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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: .. Home.. Contact / Imprint.. Company.. Joint Venture.. Product Data Sheets.. Bromelain.. Chymotrypsin.. Pancreatin.. Papain.. Pepsin.. Trypsin.. Trypsin-Chymotrypsin.. Product Specifications.. Welcome.. BIOZYM`s object is production, wholesale and foreign trade of enzymes and biological products to be used as Active Pharmaceutical Ingredients (API) as well as Food Ingredients (FI).. BIOZYM holds a manufacturing and import permit according  ...   with the Principles of GMP for active substances referred to in Article 47 of Directive 2001/83/EC.. BIOZYM standardizes enzymes to defined activities upon customer's request.. Quality control, development of production processes and research on application of enzmes are further engagements.. BIOZYM Gesellschaft für Enzymtechnologie mbH Pinkertweg 50 D-22113 Hamburg Phone +49 (0)40 - 73 20 131..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Imprint.. BIOZYM Gesellschaft für Enzymtechnologie mbH.. Pinkertweg 50.. D-22113 Hamburg.. Phone +49 (0)40 - 73 20 131.. Fax +49 (0)40 - 73 20 134.. VAT Tax Identification Number in accordance with § 27 a VAT Tax Act.. : DE 118702669.. Register court.. : (district court): Hamburg.. Registered association.. : HRB 45240.. Managing Director.. : Rolf Peters.. Usage Rights.. Copyright © by BIOZYM.. All rights reserved.. Copyrights of this website are owned in full by BIOZYM.. It is hereby expressly permitted - in accordance with the following restrictions - to copy, print, and distribute these Web pages as well as the attached documents.. The Web pages and documents provided herein may only be used for informational purposes.. These Web pages and documents may not be used for commercial purposes.. Each copy (this also applies to excerpts) must include this proof of copyright.. Disclaimer.. Despite careful checking of contents when setting links to the contents  ...   that it is accurate and complete.. Nevertheless, the possibility of errors cannot be entirely ruled out.. BIOZYM does not give any warranty in respect of the timeliness, accuracy or completeness of material published on its website, and disclaims all liability for (material or non-material) loss or damage incurred by third parties arising from the use of content obtained from the website, except in such cases where it can be proven that misleading or incorrect information was posted with deliberate intention to harm or through gross negligence.. Registered trademarks and proprietary names, and copyrighted text and images, are not generally indicated as such on our webpages.. But the absence of such indications in no way implies that these names, images or text belong to the public domain in the context of trademark or copyright law.. Design and Onlinerealisation.. Karen Floss,.. www.. floss-design.. de.. Fotos.. Bernd Kusber,.. kusber.. Foto Rind.. :.. istockphoto.. com.. , Ingmar Wesemann..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: BIOZYM.. 1990.. Establishment of BIOZYM in Hamburg as associate company of DANIMEX in Hamburg.. 1991.. Completion of laboratory especially for quality control of pharmaceutical enzymes.. 1994.. Factory extension to increase capacity of enzyme standardization.. 1995.. BIOZYM becomes subsidiary company of DANIMEX in Hamburg by share transfer.. 1997.. Manufacturing permit according to German Drug Act (AMG) and GMP status complying with the principles of..  ...   and liabilities over from DANIMEX in Hamburg.. DANIMEX.. 1969.. Establishment of DANIMEX in Hamburg as subsidiary company of a Danish enterprise.. 1976.. German management accepts takeover bid from Danish shareholder.. 1984.. Construction of todays factory and office.. BIOZYM becomes subsidiary company of DANIMEX Hamburg by share transfer.. DANIMEX becomes a holding company having all shares, making the investments and superintends its subsidiary.. companies..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Deyang Sinozyme Pharmaceutical Co.. , Ltd.. was established as a Sino-German joint venture company in 2003.. Seat of the company is in the Industrial District of Xiaohan Town, Guanghan City, Sichuan Province, Peoples Republic of China.. Sinozyme’s joint venture partners are Deyang Biochemical Products, Deyang, the holding company of Biozym Gesellschaft für Enzymtechnologie mbH, Hamburg and Changzhou Qianhong Biochemical, Changzhou.. Sinozyme’s object is the manufacture of enzymes particularly of porcine origin which  ...   as Active Pharmaceutical Ingredients (API) and as Food Ingredients (FI).. Dovelopment of production processes of enzymes and research on application of enzymes are further engagements.. Sinozyme holds a manufacturing and export permit and complies with the principles of Good Manufacturing Practice (GMP) for active substances of the European Union (EU), the World Health Organization (WHO) and of the Pharmaceutical Inspection Convention (PIC).. Sinozyme has been inspected and certified by the German Health Authorities..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Data Sheet - Bromelain.. Source:.. Stem of pineapple (Ananas comosus (L.. ) MERR.. ).. Systematic name:.. Peptidyl peptide hydrolase; EC 3.. 4.. 22.. 32.. Other names:.. Bromelin, Ananase.. CAS-No.. 37189-34-7.. E.. C.. -No.. 3.. 32.. EG-Indexnummer.. 647-005-00-X.. Occurrence.. Bromelain is the collective name for proteolytic enzymes found in various members of the family Bromeliaceae.. Bromelain from the pineapple (Ananas comosus) is the most studied.. The highest enzyme concentration occurs in the lower portion of mature pineapple plant stems, appreciable quantities are also present in fruits and leaves.. Characteristics.. Specificity:.. Two forms (A and B) of Bromelain with similar specificity have been isolated from pineapple stem (1).. Bromelain hydrolyzes proteins, peptides, amides and esters of amino acids and peptides; preferential cleavage site is the carbonyl end of lysine, alanine, tyrosine and glycine (1,2).. For relative activities on a number of amino acid substrates see Barman (3).. The following side activities can be detected in Bromelain preparations: amylase, phosphatase, peroxidase, the latter being very labile upon storage.. Effectors:.. Bromelain is a thiol proteinase, i.. e.. it contains in the active centre a highly reactive cysteine which is essential for catalysis.. Therefore, the enzyme can be activated by reducing compounds, e.. g.. cysteine, 2-mercaptoethanol, dithiothreitol, KCN (4).. On the other hand, Bromelain is irreversibly inhibited by alkylating agents such as N-ethylmaleimide (NEM), iodoacetic acid and 1,3-dibromoacetone (4).. Reversible inhibition is caused by inorganic Hg ion, organic Hg compounds and tetrathionate (4).. Catalytic optima:.. The optimum pH for catalytic activity depends on the nature of substrate, type and concentration of buffer and the presence or absence of a reducing agent.. The optimum pH range is about 4.. 5-7.. 5.. The optimum temperature is 35-45 C (5), the maximum operating temperature for industrial application (reaction time 4h) is 50 C (2).. Stability:.. Bromelain is stable at pH 3-6 and at temperatures up to 60 C (5).. Bromelain powder can be stored originally packed and tightly closed up to two years at 8 C without loss of activity.. Upon opening the activity gradually decreases, unless the enzyme is immediately mixed with diluent (lactose) which prevents the protein from self-inactivation.. Activity losses caused by unproper storage or preparation conditions can be abolished to an appreciable extent by the addition of cysteine.. Solubility:.. Bromelain powder is partly soluble in water, insoluble in most organic solvents.. Molecular weight:.. 33,200 daltons.. Composition:.. Bromelain is a glycoprotein constituted of one single polypeptide chain with 1 glycan per molecule.. The number of amino acids per molecule is not yet unequivocally established.. The NH2-terminal amino acid is valine, the COOH-terminal is glycine.. Isoelectric point:.. 9.. 55.. Spectral data:.. 280.. (1%, 1cm) = 20.. 1.. Assay.. The method described here is the one given by Lauwers and Scharpé (4).. Principle.. Bromelain hydrolyzes casein into small peptides, which cannot be precipitated with a specific reagent.. Thus, after incubation (10 min at 35 C) undigested casein is removed  ...   Tris-buffer (A).. Enzyme activation solution:.. 5 mM cysteine, 1 mM EDTA, 10 mM KCl in tris buffer (A).. Dissolve 60.. 5 mg cysteine, 37.. 2 mg EDTA x 2H2O and 74.. 6 mg KCl in Tris-buffer (A), adjust to pH 7.. 15 and make up to 100 ml with Tris-buffer (A).. Enzyme solution:.. Dissolve Bromelain in enzyme activation solution (D).. The solution should contain 0.. 1-0.. 12 FIP units/ml.. Before use the solution is equilibrated to 35 C.. Enzyme Reference Solution:.. Dissolve a suitable amount of bromelain FIP standard in enzyme-activation solution to obtain a solution with an activity of apparently 0.. Bromelain FIP Reference Standard is issued by:.. Centre for Standards FIP, Harelbekestraat 72, B-9000 Ghent, Belgium.. Protein precipitation reagent:.. 11 M TCA, 0.. 364 M sodium acetate, 0.. 688 M acetic acid.. Dissolve 9.. 0 g TCA (trichloroacetic acid) p.. a.. , 24.. 8 g sodium acetate x 3H2O and 19.. 5 ml acetic acid in distilled water to a final volume of 500 ml.. Filter paper control:.. Determine the suitability of the filter paper by filtering through the paper 5 ml of protein precipitation reagent.. Measured at 275 nm against an unfiltered aliquot of the reagent the absorbance of the filtered reagent should not exceed 0.. 04.. Procedures.. Transfer into test tubes.. Blank.. Substrate solution (C).. equilibrate to 35 C in a water bath.. 2.. 5 ml.. 5 ml.. Enzyme solution (E F).. pre-equilibrated to 35 C.. mix, incubate for exactly 10 min (stop watch).. ----.. Protein precipitation reagent (B).. 5.. 0 ml.. 0 ml.. mix, incubate for 30 min at 35 C,.. filter through filter paper;.. measure absorbance of filtrate.. against blank (1 cm cuvette, 275 nm).. Calculation.. The specific activity of the enzyme is calculated as:.. m x E x A.. m x E.. m = mass in milligrams of the substance to be examined,.. m = mass in milligrams of Bromelain FIP standard,.. E = absorbance of the filtrate of the substance to be examined,.. E = absorbance of the filtrate of the Bromelain FIP standard.. A = activity of Bromelain FIP standard in FIP units/mg.. Availability.. Standard qualities.. Bromelain 5.. 0 FIP units/mg 2500 GDU/g 1560 CDU/mg.. Bromelain 4.. 0 FIP units/mg 2000 GDU/g 1250 CDU/mg.. Bromelain 3.. 2 FIP units/mg 1600 GDU/g 1000 CDU/mg.. Bromelain 2.. 4 FIP units/mg 1200 GDU/g 750 CDU/mg.. Customized qualities.. are available upon request.. References.. International Union of Biochemistry.. Nomenclature Committee: Enzyme Nomenclature.. Academic Press, Inc.. , London 1984.. Godfrey, T.. , Reichelt, J.. : Industrial Enzymology.. Macmillan Publishers Ltd.. , Surrey (U.. K.. ), 1983.. Barman, T.. E.. : Enzyme Handbook, Vol.. II.. Springer, New York-Heidelberg-Berlin-Tokyo, 1985.. Lauwers, A.. ; Scharpé, S.. : Pharmaceutical Enzymes, drugs and pharmaceutical sciences.. , Volume 84, Marcel Dekker, Inc.. , New York-Basel-Hong Kong, 1997.. Stellmach, B.. : Bestimmungsmethoden Enzyme für Pharmazie, Lebensmittel-chemie, Technik, Biochemie, Biologie, Medizin.. Steinkopff-Verlag, Darmstadt, 1988.. top of the page..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Data Sheet - Chymotrypsin.. Bovine or porcine pancreas.. Peptidyl peptide hydrolase.. 9004-07-3.. 21.. 1.. 647-011-00-2.. Unless otherwise specified, this paragraph will deal with literature data on bovine Chymotrypsin Aα.. Chymotrypsin hydrolyzes peptides, amides and esters at bonds involving the carboxyl group of L-tyrosine, L-tryptophan and L-phenylalanine.. Proteolysis also occurs, although much slower, adjacent to other large hydrophobic amino acid residues, such as L-leucine,.. L-methionine.. and L-histidine (1,2).. In assays for Chymotrypsin activity the synthetic substrates N-acetyl-L-tyrosine ethyl ester, N-benzoyl-L-tyrosine ethyl ester and N-acetyl-L-tryptophan amide are most frequently used (3).. Ca.. + ions have been shown to enhance the activity of Chymotrypsin and also to stabilize the enzyme against denaturation (3,4).. Chymotrypsin is inhibited by several low molecular weight and high molecular weight substances.. Indole, b-indolepropionate, p-iodo-phenylacetate as well as D stereoisomers of substrates, e.. N-acetyl-D-tyrosine ethyl ester or N-acetyl-D-tryptophan methyl ester, behave as competitive inhibitors (1).. Like other serine proteases, Chymotrypsin is inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylphosphofluoridate (DFP) (1,5).. Tosylphenylalanylchloromethane (TPCK) irreversibly inhibits Chymotrypsin (but not Trypsin) (3).. Heavy metals like Cu2.. +.. and Hg2.. are also inhibitory (3,4).. Among the most important high molecular weight inhibitors are proteins like α1-antichymotrypsin, α1- proteinase inhibitor (formerly: a1-antitrypsin), α2-macroglobulin, aprotinin (pancreatic Trypsin inhibitor, Kunitz inhibitor), soybean Trypsin inhibitor and Ascaris inhibitors (3,5; see also: Methods in Enzymology, Vol.. XIX, pp.. 844-905).. Chymotrypsin acts optimally at pH values around 8 (6).. As a lyophilized powder, Chymotrypsin is stable almost indefinitely provided it is stored dry in a cool place (7).. In solution Chymotrypsin is most stable at pH 3.. Below pH 3 the enzyme is reversibly denatured.. Above pH 10 Chymotrypsin becomes inactive.. The enzyme is stabilized by Ca2.. ions (3, 4).. Chymotrypsin is sparingly soluble in water.. approx.. 25,000 (2,3,6).. Chymotrypsin Aa consists of 241 amino acid residues in 3 poly-peptide chains, A (13 residues), B (131 residues) and C (97 residues), which are held together by 5 disulfide bridges (3,4).. Chymotrypsin Aα arises from the inactive single-chain precursor Chymotrypsinogen A by the successive cleavage of 4 peptide bonds with a concomitant removal of 2 dipeptides, Ser14-Arg15 and Thr147-Asn148.. Only one of the 4 cleavages is catalyzed by Trypsin (Arg15-Ile16) and it is basically this one which renders Chymotrypsinogen active; the other 3 are autolytic cleavages by Chymotrypsin (2,3,4,8).. The activation of Chymotrypsinogen A to Chymotrypsin Aa proceeds via various intermediate forms.. The amount of Trypsin present during the activation governs the appearance either of several.. active.. interme-diate forms (Chymotrypsin Api, Adelta, Akappa, Agamma) or of.. inactive.. forms, called neo-chymotrypsinogens (3,4,8).. 8.. 4 (4).. 282.. 0 (4,6).. Assay (according to current FIP-Method).. The method described here is the one given in the monograph on Chymotrypsin in the Ph.. Eur.. It is identical to the method given by FIP (Fédération International Pharmaceutique; see 3) which originally developed this assay method.. Chymotrypsin hydrolyzes the synthetic substrate N-acetyl-L-tyrosine ethyl ester (ATEE).. The amount of acid liberated (at pH 8.. 0 and 25 C) is measured by titration with sodium hydroxide, recorded as a function of time.. The activity of Chymotrypsin is determined by comparing the rate at which it hydrolyzes acetyltyrosine ethyl ester with the rate at which Chymotrypsin BRP hydrolyzes the same substrate under the same conditions.. Apparatus.. Use a reaction vessel of about 30 ml capacity provided with:.. a device that will maintain a temperature of 25.. 0 ± 0.. 1 C,.. a stirring device, for example a magnetic stirrer,.. a lid with holes for the insertion of electrodes, the tip of a burette, a tube for the admission of nitrogen and the introduction of reagents.. An automatic or manual titration apparatus may be used.. For the latter, the burette is graduated in 0.. 005 ml and the pH meter is provided with a wide scale and glass-calomel electrodes.. ATEE solution (0.. 2 M):.. Dissolve 0.. 539 g of N-acetyl-L-tyrosine ethyl ester monohydrate (ATEE x H.. O) in ethanol 96% and dilute to 10.. 0 ml with the same solvent.. Calcium chloride solution (0.. 01 M):.. 147 g of CaCl.. x 2H.. O in water and dilute to 100.. 02 N Sodium hydroxide.. 001 N Hydrochloric  ...   activity according to USP 24 the rate of hydrolysis of ATEE at pH 7.. 0 is measured spectrophotometrically at 237 nm.. Assay (according to USP-Monograph).. Definition.. One USP-Unit of Chymotrypsin correspond the amount of Enzyme, that under the test conditions hydrolysed 1 mol N-Acetyl-L-tyrosinethylester (= ATEE), that makes a Change of Absorbance of 0.. 0075 pro Minute at 237 nm.. Preparation of solutions.. Temperature equalisation.. : Chymotrypsin Reference Standard USP, the Samples and N-Acetyl-L-tyrosinethylester from 4 C to Room temperature.. 1 mM HCl-Solution:.. 1.. 0 ml 1 N HCl-Solution + pure H.. O add 1000 ml.. N-Acetyl-L-tyrosinethylester-Solution:.. 61.. 0 mg ATEE*H.. O (M = 269.. 30 g/mol) + 96%ige EtOH add to 1.. 067 M KH2PO4-Solution:.. 4.. 56 g KH.. PO.. (M=136.. 09 g/mol, MERCK 4873) + pure H2O add 500 ml.. 067 M Na2HPO4-Solution:.. 4,73 g Na2HPO4 (M = 141,96 g/mol, MERCK 6586) + pure H.. O add 500 ml.. 067 M Phosphat-Buffer, pH 7.. 00:.. Give to 200 ml KH.. -Solution the amount of Na.. HPO.. -Solution until the pH-value adjust to 7.. Enzyme-Solution:.. Test the Activity of each Sample with two Initial weight.. Weight the amount of Enzyme Sample or the Standard and solve in 1 mM HCl-Solution.. The Dilution of this Enzyme soluion in 1.. 2 mM HCl-Solution give a change of absorbance (= ΔA/min) of maximal 0.. 02 in „ml in Test“ of 0.. 200.. Formula:.. mg Initial weight = [(1.. 9 USP-U/„0.. 200 ml/Test“)* dilution factor*Enzyme-Solution] / declared Activity of Enzyme.. If the Sample is a Trypsin- und Chymotrypsin-Mixture, make an initial weight of the Sample that the Try- and Chy-Activity can be determined according to USP from the same Enzyme Solution (50 ml) with a „ml in Test“ of 0.. 080-0.. 200 ml.. Procedure.. Give in 100 ml Phosphat-Buffer 0.. 42 ml of ethanolic ATEE-Solution.. Put Phosphat-Buffer-Solution in Bath thermostat to adjust the temperature to 25 C.. Shortly before to beginning with the test give the ATEE-Solution into buffer.. This Solution is stabile at 25 C for about 1.. 5 h.. To reach a homogeny temperature, mix the solution before using each time.. Calibrate the UV-spectral photometer at 237 nm with pure H.. O.. For each test measure a Blank (200 l of 1.. 2 mM HCl-Solution).. Calibrate the UV-Spectral photometer with this Blank.. For testing of Enzyme-solution: E.. give in a cuvette 80 l of Enzyme-dilution and 120 l of 1.. 2 mM HCl-solution.. Mix the Buffer-ATEE-Solution each time before using.. Give into this cuvette 3 ml of ATEE-Buffer-Solution, mix and put it immediately in spectral photometer and start the measurement.. Measure the absorbance of the reaction every 30 seconds for 5 Minutes.. Is the Change of Absorbance (= D A/min) higher than 0.. 02 or lower than 0.. 009 then change for the next measurement the amount of “ l in Test” 80 - 200 l.. Print the Rates of absorbance and the tabulate of „ΔA/min“.. Prepare the UV-Spectral photometer according the manufacturer instruction to starting a new measurement.. Measure each Enzyme dilution 4 times.. Clear the cuvette for each using with pure H.. Calculation:.. Calculate the average of D A/min of each Enzyme-Solution.. Factor =.. 1.. {[(Initial weight in mg/Enzyme Solution in ml)/(Dilution factor)] * (ml in Test)} * 0.. 0075.. USP-unit/mg (abs.. = Δ A/min * Factor.. Standard: USP-unit/mg (rel.. = r = USP-unit/mg (abs.. ) / declared Activity of reference Standard.. Sample: USP-unit/mg (rel.. = USP-unit/mg (abs.. ) / r.. Chymotrypsin 1400 USP units/mg.. Chymotrypsin 1000 USP units/mg.. Chymotrypsin Ph.. Eur.. (5 mkatal/mg).. Chymotrypsin 300 FIP units/mg.. Blow, D.. M.. in: The Enzymes (P.. D.. Boyer, ed.. ) 3rd ed.. , Vol.. III, p.. 185.. Academic Press, New York, 1971.. Hess, G.. P.. 213.. , Scharpé, S.. : Pharmaceutical Enzymes.. drugs and pharmaceutical sciences.. Wilcox, P.. in: Methods in Enzymology (G.. Perlmann, L.. Lorand, eds.. ) Vol.. XIX, p.. 64.. , Orlando, Florida, 1970.. Geiger, R.. in: Methods of Enzymatic Analysis (Bergmeyer, J.. , Graál, M.. , eds.. V, p.. 99.. Verlag Chemie, Weinheim, 1984.. Walsh, K.. A.. , Wilcox, P.. 31.. : Bestimmungsmethoden Enzyme.. Steinkopff Verlag Darmstadt, 1988.. Kraut, J.. 165..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Data Sheet - Pancreatin.. Pancreatin.. is the name of an extract from porcine pancreas.. 8049-47-6.. The pancreas is a gland with a small endocrine part (Islets of Langerhans), and a large exocrine part.. The Islets of Langerhans secrete insulin and glucagon, whilst the cells of the exocrine part produce various hydrolytic enzymes most of which have digestive functions.. Upon synthesis, the proteins are deposited in granules, either as active enzymes or as inactive precursors (zymogens).. When the secretion is induced, the contents of the granules are released into a system of ducts and finally reach the duodenum where the zymogens are being transformed into active enzymes.. The pancreatic enzymes can be classified in four groups:.. peptide hydrolases/proteases: e.. Trypsin, Chymotrypsin, Elastase, Carboxy-peptidase A, Carboxypeptidase B, Kallikreins; (.. Note.. : all of these enzymes are produced and stored as zymogens.. In the intestine, Trypsinogen is activated by enterokinase, and the other zymogens are activated by the Trypsin formed).. lipolytic enzymes: e.. Lipase, Phospholipase A2, Phospholipase B, cholinester-ase, cholesterol esterase;.. Glycosidases: e.. α-Amylase, Glucosidase;.. nucleases: e.. Deoxyribonuclease I, Deoxyribonuclease II, Ribonuclease.. Pancreatin contains many of these enzymes.. The quality of any Pancreatin preparation is defined by three enzyme activities, Protease, Amylase and Lipase.. The pharmacopoeias specify minimum activity levels (in units per mg) for each of these three enzymes in Pancreatin.. In both absolute and relative terms, the activities of these enzymes vary with, and depend upon, the origin of the pancreas and the treatment of the glands.. Porcine pancreas is preferred for the industrial manufacture of Pancreatin for the following reasons:.. porcine pancreas is commercially available in substantial quantities;.. porcine pancreas contains high Protease, Amylase and Lipase activity (provided the glands were frozen immediately after slaughtering);.. the enzyme composition is similar to that of human pancreas (1).. The following sections will deal with some characteristics of the Pancreatin enzymes which are relevant for their biological function and for their assay.. Pancreas proteases.. The pancreas contains a mixture of endo- and exopeptidases.. Trypsin (EC 3.. 4), Chymotrypsin (EC 3.. 1) and Elastase (EC 3.. 36) are specific endopeptidases which cleave peptide bonds preferentially at the carboxyl end of basic, aromatic and aliphatic L-amino acids, respectively.. Carboxypeptidase A (EC 3.. 17.. 1) and Carboxypeptidase B (EC 3.. 2) are exopeptidases which cleave L-amino acids from the free carboxyl terminus of peptides with a preference for C-terminal hydrophobic and basic amino acids, respectively.. This broad spectrum of specificities has two important implications.. Physiologically.. , it provides an efficient tool for the degradation of proteinaceous food components into resorbable fragments.. Biochemically.. , in any assay for Protease activity in Pancreatin only a protein substrate, e.. casein, can supply a sufficient range of susceptible bonds for all Proteases present.. In the assay, the protein substrate should be denatured prior to use because Trypsin and Chymotrypsin which together contribute the most abundant part of the endopeptidases in Pancreatin both display a preference for unfolded proteins.. In Pancreatin the major amount of each proteolytic enzyme is present as the corresponding Zymogen, i.. as Trypsinogen, Chymotrypsinogen, procarboxypeptidase A, procarboxypeptidase B, and proelastase.. Therefore an assay performed with Pancreatin as such will simply determine any.. free.. Protease activity.. The.. total.. Protease activity can only be measured after preincubation of a Pancreatin solution with an activating compound.. Enterokinase is routinely used for this purpose.. This enzyme renders Trypsin active and thus triggers the activation of the other zymogens.. The catalytic optima of the pancreas proteases are in good agreement with the physiological requirements, i.. about 37 C and between pH 7 and 9.. Due to the nature of the enzymes a Pancreatin solution is not very stable under these conditions because autolytic and proteolytic degradation of the enzymes takes place.. Calcium ions increase the activity and stabilize Trypsin against the autolytic attack.. The same holds true for Chymotrypsin though the stabilizing effect of calcium is less pronounced than with Trypsin (1).. Pancreas Lipase.. Pancreatic Lipase (EC 3.. 3) can be characterized both by its ability to liberate fatty acids from emulsified long-chain triglycerides and by its action at the oil-water interface (2, 3).. Like other esterases, pancreatic Lipase has a low substrate specificity.. The Enzyme hydrolyzes not only tri-, di- and monoglycerides but also esters of n-alcohols and fatty acids with short to long carbon chains.. The rate of hydrolysis decreases in the order tri-, di- and monoglyceride (1, 3, 4).. The reaction velocity also decreases with increasing fatty acid chain lengths of triglycerides, that is e.. from tributyrin to triolein.. On the other hand, in the same order triglycerides become more specific substrates (2).. Therefore triolein (or olive oil as its natural substitute) is used as a substrate in most of the assays on pancreatic Lipase.. Several factors are known to affect the activity of pancreatic Lipase.. Since pancreatic Lipase acts at the oil-water interface, the reaction rate depends on the surface area of the substrate and thus on the degree of emulsification (3).. Bile salts promote emulsification and they are therefore used - together with gum arabic - for the preparation of stable substrate emulsions.. Colipase, a small protein (molecular weight approx.. 10,000), is a specific cofactor of Pancreatin Lipase.. It is synthesized and secreted by the Pancreas and serves to anchor pancreatic Lipase on the substrate surface in the presence of bile acids or other amphipaths like proteins and fatty acids (2).. Sodium chloride is absolutely required for and increases the activity of pancreatic Lipase (1).. Calcium ions improve the thermal stability of pancreatic Lipase and also enhance the activity of the enzyme (3).. Pancreatic Lipase is active in the range of pH 6.. 5 to 9.. 0 (1).. Pancreas Amylase.. α-Amylase (EC 3.. 2.. 1; 1,4-α-D-glucan Glucanohydrolase) hydrolyzes 1,4-α-D-glucosidic linkages in polysaccharides containing 3 or more 1,4-α-linked D-glucose units (5).. The enzyme from porcine pancreas is composed of 2 subunits and has a molecular weight of 50,000 (6).. Three types of amylases can be distinguished:.. a-Amylase is an endoamylase which is found in all living organisms.. a-Amylase acts in a random fashion by a multiple-attack mechanism on starch, glycogen and related polysaccharides and oligosaccharides ultimately yielding glucose and maltose as well as larger oligosaccharides.. Reducing groups are in the α-configuration.. The Enzyme cannot hydrolyze 1,6-α-bonds in glycogen and amylopectin, but it is able to bypass these branch points (1, 5).. β-Amylase and g-Amylase are exoamylases which are exclusively found in plants and microorganisms.. β-Amylase (EC 3.. 2; 1,4-α-D-glucan maltohydrolase) can neither hydrolyze 1,6-α-bonds nor bypass these branch points.. This enzyme acts on the same substrates as a-Amylase but it removes successive maltose units from the non-reducing end; by inversion the maltose units released are in the β-configuration (5).. γ-Amylase (EC 3.. 3; 1,4-α-D-glucan Glucohydrolase) releases β-D-glucose successively from the non-reducing end of the polysaccharide chains (5).. Calcium and chloride ions are essential for a-Amylase.. One Ca2.. is tightly bound by each enzyme molecule thereby keeping it in the correct conformation for activity.. Excess calcium stabilizes a-Amylase towards heat (1).. Thus, chelating agents are inhibitory.. Based on their strong effect on α-Amylase chloride ions have been regarded as natural activators of the enzyme (6).. NaCl activates the enzyme when added to the assay in a concentration of 10-100 mM.. At higher concentrations NaCl is inhibitory (1, 7).. The catalytic optima are a temperature between 40 C and 45 C and a pH about neutral (1).. Assays.. The methods described here are those given in the monography on Pancreas powder in the Ph.. (Pancreatis pulvis).. They are fairly identical to the methods given by the FIP (see: Lauwers Scharpé (1)) which originally developed these assay methods.. Note:.. The biological reference preparations Pancreas powder (Protease) BRP and Pancreas powder (Amylase and Lipase) BRP are issued by: Technical Secretariat, European Pharmacopoeia Commission, Council of Europe, P.. Assay for proteolytic activity.. Pancreatin proteases hydrolyze casein into peptides.. The amount of peptides which cannot be precipitated with trichloroacetic acid is determined spectrophotometrically at 275 nm.. The total proteolytic activity of Pancreas powder is determined by comparing the quantity of non-precipitable peptides released per minute from a casein substrate solution with the quantity of such peptides released by Pancreas powder (Protease) BRP from the same substrate in the same conditions.. 1 N Sodium hydroxide.. 1 N Hydrochloric acid.. 02 M Calcium chloride:.. Dissolve 2.. 94 g of CaCl.. x 2 H.. O in 900 ml of water, adjust the pH to 6.. 0-6.. 2 and add water to a final volume of 1000.. Store at 5 ± 3 C.. Borate buffer solution pH 7.. 5:.. 5 g of NaCl, 2.. 85 g of disodiumtetraborate  ...   pH 7.. 0.. Dissolve 10.. 0 g sodiumchloride, 6.. 06 g tris(hydroxymethyl)aminomethane and 4.. 90 g maleic anhydride in 900 ml of water.. Adjust with 4 N sodium hydroxide to pH 7.. 0 and add water to a final volume of 1000 ml.. In a small mortar cooled to 0 C to 4 C, triturate carefully a quantity of the substance to be examined equivalent to about 2500 Ph.. units of lipolytic activity with 1 ml of cooled Lipase solvent (G) until a very fine suspension is obtained.. Dilute with cold Lipase solvent (G), transfer quantitatively to a volumetric flask and add cold Lipase solvent (G) to a final volume of 100.. Equilibrate the bottle with standard to room temperature before opening to prevent moistening by condensing water.. Prepare a suspension of standard (Pancreas powder (Amylase and Lipase) BRP) as described for the test suspension using a quantity equivalent to about 2500 Ph.. units.. Keep the test and reference suspensions in iced water during the test!.. Apparatus.. Use a reaction vessel of about 50 ml capacity provided with:.. a device that will maintain a temperature of 37 ± 0.. 5 C,.. a magnetic stirrer,.. a lid with holes for the insertion of electrodes, the tip of a burette,.. a tube for the admission of nitrogen and the introduction of reagents.. In the latter case, the burette is graduated in 0.. 005 ml and the pH-meter is provided with a wide reading scale and glass-calomel electrodes.. After each test the reaction vessel is evacuated by suction and washed several times with water, the washings being removed each time by suction.. Procedure.. Carry out the titrations immediately after the preparation of the test suspension and the reference suspension.. Place 29.. 5 ml of substrate emulsion (F) in the reaction vessel equilibrated at 37 ± 0.. Fit the vessel with the electrodes, a stirrer and a burette (the tip being immersed in the olive oil suspension), put the lid in place and switch on the apparatus.. Carefully add 0.. 1 N sodium hydroxide (D) with stirring to adjust to pH 9.. Using a rapid-flow graduated pipette transfer about 0.. 5 ml of the previously homogenized reference suspension (I), start the chronometer and add continuously 0.. 1 N sodium hydroxide (D) to maintain the pH at 9.. Read the volume of 0.. 1 N sodium hydroxide (D) used after exactly 1 min and repeat this reading four times.. Determine the average of the last four readings (S1).. Make 2 further determinations (S2 and S3) and calculate the average n1 of the values S1, S2 and S3.. The average volume of 0.. 1 N sodium hydroxide used should be about 0.. 12 ml/min with limits of 0.. 08 and 0.. 16 ml/min.. Carry out 3 determinations in the same manner for the test suspension (T1, T2 and T3) and calculate the average n1 of the values T1, T2 and T3.. If the quantity of 0.. 1 N sodium hydroxide used is outside the limits of 0.. 08 to 0.. 16 ml/min, the assay should be started again with a quantity of test suspension which is more suitable but situated between 0.. 4 and 0.. 6 ml.. Otherwise the quantity of the substance to be examined should be adjusted to comply with the conditions of the test.. Calculate the activity in Ph.. units/mg from the expression:.. n * m1.. n1 * m.. n = average volume of 0.. 1 N sodium hydroxide used per min during the titration of the test suspension,.. n1 = average volume of 0.. 1 N sodium hydroxide used per min during the titration of the reference suspension,.. m = mass in mg of the substance to be examined,.. m1 = mass in mg of the reference preparation,.. A = activity of the Pancreas powder (Amylase and Lipase) BRP in Ph.. units/mg.. Assay for amylolytic activit.. y.. α-Amylase liberates from starch reducing groups which in alkaline solution react with iodine which is added in excess.. The amount of iodine not used by the reaction is determined titrimetrically with thiosulfate.. The amylolytic activity is determined by comparing the rate at which a suspension of Pancreas powder hydrolyzes a starch substrate solution with the rate at which a suspension of Pancreas powder (Amylase and Lipase) BRP hydrolyzes the same substrate under the same conditions.. Phosphate buffer solution pH 6.. 8 (0.. To 51 ml of 0.. 2 M KH.. (2.. 72 % m/V) add about 49 ml of 0.. 2 M Na.. x 12 H.. O (7.. 16 % m/V) to give a pH of 6.. 8.. Starch solution:.. To a quantity of starch BRP equivalent to 2.. 0 g of dried substance add 10 ml of water and mix (determine the water content of starch BRP prior to the test by heating at 120 C for 4 h).. Add this suspension with continuous stirring to 160 ml of boiling water.. Rinse the starch container several times with water, 10 ml each, and add the washings to the hot starch solution.. Heat to boiling, stirring continuously.. Cool to room temperature and dilute to 200 ml with water.. 2 M Sodium chloride (1.. 17 % m/V).. 1 N Hydrochloric acid.. 1 N Iodine.. Sulfuric acid (96 %).. 1 N Sodium thiosulfate.. Triturate an amount of the substance to be examined equivalent to about 1500 Ph.. units of amylolytic activity with 60 ml of phosphate buffer solution pH 6.. 8 (A) for 15 min.. Transfer quantitatively to a volumetric flask and dilute to 100.. 0 ml with phosphate buffer solution pH 6.. 8 (4 C) (A).. Prepare a suspension of standard (Pancreas powder (Amylase and Lipase) BRP) as described for the test suspension, using a quantity equivalent to about 1500 Ph.. Keep all suspensions in iced water during the test!.. In a test tube (200 mm long, 22 mm diameter; with ground-glass stopper) place 25.. 0 ml of starch solution (B), 10.. 0 ml of phosphate buffer solution pH 6.. 8 (A) and 1.. 0 ml of 0.. 2 M sodium chloride (C).. Stopper the tube, mix the contents and place in a waterbath at 25.. 1 C.. When the temperature equilibrium has been reached, add 1.. 0 ml of the test suspension (I) and start the chronometer.. Mix thoroughly and place the tube in the waterbath.. After exactly 10 min, add 2 ml of 1 N hydrochloric acid (D) to stop the reaction.. Transfer the mixture quantitatively to a 300 ml conical flask fitted with a ground-glass stopper.. While shaking continuously, add 10.. 1 N iodine (E) and immediately 45 ml of 0.. 1 N sodium hydroxide (F).. Allow to stand in the dark at 15 C to 25 C for 15 min.. Add 4 ml of a mixture of 4 volumes of water and 1 volume of sulfuric acid (G).. Titrate the excess of iodine with 0.. 1 N sodium thiosulfate (H) using a microburette.. Carry out a blank titration adding 2 ml of 1 N hydrochloric acid (D) before introducing the test suspension (I).. Carry out the titration of the reference suspension (J) in the same manner.. Calculate the amylolytic activity in Ph.. (n' - n ) * m1.. (n'1 - n1) * m.. n = number of ml of 0.. 1 N sodium thiosulfate used in the titration of the test suspension,.. n1 = number of ml of 0.. 1 N sodium thiosulfate used in the titration of the reference suspension,.. n' = number of ml of 0.. 1 N sodium thiosulfate used in the blank titration of the test suspension,.. n'1 = number of ml of 0.. 1 N sodium thiosulfate used in the blank titration of the reference suspension,.. A = activity of Pancreas powder (Amylase and Lipase) BRP in Ph.. 1 x USP (powder).. 2 x USP (powder).. 3 x USP (powder).. 4 x USP (powder or granules).. 5 x USP (powder or granules).. 6 x USP (powder or granules).. 7 x USP (powder or granules).. 8 x USP (powder or granules).. 8 x USP (powder or granules) „high Lipase“.. 8 x USP (powder or granules) „high Protease“.. 1 x Ph.. / FIP (powder or granules).. 2 x Ph.. 3 x Ph.. Concentrate (powder or granules) very high activities.. Customized qualities are available upon request.. : Pharmaceutical Enzymes, Drugs and Pharmaceutical Sciences, Volume 84, Marcel Dekker, Inc.. Junge, W.. IV, p.. Neumann, U.. , Kaspar, P.. , Ziegenhorn, J.. 26.. Springer, New York - Heidelberg-Berlin-Tokyo, 1985.. , London, 1984.. Dixon, M.. , Webb, E.. : Enzymes.. 3rd ed.. Academic Press, New York, San Francisco, 1979.. Rauscher, E.. ) 3rd.. 152..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Data Sheet - Papain.. Latex from the fruit of Carica papaya L.. Peptidyl peptide hydrolase;.. 9001-73-4.. Papain is one of the proteolytic enzymes found in the latex of the leaves and of the green fruit of the papaya tree (Carica papaya L.. , Caricaceae).. In industrial preparations, referred to as.. refined Papain.. , Papain EC 3.. 2 represents only a minor part when compared to the amount of the other proteinases, papaya proteinase Ω and Chymopapain (EC 3.. 6), the latter being by far the most abundant enzyme.. Some other hydrolytic enzymes, e.. papaya lysozyme, are also present in refined Papain though only in minute amounts.. Note: Papain EC 3.. 2 contributes less than 10 % to the total proteinase content of industrial preparations of papaya latex, in the following referred to as refined Papain.. The characteristics of refined Papain are largely influenced by the two other proteinases, papaya proteinase W and Chymopapain, because these two make up about 15 % and 75 %, respectively, of the total proteinase content of refined Papain.. Therefore, the data below consider the respective values for.. all three.. proteinases of refined Papain, where appropriate.. Papain EC 3.. 2 hydrolyzes a wide variety of proteins and peptides.. It has also esterase, thiolesterase, transesterase and transamidase activity.. It has a preference for bonds involving the carbonyl end of α-NH2 substituted arginine and lysine, and to a lesser extent, histidine, glycine, glutamine and tyrosine (1,2,3).. Phenylalanine in a position Phe-X- enhances the susceptibility of a peptide to hydrolysis at the C-terminal end of X (1,2).. Chymopapain and papaya proteinase Ω have substrate specificities close but not identical to that of Papain.. The presence of these enzymes in refined Papain therefore extends the range of bonds susceptible to hydrolysis and hence the degree of digestion of protein by refined Papain when compared with Papain EC 3.. 2 alone.. All three aforementioned proteinases are thiol enzymes, containing in the active centre a highly reactive cysteine which is essential for catalysis.. Alkylating reagents such as p-chloro-mercuribenzoate (PCMB), N-ethylmaleimide (NEM), and iodoacetate cause irreversible inactivation.. Heavy metals (e.. Hg.. 2+.. , Zn.. , Fe.. , Cu.. ), air and H.. are also inhibitory.. The papaya proteinases can on the other hand be activated by the application of mild reducing agents like cysteine, sulfide, cyanide or thioglycolic acid, supplemented by a heavy metal chelator such as EDTA, or by the addition of 2,3-dimercaptopropanol, a compound which combines the functions of both a thiol reagent and a metal binder (1,4).. Depending on the nature of the substrate, refined Papain shows maximum activity at pH 4.. 0-7.. The optimum temperature is in the range of 60-70 C.. 2 and papaya proteinase Ω are unstable under acidic conditions.. At pH values lower than pH 2.. 8 rapid and irreversible inactivation occurs even at ambient temperature (1,4).. In contrast, Chymopapain is extremely stable at pH 2.. 2 shows high stability towards urea and organic solvents; exposure to up to 9 M urea, 70% methanol, or 15% dimethylsulfoxide (DMSO) cause no loss of activity (1,4).. All three papaya proteinases are particularly resistant to heat.. Refined Papain is almost completely soluble in water, insoluble in most organic solvents.. Chymopapain exhibits high solubility in saturated NaCl above pH 3 and is more soluble in aquatic solution than Papain EC 3.. 2: approx.. 23,400, Chymopapain: approx.. 24,000, papaya proteinase Ω: approx.. 24,000.. Papain, Chymopapain and papaya proteinase W consist of single polypeptide chains of 212, 216, and 218 amino acids, respectively.. The amino acid sequences, the secondary structures, and the three-dimensional structures of the three papaya proteinases show a lot of significant similarities, suggesting that these enzymes are closely related to each other (5).. On the other hand, based on the differences in the amino acid composition and sequence it can be ruled out that the three proteinases arose from each other or from a common ancestor by proteolysis (5).. 2: 8.. 7-8,9,.. Chymopapain: 10.. 3 - 10.. 7,.. papaya proteinase Ω: 11.. 2: E.. 278.. (1%, 1cm) = 25.. 0 (6),.. Chymopapain: E.. (1%, 1cm) = 18.. 4 - 18.. 7 at pH 7.. 0 (6).. Assay according to FIP.. (1 FIP unit ≈ 10,000-13,000 USP units).. The method described here is the one given by Lauwers Scharpé (1).. The assay reflects the activity of all three proteinases contained in refined Papain.. Papain hydrolyzes the synthetic substrate N-benzoyl-L-arginine ethyl ester (BAEE).. The amount of acid liberated (at pH 7.. 1 FIP unit of Papain is the amount of Enzyme that hydrolyzes under the standard conditions 1 mmole of BAEE per minute.. 06 M N-benzoyl-L-arginine ethyl ester (BAEE).. Dissolve 514 mg BAEE  ...   conditions of the Assay, using the enzymes concentration that liberates 40 µg of tyrosine per ml of test solution.. 05 M Dibasic sodium Phosphate solution:.. 71 g Na.. (M = 141.. 96 g/mol) in pure H.. O to make 100 ml.. 05 M Citric acid solution:.. Dissolve 1.. 05 g Citric acid monohydrate (M = 210.. 14 g/mol) in pure H.. Buffer solution (pH 6.. 00):.. 78 g Na.. 96 g/mol) in 200 ml pure H2O.. Give 3.. 5 g Disodium edetate (M = 372.. 24 g/mol) and 1.. 53 g Cysteine x HCl x H.. O (M = 175,64 g/mol).. Adjust the pH with 1 N NaOH or HCl to pH 6.. 00 and fill with pure H.. O to 250 ml.. (Prepare fresh daily).. Casein Substrate solution:.. Give 0.. 5 g Casein (Hammersten-type or BRP) into 25 ml 0.. 05 M Na.. solution.. Place in a boiling water bath for 30 minutes with occasional stirring.. Cool to room temperature and add.. 05 M Citric acid solution.. under rapidly stirring to adjust a pH of 6.. Dilute with pure water to 50 ml.. 30% (m/V) Trichloroacetic acid solution:.. Dissolve 30 g TCA (M = 163.. 39 g/mol) with pure H.. Preparation of Enzyme solution:.. Prepare an enzyme solution in that kind that the release of 40 g Tyrosine in test does not exceed.. {400 USP-U/10 ml 40 USP-u/ml (= 40 g Tyrosine)} Use the enzyme dilution within.. 60 Minutes.. Initial weight of Enzyme = [(40 USP-E*10 ml/1.. 5 ml)*dilution factor*ml Enzyme solution] ÷ declared Activity.. Label test tubes in duplicate Ta, Tb, S1a, S1b, S2a, S2b, S3a, S3b.. For.. as follow: BTa, BTb, BS1a, BS1b, BS2a, BS2b, BS3a, BS3b Add to the test tubes in the order given:.. Give from 0.. 5 M Phosphat-buffer solution pH 6.. 00 (.. C.. ) in S1a, S1b BS1a, BS1b.. , in S2a, S2b BS2a, BS2b, Ta and Tb.. and in S3a, S3b and BS3a, BS3b.. ;.. Give in each tube 5.. 0 ml of Casein solution (.. D.. ).. Place all tubes in a water bath at 40 ± 0.. When temperature equilibrium is reached, start the stop watch and at t = 0 add.. of Papain Reference Standard dilution into the tubes S1a, S1b, and.. of Papain Reference Standard dilution into the tubes S2a, S2b and.. of Papain Reference Standard dilution into the tubes S3a, S3b.. Also add.. of Papain Test dilution into the tubes T1a, T1b.. Shake the tubes and let at 40 C for exactly 60 Minutes.. Exactly 60 min after addition of casein solution, taking into account the regular interval adopted, add.. 0 ml of 30%.. trichloroacetic acid (E) to tubes S1a, S1b, S2a, S2b, S3a, S3b and Ta, Tb.. For 30-40 Minutes allow to coagulate fully the precipitated protein at 40 C.. For the Blanks add at first.. trichloroacetic acid (E) to the tubes BTa, BTb, BS1a, BS1b, BS2a, BS2b, BS3a, BS3b.. Mix by shaking and than give as follows:.. of Papain Reference Standard dilution into the tubes BS1a, BS1b,.. of Papain Reference Standard dilution into the tubes BS2a, BS2b and.. of Papain Reference Standard dilution into the tubes BS3a, BS3b.. of Papain Test dilution into the tubes BT1a, BT1b.. Withdraw all the tubes from the water bath and allow to stand at room temperature for 30 min.. Filter the contents of each tube twice through the same suitable filter paper previously washed with 30% trichloroacetic acid (E), then with water and dried.. Measure the absorbance of the clear filtrates at 280 nm against the filtrate of their respective Blanks.. Plot the absorbance reading for S1, S2 and S3 (= E.. -correc.. ) against the enzymes concentration of each corresponding level (mg/ml).. By interpolation from this curve, taking into consideration dilution factors, calculation the potency in the weight of Papain taken by the formula:.. = E.. -Test - E.. -Blank.. mg / ml = { [ (Initial weight of enzyme.. /.. Enzyme volume).. dilution factor].. *.. „ml in Test“} / 10 ml.. Factor = 1 / (mg/ ml).. USP-u/mg for Papain sample = Factor.. [declared Activity of Reference Standards.. (mg/ml).. read from the curve.. ].. Papain BPC 1954 (3000 - 4000 USP units/mg).. Papain 1 x USP 6,000 USP units/mg.. Papain 5 x USP 30,000 USP units/mg.. Papain Concentrate 40,000 USP units/mg.. Papain 2.. 5-3.. 5 FIP-units/mg.. Papain 0.. 5-2.. Glazer, A.. N.. , Smith, E.. L.. 502.. Arnon, R.. in: Methods of Enzymology (G.. 226.. Dubois, T.. , Kleinschmidt, T.. , Schnek, A.. , Looze, Y.. , Braunitzer, G.. , Biol.. Chem.. Hoppe-Seyler.. 369.. (1988) 741.. Merck Index, 11th ed.. , Merck Co.. , Inc.. , Rahway, USA, 1989..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Data Sheet - Pepsin.. Porcine gastric mucosa.. Other name.. Pepsin A.. 9001-75-6.. 23.. Acid proteinases are found in the gastric juice of mammals and have been reported in the juice of birds, amphibia and fishes.. The major enzyme from the pig, pepsin A, is a single polypeptide of 327 residues and is formed by cleavage of 44 residues from the amino terminus of pepsinogen A; one or more of the peptide fragments removed inhibit the activity of pepsin A and other acid proteinases at pH values above 5.. Besides having proteinase and peptidase activity, pepsin can catalyze the hydrolysis of suitable depsipeptids (ester analogues of peptides) and even of sulphite esters.. (1).. Pepsin has a law alpha-helical structure content.. The protein configuration of the crystalline form is that of a compact nearly spherical molecule, the structure being maintained by hydrophobic bonds.. The amino acid analysis shows a high content of aspartic acid, glutamic acid, and serine whereas the content of the basic amino acids is unusually low.. This explains that pepsin even in 0.. 1 N HCl is still a negative ion.. (2).. Specificity.. : Almost all high molecular weight proteins are attacked by Pepsin; exceptions are keratins, mucins, ovomucoid, silk fibroin, spongin, protaminases and protein hydrolysis products of low molecular weight.. Certain chemical substrates of low molecular weight are hydrolysed, particularly peptides containing in close proximity aromatic residues, tyrosine and cysteine, including for instance carbobenzoxy-L-glutamyl-L-tyrosine and L-histidine.. The presence of two carboxyl groups is necessary for pepsin action on synthetic substrates.. If both of the carboxyl groups are masked then pepsin will not split the resulting compound.. Pepsin reacts more slowly than other proteinases on protein substrates and even more slowly on peptides.. Generally, proteolysis by pepsin is greatly favoured if an aromatic ring is present in the side-chain of either of the amino acids on both sides of the bond hydrolyzed.. The amino group may involve a tyrosine or a phenylalanine residue.. When both involved residues are aromatic, these are split even more rapidly than any others.. The optimum pH of activity on proteins is between 1.. 6 and 1.. 8 and is narrower for crystalline pepsin than for a crude preparation.. Below pH 5, the conversion of pepsinogen is effected by pepsin in an autocatalytic process that involves the removal of the 41-residue amino terminal portion of the zymogene.. Several low molecular weight products are formed, including an inhibitory peptide composed of 29 amino acid residues.. This inhibitor, which was obtained in crystalline form, is bound to pepsin at pH 5; at more acid pH values, dissociation of the pepsin-inhibitor complex is favoured, and the peptide is cleaved.. The rate of activation is optimal at pH 2, is first-order with respect to both pepsinogen and pepsin, and is increased by the addition of inorganic salts.. Stability of solution:.. Pepsin is in aqueous solution at pH 5-5.. 5 relative stable.. The stability is higher in Glycerine.. Pepsin is irreversible inactivated in alkaline medium.. freely soluble in water, insoluble in most organic solvents.. 34500 Daltons.. (4).. pH 1.. 0 (3).. Isoionic point.. : 2.. 2-3.. (1%, 1cm) = 14.. 7 (3)..  ...   at 5 ± 0.. 1 C and readjust to pH 1.. 6 before use.. Folin-Ciocalteus-Phenol reagent:.. Merck; Order-N 1.. 09001.. Trichloroacetic acid solution (5%):.. Dissolve 50.. 0 g trichloroacetic acid R (C.. HCl.. ; Mr = 163.. 4 g/mol) in distilled water and dilute to 1000 ml with the same solvent.. Sodium hydroxide Solution (20%).. : Dissolve 20.. 0 g of NaOH in distilled water and dilute to 100 ml with the same solvent.. Immediately before use, prepare a solution of pepsin Reference Standard BRP and of the substance to be examined expected to contain 0.. 5 Ph.. U.. per millilitre in.. ; before dilution to volume, adjust to pH 1.. 1, if necessary , using 1 N HCl.. Pepsin Reference Standard is issued by:.. Or European Pharmacopoeia, Council of Europe; BP 907; 67029 Strasbourg Cedex 1 (France).. Place flasks of.. and.. in the water bath to equilibrate at 25 ± 0.. 1 C.. BS1.. BS2.. BS3.. BT.. ml.. 25.. ml standard.. 75.. ml sample.. ml 5% TCA.. 10.. Equilibrate to 25 C in a water bath 5 Minutes.. ml Haemoglobin.. 25 C.. Mix and incubate at 25 C in a water bath for exactly 10 min.. Remove from water bath after 30 minutes and allow standing at room temperature for 20 min, centrifuge or filtering twice through the same suitable filter paper to remove any insoluble material remaining.. In other tubes with the same name give from the clear solutions.. distilled water.. 20.. +ml NaOH-Solution.. F.. + ml.. After 15 minutes measure the absorbance of solutions at 540 nm in 1 cm cells using the solution of Blank (B) as the compensation liquid.. Correct the average absorbance values for the filtrates obtained from tubes S1, S2, S3 by subtracting the average values obtained for the filtrates from tubes BS1, BS2, BS3 respectively.. Draw a calibration curve of the corrected values against volume of reference solution used.. Determine the activity of the substance to be examined using the corrected absorbance for the test solution (T-BT) together with the calibration curve and taking into account the dilution factors.. Other assays (1, 6).. Anson.. : The rate of hydrolysis of denatured haemoglobin by pepsin is measured by the increase in absorption at 280 nm.. A unit is the enzyme activity, which causes an increase in absorption of 0.. 001 per min.. under the condition of the assay.. (37 C, 10 minutes).. NF XII.. : Pepsin, when assayed under standard condition digests not less than 3000 and not more than 3500 times its weight of coagulated egg albumen.. BP:.. Pepsin, when assayed under standard condition digests 2500 times its weight of coagulated egg albumen.. Pepsin 1:15000 NF.. Pepsin 1:15000 BP.. Pepsin 1:10000 NF.. Pepsin 1:10000 BP.. Pepsin 1:3000 NF.. Pepsin 1:3000 BP.. Pepsin Ph.. Bergmeyer, Methods of enzymatic analysis; third edition, volume V; Verlag Chemie; Weinheim Deerfield, Florida Basel, 1974.. Ruyssen, R.. ; Lauwers, A.. : Pharmaceutical Enzymes, Properties and assay methods, Story-Scientia, Gent 1978.. Enzymes and related biochemicals; Biochemical products devision; Worthington Diagnostic systems, inc.. ; Freehold, New Jersey 07728 U.. S.. ; Printed 1982.. Pharmaceutical Substances; 13th edition; P-Pk; ABDATA Pharma-Daten Service; Eschborn/Taunus; 2003..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Data Sheet - Trypsin.. 9002-07-7.. 4.. Trypsin is a proteolytic enzyme found in many animals and even bacteria.. In vertebrates an inactive precursor, the Trypsinogen, is formed in the pancreas.. By the action of Enterokinase or of Trypsin itself, Trypsinogen is transformed into active Trypsin.. Trypsin hydrolyzes peptides, amides and esters at bonds involving the carboxyl group of L-arginine and L-lysine.. Trypsin acts on numerous synthetic substrates, the best being esters of L-arginine, e.. N-benzoyl-L-arginine ethyl ester.. Proteins are only slowly degraded unless denatured (1).. In the process of Trypsinogen activation Ca.. ions have an accelerating effect and they are also essential for complete activation (1,2,3).. Trypsin is inhibited by a wide variety of substances.. Trypsin is a serine proteinase (EC 3.. 21) and is therefore inhibited by compounds such as phenylmethyl-sulfonylfluoride (PMSF) and diisopropylphosphofluoridate (DFP).. Aromatic and aliphatic amidines and amines are also inhibitory, the strongest low molecular weight competitive inhibitor of Trypsin being p-aminobenzamidine (1,4).. Tosyl-L-lysine chloromethyl ketone (TLCK) irreversibly inhibits Trypsin (but not Chymotrypsin) (1,2,5).. High molecular weight inhibitors of Trypsin comprise polypeptides and proteins from animal origin (aprotinin, a2-macroglobulin, a1-antitrypsin, bovine pancreatic Trypsin inhibitor etc.. , as well as those isolated from plants e.. from soybean, lima bean, and barley (see also: Methods in Enzymology, Vol.. 840-915).. Trypsin acts optimally at pH values between 7 and 9 (1,6).. Trypsin is most stable at pH 3.. At this pH at low temperature it retains its activity for weeks (1,2).. Autolysis of Trypsin at pH values well above 3 can be retarded by the addition of calcium ions.. This protective effect is much more pronounced with bovine Trypsin than with porcine Trypsin (2).. Heat denaturation is dependent on the pH: below pH 8 an increased temperature leads to reversible denaturation, but above pH 8 a temperature rise induces irreversible denaturation (2,3).. Precipitation with trichloroacetic acid or the presence of high concentrations of urea (8 M) leads to a reversible loss of Trypsin activity (1).. Lyophilized Trypsin, when stored cool and dry in the dark, is stable almost indefinitely (7).. Trypsin is soluble in water and in isotonic saline solution.. 23,500 (4).. Bovine Trypsinogen consists of a single polypeptide chain with 229 amino acid residues stabilized by 6 disulfide bridges.. Upon activation, a hexapeptide (Val-(Asp)4-Lys-) is split off from the amino terminus leading to the formation of b-Trypsin, the first active enzyme form.. Sequential cleavage at two other sites leads to a-Trypsin, and Pseudotrypsin, respectively, both of which are also active forms of Trypsin (1,2,3).. 8 (bovine and porcine Trypsin) (1,3).. (1%, 1cm) = 15.. 4 (1).. The method described here is the one given in the monography on Trypsin in the Ph.. is identical to the method given by FIP (Fédération International Pharmaceutique; see 3) which originally developed this assay method.. Trypsin hydrolyzes the synthetic substrate N-benzoyl-L-arginine ethyl ester (BAEE).. The activity of Trypsin is determined by comparing the rate at which it hydrolyzes benzoylarginine ethyl ester with the rate at which Trypsin BRP hydrolyzes the same substrate under the same conditions.. BAEE solution (0.. 02 M):.. 686 % (m/V).. 1714 g of N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE x HCl) in distilled water and dilute to 25.. Borate buffer solution  ...   katal/mg = 30 FIP units/mg.. In the assay for Trypsin activity according to USP 29 the rate of hydrolysis of BAEE at pH 7.. 6 is measured spectrophotometrically at 253 nm.. Assay (according to USP-Monograph).. :.. One USP Trypsin Unit is the activity which hydrolses 1 µmol Na- Benzoyl-L-argininethylesterhydrochlorid (= BAEE*HCl) such that causing a change in absorbance of 0.. 003 per minute under the conditions specified in this assay at 253 nm.. : Trypsin Reference Standard USP, the Samples and BAEE*HCl (= Na- Benzoyl-L-argininethylesterhydrochlorid) from 4 C to Room temperature.. O add.. to 1000 ml.. BAEE-Solution:.. 85.. 7 mg BAEE*HCl (M = 342.. 83 g/mol, Calbiochem 2645-08-1) + pure H.. O add to 100.. 067 M KH2PO4-Solution:.. 09 g/mol, MERCK 4873) + pure H.. O add to 500 ml.. 4,73 g Na.. (M = 141,96 g/mol, MERCK 6586) + pure H.. 60:.. Give to 200 ml Na.. -Solution an amount of KH.. -Solution until it adjust a pH of 7.. Weight an amount of Enzyme Sample or Standard that solved and diluted in 1 mM HCl-Solution give a change of absorbance (=.. A/min) of.. maximal 0.. 02.. in.. „ml in Test“ of 0.. Formular.. Initial weight (mg) =.. [(5.. 0 USP-U / “0.. 200“ ml in Test) * dilution factor * Enzyme solution].. Declared activity of enzyme.. Because in the most of cases the product is a mixture of Trypsin and Chymotrypsin the initial weight should be chosen such that you can use the same Enzyme solution for determining the activity of Trypsin and Chymotrypsin with a.. For each 90 ml of Phosphat-Buffer use 10 ml BAEE-Solution (1:10).. The Absorbance should be at 253 nm 0.. 575 - 0.. 585.. If necessary add more Phosphat-Buffer or BAEE-Solution.. Put the amount Phosphate-Buffer-Solution in Bath thermostat to adjust the temperature to 25 C.. Shortly before test begin give the necessary amount of BAEE-Solution.. The Solution at 25 C is stable.. for 2 h.. Adjust the UV-Spectral photometer such that the change of absorbance for each reaction in the cuvettes can be reading.. every 30 seconds for 5 minutes.. Calibrate the UV-Spectral photometer at 253 nm using pure H.. Blank:.. Give in a cuvette 200 l of 1 mM HCl-Solution and 3 ml of.. Phosphate-Buffer-Solution and mix.. Calibrate the UV-Spectral photometer with this solution.. After calibration give in the cuvette e.. 100 l of Enzyme dilution, 100 l of 1 mM HCl-Solution and 3 ml of.. Phosphate-Buffer-Solution.. Mix immediately put in UV-Spectral photometer and start reading the absorbance.. Is the Change of Absorbance (= Δ A/min) higher than 0.. 009 then change for the next measurement the amount of “.. l in Test” of 80-200 l.. Print the.. Rates.. of absorbance and the tabulate of „.. Δ A/min“.. Measure each Enzyme dilution.. 4 times.. Calculate the average of.. Δ A/min.. of each Enzyme-Solution.. Factor =.. {[(Initial weight (mg).. /Enzyme solution (ml))/.. (dilution factor)]*(ml in Test)]} * 0.. 003.. = [Δ A/min]* Factor.. =.. ) / declared activity of reference Standard.. ) /.. Trypsin 2500 USP units/mg.. Trypsin 3200 USP units/mg.. Trypsin 4500 USP units/mg.. Trypsin Ph.. (0.. 5 katal/mg).. Trypsin 30 FIP units/mg.. Keil, B.. 250.. 41.. Springer, New York-Heidelberg-Tokyo, 1985.. Steinkopff Verlag Darmstadt 1988..

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  • Title: BIOZYM Gesellschaft für Enzymtechnologie mbH
    Descriptive info: Data Sheet - Trypsin Chymotrypsin Mixture.. Peptidyl peptide hydrolase.. Chymotrypsin is a proteolytic enzyme which has been isolated from many vertebrates.. It is synthesized in the pancreas in the form of an inactive precursor or zymogen, named Chymotrypsinogen.. This precursor is transformed into the active enzyme by Trypsin and by Chymotrypsin itself.. Unless otherwise specified, this paragraph will deal with literature data on bovine Chymotrypsin Aα.. ions have been shown to enhance the activity of Chymotrypsin and also to stabilize the enzyme against denaturation (3,4).. Heavy metals like Cu.. and Hg.. Among the most important high molecular weight inhibitors are proteins like a1-antichymotrypsin, a1- proteinase inhibitor (formerly: a1-antitrypsin), a2-macroglobulin, aprotinin (pancreatic Trypsin inhibitor, Kunitz inhibitor), soybean Trypsin inhibitor and Ascaris inhibitors (3,5; see also: Methods in Enzymology, Vol.. The enzyme is stabilized by Ca.. Chymotrypsin Aa arises from the inactive single-chain precursor Chymotrypsinogen A by the successive cleavage of 4 peptide bonds with a concomitant removal of 2 dipeptides, Ser14-Arg15 and Thr147-Asn148.. Isoelectric point:.. High molecular  ...   also active forms of Trypsin (1,2,3).. Assay (Activity of.. according to current USP-Monograph).. 067 M KH.. -Solution:.. 067 M Na.. Give to 200 ml KH2PO4-Solution the amount of Na.. For Trypsin- und Chymotrypsin-Mixture, make an initial weight of the Sample that the Try- and Chy-Activity can be determined according to USP from the same Enzyme Solution (50 ml) with a „ml in Test“ of 0.. Calibrate the UV-spectral photometer at 237 nm with pure H2O.. Print the Rates of absorbance and the tabulate of „Δ A/min“.. {[(Initial weight in mg/Enzyme Solution in ml)/(Dilution factor)]*(ml in Test)} * 0.. Δ A/min) of.. 0-10.. 0 USP-U/“0.. 200“ ml in Test)*dilution factor*Enzyme solution].. Calibrate the UV-Spectral photometer at 253 nm using pure H2O.. Is the Change of Absorbance (=.. Δ.. A/min) higher than 0.. of absorbance and the tabulate of „Δ.. A/min“.. Clear the cuvette for each using with pure H2O.. Calculate the average of Δ.. A/min.. ) = r.. Chymotrypsin 1000/1000.. Trypsin 2400:400.. Trypsin 900:150.. Trypsin 1200:300.. Trypsin 800:330..

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