Characterization of "lettucine", a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting

In this work we focused on the characterization of a novel plant rennet purified from lettuce leaves (Lactuca sativa L. cv Romana). The lettuce protease, lettucine, showed trypsin-like, SV8-like, and caseinolytic activities. Although the enzyme did not recognize peptides having hydrophobic amino acid residues in the P(1) position of the target bond, it did show milk-clotting activity, suggesting that different bonds rather than the Phe(105)-Met(106) of the kappa-casein might be cleaved, still inducing milk-clotting. The enzyme exhibited proteolytic activity toward alpha-casein, beta-casein, kappa-casein, and milks with different fat contents, with the highest activity observed with partially skimmed milk, total casein, and alpha- and kappa-casein. SDS-PAGE studies showed that lettucine cleaved alpha-casein, beta-casein, and kappa-casein. In particular, we showed that alpha-casein breakdown occurred even though total casein or milks were supplied, suggesting that the lettuce enzyme is able to operate a significant disorganization of the casein's micellar structure. Moreover, the proteolytic activity of the enzyme analyzed under various technological parameters, such as temperature and pH, indicated that the lettuce enzyme is highly consistent with the milk-clotting process.     

Purification, characterization, and solvent-induced thermal stabilization of ficin from Ficus carica

Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur in multiple forms. Although crude ficin is of considerable commercial importance, ficin as such has not been fully characterized. A major ficin from the commercial crude proteinase mixture preparation of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography and is a single polypeptide chain protein with a molecular mass of 23 100 +/- 300 Da as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core sequence of ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme contains three disulfide bonds and a single free cysteine residue at the active site. The effect of co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of ficin was determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent thermal denaturation temperature (T(m)) of ficin was significantly increased from the control value of 72 +/- 1 degrees C in the presence of all co-solvents. However, the maximum stabilization effect was observed in terms of thermal stabilization by the co-solvent trehalose.     

棉花黄萎病拮抗细菌DS45-2菌株抗菌蛋白的分离纯化

枯草芽孢杆菌(Bacillus subtilis) DS45-2菌株产生的抗菌蛋白对棉花黄萎病有较强的抗性。用NB培养基摇床振荡培养DS45-2菌株(30℃,180r/min,48h),发酵液经硫酸铵盐析得到抗菌蛋白。经分析,抗菌蛋白对热稳定;对胃蛋白酶、胰蛋白酶均不敏感,对蛋白酶K部分敏感;在碱性条件下稳定,酸性条件下抑菌活性减弱。抗菌蛋白经DEAE Sepharose Fast Flow阴离子交换层析和反相层析后,分离纯化出一个抗菌蛋白0组分,经SDS-PAGE检测分子.质量约为23 kDa。     

A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria

A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.     

Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme

This work was aimed at the isolation, purification, and characterization of novel antimicrobial peptides from chicken egg white lysozyme hydrolysate, obtained by peptic digestion and subsequent tryptic digestion. The hydrolysate was composed of over 20 small peptides of less than 1000 Da, and had no enzymatic activity. The water-soluble peptide mixture showed bacteriostatic activity against Gram-positive bacteria (Staphylococcus aureus 23-394) and Gram-negative bacteria (Escherichia coli K-12). Two bacteriostatic peptides were purified and sequenced. One peptide, with the sequence Ile-Val-Ser-Asp-Gly-Asp-Gly-Met-Asn-Ala-Trp, inhibited Gram-negative bacteria E. coli K-12 and corresponded to amino acid residues 98-108, which are located in the middle part of the helix-loop-helix. Another novel antimicrobial peptide inhibited S. aureus 23-394 and was determined to have the sequence His-Gly-Leu-Asp-Asn-Tyr-Arg, corresponding to amino acid residues 15-21 of lysozyme. These peptides broadened the antimicrobial activity of lysozyme to include Gram-negative bacteria. The results obtained in this study indicate that lysozyme possesses nonenzymatic bacteriostatic domains in its primary sequence and they are released by proteolytic hydrolysis.     

Modeling the kinetics of whey protein hydrolysis brought about by enzymes from Cynara cardunculus

The purpose of this research work was to study the proteolytic activity of aqueous crude extracts of flowers of the plant Cynara cardunculus on the major whey proteins, namely, beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-La). These extracts, containing a mixture of cardosins A and B (i.e., two distinct aspartic proteases), have been employed for many years in traditional cheese-making in Portugal and Spain. Cow's milk sweet whey was incubated for up to 24 h at various ratios of addition of crude enzyme extract, under controlled pH (5.2 and 6.0) and temperature (55 degrees C). The samples collected were assayed by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A mechanistic model was proposed for the kinetics of the hydrolysis process, which is basically a double-substrate, double-enzyme Michaelis-Menten rate expression; the kinetic parameters were estimated by multiresponse, nonlinear regression analysis. The best estimates obtained for the specificity ratio (i.e., k(cat)/K(m)) of each cardosin within the mixture toward each whey protein indicated that said aspartic proteases possess a higher catalytic efficiency for alpha-La (0.42-4.2 mM(-1).s(-1)) than for beta-Lg (0-0.064 mM(-1).s(-1)), at least under the experimental conditions used. These ratios are below those previously reported for caseins and a synthetic hexapeptide. Cardosins are more active at pH 5.2 than at pH 6.0 and (as expected) at higher enzyme-to-substrate ratios.     

Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability

A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. Imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.     

Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio)

A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.     

PsoP1, a milk-clotting aspartic peptidase from the basidiomycete fungus Piptoporus soloniensis

The first enzyme of the basidiomycete Piptoporus soloniensis, a peptidase (PsoP1), was characterized after isolation from submerged cultures, purification by fractional precipitation, and preparative native-polyarylamide gel electrophoresis (PAGE). The native molecular mass of PsoP1 was 38 kDa with an isoelectric point of 3.9. Similar to chymosin from milk calves, PsoP1 showed a maximum milk-clotting activity (MCA) at 35-40 °C and was most stable at pH 6 and below 40 °C. The complete inhibition by pepstatin A identified this enzyme as an aspartic peptidase. Electrospray ionization-tandem MS showed an amino acid partial sequence that was more homologous to mammalian milk clotting peptidases than to the chymosin substitute from a fungal species, such as the Zygomycete Mucor miehei. According to sodium dodecyl sulfate-PAGE patterns, the peptidase cleaved κ-casein in a way similar to chymosin and hydrolyzed β-casein slowly, as it would be expected from an efficient chymosin substitute.     

Proteolytic Enzymes in Germinating Rye Grains

The proteolytic activities during rye (Secale cereale L. ‘Humbolt’) grain germination were monitored using in‐solution methods and one‐ and two‐dimensional PAGE with gels that contained incorporated substrate proteins. The total proteolytic activity increased during the first three days of germination, but not after that. The proteinase activity was measured at pH 3.8, 6.0, and 8.0 in the presence and absence of class‐specific proteinase inhibitors. This indicated that enzymes from all four proteinase classes were present during the germination process. Germinated rye grain contained mainly aspartic and cysteine proteinase activities that are especially active at pH 3.8. Serine‐ and metallo‐proteinases were less abundant. Overall, the pattern of hydrolysis was very similar to that observed during barley and wheat germination.     

Cathepsin L: a predominant heat-activated proteinase in arrowtooth flounder muscle

Characterization of the autolytic profile of arrowtooth flounder (ATF) muscle indicated the involvement of heat-activated proteinases active at both acidic and alkaline pH values. Further assay of fish extract exhibited the maximum activity at 60 degrees C against casein used as a substrate at both pH 5.5 and 8.0. The maximum activity shifted to lower temperatures by the addition of urea with two distinctive patterns: activity reduction at pH 5.5 and activity enhancement at pH 8.0. The highest inhibition by E-64 indicated the proteinase belongs to the cysteine proteinase class. At pH 5.5, the proteinase hydrolyzed Z-Phe-Arg-NMec and all types of protein substrates tested at higher rate than that at pH 8.0. Activity bands, observed on the activity-stained substrate gels, indicated similar proteinases are responsible for the proteolytic activity observed at both pH values. When proteins of fish extract were separated by HPLC-SEC, only one proteolytic peak was observed at the retention time of 26 min with an estimated molecular weight of 39800 Da. The results implied cathepsin L is a predominant proteinase responsible for autolysis of ATF muscle at elevated temperatures.     

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.     

Characterization of an aspartic proteinase activity in buckwheat (Fagopyrum esculentum Moench) seeds

The pepstatin A sensitive acidic proteolytic activity of total protein extracts of buckwheat seeds has been analyzed in developing, mature, and germinating seeds by activity measurements as well as by electrophoretic and immunochemical techniques. Immunoblot analysis using cross-reactive antibodies raised against barley phytepsin suggested that specific proteolytic activity could be attributed to a 47 kDa heterodimeric polypeptide, composed of two subunits: 31 and 16 kDa polypeptides. The analysis of time course expression revealed that the 47 kDa heterodimer accumulated during seed maturation starting from 12 days after pollination and was also present at the beginning of germination. Milk-clotting activity of this proteinase was also indicated.     

Degradation of Secalins During Rye Sourdough Fermentation

Rye sourdough (RSD) gives rye bread mildly acidic taste and desired flavor. Flavor precursors (amino acids and small peptides) are generated in the proteolytic breakdown of rye proteins. Our aim was to study the protein degradation during RSD fermentations. Two sourdoughs were prepared of flours derived from two rye cultivars (Amilo and Akusti). RSD samples were collected during fermentations. Three protein fractions were obtained by sequential protein extraction and these were analyzed by SDS‐PAGE. Free amino nitrogen (FAN) was measured with a ninhydrin method. In addition, two rye incubations without starter microorganisms (with antibiotics) were made at pH 3.6 and 6.1, and proteinase profiles of the rye cultivars were analyzed at pH 4.3. SDS‐PAGE analysis showed that during RSD fermentations, rye proteins, especially the alcohol‐soluble secalins, were degraded. Secalins also evidently degraded during the incubation without starter microorganisms at pH 3.6. Aspartic proteinases were in the major proteinase group in both rye cultivars. This study confirms that endogenous proteinases of rye, mainly aspartic proteinases, hydrolyze rye proteins, especially secalins, during RSD fermentation. Protein degradation in rye sourdoughs may thus be enhanced by selecting rye flours with high proteolytic activity toward secalins.     

Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae)

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.     

Heat-Induced Fragmentation of the Maize Waxy Protein During Protein Extraction from Starch Granules

The starch granule of maize contains a characteristic set of tightly bound polypeptides. Granule-associated polypeptides are typically extracted from starch granules by heating starch granule suspensions at 90–100°C in a detergent such as SDS. Solubilized proteins are recovered by centrifugation and analyzed by gel electrophoresis. Previously identified tightly bound granule intrinsic proteins consist of the 85-kDa starch-branching enzyme IIb, the 76-kDa starch synthase I, and the 60-kD waxy (Wx) protein, also known as granule-bound starch synthase I. However, SDS extracts from starch granules of maize also contain a cluster of proteins ranging in mass between 47 and 32 kDa In this study, we analyzed this group of granule-associated proteins and found that each was recognized by the Wx antibody. A 15 amino acid N-terminal sequence from the 47-kDa polypeptide was identical to the predicted N-terminus of the Wx protein. Further analysis revealed that each immunoreactive polypeptide between 47 and 32 kDa was a heat-induced fragmentation product of the Wx protein. Conditions for the extraction of granule proteins were evaluated. Our results demonstrate that granule proteins are effectively released by mild extraction (10-min incubation at 72°C). Relative to the Wx protein, starch synthase I and starch branching enzyme IIb were less susceptible to thermal fragmentation. These results demonstrate that the 85-, 76-, and 60-kDa polypeptides are authentic granule-intrinsic proteins, and that the majority of polypeptides between 47 and 32 kDa are artifacts of high-temperature granule extraction procedures.     

陆地棉巯基蛋白酶抑制剂基因克隆及其氨基酸序列分析

通过对植物中已知巯基蛋白酶抑制剂(cystatin)的保守性分析,设计1对简并引物,从陆地棉栽培种中棉所29(GossypiumhirsutumL.cv.Zhongmiansuo29)cDNA中克隆出1条巯基蛋白酶抑制剂基因片断,经测序和对测序结果在有关数据库中检索分析,发现该片段与1条中棉(G.arboreumL.)EST及1条雷蒙德氏棉(G.raimondiiL.)cDNA同源性高达96%。三者编码蛋白的氨基酸同源性达100%,且完全符合CPI的特征;所克隆基因片段的氨基酸序列与NBCI蛋白质数据库中登录的豇豆、向日葵、玉米、水稻中CPI均有高度同源性,表明该片段包含编码陆地棉CPI的完整序列。     

Purification, characterization, and cDNA cloning of a myofibril-bound serine proteinase from the skeletal muscle of crucian carp (Carassius auratus)

A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.     

Trypsin-like proteinase produced by Fusarium culmorum grown on grain proteins

The fungal disease Fusarium head blight occurs on wheat (Triticum spp.) and barley (Hordeum vulgare L.) and is one of the worldwide problems of agriculture. It can be caused by various Fusarium species. We are characterizing the proteinases of F. culmorum to investigate how they may help the fungus to attack the grain. A trypsin-like proteinase has been purified from a gluten-containing culture medium of F. culmorum. The enzyme was maximally active at about pH 9 and 45 degrees C, but was not stable under those conditions. It was stabilized by calcium ions and by the presence of other proteins. The proteinase was most stable at pH 6-7 at ambient temperatures, but was quickly inactivated at 50 degrees C. It was strongly inhibited by p-amidino phenylmethylsulfonyl fluoride (p-APMSF), and soybean trypsin and Bowman-Birk inhibitors, and it preferentially hydrolyzed the peptide bonds of the protein substrate beta-purothionin on the C-terminal side of Arg (mainly) and Lys residues. These characteristics show that it is a trypsin-like proteinase. In addition, its N-terminal amino acid sequence was 88% identical to that of the F. oxysporum trypsin-like enzyme. The proteinase hydrolyzed the D hordein and some of the C hordeins (the barley storage proteins). This enzyme, and a subtilisin-like proteinase that we recently purified from the same organism, possibly play roles in helping the fungus to colonize grains.     

枯草芽孢杆菌SO113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化

枯草芽孢杆菌（Bacillus subtilis)SO113对水稻白叶枯病菌（Xanthomonas oryzaepv.oryzae）有强烈的抑菌作用（表现出一透明抑菌圈）。培养液上清中加入60％饱和度的硫酸铵沉淀所得的蛋白粗提液对热稳定，对链霉蛋白酶E不敏感，对蛋白酶K部分敏感，对中国各稻区白叶枯病菌的7种致病型都有强烈的抑杀作用，蛋白粗提液100℃加热15min后脱盐，经DEAE0－Sepharose Fast Flow柱层析得3个未分开的抗菌活性峰，示吸附部分经CM－Sepharose Fast Flow柱层析，得到1个主活性峰，由于B.subtilis SO113的分泌蛋白对水稻白叶枯病菌表现出良好的广谱抗性以及丰富的抗菌活性峰，因此对其进一步的研究和设法隆编码抗菌蛋白的基因都具有重要意义。