Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties

Three gastric proteases were isolated from the stomach mucosa of the Greenland cod (Gadus ogac). The cod proteases were all less stable to heating and protease 1 retained less activity at 5 degrees C when the pH was greater than 5 in comparison with porcine pepsin. The activities of cod proteases 1 and 2, with hemoglobin as the substrate, were doubled in the presence of 25 mM NaCl, while cod protease 3 and porcine pepsin were not stimulated by the salt. The cod proteases did not cross-react with antibodies raised against porcine pepsin. However, some cross-reactivity was noted with antibodies raised against proteases from psychotrophic pseudomonads. The molecular weights of all the cod proteases were in the range of 36,000-38,000. The amino acid compositions of the cod proteases as compared by the Metzger difference index differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin, and chymosin differ from each other. Of the cod enzymes, protease 1 differed from mammalian gastric proteases, while cod proteases 3 was more like chymosin with respect to amino acid composition. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod proteases 2 and 3 were intermediate between that of porcine pepsin and bovine chymosin. It is suggested that the Greenland cod proteases represent less differentiated forms of gastric proteases than the mammalian pepsins, gastricsins, and chymosins.     

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.     

微小毛霉凝乳酶的分离纯化研究

研究微小毛霉(HL-1)凝乳酶的分离纯化条件及方法。研究酶的最适浸提温度、酶的浸提pH值和最适浸提时间,探讨离子浓度、加水量对浸提效率的影响,利用高速冷冻离心法、有机溶剂沉淀法,膜分离法和层析法等对粗酶液进行了分离。利用光谱法对纯化样品进行检测。酶的最适浸提温度为30℃;最适pH为6.0;浸提10 h活力最高;1%的氯化钠有利于酶的分离,加水比例为15时有利于提取,在10 000 r/min下离心10min澄清效果最好,95%的酒精沉淀效果最好,利用0.2μm的微滤膜可除去发酵液中的菌体,8 000的超滤膜可拦截凝乳酶蛋白,S300的填料可有效分离凝乳酶,纯度达95%以上。     

Effects of glycosylation on the secretion and enzyme activity of Mucor rennin, an aspartic proteinase of Mucor pusillus, produced by recombinant yeast

The Mucor rennin gene encoding a prepro form of the fungal aspartic proteinase from Mucor pusillus was expressed under the control of the yeast GAL7 promoter in Saccharomyces cerevisiae. The mature M. pusillus rennin secreted efficiently by yeast was a highly glycosylated protein. Analysis by a combination of site-directed mutagenesis of each of the three possible glycosylation sites and treatment of the secreted M. pusillus rennins with endo-beta-N-acetylglucosaminidase H revealed that the mature yeast M. pusillus rennin contained two asparagine-linked glycosylation sites among the three possible glycosylation sites. A mutation of the 2 glycosylated asparagine residues of M. pusillus rennin resulted in significant decreases in the level of secretion by yeast cells. In addition, the extent of glycosylation of M. pusillus rennin was found to affect the enzyme properties such as milk-clotting and proteolytic activities.     

Purification and characterization of milk clotting enzyme from goat (Capra hircus)

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 kappa-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 degrees C with maximum activity at 30 degrees 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 degrees C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.     

Detection of the cholera toxin-binding activity of kappa-casein macropeptide and optimization of its production by the response surface methodology

The cholera toxin (CT)-binding activity of purified kappa-casein macropeptide (CMP) from bovine kappa-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of kappa-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5 degrees C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.     

Isolation and partial characterization of prochymosin and chymosin from cat

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.     

Kinetic study of the action of bovine chymosin and pepsin A on bovine kappa-casein

A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine kappa0-casein (carbohydrate-free fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 degrees C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106-169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: kcat = 68.5 s-1, Km = 0.048 mM, kcat/Km = 1,413 mM-1 s-1 for chymosin, and kcat = 45 s-1, Km = 0.018 mM, kcat/Km = 2,439 mM-1 s-1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 degrees C, using fragment 98-111 of kappa-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of kappa-casein (our conditions) significantly affect the action of chymosin on fragment 98-111, which seems to contain all information that makes bond 105-106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103-108 appears to be required.     

Extracellular Proteases of Mucor pusillus

Mucor pusillus was grown in different media for a period of 92 h, and the media were investigated for both milk-clotting and protease activities. It was observed that the ratio of extracellular milk-clotting activity to protease activity was the highest for 3% corn steep liquor containing 1% glucose as the source of carbon. Variation of both milk-clotting and protease activities was studied during the growth of the organism in the medium stated above. Separation of protease was carried out by ion-exchange chromatography at pH 8.0. Fractions collected were assayed for both activities simultaneously. The findings suggested that, instead of only one major acid protease, as reported by previous workers, two major acid proteases were produced. One of them had significant rennin-like activity, and the other lacked it. The former could be assumed to be the enzyme reported and studied by previous workers. The existence of two proteases was further confirmed by the appearance of two protease activity bands on polyacrylamide gels after electrophoresis. An attempt was made to separate the rennin-like enzyme from nonspecific protease activity by ammonium sulfate fractionation followed by ion-exchange chromatography at pH 6.0. The results indicated that the nonspecific protease activity due to the enzyme that lacked rennin action was substantially removed by the ammonium sulfate fractionation.     

Intranuclear growth of Rickettsia rickettsii.

The acid protease synthetized by Mucor pusillus in chemically defined medium displayed a gel filtration pattern and optimal pH similar to that shown by protease isolated from complex medium. Protease synthesis was influenced by the concentration of sulfur in the medium. At sulfur concentrations greater than 10(-4)m, protease synthesis was suppressed, indicating a controlling role of sulfur compounds in the synthesis of extracellular protein. Inorganic phosphate was found to be without negative effect on enzyme synthesis. Growth studies showed that M. pusillus had the capacity to utilize ammonium N, nitrate N, amino N, and urea.     

Bovine chymosin: production by rDNA technology and application in cheese manufacture

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.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties

AIMS: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance. METHODS AND RESULTS: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l-1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4.7H2O, 0.1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60 degrees C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases. CONCLUSIONS: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.     

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose.

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5-8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65000 and 52000 were isolated, the 65000 molecular weight species apparently representing a protomer of concanavalin A (24000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity. At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including alpha-methyl D-glucoside and NaC1 in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 degrees C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor renin did not react with concanavalin A and were not bound to the affinity column.     

Purification and Properties of Mucor pusillus Acid Protease

The protease produced by Mucor pusillus was recovered from a wheat bran medium by treatment with ammonium sulfate, ethyl alcohol, gel filtration and ion-exchange chromatography. The yield of the enzyme was 55%. The overall increase in the specific activity of the protease was 34-fold. The purified protease was most active at pH 3.8 and 5.6 against hemoglobin and casein, respectively. Optimal hydrolysis of casein was observed at 55 C. The enzyme was stable from pH 3.0 to 6.0. Enzyme inactivated by metal ions was reactivated by ethylenediaminetetraacetate and o-phenanthroline. Reducing agents and thiol poisons had no effect on the protease, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inhibit the protease, indicating the probable absence of serine in the active center. The Michaelis-Menten constant for casein was 0.357%. Electrophoretic analysis of active protein recovered by ion-exchange chromatography showed that the protease preparation was homogeneous.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21,000 and 36,000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10-12 (protease I) and pH 10.5 (protease II). The optimum temperture for the activity of protease I was 70 degrees C and that for protease II was 60 degrees C. Protease I was stable in the range of pH 4.0-8.0 up to 60 degrees C and protease II was stable in the range of pH 6.0-12.0 up to 50 degrees C.     

Protein chemical characterization of Mucor pusillus aspartic proteinase. Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment

The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.     

Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70 degrees C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes.     

Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk

Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in kappa-casein by an aspartic acid protease. Bovine chymosin is the preferred enzyme, combining a strong clotting activity with a low general proteolytic activity. In the present study, we report expression and enzymatic properties of recombinant camel chymosin expressed in Aspergillus niger. Camel chymosin was shown to have different characteristics than bovine chymosin. Camel chymosin exhibits a 70% higher clotting activity for bovine milk and has only 20% of the unspecific protease activity for bovine chymosin. This results in a sevenfold higher ratio of clotting to general proteolytic activity. The enzyme is more thermostable than bovine chymosin. Kinetic analysis showed that half-saturation is achieved with less than 50% of the substrate required for bovine chymosin and turnover rates are lower. While raw camel milk cannot be clotted with bovine chymosin, a high clotting activity was found with camel chymosin.     

毛霉的产蛋白酶发酵条件优化

从发酵豆制品中分离出1株产蛋白酶性质优良的毛霉M2, 并研究M2菌株的产蛋白酶条件。研究优化得出该毛霉菌株的产酶培养基条件为：氮源为大豆分离蛋白, 碳源为葡萄糖, 无机盐为磷酸二氢钾、氯化钙和氯化镁; 它的适宜的产酶发酵条件为：培养温度为28°C, 接种量为2%, 300 mL三角瓶的装液量为100 mL, 初始pH为5, 摇床转速为150 r/min, 培养时间为48 h; 发酵液蛋白酶酶活力可达4.35 U/mL。凝胶电泳法测定出毛霉所分泌蛋白酶的分子量为36.4 kD。