Process development for the enzymatic hydrolysis of food protein: Effects of pre-treatment and post-treatments on degree of hydrolysis and other product characteristics

An enzymatic process was developed to produce protein hydrolysate from defatted soya protein. Various unit operations were tried, and the effects of pre- and post-treatments on the product characteristics such as degree of hydrolysis (DH), free amino acid content (%FAA) and average molecular weight (MW) were investigated. The use of acid washes showed no difference in %DH. Increasing pH during pre-cooking gave lower %DH. Alkaline cooking made too much insoluble protein, thus the protein yield was too small. A better hydrolysis with more acceptable taste was obtained when the combination of Neutrase/Alcalase/Flavourzyme was used in place of Alcalase/Flavourzyme combination. Untoasted defatted soya was more effective on the proteolysis than toasted one. The MW of the evaporated and spray dried product was higher than that of undried product, due to precipitation of low-solubility components. When the product separation was carried out by ultrafiltration and the product concentration by reverse osmosis, the solubility and the taste of the product were improved. The difference between enzyme hydrolysate and acid hydrolysate was significant in free amino acid composition, especially in tyrosine, phenylalanine, glutamine and asparagine.     

Continuous hydrolysis of goat whey in an ultrafiltration reactor: generation of alpha-lactorphin

Bovine whey hydrolysate has been developed and applied to areas such as nutrition, culture media, and isolation of bioactive peptides. In order to produce such a type of hydrolysate, it is possible to use goat whey, which constitutes also a food processing by-product. Enzymatic hydrolysis of goat whey by pepsin was carried out in a continuous ultrafiltration reactor. The permeate contained peptide hydrolysate that was resolved by RPHPLC. Second order derivative spectroscopy, amino acid analysis, and mass spectrometry revealed the presence of a biologically active peptide called alpha-lactorphin. This constitutes preliminary information about goat whey enzymatic degradation for future applications.     

Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten

Hydrolysate of extruded corn gluten with higher solubility and antioxidative property was prepared. Extrusion and starch removal of corn gluten were applied as pretreatment before enzymatic hydrolysis by Alcalase. The amylase hydrolysis of starch at 70°C for 3 h resulted in the removal of the starch from the extruded corn gluten. The best hydrolysis results can be obtained by conducting the hydrolysis at 60°C with water addition 20 g/g protein, enzyme addition 0.048 Ansen units/g protein, pH 8.5, and 120 min. Degree of hydrolysis of extruded and nonextruded corn gluten reached 39.54 and 31.16%, respectively, under the optimal condition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the optimal hydrolysate revealed that proteolysis of extruded corn gluten was more extensive than proteolysis of its counterpart which was not subjected to extrusion. The molecular weight of the peptides in the optimal hydrolysate was mainly over 3,710–660 Da as determined by gel filtration chromatography. The hydrolysates displayed good solubility and antioxidative activity. The separation profile of the hydrolysate on an ion exchange chromatography of Q-Sepharose Fast Flow showed that many kinds of peptides had antioxidative effect. A new peptide with antioxidative activity was purified, and its amino acid sequence was Phe-Pro-Leu-Glu-Met-Met-Pro-Phe, which was identified by Q-TOF2 mass spectrometry.     

一种米曲霉蛋白酶的酶学性质及其在酪蛋白磷酸肽制备中的应用

【目的】分离纯化米曲霉蛋白酶的主要组分,分析其酶学特性,并应用于酪蛋白磷酸肽(Casein phosphopeptides,CPPs)的制备。【方法】采用硫酸铵盐析、DEAE-Sepharose FF阴离子交换层析和Butyl-sepharose HP疏水层析对米曲霉蛋白酶进行分离纯化,SDS-PAGE检测分子量与纯度,MALDI-TOF-MS检测酶切位点。【结果】得到一种蛋白酶组分(命名为PE),分子量大小为58 kD左右。该酶最适反应条件为55 °C,pH 8.0,酶活被Fe3+抑制,被Mn2+激活。以酪蛋白为底物时,Km=0.36 g/L,最大反应速率Vm=18.18 mg/(L?min)。蛋白酶PE对牛胰岛素B链上-Leu-Cys-、-Val-Glu-、-Tyr-Leu-和-Arg-Gly-组成的肽键有较高的切割能力,酶切位点较多。利用其水解酪蛋白,通过钡-乙醇沉淀法得到CPPs,产率为15.87%,摩尔氮磷比r (N/P)为6.17,得到的CPPs可以使钙沉淀推迟35 min。【结论】利用米曲霉蛋白酶水解酪蛋白产生CPPs,为其在功能性食品加工方面的应用提供有利的参考。     

Purification and evaluation of a novel antioxidant peptide from corn protein hydrolysate

In the present study, corn protein hydrolysate (CPH) with antioxidant activity was obtained by enzymatic hydrolysis. Corn gluten meal (CGM) was hydrolyzed using two proteases (Alcalase and Protamex) to produce the antioxidant peptide. Extrusion and starch removal of corn protein were used as pretreatment procedures before proteolysis. Hydrolysis by Alcalase has more remarkable digesting efficiency on corn protein than that by Protamex. Therefore, the hydrolysate catalyzed by Alcalase was fractionated by ultrafiltration, and peptide with the highest antioxidant activity was purified from <6 kDa molecular weight fraction. The amino acid sequence of the novel peptide was Gln-Gln-Pro-Gln-Pro-Trp as identified by a quadrupole time-of-flight mass spectrometer (Q-TOF2), with molecular weight of 782.34 Da, which was matched to γ-zein f (50–55). The new peptide was further synthesized by Fmoc solid-phase method. It showed scavenging activity against DPPH, ABTS, and hydroxyl free radicals in dose dependent manner with EC₅₀ values of 0.95, 0.0112 and 4.43 mg/mL, respectively. It also exhibited notable reducing power of 0.54 at 2.0 mg/mL, but showed weaker Fe²⁺-chelating capacity with EC₅₀ value of 6.27 mg/mL. These results suggest that the hexapeptide is a potential natural antioxidant that can be used as drug or functional food ingredient.     

The occurence of a neutral protease and its inhibitor in rat peritoneal macrophages.

The lysate of the glycogen-induced macrophages in rat peritoneal exudate was fractionated by centrifugation and extraction into a water extract, 1 M KCl extract and residue fractions. Approximately 50% of the neutral protease activity toward casein in the lysate was recovered in the KCl extract fraction, which was practically devoid of acid protease, cathepsin D. The pH optimum of the neutral protease toward casein and urea-denatured hemoglobin was pH 8.5. The activity was inhibited strongly by DFP or chymostatin and only partially by HgCl2 or PCMB. Addition of a salt to the reaction medium caused enhancement of the activity with an optimum concentration of 0.25 M: KCl, KBr, KI, NaCl, NaBr, NaI, and MgCl2 were all almost equally effective. When the enzyme preparation was filtered through a column of Sephadex G-75 gel in the presence of 1 M KCl, a larger molecular weight fraction at the void volume was obtained in addition to a smaller molecular weight fraction showing a caseinolytic activity insensitive to KCl concentration. The former was found to have a specific inhibitory effect on the latter activity.     

GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Optimization of Enzymatic Hydrolysis Conditions of Caspian kutum (Rutilus frisii kutum)ˮ By-product for Production of Bioactive Peptides with Antioxidative Properties

The enzymatic hydrolysis was performed by Alcalase to recover the fish protein hydrolysate from Caspian kutum by-product (CB). The degree of hydrolysis (DH) was applied for monitoring the hydrolysis reaction of CB. The response surface methodology was applied based on a D-optimal design to perform the optimization process for obtaining the high yield of CB protein hydrolysate. The effect of four independent variables including pH (7.5–8.5), temperature (45–55 °C), time (1–3 h), and enzyme concentration (0.5–1.5% w/w) on DH was studied. The results indicated that the predicted and actual values of the optimum condition had no significant difference. The optimum enzymatic hydrolysis conditions were achieved at pH 8.5, temperature of 55 °C, enzyme concentration of 1.5% w/w, and time of 3 h, which resulted in the maximum value of DH (19.08%). Antioxidant assays including DPPH scavenging and metal chelating activities showed that Caspian kutum protein hydrolysates had antioxidant properties.

     

The nature of the unhydrolysed fraction of alpha-globulin, the major protein component of Sesamum indicum L. hydrolysed by alpha-chymotrypsin.

Alpha-globulin, the high molecular weight protein fraction from sesame (Sesamum indicum L.) seed, was hydrolysed by alpha-chymotrypsin. The hydrolysate was resolved into two fractions, the hydrolysed part and the unhydrolysed part of alpha-globulin using gel filtration on Sepharose 6B-100. The unhydrolysed alpha-globulin residue was characterized for its sedimentation coefficient, subunit composition, fluorescence emission spectrum, secondary structure, and other biophysical properties. The results indicated a decrease in the size of the protein molecule upon hydrolysis to a very small extent. The effect of hydrolysis products on hydrolysis of native alpha-globulin as well as on a standard substrate, casein, was also investigated. The results indicated that the hydrolysis products contribute to the resistance of alpha-globulin to proteolysis by alpha-chymotrypsin to the extent of 40%.     

Alcalase rapeseed inhibitors: purification and partial characterization

Extensive rapeseed protein hydrolysate obtained sequentially with Alcalase and Flavourzyme showed inhibitory activity towards Alcalase. Inhibitory activity decreased as the hydrolytic process progressed probably by heat denaturation and/or partial protease degradation. Alcalase rapeseed inhibitors were purified by gel filtration and subsequent ion exchange chromatography. They are composed of peptides of 8.4 and 6.1 kDa linked by interchain disulphide bonds, as observed by reducing SDS-PAGE, with a native molecular weight of 18 kDa. Aminoacid composition of the inhibitors was characterized by the high proportion of methionine (4.2%) and cysteine (4.6%). Alcalase inhibitors were partially resistant to heat treatment; after heating at 70 degrees C for 45 minutes more than 50% of the original inhibitory activity remained in the purified protein but after heating at 90 degrees C for 5 minutes, inhibitory activity decreased very fast to a basal level. The possible relation of these protease inhibitors with the 2S albumin storage proteins is discussed.     

Reticulocyte lipoxygenase, ingensin, and ATP-dependent proteolysis

Lipoxygenase purified from rabbit reticulocyte lysate has a molecular mass of 68 kDa on SDS gel and a pI of 5.97. Lipoxygenase is inhibited by nordihydroguaiaretic acid (NDGA), 3-amino-1-(m-(trifluoromethyl)phenyl)-2-pyrazoline (BW755C), 5,8,11,14-eicosatetraynoic acid (ETYA), salicylhydroxamate (SHAM) or hemin. Metal ions or nucleotides do not affect its activity. The addition of certain of these inhibitors to the reticulocyte extract also inhibited the ATP-dependent proteolysis of casein, one of the distinct characteristics of reticulocytes. No clear correlation between lipoxygenase activity and ATP-dependent proteolysis could be detected. Hemin and NDGA inhibited both processes, but the concentrations necessary for inhibition were quite different. SHAM completely inhibited lipoxygenase, but not proteolysis. o-Phenanthroline inhibited ATP-dependent proteolysis, but had no effect on lipoxygenase activity. We have also purified a high-molecular-mass protease, ingensin, from reticulocyte extract. This protease accounted for more than 90% of the casein-degrading activity in reticulocyte extract. NDGA inhibited ingensin at the same concentrations required for inhibition of ATP-dependent proteolysis. These results suggest that lipoxygenase is not indispensable for the ATP-dependent proteolysis and the novel high-molecular-mass protease, ingensin, may be involved in the process.     

Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry

The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phasoleus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 microg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule-glucosamine and N-acetylglucosamine--exhibited no antiviral activity.     

Phosphorylation and proteolytic degradation of nucleolin from 3T3-F442A cells

The effect of phosphorylation on the proteolysis of nucleolin has been investigated. Nucleolin is readily phosphorylated both in vitro and in vivo. Utilizing phosphorylation assays and immunoblotting with anti-nucleolin serum, we have observed that phosphorylation enhances nucleolin as a substrate for a protease. This protease activity cleaves the protein into a highly phosphorylated 30 kDa peptide and a 72 kDa peptide. The involvement of casein kinase II is suggested since this cleavage is promoted by spermine and inhibited by heparin, which are, respectively, a stimulator and an inhibitor of casein kinase II activity. The molecular identity of the protease and the physiologic significance of the proteolytic cleavage of nucleolin remain to be studied.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

Biochemical characterization of a halophilic,alkalithermophilic protease from <Emphasis Type="Italic">Alkalibacillus</Emphasis> sp. NM-Da2

An extracellular, halophilic, alkalithermophilic serine protease from the halo-alkaliphilic Alkalibacillus sp. NM-Da2 was purified to homogeneity by ethanol precipitation and anion-exchange chromatography. The purified protease was a monomeric enzyme with an approximate molecular mass of 35 kDa and exhibited maximal activity at 2.7 M NaCl, pH^55 °C 9 and 56 °C. The protease showed great temperature stability, retaining greater than 80 % of initial activity after 2 h incubation at 55 °C. The protease was also extremely pH tolerant, retaining 80 % of initial activity at pH^55 °C 10.5 after 30 min incubation. Protease hydrolyzed complex substrates, displaying activity on yeast extract, tryptone, casein, gelatin and peptone. Protease activity was inhibited at casein concentrations greater than 1.2 mg/mL. The enzyme was stable and active in 40 % (v/v) solutions of isopropanol, ethanol and benzene and was stable in the presence of the polysorbate surfactant Tween 80. Activity was stimulated with the oxidizing agent hydrogen peroxide. Inhibition with phenyl methylsulfonylfluoride indicates it is a serine protease. Synthetic saline wastewater treated with the protease showed 50 % protein removal after 5 h. Being halophilic, alkaliphilic and thermophilic, in addition to being resistant to organic solvents, this protease has potential for various applications in biotechnological and pharmaceutical industries.     

Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits

In addition to protease La (the lon gene product), Escherichia coli contains another ATP-dependent protease, Ti. This enzyme (approximately 340 kDa) is composed of two components, both of which are required for proteolysis. Both have been purified to homogeneity by conventional procedures using [3H]casein as the substrate. The ATP-stabilized component, A, has a subunit molecular weight of 80,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate, but it behaves as a dimer (140 kDa) upon gel filtration. Component P, which is relatively heat stable, is inactivated by diisopropyl fluorophosphate and can be labeled with [3H] diisopropyl fluorophosphate. It has a subunit size of 23 kDa, but the isolated component behaves as a complex (260 kDa) of 10-12 subunits. The isoelectric point of component A is 7.0 and that of P is 8.2, and their amino acid compositions differ considerably. The purified enzyme has an ATPase activity that is stimulated 2-4-fold by casein and other protein substrates but not by nonhydrolyzed proteins. Component A also shows ATPase activity which can be stimulated by casein. Addition of component P (which lacks ATPase activity) inhibits basal ATP hydrolysis by A and makes this ATPase more responsive to casein. Although component P contains the serine active site for proteolysis, it shows no proteolytic activity in the absence of component A, Mg2+, and ATP or dATP. Other nucleoside triphosphates are not hydrolyzed and do not support proteolysis. Protease Ti has a Km for ATP of 210 microM for hydrolysis of both casein and ATP. Casein increases the Vmax for ATP without affecting the Km. A Mg2+ concentration of 5 mM is necessary for half-maximal rates of ATP and casein hydrolysis. Ca2+ and Mn2+ partially support these activities. Thus, protease Ti shares many unusual properties with protease La (e.g. coupled ATP and protein hydrolysis and protein-activated ATPase), but these functions in protease Ti are associated with distinct subunits that modify each other's activities.     

Proteolytic Activities of the Starch-Fermenting Ruminal Bacterium, Streptococcus bovis

The objective of this study was to characterize the extracellular proteolytic activity of Streptococcus bovis. Strains KEG, JB1, NCFB 2476, and K11.21.09.6C produced very similar large molecular weight (160–200 kDa) extracellular proteases that were specifically inhibited by PMSF, a serine protease inhibitor. Further experiments with S. bovis KEG indicated that cultures grown with casein as the sole added N source produced the greatest level of proteolytic activity, and the level of proteolytic activity was independent of growth rate. Clarified ruminal fluid (CRF) decreased proteolytic activity by 54% compared with cultures grown with casein alone, and addition of exogenous peptides and carbohydrates (CHO) to the CRF further reduced the level of proteolytic activity by 44% and 52%, respectively. These results suggested that the proteolytic activity of S. bovis KEG was modulated by available N source and that the proteolytic activity was present for reasons other than providing N for growth. The role of S. bovis in ruminal proteolysis requires further definition, but phenotypic similarity among some ruminal strains would suggest a common niche in ruminal proteolysis. The uniformity of proteolytic activities could make S. bovis a prime candidate for manipulation in ruminal proteolysis control strategies. Received: 12 January 1999 / Accepted: 19 May 1999     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.