Academic literature on the topic 'Microbial clotting enzymes'

Milk-clotting enzymes are the primary active agents in the manufacture cheeses. Animal rennet, microbial coagulant and plant coagulant are used as milk coagulants in cheese making. However, alternative milk coagulants are investigated instead of animal enzymes due to slaughtering of young ruminant. Manufacturing of milk clotting enzyme in farmhouse have been employed successfully for many centuries in Mediterrenean region and Toros mountain villages of Turkey for the production of traditional Tulum cheese. Figs, raisins, white beans, chickpeas, carob, granulated sugar, salt, yoghurt and home-made rennet (sarkanak) are found in the content of this enzyme. This mixture is left at room temperature for 5-6 days. The enzyme is filtered from using cloth bag and added into milk for coagulation. In this research; chemical composition of cow’s milk, goat’s milk and ewe’s milk were determined and obtained enzymes from different manufacturers were investigated of clotting effects on cow’s milk, goat’s milk and ewe’s milk. Four different coagulants had a strong coagulating effect on raw and pasteurized ewe's milk. The highest milk clotting activity of all coagulant samples were seen in ewe's milk, followed by cow's milk and goat's milk.

Milk-clotting enzymes are used during the production of cheese to coagulate the casein, allowing the formation of a three-dimensional network that entraps the milk fat. Commercially available milk-clotting enzymes differ with respect to source, specificity, optimum pH and thermostability. All are acid proteinases that can cleave κ-casein resulting in the coagulation of milk. Chymosin (EC 3.4.23.4) is specific for the Phe–Met bond in κ-casein at the natural pH of milk (6·7). Recombinant chymosin is available commercially from a variety of sources and has a maximum activity at 40 °C. Recombinant chymosins are purified from the fermentation of recombinant strains of Aspergillus niger, Asp. oryzae or Kluyveromyces marxianus. These enzyme preparations are chemically and functionally identical to calf chymosin. Rennets are purified from the abomasum of bovines and can contain from 60 to 100% chymosin with the remainder being primarily bovine pepsin (Wigley, 1996). Microbial proteinases (EC 3.4.23.6) are generally more proteolytic than chymosin, with varying heat stability. These enzymes liberate more non-protein N from casein and can cleave α- and β-casein as well as κ-casein at the natural pH of milk. Acid proteinases from Cryphonectria parasitica are more heat labile than those from Rhizomucor miehei, which are characterized as thermostable (Ernstrom & Wong, 1974).The objective of this research was to characterize milk-clotting enzymes with respect to thermal inactivation in skim milk. This information has applications in milk and whey processing.

Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total global enzymes sale. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases are classified in enzymes of class 3, the hydrolases, and the subclass 3.4, the peptide hydrolases or peptidase. Proteases are generally grouped into two main classes based on their site of action, that is, exopeptidases and endopeptidases. Protease has also been grouped into four classes based on their catalytic action: aspartic, cysteine, metallo, and serine proteases. However, lately, three new systems have been defined: the threonine-based proteasome system, the glutamate-glutamine system of eqolisin, and the serine-glutamate-aspartate system of sedolisin. Aspartic proteases (EC 3.4.23) are peptidases that display various activities and specificities. It has two aspartic acid residues (Asp32 and Asp215) within their active site which are useful for their catalytic activity. Most of the aspartic proteases display best enzyme activity at low pH (pH 3 to 4) and have isoelectric points in the pH range of 3 to 4.5. They are inhibited by pepstatin. The failure of the plant and animal proteases to meet the present global enzyme demand has directed to an increasing interest in microbial proteases. Microbial proteases are preferred over plant protease because they have most of the characteristics required for their biotechnological applications. Aspartic proteases are found in molds and yeasts but rarely in bacteria. Aspartic protease enzymes from microbial sources are mainly categorized into two groups: (i) the pepsin-like enzymes produced byAspergillus,Penicillium,Rhizopus, andNeurosporaand (ii) the rennin-like enzymes produced byEndothiaandMucorspp., such asMucor miehei,M. pusillus, andEndothia parasitica. Aspartic proteases of microbial origin have a wide range of application in food and beverage industries. These include as milk-clotting enzyme for cheese manufacturing, degradation of protein turbidity complex in fruit juices and alcoholic liquors, and modifying wheat gluten in bread by proteolysis.

The bacterium Porphyromonas gingivalis is a major etiologic agent in the pathogenesis of adult periodontitis in humans. Cysteine proteinases produced by this pathogen, termed gingipains, are considered to be important virulence factors. Among many other potentially deleterious activities, arginine-specific gingipains-R (RgpB and HRgpA) efficiently activate coagulation factors. To further expand knowledge of the interaction between gingipains and the clotting cascade, this study examined their effects on cellular components of the coagulation system. The enzymes induced an increase in intracellular calcium in human platelets at nanomolar concentrations and caused platelet aggregation with efficiency comparable to thrombin. Both effects were dependent on the proteolytic activity of the enzymes. Based on desensitization studies carried out with thrombin and peptide receptor agonists, and immunoinhibition experiments, gingipains-R appeared to be activating the protease-activated receptors, (PAR)-1 and -4, expressed on the surface of platelets. This was confirmed by the finding that HRgpA and RgpB potently activated PAR-1 and PAR-4 in transfected cells stably expressing these receptors. Cumulatively, the results indicate the existence of a novel pathway of host cell activation by bacterial proteinases through PAR cleavage. This mechanism not only represents a new trait in bacterial pathogenicity, but may also explain an emerging link between periodontitis and cardiovascular disease.

Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genusPleurotushas been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases ofPleurotusspp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine.

The toxic free radical NO (nitric oxide) has diverse biological roles in eukaryotes and bacteria, being involved in signalling, vasodilation, blood clotting and immunity, and as an intermediate in microbial denitrification. The predominant biological mechanism of detecting NO is through the formation of iron nitrosyl complexes, although this is a deleterious process for other iron-containing enzymes. We have previously applied techniques such as UV–visible and EPR spectroscopy to the analysis of protein Fe–NO complex formation in order to study how NO controls the activity of the bacterial transcriptional regulators NorR and NsrR. These studies have analysed NO-dependent biological activity both in vitro and in vivo using diverse biochemical, molecular and spectroscopic methods. Recently, we have applied ultrafast 2D-IR (two-dimensional IR) spectroscopy to the analysis of NO–protein interactions using Mb (myoglobin) and Cc (cytochrome c) as model haem proteins. The ultrafast fluctuations of Cc and Mb show marked differences, indicating altered flexibility of the haem pockets. We have extended this analysis to bacterial catalase enzymes that are known to play a role in the nitrosative stress response by detoxifying peroxynitrite. The first 2D-IR analysis of haem nitrosylation and perspectives for the future are discussed.

This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (<300 000 cells/ml), SC500 (from 301 000 to 500 000 cells/ml), SC1000 (from 501 000 to 1 000 000 cells/ml), SC2000 (from 1 001 000 to 2 000 000 cells/ml) and SC>2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.

We tested the hypothesis that hemolymph contains alarm substances in the crayfish Procambarus clarkii (Girard, 1852) and collected preliminary information on their chemical nature in this species. We analyzed crayfish responses in the presence of different test solutions in four experiments. The crayfish displayed an alerted behavior (i.e., feeding and locomotion were inhibited) in the presence of solutions containing different concentrations of hemolymph combined with food odor. However, hemolymph lost its bioactivity when tested 24 h after its extraction but maintained its ability to elicit alerted responses when diluted in a solution containing L-ascorbic acid. This may suggest that crayfish alarm molecules are degraded with time by oxidation. Microbial activity did not lead to the degradation of alarm substances, since hemolymph activity still declined after 24 h even if extracted and preserved in sterile conditions. Hemolymph molecules less than 5 kDa fractionated from hemolymph showed a strong bioactivity and were still bioactive after 24 h at 20 °C. As the 5 kDa fractioning eliminates all enzymes, we hypothesize that alarm substances are degraded through enzymatic reactions. Finally, we propose that alarm substances are peptides involved in the hemolymph clotting process.

<p><strong>Título en ingles: Evaluation of experimental production of milk-clotting enzymes using <em>Rhizomucor</em> spp<em> </em>strains</strong><strong></strong><strong></strong></p><p><strong>Título corto: Producción de enzimas coagulantes de leche</strong></p><p><strong>Resumen</strong>: La producción experimental de enzimas coagulante de leche se llevó a cabo<em> </em>en un medio de cultivo de laboratorio durante 190 h de incubación, utilizando tres cepas certificadas de <em>Rhizomucor pusillus, </em> <em>R.</em> <em>miehei</em> y dos especies nativas de <em>Rhizomucor spp. </em>BIOMI-12 y 13. La evaluación se realizó midiendo la concentración de glucosa y proteína durante la incubación, estimación de la productividad, actividad específica, índice fuerza de cuajo/actividad proteolítica en los extractos enzimáticos crudos, determinación de los pesos moleculares y actividad proteolítica en los extractos enzimáticos parcialmente purificado. Todas las cepas mostraron un consumo de glucosa similar, el mismo comportamiento se observó en el contenido de proteína, excepto la cepa BIOM-13. Los incrementos en el contenido de proteínas después del descenso, coincidieron con la máxima actividad coagulante registrada por cada cepa, siendo el extracto crudo de la cepa BIOMI-13 la de mayor actividad coagulante (148,15 FC), productividad (3,09 FC/h), índice fuerza de cuajo/actividad proteolítica (142,60 FC/U) y actividad específica (1.062,00 FC/mg). Los extractos enzimáticos parcialmente purificados de las cepas <em>R miehei</em> 37, <em>Rhizomucor spp</em> BIOM-12 y 13, presentaron proteínas con pesos moleculares en aproximadamente 22,6 y 46,52 KDa, mientras el extracto <em>R pusillus</em> 39 presentó una banda adicional de 39,6 KDa. En el zimograma se observó para todas las cepas actividad proteolítica en las bandas comprendidas entre 40-50 KDa y 20-22 KDa, no así para el <em>R pusillus</em> 36, donde fue escasa. Finalmente se determinó que la cepa<strong> </strong>BIOMI-13, tiene la mayor capacidad para producir enzimas coagulantes de la leche.</p><p><strong>Palabras clave: </strong>renina microbiana, fuerza de<strong> </strong>cuajo, actividad proteolítica<em>, </em>productividad.</p><p><strong>Abstract: </strong>Experimental production of milk clotting enzymes was conducted on a laboratory culture medium for 190 h incubation, using three certified strains of <em>Rhizomucor pusillus</em>, <em>miehei </em>and two<em> </em>native <em>Rhizomucor </em>spp. BIOMI-12 and 13. The evaluation was performed by measuring the concentration of glucose and protein during incubation, estimate productivity, specific activity, rennet strength/proteolytic activity index in the crude enzyme extracts, determining the molecular weights and proteolytic activity in the partially purified enzyme extracts. All strains showed consumption rates of glucose, the same behavior observed protein content, except strain BIOM-13. The increase in protein content after descent coincided with the recorded maximum coagulant activity each strains, being the crude extract of strain BIOMI-13 higher coagulant activity (148,15 FC), productivity (3.09 HR / h), rennet strength/proteolytic activity index (142,60 FC/U) and specific activity (1,062 FC/mg). The partially purified enzyme extracts from strains <em>R miehei</em> 37, <em>Rhizomucor </em>spp BIOM-12 and 13, presented proteins with molecular weights in approximately 22,6 kDa and 46.52, while the extract <em>R pusillus</em> 39 present an additional band of 39,6 KDa. In the zymogram was observed for all strains, proteolytic activity in the bands between 40-50 KDa and 20-22 KDa, but not for the <em>R pusillus</em> 36, where activity was very dim. Finally it was determined that the strain BIOMI-13, has the greatest capacity to produce milk clotting enzymes.</p><p><strong>Key words</strong>: microbial rennet, rennet strength, proteolitic activity,<strong> </strong>productivity.</p><p><strong>Recibido: </strong>mayo 14 de 2014<strong> Aprobado: </strong>abril 21 de 2015</p>

Die Dicklegung der Milch, ausgehend von der hydrolytischen Spaltung des κ-Caseins, stellt den ersten essentiellen Schritt in der Käseherstellung dar. Dabei finden Gerinnungsenzyme verschiedener Herkunft Anwendung, deren neueste Varianten auf Grundlage des aktuellen Forschungsstandes umfassend charakterisiert werden. Verschiedene Kälberlabpräparate, mikrobielle Gerinnungsenzyme aus Rhizomucor miehei und mit Hilfe von gentechnisch modifizierten Mikroorganismen gewonnenes Chymosin (FPC) aus Rind und Kamel werden mittels HPLC und Elektrophorese hinsichtlich ihrer Zusammensetzung analysiert. Die neueste Generation mikrobieller Enzyme weist im Gegensatz zur herkömmlichen Variante keine Nebenkomponenten und damit einen höheren Aufreinigungsgrad auf. Die unspezifische proteolytische Aktivität wird durch fluorimetrische Quantifizierung der in 12 % TCA löslichen Stickstoffkomponenten bestimmt, die nach Inkubation rekonstituierter Magermilch bei 32 °C und pH 6,5 über 24 h mit den Enzymen freigesetzt werden. Mikrobielle Gerinnungsenzyme besitzen eine signifikant höhere unspezifische proteolytische Aktivität gegenüber chymosinbasierten Präparaten, deren Auswirkung sich bei Erhöhung der zugegebenen Enzymmenge besonders ausgeprägt darstellen. Oszillationsrheometrische Analysen lassen bei gleicher Enzymaktivität eine geringere Gelfestigkeit nach 40 min bei Einsatz von mikrobiellen Präparaten im Vergleich zu Kälberlab und bovinem FPC erkennen. Zusätzlich wird eine Abhängigkeit der Flockungszeit und der Gelfestigkeit vom eingesetzten Substrat beobachtet, die für Chymosin aus Kamel besonders stark ausgeprägt ist. Die Substratspezifität dieses Enzyms ist weder mit der des bovinen Chymosins noch mit der der mikrobiellen Gerinnungsenzyme identisch. Im Rahmen von Käsungsversuchen im Labor-, Pilot- und Industriemaßstab wird eine signifikant höhere Käseausbeute (0,50 - 1,19 %) bei Verwendung vom traditionellem Kälberlab im Vergleich zur neuesten Generation der kommerziellen mikrobiellen Substitute ermittelt. Im Verlaufe der Reifung von Schnittkäse wird durch mikrobielles Gerinnungsenzym gegenüber Kälberlab eine signifikant höhere Menge an Nichtproteinstickstoff freigesetzt sowie ein unterschiedliches Profil an Proteinabbauprodukten gebildet. Die höhere proteolytische Aktivität resultiert in einer signifikant stärker ausgeprägten Bitterkeit der mit mikrobiellem Gerinnungsenzym hergestellten Käse nach 12 Wochen Reifungszeit
Clotting of milk caused by hydrolytic cleavage of κ-casein is the first important step in cheese milk processing. This cleavage is caused by clotting enzymes of different origin, which are comprehensively characterized by considering results of latest investigations. The composition of selected calf rennets, microbial coagulants derived from Rhizomucor miehei and genetically engineered chymosin (FPC) derived from cow and camel is analyzed by HPLC and electrophoresis. In contrast to conventional products, the latest generation of microbial coagulants does not show minor components in a detectable amount because of a sufficient purification. The unspecific proteolytic activity is determined by fluorimetric quantification of 12 % tricloric-acid-soluble nitrogen, which is released by the enzymes from reconstituted skim milk, pH 6.5, after incubation at 32 °C for 24 h. Microbial coagulants show a significantly higher unspecific proteolysis as compared to chymosin-based clotting enzymes, especially when the enzymes are added in amount higher than used during cheese-making. Small amplitude oscillation rheometry analysis showed a lower gel firmness after 40 min of gelling when microbial coagulants were applied instead of calf rennet or FPC. Furthermore, flocculation time, gel formation time and gel firmness additionally depends on the test substrate, and this dependency is exceptionally pronounced when camel chymosin was used. The substrate specificity of this enzyme is neither identical to that of bovine chymosin nor to that of microbial coagulants. Cheese making experiments in laboratory-, pilot- and commercial-scale revealed a significantly higher cheese yield (0.50 - 1.19 %) when using calf rennet instead of microbial coagulant of the latest generation. During ripening of semi-hard cheese a higher amount of non-protein-nitrogen was released and a different electrophoretic casein degradation profile was generated when using microbial enzymes. Enhanced proteolysis is responsible for a significantly higher pronounced bitterness of microbial produced cheese after 12 weeks of maturation

Commercial microbial milk clotting enzyme preparations were purified by immunoaffinity chromatography using purified antibody covalently coupled to porous glass beads as the column matrix. Commercial enzyme preparation diluted in 1 mM sodium acetate buffer at pH 5.0 was then biospecifically adsorbed to the column matrix by end-over-end mixing of the glass-antibody complex in the enzyme solution for 12 h at 5°C. The antibody bound enzyme adsorbed glass beads were soaked in .2 M glycine or ethanolarnine at pH 7.0 to block uncoupled reactive sites on the matrix. Following this, the column was washed with 1 mM sodium acetate buffer at pH 7 .0, followed by additional wash with .5 M NaCl, until absorbance at 280 nm returned to baseline. Elution of adsorbed enzyme was achieved with .2 M sodium acetate at pH 3.0, .2 M acetate at pH 3.5, containing .15 M NaCl and .5 M acetate at pH 4.0 containing .5 M NaCL At the same protein concentration, immunoaffinity chromatography purified enzymes had higher clotting activity than the commercial enzyme preparations. Amino acid analysis and OPA proteolysis tests of TCA soluble peptides liberated from casein hydrolysis showed purified enzymes to exhibit lower general proteolytic activity. Immunological reactivity of Mucor enzymes with calf rennet was determined with antibodies produced by intramuscular injections of Mucor miehei protease, Mucor pusillus protease and calf rennet emulsified in Freund's adjuvant into three New Zealand White rabbits . Harvested antisera were heated at 56°C for 30 min to inactivate complement factors and contaminating proteins then centrifuged at 1700 x g for 30 min. Ouchterlony double immunodiffusion method was used to test for presence of antibodies in the antisera, and for cross immunoreactivity. Antibodies against M. miehei were cross reactive with M. pusillus antigen and M. pusillus antibodies cross reacted with M. miehei antigen. Immunodiffusion assay did not show cross reactivity of calf rennet antibodies with either M. miehei antigen or M. pusillus antigen. Antibodies against the Mucor enzymes did not show cross reactivity with calf rennet antigen. Although their actions in milk differ, proteolytic enzyme preparations from M. miehei and M. pusillus are both used as calf rennet substitutes in cheese manufacture. Differences in the characteristics of the two Mucor enzyme preparations exist, even though they exhibit some immunological homology . From our results, at least one antigenic factor is common to both enzyme preparations.