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1.
The initial site of cleavage towards the oxidized B-chain of insulin by thermophilicStreptomyces alkaline proteinase (EC 3.4.21.14) was investigated. The most susceptible bond was between Leu15 and Tyr16. Additional cleavage of the bonds Leu11-Val12, Leu17-Val18, and Gln4-His5 were noted. The results suggested that the specificity of bond, cleavage of thermophilicStreptomyces alkaline proteinase is close to that of subtilisin Carlsberg. 相似文献
2.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1986,869(2):178-184
The specificities of extracellular and ribosomal serine proteinase from Bacillus natto, a food microorganism, were investigated. Both proteins have highly restricted and characteristic specificities. With the extracellular serine proteinase, initial cleavage site was observed at Leu15-Tyr16, secondary site at Ser9-His10 and additional cleavage sites at Gln4-His5 and His5-Leu6 in the oxidized insulin B-chain. Hydrolysis of proangiotensin with the extracellular serine proteinase was observed primarily at Phe8-His9 and secondary at Tyr4-Ile5. The extracellular serine proteinase has a Km of 0.08 mM and kcat of 3 s−1 for angiotensin hydrolysis. With the ribosomal proteinase, initial cleavage site of the oxidized insulin B-chain was observed at Leu15-Tyr16 and additional cleavage site at Phe24-Phe25. Hydrolysis of proangiotensin was observed at Tyr4-Ile5 bond with the ribosomal proteinase. 相似文献
3.
Masashi Matsue Eiji Majima Eiji Ichishima 《Bioscience, biotechnology, and biochemistry》2013,77(10):2485-2490
The specificity of highly purified alkaline proteinase B (EC 3.4.21.14) from thermophilic Streptomyces rectus var. proteolyticus was investigated with an oxidized insulin B chain. Hydrolysis of the oxidized insulin B chain in a 4-hr incubation was observed mainly at three peptide bonds (Phe24-Phe25, Leu15-Tyr16 and Leu11-Val12) and additionally at six others (Leu6-CySO3H7, Gln4-His5, Leu17-Val18, His5-Leu6, Glu13-Ala14, Asn3-Gln4).Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr4-Ile5 bond. Hydrolysis of proangiotension (formerly designated angiotensin I) was observed at the Tyr4-Ile5 and Phe8-His9 bonds. 相似文献
4.
A carboxyl proteinase was purified from submerged-culture filtrate of a wood-deteriorating basidiomycete,Pycnoporus coccineus. The purified enzyme was found to be essentially homogeneous in disc gel electrophoresis tests at pH 9.4 and 2.3. The specificity
and mode of action ofP. coccineus carboxyl proteinase Ia were investigated with the oxidized B-chain of insulinP. coccineus carboxyl proteinase Ia hydrolyzed primarily three peptide bonds, Ala14-Leu15, Tyr16-Leu17, and Phe24-Phe25 bonds, in the oxidized B-chain of insulin. 相似文献
5.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1987,925(3):272-281
A proteinase from the venom of Vipera lebetima was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37 000±1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr16-Leu17, Phe24-Phe25 and Phe25-Tyr26, and glucagon at the bonds Tyr10-Ser11, Leu14-Asp15 and Leu26-Met27. The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA. 相似文献
6.
Takeshi Kimura Yasuhiro Mayumi Michio Takeuchi Kazuya Hayashi Eiji Ichishima 《Current microbiology》1979,3(3):153-156
The specificity and mode of action ofAspergillus sojae carboxyl proteinase I were investigated with the oxidized B-chain of insulin.A. sojae carboxyl proteinase I hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu15-Tyr16 bond and the Phe24-Phe25 bond. Additional cleavage of the bond Tyr16-Leu17 was also noted. 相似文献
7.
Hiroyuki Kumagai Masashi Matsue Eiji Majima Katsumi Tomoda Eiji Ichishima 《Bioscience, biotechnology, and biochemistry》2013,77(4):981-985
The specificity of highly purified carboxyl proteinase from Pycnoporus coccineus (formerly designated Trametes sanguined) was investigated with oligopeptides at pH 2.7. Hydrolysis of oxidized insulin peptide Bl ~ B16 was observed at two peptide bonds (His10-Leu11 and Ala14-Leu15) during 3-hr incubation. The enzyme did not hydrolyze oxidized insulin peptide B15 ~ B24. Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr4-Ile5 bond. Hydrolysis of proangiotensin (formerly designated angiotensin I) was also at the Tyr4-Ile5 bond. In conclusion, peptide bonds which have a hydrophobic amino acid in the P′1 position (as defined by Schechter and Berger, Biochem. Biophys. Res. Commun., 27, 157 (1967)) are preferentially cleaved by the trypsinogen activating carboxyl proteinase of Pycnoporus coccineus. 相似文献
8.
Dr. Eiji Ichishima Yasuhiro Kawai Michio Takeuchi Kenkichi Ahiko 《Current microbiology》1986,13(1):47-49
The unique specificity of a ribosomal proteinase fromStreptococcus lactis, a food microorganism, was investgated with the oxidized insulin B-chain. The initial cleavage site of the enzyme was observed at Leu15-Tyr16 bond, and additional site at Arg22-Gly23 相似文献
9.
The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu5-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln4-His5, Ser9-His10, Leu11-Val12, Leu15-Tyr16 and in the oxidized insulin A chain at Cys SO3H11-Ser12, Leu13-Tyr14, and Leu16-Glu17 were observed. Additional cleavages of the bonds His5-Leu6, Arg22-Gly23, Phe24-Phe25, Phe25-Tyr26, and Tyr26-Thr27 in the oxidized B chain and Cys SO3H6-Cys SO3H7 and Tyr19-Cys SO3H20 in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg5-Ser6, Ser6-Ser7, Arg18-Val19, and Gly27-Gly28. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins. 相似文献
10.
T. Kobayashi Y. Hakamada S. Adachi J. Hitomi T. Yoshimatsu K. Koike S. Kawai S. Ito 《Applied microbiology and biotechnology》1995,43(3):473-481
Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like cyrstals. 相似文献
11.
Makoto Abe Soichi Arai Masao Fujimaki 《Bioscience, biotechnology, and biochemistry》2013,77(10):1813-1817
Substrate specificity of a sulfhydryl protease (P-Ia) purified from germinating corn was investigated by using synthetic substrates and oxidized insulin B-chain. P-Ia showed a potent activity for p-nitrophenyl esters of various amino acid derivatives, except for those of carbo- benzoxy-L-proline and carbobenzoxy-L-valine. Benzoylarginine-β-naphthylamide, a good substrate for papain and cathepsin Bl, was not hydrolyzed by P-Ia. An investigation with acyl dipeptides showed that P-Ia hydrolyzed preferably the peptide bond adjacent to the carboxyl group of the aromatic amino acid. Oxidized insulin B-chain was hydrolyzed at the peptide bonds Gln4-His5, Glu13-Ala14, Ala14-Leu15, Leu15-Tyr16, Tyr16-Leu17 and Tyr26- Thr27. P-Ia, in spite of a sulfhydryl protease, seems to be characterized by its similarity to pepsin rather than papain, as far as the substrate specificity studied in the present work is concerned. 相似文献
12.
Lakshman PL Tachibana S Toyama H Taira T Suganuma T Suntornsuk W Yasuda M 《Journal of industrial microbiology & biotechnology》2011,38(9):1485-1492
An acid proteinase from Monascus purpureus No. 3403, MpuAP, was previously purified and some characterized in our laboratory (Agric Biol Chem 48:1637–1639, 1984). However, further information about this enzyme is lacking. In this study, we investigated MpuAP’s comprehensive substrate
specificity, storage stability, and prospects for reducing antigenicity of whey proteins for application in the food industry.
MpuAP hydrolyzed primarily five peptide bonds, Gln4–His5, His10–Leu11, Ala14–Leu15, Gly23–Phe24 and Phe24–Phe25 in the oxidized insulin B-chain. The lyophilized form of the enzyme was well preserved at 30–40°C for 7 days without stabilizers.
To investigate the possibility of reducing the antigenicity of the milk whey protein, enzymatic hydrolysates of the whey protein
were evaluated by inhibition ELISA. Out of the three main components of whey protein, casein and α-lactalbumin were efficiently
degraded by MpuAP. The sequential reaction of MpuAP and trypsin against the whey protein successfully degraded casein, α-lactalbumin
and β-lactoglobulin with the highest degree of hydrolysis. As a result, the hydrolysates obtained by using the MpuAP–trypsin
combination showed the lowest antigenicity compared with the single application of pepsin, trypsin or pepsin–trypsin combination.
Therefore, the overall result suggested that the storage-stable MpuAP and trypsin combination will be a productive approach
for making hypoallergic bovine milk whey protein hydrolysates. 相似文献
13.
Tetsuo Muro Yoshio Tominaga Shigetaka Okada 《Bioscience, biotechnology, and biochemistry》2013,77(2):283-289
The substrate specificity and the mode of action of the protease from Streptomyces cellulosae were investigated, using many kinds of peptides and proteins as substrates. The protease hydrolyzed peptides consisting of hydrophobic amino acids such as L-Phe-L-Leu-NH2, L-Pro-L-Phe-NH2, l-Leu-L-Met, L-Leu-L-Leu, Gly-L-Ile, L-Phe-L-Phe, L-Pro-L-Leu-Gly-NH2, etc. The protease hydrolyzed zein best among the proteins tested, but weakly hydrolyzed gelatin, myoglobin, bovine serum albumin, γ-globulin, and collagen. The protease mainly hydrolyzed Ser12-Leu13, Leu13-Tyr14, and Tyr14-Gln15 bonds in the oxidized A-chain of insulin and at least the Leu15-Tyr16 bond in the oxidized B-chain of insulin. 相似文献
14.
《Bioscience, biotechnology, and biochemistry》2013,77(4):710-715
We have discovered a novel metalloproteinase, which has high activity at low temperatures, from the culture supernatant of a marine bacterium. The strain was identified as Alteromonas sp. No. 3696. The metalloproteinase, named almelysin, was purified to homogeneity from the cultured supernatant at 10°C by two column chromatographies. About 20 mg of purified almelysin was obtained from 18.4 liters of the culture supernatant. The molecular mass of almelysin was estimated to be 28 kDa by SDS–PAGE and the isoelectric point was 4.3. The optimum pH for activity of almelysin was pH 8.5–9.0 and 6.5 using casein and (7-methoxycoumarin-4-yl)acetyl(MOCAc)-Pro-Leu-Gly-Leu-(N3-[2,4-dinitrophenyl]-L-2,3-di-aminopropionyl)[A2pr(Dnp)]-Ala-Arg-NH2 as substrates, respectively. Almelysin was stable between pH 7.5–8.0 and below 40°C. The optimum temperature for the activity was observed to be 40°C using both casein and MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 as substrates. The activity of almelysin was inhibited by such metallo chelators as EDTA and o-phenanthroline, while talopeptin, phosphoramidon, and SMPI, typical metalloproteinase inhibitors, had no effect. Almelysin primarily cleaved the Ala14-Leu15 bond and Phe24-Phe25 bond, and secondarily the Tyr16-Leu17 bond in oxidized insulin B-chain. However, almelysin could not cleave the His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds, which were cleaved by other metalloproteinases. These results indicate that the substrate specificity of almelysin is different from other metalloproteinases. Interestingly, Alteromonas sp. No. 3696 strain produced another proteinase as well as almelysin at 25°C. 相似文献
15.
Toshihiro Nakanishi Takehiko Yamamoto 《Bioscience, biotechnology, and biochemistry》2013,77(12):2391-2397
Action of the crystalline alkalophilic proteinase of a Streptomyces sp. which was active at pH values around 13, was investigated. The enzyme attacked various keratinous proteins such as wool, hair, feather etc., dissolving them with hydrolysis degrees from 2 to 6%, and the degradation of several keratins was observed under a microscope and scanning electron-microscope. Specificity of the enzyme was also studied using oxidized insulin B-chain. The enzyme cleaved most easily the peptide linkages between -Ser · His-, -Leu · Val-, -Phe · Tyr- and -Lys · Ala-, though it split several other sites of oxidized insulin B-chain, too, indicating that the action pattern of the proteinase is distinguished from those of alkaline proteinases so far reported. 相似文献
16.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1986,883(3):439-447
The alkaline elastase produced by alkalophilic Bacillus Ya-B was a new type of proteinase which had a very high optimum pH and high elastolytic activity. It also had a high hydrolyzing activity against keratin and collagen. The molecular weight was determined to be 23 700 and 25 000 by ultracentrifugation analysis and SDS-polycrylamide gel electrophoresis, respectively. The isoelectric point was 10.6. The optimum reaction temperature was 60°C. Like many alkaline proteinases, this enzyme required Ca2+ for stability. The optimum reaction pH was 11.75 toward casein and elastin-orcein. The Kcat/Km values of the enzyme to synthetic substrates were constant from pH 8.5 up to 12.75. The enzyme was stable in the pH range 5.0–10.0. The enzyme was inhibited by alkaline proteinase inhibitors Streptomyces subtilisin inhibitor and microbial alkaline proteinase inhibitor, but not by elastatinal or the metalloproteinase inhibitor metalloproteinase inhibitor. Sodium chloride inhibited the elastolytic activity but not the caseinolytic activity at a concentration below 0.2 M. The inhibitory effect of sodium chloride to elastolytic activity was much more prominent at pH 9.0 than at pH 11.5. More than 50% of the enzyme bound onto elastin in the pH range below the isoelectric point of this enzyme. The amino-terminal sequence of the enzyme was determined, and compared with those of subtilisin BPN′ and subtilisin Carlsberg. Extensive sequence homology was noted among these three enzymes. 相似文献
17.
An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 °C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H2O2, 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P1 position, in descending order. The k cat, K m and k cat/K m values for Z-Val-Lys-Met-MCA were 16.8±0.14 min?1, 5.1±0.28 μM, and 3.3±0.28 μM?1 min?1 respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS. 相似文献
18.
Senarath B. P. Athauda Masaaki Nishigai Hideo Arakawa Atsushi Ikai Masanori Ukai Kenji Takahashi 《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):219-224
The inhibitory effects of human α2-macroglobulin (α2-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by α2-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved α2-M mainly at the His694-Ala695 bond and Leu697-Val698 bond, respectively, in the bait regions sequence of α2-M. The conformation of α2-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by α2-M. 相似文献
19.
《Bioscience, biotechnology, and biochemistry》2013,77(10):1651-1654
Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp. T1800. The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates. Vimelysin cleaved mainly Pro7-Phe8 bond and slightly Tyr4-Ile5 bond in human angiotensin I. Vimelysin also cleaved mainly Phe24-Phe25 and Tyr16-Leu17 bonds, and slightly His5-Leu6, His10-Leu11, Ala14-Leu15, and Gly23-Phe24 bonds in oxidized insulin B-chain. The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied. The ratio of kcat/Km for Fua-Gly-Phe-NH2/Fua-Gly-Leu-NH2, Fua-Phe-Leu-NH2/Fua-Gly-Leu-NH2, and Fua-Phe-Phe-NH2/Fua-Gly-Leu-NH2 were 15.9, 27.8, and 59.0, respectively. These results indicate that vimelysin easily recognizes phenylalanine in P1′ positions, which is different from thermolysin. 相似文献
20.
Snake venom from Agkistrodon acutus consists of a number of compounds which may potentially be used as drugs. However, it is hard to obtain enough pure protein for drug development. Recently, we reported expression and purification of a novel recombinant fibrinogenase which was named rFII. Here we reported for the first time the enzymatic activities and functional characterization of rFII. Circular dichroism spectra showed the gross conformation of FIIa and rFII to be notably similar. It is an alkaline proteinase and the amino acid sequence exhibits a high degree of sequence identity with other snake venom metalloproteinases. rFII also exhibits amidase activity against N-(p-Tosyl)-Gly-Pro-Lys-p-nitroanilide, which is specified synthetic substrate for plasmin. Functional characterization showed that rFII possesses both fibronectin and type IV collagen cleaving activities. In addition, rFII preferentially cleaved the Aalpha-chain of fibrinogen, followed by the Bbeta-chain and finally, the gamma(γ) chain was affected. Furthermore, rFII was also capable of cleaving fibrin without plasminogen activation and suppressing ADP-induced platelet aggregation. The proteolytic activity of rFII was inhibited completely by PMSF and mostly by EDTA. The cations Ca2+, Mg2+, Na+, K+ didn't affect its proteolytic activity, while Cu2+ and Zn2+ slightly inhibited this activity. Study of hydrolysis of oxidized insulin B-chain reveals that rFII preferentially cleaved oxidized insulin B-chain at the site of Val12-Glu13, Leu15-Tyr16, and Phe24-Phe25. 相似文献