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Jesper Langholm Jensen - One of the best experts on this subject based on the ideXlab platform.
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the function of the milk clotting enzymes bovine and camel Chymosin studied by a fluorescence resonance energy transfer assay1
Journal of Dairy Science, 2015Co-Authors: K B Qvist, Jesper Langholm Jensen, Jonas Jacobsen, Marcia L Moss, Fred H Rasmussen, Sine Larsen, Johannes Maarten Van Den BrinkAbstract:Abstract Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel Chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study Chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel Chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine Chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98–108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine Chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 m M ). The results obtained show that the catalytic efficiency of camel Chymosin is 3-fold higher than bovine Chymosin. The substrate affinity and catalytic activity of bovine and camel Chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel Chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel Chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method.
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camel and bovine Chymosin the relationship between their structures and cheese making properties
Acta Crystallographica Section D-biological Crystallography, 2013Co-Authors: Jesper Langholm Jensen, Anne Molgaard, J C N Poulsen, Marianne Kirsten Harboe, Jens B Simonsen, Andrea Lorentzen, Karin Hjerno, Johannes Maarten Van Den Brink, Karsten B QvistAbstract:Bovine and camel Chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel Chymosin shows a 70% higher milk-clotting activity than bovine Chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel Chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N-terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel Chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel Chymosin compared with bovine Chymosin. The crystal structure of a doubly glycosylated variant of camel Chymosin was determined at a resolution of 1.6 A and the crystal structure of unglycosylated bovine Chymosin was redetermined at a slightly higher resolution (1.8 A) than previously determined structures. Camel and bovine Chymosin share the same overall fold, except for the antiparallel central β-sheet that connects the N-terminal and C-terminal domains. In bovine Chymosin the N-terminus forms one of the strands which is lacking in camel Chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids delineating the substrate-binding cleft suggest a greater flexibility in the ability to accommodate the substrate in camel Chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel Chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel Chymosin towards bovine milk.
V K Batish - One of the best experts on this subject based on the ideXlab platform.
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expression of buffalo Chymosin in pichia pastoris for application in mozzarella cheese
Lwt - Food Science and Technology, 2017Co-Authors: Ashish Tyagi, Ashwani Kumar, Sunita Grover, Ashok Kumar Mohanty, Jai K Kaushik, V K BatishAbstract:Abstract Chymosin, a major component of rennet, is used extensively as a milk clotting enzyme in cheese making industry. Increased demand coupled with scarce availability of native animal rennet has encouraged use of recombinant Chymosin (rChymosin) in the dairy industry. Better stability of buffalo Chymosin as compared to its other sources such as lamb, calf etc. makes it a desirable source for recombinant enzyme production. In the present investigation, we report the inducible expression of recombinant buffalo proChymosin in P. pastoris, its chromatographic purification and mozzarella cheese preparation. RNA extracted from the abomasum of a suckling calf buffalo was used to synthesize proChymosin cDNA and expressed in P. pastoris X-33 as well as SMD1168. We could observe 68% higher expression of recombinant buffalo Chymosin in X-33 in comparison to SMD1168 strain. The enzyme was subsequently purified to approximately 28 fold. The new recombinant yeast strain could represent a novel and excellent source of high level expression of Chymosin for application in cheese making industry.
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Chymosin and other milk coagulants sources and biotechnological interventions
Critical Reviews in Biotechnology, 2010Co-Authors: Anil Kumar, Jitender Sharma, Sunita Grover, V K BatishAbstract:Calf rennet, which consists of over 90% Chymosin, is commonly used in cheese industries for the curdling of milk. Various animal, plant and microbial sources have been exploited as possible alternatives to calf rennet. The coagulating properties of the enzymatic preparations (coagulants) from these sources differ in terms of their physicochemical factors. The cheese industry has always sought out novel and stable enzyme sources, and recombinant Chymosin has been found to be an effective alternative since it possesses several advantages over plant and microbial milk-clotting enzymes. This paper reviews the use of various milk coagulants, especially animal coagulants, for cheese making. Advancements in genetic and protein engineering to produce recombinant Chymosin are discussed in addition to evaluating its identity to the rennet available from natural sources.
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purification and characterization of milk clotting enzyme from goat capra hircus
Comparative Biochemistry and Physiology B, 2006Co-Authors: Ashwani Kumar, Jitender Sharma, A K Mohanty, Sunita Grover, V K BatishAbstract:Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor proChymosin. It is responsible for hydrolysis of κ-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat (Capra hircus) Chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat Chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 °C with maximum activity at 30 °C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat Chymosin increased with incubation time at 37 °C. Goat Chymosin was found to be more thermostable than cattle Chymosin and equally stable to buffalo Chymosin.
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isolation purification and characterization of Chymosin from riverine buffalo bubalos bubalis
Journal of Dairy Research, 2003Co-Authors: Ashok Kumar Mohanty, Sunita Grover, Jai K Kaushik, Utpal Kumar Mukhopadhyay, V K BatishAbstract:Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of Chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo Chymosin was identical to the first eight amino acid sequences of cattle Chymosin. Buffalo Chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle Chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo Chymosin.
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bovine Chymosin production by rdna technology and application in cheese manufacture
Biotechnology Advances, 1999Co-Authors: Ashok Kumar Mohanty, Sunita Grover, Utpal Kumar Mukhopadhyay, V K BatishAbstract:Bovine Chymosin, an aspartyl protease extracted from abomasum of suckling calves, is synthesized in vivo as preproChymosin and secreted as proChymosin which is autocatalytically activated to Chymosin. Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues. Chymosin A and Chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving kappa-casein between Phe 105 and Met 106. The gene encoding Chymosin has been cloned and expressed in suitable bacteria and yeast hosts under the control of lac, trp, trp-beta, gly A genes, and serine hydroxymethyl-transferase promoters. Protein engineering of Chymosin has also been attempted. A number of companies are now producing recombinant Chymosin for commercial use in cheese manufacture.
David A. Long - One of the best experts on this subject based on the ideXlab platform.
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Loss of endogenous thymosin β4 accelerates glomerular disease
Kidney international, 2016Co-Authors: Elisavet Vasilopoulou, Maria Kolatsi-joannou, Maja T. Lindenmeyer, Kathryn White, Michael G. Robson, Clemens D. Cohen, Neil J. Sebire, Paul R. Riley, Paul J.d. Winyard, David A. LongAbstract:Glomerular disease is characterized by morphologic changes in podocyte cells accompanied by inflammation and fibrosis. Thymosin β4 regulates cell morphology, inflammation, and fibrosis in several organs and administration of exogenous thymosin β4 improves animal models of unilateral ureteral obstruction and diabetic nephropathy. However, the role of endogenous thymosin β4 in the kidney is unknown. We demonstrate that thymosin β4 is expressed prominently in podocytes of developing and adult mouse glomeruli. Global loss of thymosin β4 did not affect healthy glomeruli, but accelerated the severity of immune-mediated nephrotoxic nephritis with worse renal function, periglomerular inflammation, and fibrosis. Lack of thymosin β4 in nephrotoxic nephritis led to the redistribution of podocytes from the glomerular tuft toward the Bowman capsule suggesting a role for thymosin β4 in the migration of these cells. Thymosin β4 knockdown in cultured podocytes also increased migration in a wound-healing assay, accompanied by F-actin rearrangement and increased RhoA activity. We propose that endogenous thymosin β4 is a modifier of glomerular injury, likely having a protective role acting as a brake to slow disease progression.
Birgit Schiott - One of the best experts on this subject based on the ideXlab platform.
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solvent binding analysis and computational alanine scanning of the bovine Chymosin bovine κ casein complex using molecular integral equation theory
Journal of Chemical Theory and Computation, 2013Co-Authors: David S Palmer, Birgit Schiott, Jesper Sorensen, Maxim V FedorovAbstract:We demonstrate that the relative binding thermodynamics of single-point mutants of a model protein-peptide complex (the bovine Chymosin-bovine κ-casein complex) can be calculated accurately and efficiently using molecular integral equation theory. The results are shown to be in good overall agreement with those obtained using implicit continuum solvation models. Unlike the implicit continuum models, however, molecular integral equation theory provides useful information about the distribution of solvent density. We find that experimentally observed water-binding sites on the surface of bovine Chymosin can be identified quickly and accurately from the density distribution functions computed by molecular integral equation theory. The bovine Chymosin-bovine κ-casein complex is of industrial interest because bovine Chymosin is widely used to cleave bovine κ-casein and to initiate milk clotting in the manufacturing of processed dairy products. The results are interpreted in light of the recent discovery that camel Chymosin is a more efficient clotting agent than bovine Chymosin for bovine milk.
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hot spot mapping of the interactions between Chymosin and bovine κ casein
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Jesper Givskov Sorensen, David S Palmer, Birgit SchiottAbstract:Chymosin is a commercially important enzyme in the manufacturing of cheese. Chymosin cleaves the milk protein κ-casein, which initiates the clotting process. Recently, it has been shown that camel Chymosin has superior enzymatic properties toward cow's milk, compared to bovine Chymosin. The two enzymes possess a high degree of homology. There are only minor differences in the binding cleft; hence, these must be important for binding the substrate. Models for the binding of a 16 amino acid fragment, consisting of the Chymosin-sensitive region of bovine κ-casein (97-112), to both enzymes have previously been presented. Computational alanine scanning for mutating 39 residues in the substrate and the bovine enzyme are presented herein, and warm- (ΔΔG > 1 kcal/mol) and hot-spot (ΔΔG > 2 kcal/mol) residues in the bovine enzyme are identified. These residues are relevant for site-directed mutagenesis, with the aim of modifying the binding affinity and in turn affecting the catalytic efficacy of the enzyme.
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initial stage of cheese production a molecular modeling study of bovine and camel Chymosin complexed with peptides from the Chymosin sensitive region of κ casein
Journal of Agricultural and Food Chemistry, 2011Co-Authors: Jesper Givskov Sorensen, K B Qvist, David S Palmer, Birgit SchiottAbstract:Bovine Chymosin has long been the preferred enzyme used to coagulate cow's milk, in the initial stage of cheese production, during which it cleaves a specific bond in the milk protein κ-casein. Recently, camel Chymosin has been shown to have a 70% higher clotting activity toward cow's milk and, moreover, to cleave κ-casein more selectively. Bovine Chymosin, on the other hand, is a poor clotting agent toward camel's milk. This paper reports a molecular modeling study aimed at understanding this disparity, based on homology modeling and molecular dynamics simulations using peptide fragments of κ-casein from cow and camel in both bovine and camel Chymosin. The results show that the complex between bovine Chymosin and the fragment of camel κ-casein is indeed less stable in the binding pocket. The results also indicate that this in part may be due to charge repulsion between a lysine residue in bovine Chymosin and an arginine residue in the P4 position of camel κ-casein.
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molecular modelling studies of bovine and camel Chymosin κ casein complexes
Biophysical Journal, 2010Co-Authors: Jesper Givskov Sorensen, K B Qvist, David S Palmer, Anders U Christensen, Leyla Celik, Birgit SchiottAbstract:We present computational studies of two homologous mammalian aspartic proteases (calf and camel Chymosin) complexed with 16-residue fragments of their native peptide ligands (cow and camel κ-casein) and the cross-complexes. Using molecular docking calculations, homology modelling and molecular dynamics simulations, we compare the binding modes of the four systems. The complexes are of industrial interest because camel Chymosin has recently been marketed as an alternative to bovine Chymosin as an enzyme to clot milk in cheese manufacturing. The camel enzyme has been shown to have 70% higher clotting activity and only 20% of the unspecific protease activity for bovine κ-casein as compared to the bovine enzyme. Interestingly, bovine Chymosin has a very low proteolytic rate for camel κ-casein. The models provide putative atomic coordinates for these complexes, for which there are no available crystallographic or NMR structures, and help to explain some existing experimental results.View Large Image | View Hi-Res Image | Download PowerPoint Slide
Hynda K Kleinman - One of the best experts on this subject based on the ideXlab platform.
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over expression of thymosin beta4 promotes abnormal tooth development and stimulation of hair growth
The International Journal of Developmental Biology, 2010Co-Authors: Heejae Cha, Hynda K Kleinman, Deborah Philp, Soo Hyun Lee, Hye Sung Moon, Takashi NakamuraAbstract:Thymosin beta 4 has multi-functional roles in cell physiology. It accelerates wound healing, hair growth and angiogenesis, and increases laminin-5 expression in corneal epithelium. Furthermore, thymosin beta 4 stimulates tumor growth and metastasis by induction of cell migration and vascular endothelial growth factor-mediated angiogenesis. Using a construct on the skin-specific keratin-5 promoter, we have developed thymosin beta 4 over-expressing transgenic mice to further study its functional roles. Thymosin beta 4 in adult skin and in embryonic stages of the transgenic mouse was analyzed by both Western blot and immunohistochemistry. The over-expression of thymosin beta 4 was observed especially around hair follicles and in the teeth in the transgenic mice. We examined the phenotype of the thymosin beta 4 over-expressing mice. Hair growth was accelerated. In addition, the transgenic mice had abnormally-shaped white teeth and dull incisors. We found that the expression of laminin-5 was up-regulated in the skin of the transgenic mice. We conclude that thymosin beta 4 has an important physiological role in hair growth and in tooth development.
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matrigel induces thymosin beta 4 gene in differentiating endothelial cells
Journal of Cell Science, 1995Co-Authors: Derrick S Grant, Maura C. Kibbey, A L Goldstein, James L Kinsella, S Laflamme, P D Burbelo, Hynda K KleinmanAbstract:We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillary-like tubes. The mRNA for one clone, thymosin beta 4, was increased 5-fold. Immunostaining localized thymosin beta 4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin beta 4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin beta 4 inhibited tube formation on Matrigel. The results suggest that thymosin beta 4 is induced and likely involved in differentiating endothelial cells. Thymosin beta 4 may play a role in vessel formation in vivo.