production, with a specific activity of 24.49 U/mg at 40 h, was obtained in the glucose-free medium containing soybean flour 1% w/v.

Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422.

The cheese industry is seeking novel sources of enzymes for cheese production. Microbial rennets have several advantages over animal rennets. (1) They are easy to generate and purify and do not rely on the availability of animal material. (2) The production of microbial clotting enzymes may be improved by biotechnological techniques. In this work, the biochemical characterization of a novel milk-clotting extracellular enzyme from Myxococcus xanthus strain 422 and a preliminary evaluation of its cheese-producing ability are reported. Strain 422 was selected from four M. xanthus strains as the best producer of extracellular milk-clotting activity, based on both its enzyme yield and specific milk-clotting activity, which also afforded lower titration values than enzymes from the three other M. xanthus strains. The active milk-clotting enzyme from M. xanthus strain 422 is a true milk-clotting enzyme with a molecular mass of 40 kDa and a pI of 5.0. Highest milk-clotting activity was at pH 6 and 37 degrees C. The enzyme was completely inactivated by heating for 12 min at 65 degrees C. The crude enzyme preparation was resolved by anion-exchange chromatography into two active fractions that were tested in cheese production assays of compositional (dry matter, fat content, fat content/dry-matter ratio, and moisture-non-fat content) and physicochemical properties (firmness, tensile strength, pH and Aw) of the milk curds obtained. Purified protein fraction II exhibited a significantly higher milk-clotting ability than either protein fraction I or a total protein extract, underlining the potential usefulness of M. xanthus strain 422 as a source of rennet for cheese production.