PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S‐200, High Q ion‐exchange and affinity column of Arginine Sepharose‐4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l ‐3‐carboxy‐trans‐2, 3‐epoxypropionyl‐l ‐leucine‐4‐guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin‐like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.     

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55–60C as shown by SDS‐PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α‐actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril‐bound serine proteinase.     

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp (Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp (Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins

The modori phenomenon during surimi production is caused by endogenous proteinases, especially cathepsin L and myofibril-bound serine proteinase (MBSP). Cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) was purified to homogeneity by ammonium sulphate fractionation and a series of column chromatographies and revealed a single band with molecular mass of 30 kDa on SDS–PAGE. Peptide mass fingerprinting (PMF) obtained three fragments with 48 amino acid residues, which were highly identical to cathepsin L from other fish species. Its optimal pH and temperature were 5.5 and 55 °C, respectively. Meanwhile, MBSP was purified from the skeletal muscle of blue scad, and the roles of cathepsin L and MBSP in the degradation of myofibrillar proteins were compared. The results indicated that MBSP is more effective than cathepsin L in promoting the degradation of myofibrillar proteins, especially myosin heavy chain (MHC), suggesting that MBSP plays a more significant role.     

The effect of soybean trypsin inhibitor on the degradation of myofibrillar proteins by an endogenous serine proteinase of crucian carp

The effect of soybean trypsin inhibitor (STI) on the degradation of crucian carp myofibrillar proteins caused by an endogenous myofibril-bound serine proteinase (MBSP) was studied. Soybean trypsin inhibitor was purified to high homogeneity and mixed with myofibrils and its inhibitory effect on myofibrillar protein degradation was investigated. In the absence of STI, proteolysis of myofibrillar proteins including myosin heavy chain, α-actinin, actin and tropomyosin could be identified after incubation at 55 °C for 5–20 min depending on the kind of the protein. In contrast, in the presence of STI, with final concentration of 0.75 μg/ml, proteolysis of these proteins was greatly suppressed even after incubation for 1 h, suggesting STI is an effective inhibitor in preventing myofibrillar protein degradation caused by a serine proteinase that is quite possibly MBSP. Though STI has disadvantages for food digestion, as a native food grade inhibitor, it is safe as a food additive, especially at low concentration. Because in surimi production, the decrease of elasticity is always accompanied with the degradation of myofibrillar proteins, thus, the present result suggested the possibility that STI could be applicable in surimi production in order to enhance the elasticity that is the quality of the final products.     

Myofibril-Bound Serine Proteinase (MBP) and its Degradation of Myofibrillar Proteins

Proteolysis of a myofibril-bound serine proteinase (MBP) from carp Cyprinus carpio on myofibrillar proteins and their gel formation ability were investigated. MBP readily decomposed myosin heavy chain as indicated by SDS-PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBP. The optimum degradation temperatures of MBP to myosin heavy chain in myofibril and kamaboko gel were 55°C and 60°C, respectively. The degradation effects of MBP on actin, α-actinin and tropomyosin were studied by the immunoblotting method. Because of its myofibril-bound and myofibrillar protein degradation characteristics, MBP was regarded as the proteinase most probably involved in the modori effect.     

Isolation and characterisation of a kallikrein-like enzyme from Agkistrodon halys pallas snake venom

BACKGROUND: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein‐like activity. RESULTS: A kallikrein‐like proteinase of Agkistrodon halys pallas snake venom, designated AHP‐Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N‐glycosidase F (PNGase F). Sequence studies on the NH₂‐terminal region of the protein indicated that AHP‐Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP‐Ka showed high catalytic activity and kallikrein‐like activity on substrates such as arginine esterase BAEE and chromogenic H‐D‐Pro‐Phe‐Arg‐pNA·2HCl (S‐2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). CONCLUSION: The results showed that AHP‐Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein‐like enzyme. Copyright © 2011 Society of Chemical Industry     

Purification of yeast proteinase A from fresh beer and its specificity on foam proteins

Yeast proteinase A from fresh beer was first purified with Sephadex G‐100 column chromatography and the active fractions reached to 5.3‐fold purification with 7% of yield. After purification with DEAE Sephadex A50, proteinase A activity increased to be 10.1 times of the initial with 1% of yield. When identifying the sample from chromatography by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), only one protein band with 42 kDa was observed, this indicated that the enzyme was purified. The pattern of electrophoresis of hydrolysed beer by crude proteinase A did not show lipid transfer protein (LTP) on the gel. The result of SDS‐PAGE of interaction mixture of purified proteinase A and beer also indicated that LTP was decomposed.     

Identification by GeLC‐MS/MS of Trypsin Inhibitor in Sarcoplasmic Proteins of Three Tropical Fish and Characterization of Their Inhibitory Properties

Sarcoplasmic proteins from 3 fish species were fractionated by 50% to 70% ammonium sulfate precipitation. Lyophilized fractionated sarcoplasmic proteins of threadfin bream (TB‐SP), bigeye snapper (BS‐SP), and yellow croaker (YC‐SP) showed 80% to 92% trypsin inhibitory activity. Trypsin inhibitory activity staining gel electrophoresis revealed bands at 32, 33, 37, 45, 48, and 50 kDa for the 3 species, and a band at 95 kDa was observed for TB‐SP and YC‐SP. Alpha‐1‐antitrypsin with molecular mass of 45 to 50 kDa was identified in YC‐SP by gel‐based liquid chromatography‐tandem mass spectrometry (GeLC‐MS/MS). Other major protein bands appeared on trypsin activity staining included phosphorylase, glyceraldehyde‐3‐phosphate dehydrogenase, and creatine kinase with molecular mass of 95 and 35 to 40 kDa, respectively. But, these 3 proteins did not show true trypsin inhibitory activity. Trypsin inhibitory activity of fractionated sarcoplasmic proteins showed good stability, with >80% activity retained at 60 °C and up to 0.6 M NaCl. TB‐SP showed the highest inhibitory activity against autolysis of washed threadfin bream mince at 65 °C. Addition of 0.5% or 1% TB‐SP improved textural properties of threadfin bream surimi gels preincubated at 37 or 65 °C followed by heating at 90 °C. Therefore, TB‐SP could be a promising protein ingredient for enhancing surimi gel texture.     

恒温蓄冷剂冻结对大黄鱼品质的影响

为提升冷冻大黄鱼的品质,以养殖大黄鱼为研究对象,在无包装空气自然循环冻结(UAF-0)、真空薄膜包装空气自然循环冻结(PAF-1)基础上,探讨了真空薄膜包装蓄冷剂自然循环冻结(PLF-1)、无包装蓄冷剂自然循环冻结(ULF-0)等冻结条件下大黄鱼的冻结速率及理化等指标的变化。结果表明,虽然PLF-1方式下最大冰晶生成带与ULF-0相比延长了44 min,但大黄鱼肌原纤维组织微结构比较清晰,肌肉组织损伤及色差变化较小,硬度、弹性、咀嚼性、恢复性分别仅下降6.8%、1.2%、1.2%、1.9%,pH值变化不显著(P>0.05),盐溶性蛋白下降不显著( P >0.05),TVB-N上升最慢。综合比较4种冻结方式,其品质按优劣为：PLF-1>ULF-0>PAF-1>UAF-0。     

Conditioning of Meat with Raw Soy Sauce and Its Proteinases: Their Effects on the Quality of Beef

This investigation demonstrated that raw soy sauce added to beef in the process of tenderization was effective in improving its sensory quality. To estimate the contributions of proteinases occurring in the raw soy sauce, the degradation of myofibril and collagen was investigated by using the inhibitors, potato inhibitor, ethylenediaminetetraacetic acid (EDTA) and pepstatin which inhibit serine-, metallo-, and aspartic proteinases, respectively. A serine proteinase was most greatly involved in the degradations of both myofibril and collagen. Electrophoretic patterns of myofibril degraded by a purified serine proteinase coincided well with those of myofibril treated with the raw soy sauce per se.     

Identification and characterisation of the major aspartic proteinase activity in Theobroma cacao seeds

Theobroma cacao seeds contain an unusually high level of aspartic proteinase activity. Although this activity is central to the development of high‐quality cocoa flavour, the T cacao polypeptide responsible has not yet been definitively identified. Here we report the identification and characterisation of an active protein complex from T cacao seeds with an apparent molecular weight of approximately 50 kDa. This active complex contains at least two polypeptides: an approximately 30.5 kDa aspartic proteinase, the product of the TcAP2 gene, and an associated polypeptide, the 20.5 kDa trypsin inhibitor protein. The active complex co‐eluted off a size exclusion column with another complex containing the trypsin inhibitor and a putative acid chitinase. The 30.5 kDa TcAP2 proteinase is apparently a monomeric aspartic proteinase with optimal activity between 42 and 47 °C and an optimal pH of 3.0. Significant inactivation of the TcAP2 activity occurs at acid pH around 47–52 °C, a temperature potentially obtained during cocoa bean fermentation. SDS‐PAGE analysis showed that the purified TcAP2 complex efficiently degrades the cacao seed storage protein vicilin into peptides smaller than 10 kDa. In addition, high‐resolution size exclusion chromatography showed that this proteinase is capable of degrading proteins into peptides as small as di‐ and tripeptides, indicating for the first time that the main T cacao seed aspartic proteinase can produce very small peptide products. Our results demonstrate that the aspartic proteinase encoded by the TcAP2 gene plays a critical role in the production of cocoa flavour precursor peptides during cocoa bean fermentation. Copyright © 2005 Society of Chemical Industry     

Identification of a cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.     

In vitro proteolysis of myofibrillar and sarcoplasmic proteins of European sea bass (Dicentrarchus Labrax L) by an endogenous m‐calpain

The effects of m‐calpain isolated from the skeletal muscle of sea bass on sarcoplasmic and myofibrillar proteins isolated from the same tissue were examined in vitro. Incubation of sarcoplasmic proteins with m‐calpain resulted in only a slight decrease (0.7 kDa) in the molecular weight (MW) of a 26.5 kDa protein. Degradation of myofibrils, monitored by quantification of TCA‐soluble peptides generated, resulted in the maximum amount of peptides being generated after 1 h of incubation at 25 °C. Noticeable modifications in the SDS‐PAGE profile of digested myofibrils were observed, including partial denaturation of myosin heavy chain and the release of tropomyosin, ～69 and ～27 kDa doublet bands and a few polypeptides of MW lower than 20 kDa in the soluble fraction. Examination of the degradation patterns of myofibrillar proteins using Western blotting showed that α‐actinin was partially degraded, with release of native α‐actinin and its fragments from myofibrils, whereas desmin was highly degraded after 2 h of digestion. © 2002 Society of Chemical Industry     

PROPERTIES OF CYSTEINE PROTEINASE INHIBITORS FROM BLACK GRAM AND RICE BEAN

Cysteine proteinase inhibitors (CPI) were purified to 59 and 54 fold from black gram (Vignaraungo (L.) Hepper) and rice bean (Vignaumbellata Thunb.), respectively, by using heal treatment, followed by chromatography on a carboxymethyl (CM)‐papain‐Sepharose affinity column. The purified inhibitors were highly inhibitory to papain and Pacific whiting cathepsin L in a concentration dependent manner. They were detected as a dark band on tricine‐SDS‐PAGE gel stained for inhibitory activity. The apparent molecular weights of purified CPI from black gram and rice bean seeds were estimated to be 12, 000 daltons. The purified inhibitors were thermostable up to 90C and active in the neutral and alkaline pH ranges.     

DOUBLE-HEADED TRYPSIN/CHYMOTRYPSIN INHIBITORS FROM HORSE GRAM (DOLICHOS BIFLORUS): PURIFICATION, MOLECULAR AND KINETIC PROPERTIES

Four proteinase inhibitors were purified to homogeneity from horse gram (Dolichos biflorus). These inhibitors are double-headed and inhibit trypsin and chymotrypsin simultaneously and independently. Dissociation constants range between 0.87 and 4.6 × 10^?7 M. Each of the four isoinhibitors possesses a crucial lysine residue at the trypsin reactive site. These inhibitors have molecular masses of 8.5 kDa and isoelectric points of 4.6 to 5.6. They exist mainly as dimers under physiological conditions. Amino acid analysis revealed high levels of half-cystine, serine, aspartate and proline but low levels of methionine and aromatic amino acids. Amino-terminal sequence analysis revealed that each of the four isoinhibitors have a conserved core sequence but are divergent at the N-terminal end. These inhibitors belong to the Bowman-Birk (BBI) family of proteinase inhibitors as reflected by their inhibitory properties, amino acid composition and homology to other BBIs.     

Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG

The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.     

PURIFICATION OF THE CELLULAR RECEPTOR OF AN OYSTER JUICE BORNE COLIPHAGE OJ367 FROM THE OUTER MEMBRANE OF ESCHERICHIA COLI HOST

The cellular receptor of an oyster juice borne phage OJ367 was found in the outer membrane (OM) of its host Escherichia coli. The total cell envelope (TCE) was fractionated by differential extraction and it was found that the OM possesses phage neutralization ability. The OM was purified by diethylaminoethyl cellulose column chromatography to screen for the phage recognition moiety. A homogeneous, 39‐kDa OM protein (Omp) with receptor activity was eluted. It was a peptidoglycan (PG)‐associated protein which showed trypsin resistance. Lipopolysaccharide (LPS) had no effect on its receptor activity either when coexisting in the column fractions or when the isolated LPS was mixed with the Omp. Mutants resistant to lysis by phage OJ367 were isolated. The amount of the 39‐kDa and another PG‐associated 37‐kDa protein decreased significantly in the TCE of the mutant. The 39‐kDa Omp may serve as the cellular receptor for adherence of the coliphage, whereas the 37‐kDa protein is also involved in the infection mechanism.     

大黄鱼类Ⅳ型抗冻蛋白基因的克隆、表达及功能研究

目的研究大黄鱼类Ⅳ型抗冻蛋白基因编码产物的功能。方法从大黄鱼肝脏SSHcDNA文库筛选到的类似极地鱼类Ⅳ型抗冻蛋白的EST序列出发,用RACE方法克隆Ⅳ型抗冻蛋白基因全长cDNA,RT-PCR分析此基因在大黄鱼不同组织内的表达。构建原核表达载体pGEX-4T-Lycafp并表达蛋白质,亲和层析纯化GST融合蛋白,分析它的体外细菌抗冻保护作用。结果大黄鱼类Ⅳ型抗冻蛋白基因cDNA全长664bp,包含372bp开放阅读框,编码124个氨基酸,经SignalP3.0软件预测其N端前20位氨基酸为信号肽,氨基酸序列与长角杜夫鱼Ⅳ型抗冻蛋白同源性高达52%;RT-PCR分析表明其mRNA仅在肝脏表达;重组蛋白在大肠杆菌BL21（DE3）中成功表达,初步纯化后分析体外细菌抗冻保护作用表明,融合蛋白抗冻活性并不明显。结论大黄鱼类Ⅳ型抗冻蛋白不具有显著的抗冻活性,这与大黄鱼不能在低温下生存吻合。