Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.     

Participation of proteinase yscA in the in vitro formation of the smaller subunit of glycogen phosphorylase in extracts of Saccharomyces cerevisiae.

Analyses by sodium dodecyl sulphate-polyacrylamide gradient-gel electrophoresis and Western blotting of the proteins from boiled cell samples from all growth phases of yeast cultures, and from all stages of extract preparation indicate that the smaller subunit (s-monomer), which is found in purified glycogen phosphorylase (EC 2.4.1.1) from baker's yeast, is not present in the living cell. It is observed in extracts of Saccharomyces carlsbergensis and S. cerevisiae after incubation at ambient temperatures or even after storage in the frozen state at -25 degrees C. Its formation is sensitive towards pepstatin A, and it is absent from extracts of several mutants of S. cerevisiae that do not contain active proteinase yscA (EC 3.4.33.6). When purified proteinase yscA is added to the extracts of these mutants, the formation of s-monomer is restored. When the proteinase yscA-deficient strains are grown with a reduced amount of complex nitrogen compounds, the slightly smaller sc-monomer is formed in their extracts. This event must be attributed to a different proteinase, since it is sensitive towards p-hydroxymercuriphenylsulphonate, but not towards pepstatin A. The N-terminal amino acid of the sc-monomer was found to be blocked, as in the case of the native l-monomer, but not the s-monomer.     

大豆皮天冬氨酸蛋白酶的提取、纯化与鉴定

从大豆加工副产物——大豆皮的高值化利用出发,以大豆皮为原料,在纤维素酶辅助提取大豆皮蛋白质的基础上,利用CM-Sepharose柱离子交换层析纯化大豆皮蛋白粗提物,用SDS-PAGE分辨大豆皮蛋白粗提物和纯化后的蛋白质,对电泳凝胶分辨出来的蛋白条带用基质辅助激光解析电离飞行时间二级质谱(MALDI-TOF-TOF MS)进行鉴定,并测定了其在pH 2~6范围内的酶活性,结果表明首次从大豆皮中纯化鉴定出类猪笼草蛋白酶I大豆天冬氨酸蛋白酶,得率为233 mg/kg,纯化后的类猪笼草蛋白酶I大豆天冬氨酸蛋白酶在pH 3时对血红蛋白的比活力最高,为62.1 U/mg。     

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.     

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     

Trypsin-like Enzyme from Sand Crab (Portunus pelagicus): Purification and characterization

Studies with synthetic substrates and specific inhibitors indicated that a protcinase from the hepatopancreas of the sand crab (Portunus pelngicus) was a trypsin-like serine proteinase. The molecular weight of the enzyme estimated by gel filtration and mass spectrometry was ～ 25,000, whereas SDS-PAGE indicated a molecular weight of 34,800. The optimum temperature for hydrolysis of azocasein was 60°C, while inactivation of 50% enzymic activity occurred at 68°C. The enzyme, optimally active at pH 8.0 towards p-tosyl-L-arginine methyl ester and unstable at acid pH, was high in acidic amino acid residues. Under some conditions ihe knzyme readily autodigested. Our results can help understand and avoid problems of meat softening during storage of seafood products.     

The milk acid proteinase cathepsin D: a review

Cathepsin D is a lysosomal aspartic proteinase with a low pH optimum, found in many tissues and in bovine milk. It is synthesised as its inactive zymogen, procathepsin D. Procathepsin D can convert itself, by an autoproteolytic pathway, into an active intermediate, pseudocathepsin D, while other proteases are involved in the processing to mature cathepsin D. Cathepsin D is known to participate in a range of physiological processes. Pro-, pseudo- and mature cathepsin D have been purified from bovine milk, but procathepsin D is the major form present in milk. Cathepsin D activity in milk appears correlated with somatic cell count. The active forms of the enzyme readily hydrolyse α_S1-, α_S2- and β-caseins, in the case of α_S1-casein and β-casein with a specificity similar to that of chymosin. Cathepsin D can also produce para-κ-casein from κ-casein and, at high concentrations, can coagulate milk. Cathepsin D appears to be able to at least partially survive commercial pasteurisation processes. In recent years, increasing evidence has been documented indicating a role for this enzyme in proteolysis in cheese during ripening, most clearly in cheese where rennet activity is low, such as Swiss cheese, Quarg and Feta. Further research is required to evaluate the significance of this enzyme in more detail.     

Characterisation of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds

The seeds of Solanum dubium were blended and extracted using different types of buffers. The most reliable, quick, and efficient buffer was found to be 5% NaCl in acetate buffer (pH 5.0) which was used throughout the study. The extract was filtered and fractionated twice with ammonium sulphate. The partially purified enzyme was characterised by SDS–PAGE which showed a band of molecular weight of 66 kDa with the presence of other bands of low density. When compared with other plant enzymes, S. dubium enzyme was found to have higher clotting and proteolytic activities. The activity of the enzyme was steadily increased with enzyme and substrate concentration. The enzyme was found to be very stable against a wide range of pH values as well as a wide range of temperature (20–90 °C). The results of substrate specificity of the enzyme showed that the partially purified enzyme preferred both hydrophilic and hydrophobic amino acid residues at P1 position. The catalytic efficiency of the purified enzyme was enhanced by an aliphatic amino acids (Leu) compared to aromatic residue (Phe) at P1 position at the same site.     

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.     

酶法提取蛋黄油的工艺

利用酶法从鸡蛋黄粉中提取蛋黄油 ,通过正交试验选出酶解法提取蛋黄油的最佳工艺条件 :底物质量浓度为 2 0 g/dL ,先用每 10 0g底物中加入 1g蛋白酶A ,并保持其最适 pH和温度 ,反应时间为 2h ;再用每 10 0 g底物中加入 1.5 g的蛋白酶B ,并同时调节到相应的最适 pH和温度 ,反应时间为 2h ,该条件下蛋黄油得率为 5 8.89% .最后 ,对蛋黄油的主要成分进行了分析     

Catalytic Activities of Crude Enzyme Fractions from Monterey Sardine

Catalytic activities in sarcoplasmic fluid of Monterey sardine (Sardinops sagax caerulea) were identified. Hydrolysis at pH 7.6 on hippuryl-L-phenylalanine and hippuryl-L-arginine suggested the presence of carboxypeptidase A and B. Proteolysis on glutaryl-L-phenylalanine-p-nitroanilide, and inhibition by CuSO₄, phenylmethylsulfonyl fluoride and N-p-tosyl-L-lysil chloromethyl ketone, confirmed presence of a chymotrypsin-like proteinase and other serine enzymes. No hydrolysis on α-N-benzoyl-D,L-arginine-p-nitroanilide occurred. Leucine aminopeptidase was detected by hydrolysis on L-leucyl-β-naplithylamide-HCl. Activity at pH 3 proved the presence of an acid proteinase but a slight inhibition by EDTA suggested the involvement of a cathepsin D-like enzyme. Cathepsins A and B and collagenase were not detected.     

trans-Complementation assay establishes the role of proregion hydrophobic amino acid residues in the biosynthesis of Saccharomyces cerevisiae Kex2p endoprotease

The proregion of Saccharomyces cerevisiae endoprotease Kex2p is essential for the biosynthesis of an active enzyme. It has been suggested that the proregion acts in the endoplasmic reticulum to catalyse folding of the enzyme. To identify amino acid residues important for proregion function, we used an in vivo system in which the Kex2p proregion can act in trans to activate a Kex2p enzyme synthesized without its proregion. Activation of Kex2p by wild-type and mutated proregions revealed the essential role of hydrophobic residues F(37), V(39) and F(70) in enzyme activation. Further exploration of the role of these residues by in vitro inhibition of Kex2p activity by its proregion indicated that they are essential to form the proregion/enzyme bimolecular complex. In contrast, basic residues K(108) and R(109), located in the C-terminus of the proregion, are not involved in complex formation but are necessary for the biosynthesis of an active enzyme.     

Modification of the Methods for the Extraction of Carboxypeptidase and Proteinase Activities from Sorghum Malts

The effects of two extractants on the activities of carboxypeptidase and proteinase enzymes from three sorghum varieties germinated for 3 and 5 days were studied. In all three varieties, cysteine hydrochloride (Cyst. HCl) proved a better extractant than 2‐mercaptoethanol (2‐ME) as shown by the increase in carboxypeptidase and proteinase activities with 5‐day malts. A three‐way analysis of variance (ANOVA) on the effect of germination time, extractant and variety on the carboxypeptidase activities of the three malts showed that all the factors plus their possible four‐way interactions highly significantly (P < 0.001) affected the extractability/activity of the enzyme. However, while the three factors had a highly significant (P < 0.001) effect on the proteinase activity, one of their four‐way interactions, specifically, extractant/variety, had no significant effect. This suggests that, unlike the carboxypeptidase enzyme, the effectiveness of the extractants in enhancing the extractability/activity of the proteinase enzyme in all the sorghum malt samples was amongst other factors, not variety‐dependent.     

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.     

Proteinase in Pacific Whiting Surimi Wash Water: Identification and Characterization

A proteinase in Pacific whiting surimi wash water (SWW) was characterized to be cathepsin L based on molecular mass (M_r), substrate specificity, and SDS-substrate gel electrophoresis. The proteinase was highly active on Z-Phe-Arg-NMec, and the native M_r was 27,400 based on size exclusion (SEC)-HPLC. Acidification of the SEC-HPLC fractions showed a two-fold increase in activity on Z-Phe-Arg-NMec. SWW proteolytic activity was found at M_r 54,200 on SDS-substrate gel. However, acidification shifted the activity zone to M_r 39,500 corresponding to cathepsin L. No evidence of activity by calpain or cathepsin B or H was found in Pacific whiting SWW.     

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

Posttranslational activation, site-directed mutation and phylogenetic analyses of the lysine biosynthesis enzymes alpha-aminoadipate reductase Lys1p (AAR) and the phosphopantetheinyl transferase Lys7p (PPTase) from Schizosaccharomyces pombe

Alpha-aminoadipate reductase (AAR), the signature enzyme for lysine biosynthesis in fungi, catalyses the conversion of alpha-aminoadipate to alpha-aminoadipate-semiadehyde in the presence of ATP and NADPH. In Saccharomyces cerevisiae and Candida albicans, the LYS2-encoded AAR is posttranslationally activated by CoA and the LYS5-encoded PPTase. The fission yeast Schizosaccharomyces pombe is evolutionarily highly diverged from S. cerevisiae and C. albicans. We report here several unusual activation characteristics of Sz. pombe Lys1p and Lys7p, isofunctional to Lys2p (AAR) and Lys5p (PPTase), respectively. Unlike the Lys2p from S. cerevisiae and C. albicans, the Sz. pombe Lys1p was active when expressed in E. coli and exhibited significant AAR activity without the addition of CoA or the Sz. pombe Lys7p intron free PPTase. Somewhat higher AAR activity was obtained with the addition of CoA and the Sz. pombe Lys7p PPTase. Substitution of G910A, S913T or S913A in the Sz. pombe Lys1p activation domain (IGGHSI) resulted in no AAR activity. Similarly, substitutions of several amino acid residues in the Sz. pombe Lys7p PPTase domain (G79A, R80K and P81A in Core 1; F93W, D94E, F95W and N96D in Core 1a; G124A, V125I and D126E in Core 2; K172R, E173D and K177R in Core 3) also resulted in no activation of Lys1p and no AAR activity. The Sz. pombe Lys1p amino acid sequence showed a high degree of similarity to other fungal Lys2p proteins; however, the Lys7p amino acid sequence showed much less similarity to other bacterial, fungal and animal PPTases representing several phylogenetic groups.     

In silico metabolism studies of dietary flavonoids by CYP1A2 and CYP2C9

Dietary flavonoids are important contributors to cancer prevention, due to their interactions with CYP family enzymes. This study describes the application of in silico tools to study the metabolism of dietary flavonoid compounds such as quercetin, rutin, naringenin and naringin, aiming to propose their potential metabolites and chemical structures and also to investigate the abilities of these compounds to interact with cytochrome P450 CYP1A2 and CYP2C9 isoforms. The reactions founded suggest that the presence of the sugar chain may account for significant change in the position of the flavonoid in the active site, due to the steric contacts. Another important finding is the participation of the amino acid residues in the metabolism. In CYP2C9, the Asn474, Ser209, Thr304 and Arg108 residues participated almost in all of the metabolically active conformations, suggesting that these residues are important for the regioselectivity and positioning of the substrate. The same was observed for the Asp313, Phe226, Phe256 and Phe260 residues of the CYP1A2 isoform. The results presented here afford new opportunities for improving the application of dietary flavonoids for the prevention of chronic disorders, as they're capable to be readily metabolized by CYP450 family.