首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with alpha-, beta-, or kappa-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.  相似文献   

2.
Proteins of the isolated brush border membrane of Hymenolepis diminuta were hydrolyzed in vitro by chymotrypsin, papain, pepsin, subtilopeptidase A (= subtilisin Carlsberg), and trypsin. Neither proteolytic nor amidase activity was demonstrable in the isolated membrane using proteinaceous (casein and hemoglobin) or chromogenic (benzoyl-arginine-p-nitroanilide and succinyl-alanyl-alanyl-propyl-phenylalanine p-nitroanilide) substrates, and the membrane preparation did not inhibit the proteolytic and amidase activities of these enzymes. Thus, the isolated tegumental membrane of H. diminuta is not inherently resistant to the action of proteolytic enzymes, and it does not inhibit proteolytic activity. In control incubations containing only buffer, the alkaline phosphatase activity of the brush border membrane decreased in a time dependent manner, but in the presence of chymotrypsin, subtilopeptidase A, and trypsin, the membrane retained greater alkaline phosphatase activity (pepsin and papain could not be tested for this effect on alkaline phosphatase activity). A similar time dependent decrease in activity was also noted for each of the proteolytic enzymes in control assays, but subtilopeptidase A and papain retained greater activity in the presence of the isolated membrane preparation when these assays were compared to controls.  相似文献   

3.
Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with α-, β-, or κ-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.  相似文献   

4.
The flowers of cardoon (genus Cynara) are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynara cardunculus L. was characterized in terms of enzymic composition and proteolytic specificity of its proteinases (cardosin A and cardosin B). Cardosin A was found to cleave insulin B chain at the bonds Leu15-Tyr16, Leu17-Val18 and Phe25-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glu13-Ala14, Ala14-Leu15 and Phe24-Phe25 indicating that it has a broader specificity. The kinetic parameters for the hydrolysis of the synthetic peptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-oMe were also determined and compared to those of chymosin and pepsin. The results obtained indicate that in terms of specificity and kinetic parameters cardosin A is similar to chymosin whereas cardosin B is similar to pepsin. It appears therefore that the enzyme composition of cardoon rennet closely resembles that of calf rennet.  相似文献   

5.
A method has been described for the isolation and crystallization of swine pepsin inhibitor from swine pepsinogen. Solubility experiments and fractional recrystallization show no drift in specific activity. The reversible combination of pepsin with the inhibitor was found to obey the mass law. The inhibitor is quite specific, failing to act on other proteolytic and milk clotting enzymes. The inhibitor is destroyed by pepsin at pH 3.5. Chemical and physical studies indicate that the inhibitor is a polypeptide of approximately 5,000 molecular weight with an isoelectric point at pH 3.7. It contains arginine, tyrosine, but no tryptophane and has basic groups in its structure.  相似文献   

6.
Milk-clotting enzymes such as pepsin, chymosin, chymotrypsin, and M. miehei proteases were immobilized on porous, alkylamine glass and incorporated into a fluidized-bed continuous coagulation scheme. Only pepsin and calf rennet retained sufficient activity towards skim milk to warrant further studies. Comparison of kinetic data with fixed-bed reactors revealed the overall superior performance of fluidized beds; higher clotting activities were possible while avoiding plugging problems and high pressure drops common to fixed-bed reactors. Film diffusion and catalyst back-mixing appear to be significant factors in the overall kinetics. All enzymes lost activity on exposure to skim milk. The inactivation rates were lower at high substrate pH and insignificantly affected by reactor temperature. Nitrogen and sialic acid accumulation on the porous glass paralleled the loss in activity in the initial stages. Attempts to regenerate the immobilized enzymes were partially successful.  相似文献   

7.
Immunochemical Studies on the Components of the Pepsinogen System   总被引:3,自引:0,他引:3       下载免费PDF全文
Rabbit antisera to pepsin and pepsinogen were characterized by several immunological criteria. Both antisera inhibited the rennet activity of pepsin. Antipepsinogen protected pepsin from alkaline denaturation. Using antipepsinogen, precipitin analysis at pH 5.5 indicated that the native enzyme resembles the precursor more closely than did the denatured enzyme. However, all three proteins have some antigenic sites in common. Both antisera reacted more efficiently with their homologous antigens. When measured by C' fixation, the pepsinogen-antipepsinogen system was inhibited by pepsin and to a greater degree, by the activation mixture and the pepsin-inhibitor complex. Pepsin-antipepsin was inhibited by pepsinogen. The specificity of these two antibodies toward pepsin and pepsinogen conformation was used to measure the disappearance of pepsinogen and the concomitant appearance of pepsin during autocatalytic conversion at pH 4.6. The experimental results obtained during the conversion could be duplicated by using varying proportions of pepsin and pepsinogen in the model system. The potentialities of employing these antisera to detect conformational changes such as the unmasking of the pepsin moiety in pepsinogen molecules modified by physical or chemical reagents are discussed.  相似文献   

8.
Exposure of pepsinogen to acid for less than 2 min yields a product with proteolytic activity. This activity is due to intramolecular and intermolecular formation of pepsin from pepsinogen. We find no evidence for intermolecular proteolytic activity in the zymogen. These conclusions are based upon two sets of experiments. First, chemical cleavage of pepsinogen during short activation is demonstrated by quantitative analysis of the NH2-terminal 2 residues of the pepsin and pepsinogen in an activation mixture. In addition, quantitative NH2-terminal analyses after activation under different conditions confirm our previous inference that the product of unimolecular pepsinogen activation is homogeneous whereas bimolecular activation produces a pepsin product with a variety of NH2 termini. Second, spectral changes which occur upon acidification of a pepsinogen solution and are reversed by neutralization are shown to be consistent with the chemical cleavage of pepsinogen during acidification. The first order rate constant for pepsinogen activation, calculated from these spectral experiments, agrees well with the value we had determined previously.  相似文献   

9.
It was the aim of this study to evaluate the stability of two novel cystine-knot microproteins (CKM) SE-ET-TP-020 and SE-MC-TR-020 with potential clinical relevance towards luminally secreted proteases of the gastrointestinal tract in order to gain information about their potential for oral administration. Therefore, the stability of the two CKM and the model-drug insulin towards collected porcine gastric and small intestinal juice as well as towards isolated proteolytic enzymes was evaluated under physiological conditions. No intact SE-ET-EP-020 was detected after few seconds of incubation with porcine small intestinal juice. SE-ET-TP-020 was also degraded in porcine gastric juice. Furthermore, SE-ET-TP-020 was extensively degraded by isolated chymotrypsin, trypsin and pepsin. Moreover, it was degraded by elastase. SE-MC-TR-020 was degraded entirely within approximately 2 h when incubated in porcine small intestinal juice, whereas no degradation was observed within a 3 h incubation period with porcine gastric juice. In presence of the isolated proteolytic enzymes, SE-MC-TR-020 was only slightly degraded by trypsin and pepsin, whereas elastase caused no degradation to SE-MC-TR-020 at all. Chymotrypsin was the protease that caused most degradation to SE-MC-TR-020. The model drug insulin was degraded extensively by chymotrypsin, elastase, pepsin and trypsin as well as by porcine gastric and porcine small intestinal juice. In conclusion, a precise characterisation of SE-ET-TP-020 and SE-MC-TR-020 degrading luminally secreted GI enzymes has been made, which is an important and substantial prerequisite for the further optimisation of these CKM.  相似文献   

10.
In order to determine the specificity of Aspergillus Saitoi protease, the hydrolyzate of B-chain of insulin oxidized by this enzyme was investigated on paperchromatography according to the 2,4-dinitrofluorobenzene technique. Specificity was compared with pepsin and other proteolytic enzymes.  相似文献   

11.
BackgroundThe paper presents a study on the influence of different lithium carbonate and lithium citrate concentration on proteolytic enzymes, namely pepsin and trypsin, in vitro. Lithium can directly affect enzyme activity. Its influence on many bodily functions in both ill and healthy people has been proven.MethodsTo assess the influence of Li+ ions concentration and the substrate/enzyme ratio on pepsin and trypsin activity in vitro, 60 factorial experiments were conducted (each repeated 30 times).Main findingsFor both enzymes, statistically significant changes in their activity under the influence of lihium carbonate and lithium citrate were observed. The biggest increase in enzyme activity reached even 198.6 % and the largest decrease in enzyme activity reached about 50 %.ConclusionsThe study shows that both organic and inorganic forms of lithium salts cause changes in the activity of digestive enzymes. Different concentrations of lithium carbonate and lithium citrate stimulate or inhibit the activity of trypsin and pepsin.  相似文献   

12.
Radial diffusion of enzymes into a casein-containing gel is a fast and inexpensive technique for determination of proteolytic enzymes. A simple method for the preparation of substrate plates with a homogeneous distribution of caseinate and buffered at selected pH values between 2.8 and 4.8 has been described in this communication. By determination of pepsin in caseinate gels the accuracy was better than that described for photometric pepsin assays, and the sensitivity was 300 times greater.  相似文献   

13.
Following the report of Silverman and Podger (1964) that pepsin formed an association with larval receptor sites on D. viviparus and that exsheathment had an absolute requirement for pepsin, the role of pepsin was studied in greater detail. A range of enzyme incubation, pepsin labeling, histochemical and electron microscopical techniques were used. Pepsin did cause exsheathment of D. viviparus but, it was not an absolute requirement. Exsheathment occurred in a range of proteolytic enzymes each at its optimum pH. Findings suggest that the area of weakness around the anterior end of the larvae is digested by external protease and that, in vivo, exsheathment is caused by the gut enzymes of the host.  相似文献   

14.
The effects of collagenase on the immunohistochemical demonstrability of laminin, fibronectin and Factor VIII/RAg in human nervous tissue have been studied. The influence of this, and other proteolytic enzymes such as pepsin and trypsin, has been investigated in relation to different fixatives. Collagenase gave better results with Carnoy fixed material than after formalin fixation; unlike trypsin and pepsin, it did not produce tissue digestion.  相似文献   

15.
The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.  相似文献   

16.
Out of some 800 strains of microorganisms, a potent fungus for milk clotting enzyme was isolated from soil during the course of screening tests and was identified as one of strains of Mucor pusillus Lindt. Satisfactory results were obtained in cheese making experiments with this enzyme which could be produced effectively by solid culture on wheat bran at 30°C for about 70 hrs.

The balance between milk clotting activity and proteolytic activity of this enzyme resembled very much to that of rennet.

Microbial rennet from Mucor pusillus F-27 was obtained with high productivity by solid culture followed by water extraction. The enzyme could be precipitated by salting out with ammonium sulfate and also by mixing with various water-miscible organic solvents such as ethanol, methanol or acetone.

This enzyme is one of acid proteases having its optimal pH for milk casein digestion around 3.5. The ratio of milk clotting activity to proteolytic activity of this enzyme resembled that of calf rennet than those of other proteases of fungal origin. This was more heat stable and more resistant against pH changes than animal rennet. Apparent activity of milk clotting was more affected by Ca ion concentration in milk than that of calf rennet.

The liberation of 12% TCA soluble nitrogen from casein fraction was a little less specific than that of calf rennet. The optimal temperature for milk clotting lay around 56°C.

Electrophoretic patterns of α-peak of casein treated with this enzyme showed the weak proteolysis which resembled that with rennet.  相似文献   

17.
When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.  相似文献   

18.
When calf rennet containing ~ 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorhed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain > 97% pure chymosin. The fraction that passed through unadsorbed was found to contain > 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzvme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.  相似文献   

19.
Microbial keratinases and their prospective applications: an overview   总被引:1,自引:0,他引:1  
Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.  相似文献   

20.
Oxygen exchange in the amide group of leucine amide catalyzed by leucine aminopeptidase, and in leucyltyrosine amide catalyzed by porcine pepsin, was found to proceed mainly by the transfer of the leucyl residue onto the ammonia or tyrosine amide which are formed during the hydrolysis. Thus oxygen exchange in the non-hydrolyzed substrate can not be a proof of the tetrahedral intermediate formation in the course of the catalysis by proteolytic enzymes.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号