Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast,Pichia pastoris and characterization of the secreted product

Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q³⁰²) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0·47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA-Q³⁰² were attempted. Addition of Triton X-100, l-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q³⁰² and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation. Co-expression of scu-PA-Q³⁰² and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q³⁰² was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain. N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu-PA-Q³⁰² closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.     

Isolation and characterisation of four trypsin-chymotrypsin inhibitors from lentil seeds

Twenty-three proteinase inhibitors were isolated from Syrian local small lentils (Lens culinaris) by ammonium sulphate fractionation of the acidic extract followed by affinity chromatography on anhydrotrypsin-Sepharose. They all inhibited human and bovine trypsin and chymotrypsin. Three inhibitors (LCI-11·7, -3·3 and -4·6) were separated and purified to homogeneity by anion exchange chromatography and preparative isoelectric focusing (IEF) with immobilised pH gradients; a fourth (LCI-2·2) required additional reversed-phase high-pressure liquid chromatography. The four inhibitors were similar in their amino acid composition, with high cystine and aspartic acid/asparagine content, and lack of free sulphydryl groups, methionine and tryptophan. The calculated minimum number of amino acid residues per molecule, the calculated molecular masses confirmed by gel liquid chromatography, gel-permeation high-pressure liquid chromatography and sodium-dodecylsulphate polyacrylamide gel electrophoresis, and the isoelectric points determined by IEF (immobilised pH gradients and carrier ampholytes) were 84, 77, 68 and 60 residues per molecule, 9200, 8500, 7200 and 6750, and 5·26, 5·88, 6·80 and 7·80 for LCI-1·7, -2·2, -3·3 and -4·6, respectively. All four inhibitors inhibited human trypsin less than bovine trypsin, and human chymotrypsin more than the bovine enzyme. All these properties are in accordance with the classification of the four lentil inhibitors as members of the Bowman-Birk proteinase inhibitor family. © 1998 Society of Chemical Industry.     

The cytotoxic effect of Bowman–Birk isoinhibitors,IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman–Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health‐promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non‐malignant colonic fibroblast CCD‐18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9±2.3 and 48.3±3.5 μM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose‐dependent manner, with cells becoming blocked in the G0–G1 phase. Chemically inactive soybean BBI had a weak but non‐significant effect on the proliferation of HT29 cells. The anti‐proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin‐ and chymotrypsin‐like proteases involved in carcinogenesis should be considered as potential targets of BBI‐like proteins.     

Effects of Processing on the Reduction of β-ODAP (β-N-Oxalyl-L-2,3-diaminopropionic acid) and Anti-Nutrients of Khesari Dhal,Lathyrus sativus

Effects of different processing techniques on the neurotoxin, β-ODAP (β- N -oxalyl-L-2,3-diaminopropionic acid), and the anti-nutritional compounds (phytate, polyphenols, trypsin and amylase inhibitors, and lectins) within four lines of Lathyrus sativus (high-, medium- and low-ODAP, and so-called ODAP-free) were investigated. Soaking of seeds in various media reduced the contents of these compounds to a varying and significant extent; losses were higher in freshly boiled water, alkaline and tamarind solutions than after soaking in drinking water. The highest losses in boiled water (65–70%) were observed for β-ODAP, followed by trypsin inhibitors (42–48%) and polyphenols (30–37%). Ordinary cooking and pressure cooking of pre-soaked seeds were found to be most effective in reducing the levels of all the natural toxicants examined, whilst fermentation and germination were more effective in destroying both of the enzyme inhibitors (amylase inhibitors by 69–71%; trypsin inhibitors by 65–66%) than either phytates or polyphenols. Lectins were not affected by most of these processes except by pressure cooking and fermentation. Dehusking of pre-soaked seeds significantly reduced β-ODAP levels, but this reduction was lower for the anti-nutrients. These findings and the high water solubility suggest that a simple and effective means of detoxifying Lathyrus by removing this neurotoxic amino acid may be practicable.     

Immunoaffinity chromatography purification and characterisation of pea trypsin inhibitors

Trypsin inhibitors from pea (Pisum sativum (L) cultivar Progreta grown in Denmark) have been isolated and shown to consist of at least nine pea proteinase inhibitors (PPI) with inhibitor activity toward both trypsin and chymotrypsin. The isoelectric points of the inhibitors were in the range 4.9–7.8. From this PPI mixture at least four inhibitors were isolated by immunoaflinity chromatography on a column containing immobilised monoclonal antibody (mAb) with inhibitor specificity. The PPI isolated by immunoaffinity chromatography were further separated by HPLC, and subsequent SDS-PAGE analysis showed molecular weights for four of the PPI in the range 11.3–14.2 kD. Their pI were determined by isoelectric focusing, mAbs were used for immunochemical characterisation and their amino acid composition showed a high (14.7–21%) content of cysteine. Tryptophan was not present in any of the isolated PPI. The data now obtained support the resemblance of PPI with inhibitors of the Bowman-Birk class and the differences in immunochemical properties of the various PPI indicate that pea has at least two gene loci coding for the inhibitors.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF YELLOW CROAKER (PSEUDOSCIAENA CROCEA)

A myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of yellow croaker (Pseudosciaena crocea) was isolated from myofibril by heat treatment and chromatographies on Sephacryl S‐200, fast performance liquid chromatography (FPLC) on Mono Q column and high performance liquid chromatography (HPLC) using a Bio‐Sil SEC‐125 column. A single protein band with a molecular weight of 34 kDa was observed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Optimum temperature and pH of the purified protein was 55C and 8.0. Inhibitor susceptibility analysis indicated that the enzymatic activity was effectively suppressed by serine proteinase inhibitors such as Pefabloc 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride and aprotinin, while inhibitors for other proteinases (namely cysteine, asparatic and metallo) did not show any inhibitory effect. At the last purification stage, the electrophoretic study revealed that the proteinase associated with tropomyosin, as the N‐terminal amino acid sequence of the 34 kDa main band protein, exhibited high sequence homology (90%) to α‐tropomyosin from white croaker, human and rat. This result suggested that yellow croaker MBSP is an α‐tropomyosin‐binding proteinase.     

Subtilisin inhibitors in Canavalia and Vicia faba seeds. A comparative study

Subtilisin isoinhibitors (SI) were isolated from jack bean (Canavalia ensiformis L) and broad bean (Vicia faba L) seeds. Jack beans contain three isoinhibitors (pI 6.6, 6.3 and 6.0) that constitute 0.021 g per 100 g of dry seeds, while the two active proteins from broad beans (pI 5.7 and 5.1) represent 0.028%. The molecular weight, determined by gel filtration, is around 8000 D in both legumes. Large variations in specific activity of SI against subtilisin Carlsberg were detected in six species of the Canavalia genus, whereas only slight changes were found among six cultivars of C ensiformis. Antibodies raised against SI isolated from jack beans are specific for SI from different varieties of this and other species of the Canavalia genus. However, they do not recognise SI from broad beans and other legume seeds tested. In broad beans the variability between cultivars is significant. Cv Canaria has two active bands and four are detected in the ?dark’? variety, which is 2.7 times less active than the former. SI are specific towards microbial serine proteases, showing high affinity for proteinase K. No appreciable activity against animal proteases or plant thiol enzymes can be detected. SI are also inactive against 4 alpha-amylases of different origins. Reversible limited proteolysis of the reactive site bond suggests that SI interact with subtilisin by the ?standard mechanism’? usually accepted for the inhibition of trypsin by its proteinaceous inhibitors.     

Biochemical basis of insect resistance in Vigna unguiculata

The bruchid beetle Callosobruchus maculatus (F.) causes extensive damage to seeds of the cowpea, Vigna unguiculata (L.) (Walp.), when this important tropical foodstuff is stored. A variety of cowpea resistant to attack by this pest has been described. In the present work seeds of a number of cowpea varieties, including the resistant one, were tested for the presence of a physical resistance to C. maculatus, in terms of repulsion of oviposition or of failure of larvae to enter the seeds. No evidence to suggest the presence of a physical resistance was found. When seeds of cowpea varieties were tested for the presence of various antimetabolic secondary compounds, only inhibitory activity against trypsin and, to a much lesser extent, chymotrypsin, could be detected. The resistant variety of cowpea contained a significantly higher level of inhibitors, about twice as much as any other variety. A proteinase inhibitor active against trypsin was purified from cowpea varieties by affinity chromatography on trypsin-Sepharose. The purified inhibitor was shown to inhibit chyraotrypsin also, in such proportions as to account for chymotrypsin inhibition by seed extracts. The inhibitor was shown to consist of a number of isoinhibitors by gel electrophoresis and isoelectric focusing, but no qualitative differences in the inhibitor between varieties could be detected. The antimetabolic nature of the cowpea trypsin inhibitor was confirmed by insect feeding trials in which various protein fractions were added to a basic meal and the effect on larval survival noted. The albumin proteins of cowpea (containing the trypsin inhibitors) at a level of 10% were toxic to larvae of C. maculatus whereas the globulin fractions were not. Further, if cowpea trypsin inhibitor was removed from the albumin proteins they ceased to be toxic. When purified cowpea trypsin inhibitor was added to the basic meal it was shown that a level slightly less than that found in the resistant variety of cowpea caused complete mortality of larvae, whereas lower levels had lesser or no effect. It is concluded that this example of insect resistance in the cowpea is due to an elevated level of trypsin inhibitor.     

Some physicochemical and antinutritional properties of raw flours and protein isolates from Mucuna pruriens (velvet bean) and Canavalia ensiformis (jack bean)

The legumes Canavalia ensiformis and Mucuna pruriens are underexploited in tropical Mexico. Their seeds have good nutritional potential, but contain antinutritional factors. Physicochemical and antinutritional properties were determined for raw flours (RF) and protein isolates (PI) produced from these legumes. Protein content in the PI was 737 g kg^–1 for C. ensiformis and 666 g kg^–1 for M. pruriens. Protein isolation improved in vitro digestibility, while maintaining high lysine levels and adequate sulphur amino acids content. Antinutritional factors such as cyanogenic glucosides, cyanide precursors from hydrolysis, tannins and trypsin inhibitors were lower in the PIs than in the RFs. The reduction in canavanine levels, a structural analogue of arginine, in the C. ensiformis PI was noteworthy. These PIs thus have potential applications in the development of new food ingredients in tropical regions using processes that improve nutritional value.     

Cold-adapted structural properties of trypsins from walleye pollock (<Emphasis Type="Italic">Theragra chalcogramma</Emphasis>) and Arctic cod (<Emphasis Type="Italic">Boreogadus saida</Emphasis>)

Complementary DNA clones encoding trypsins were isolated from pyloric ceca of cold-adapted fish, walleye pollock (Theragra chalcogramma) (WP-T) and Arctic cod (Boreogadus saida) (AC-T). The isolated full-length cDNA clones of WP-T and AC-T were 852 and 860 bp, respectively, and both cDNAs were contained an open reading frame of 726 bp. WP-T and AC-T seemed to be synthesized as preproenzyme that contains a signal peptide, an activation peptide, and a mature trypsin. Although the amino acid sequence identities of WP-T and AC-T to that of bovine trypsin were 64 and 63%, respectively, they completely conserved the structural features for catalytic function of trypsin. On the other hand, WP-T and AC-T possessed the four Met residues (Met135, Met145, Met175 and Met242) in their molecules and the deletion of Tyr151 and substitution of Pro152 for Gly in their autolysis loops when aligned with the sequences of tropical-zone fish and bovine trypsins. In addition, the contents of charged amino acid residues at the N-terminal regions (positions 20–50) of WP-T and AC-T were extremely higher than those of other fish and bovine trypsins. Moreover, one amino acid (Asn72) and two amino acids (Asn72 and Val75) coordinating with Ca²⁺ in bovine trypsin were exchanged for another amino acids in WP-T (His) and AC-T (His and Glu), respectively, and the contents of negative charged amino acids at their Ca²⁺-binding regions were lower than those of tropical-zone fish and bovine trypsins. Therefore, it was considered that these structural characteristics of WP-T and AC-T are closely related to their lower thermostability.     

Enzymic Modification of Feather Keratin Hydrolysates with Lysine Aimed at Increasing the Biological Value

The lysine content of feather protein hydrolysate was modified via activation with alkaline proteinase and trypsin at pH 6·2 and 8·3, respectively. Lysine was used as the hydrochloride and the diacetyl derivative in the first case and as the methyl ester in the trypsin modification. The results indicate a lysine content of 26–35 g kg^-1 and 59–64 g kg^-1 after alkaline proteinase activation and 52–65 g kg^-1 after trypsin activation. Considerable changes in the molecular mass profile of peptides were observed. Thus, enzyme modification is a way to raise the nutritive value of feather protein hydrolysate by increasing its lysine content (the main limiting essential amino acid). © 1997 SCI.     

ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

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.     

Proteinase inhibitory activity of sarcoplasmic proteins from threadfin bream (Nemipterus spp.)

BACKGROUND: Thailand is the second largest surimi producer in the world and 50% of surimi is produced from threadfin bream. During surimi processing, sarcoplasmic proteins are removed through water washing and discarded in the waste stream. This study was aimed at investigating the proteinase inhibitory activity of sarcoplasmic proteins. RESULTS: Sarcoplasmic proteins from threadfin bream (TBSP) exhibited inhibitory activity toward trypsin but did not inhibit papain and chymotrypsin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis under non‐reducing condition stained by trypsin inhibitory activity revealed three protein bands of molecular mass of 95, 41 and 37 kDa. Inhibitory activity of TBSP reached a maximum when subjected to 45 °C and completely disappeared at 60 °C. The breaking force and deformation of lizardfish surimi gel with added TBSP and pre‐incubated at 37° for 20 min increased with additional levels of TBSP (P < 0.05). Trichloroacetic acid–oligopeptide content of lizardfish surimi gel with added TBSP decreased with the addition of 4 g kg^?1 TBSP (P < 0.05). Retention of myosin heavy chain (MHC) increased when TBSP concentration was increased. TBSP effectively protected MHC from proteolysis at 37 °C to a similar extent as egg white powder, but efficacy of TBSP was not observed at 65 °C. CONCLUSION: TBSP could be applied to reduce proteolytic degradation of lizardfish surimi or other surimi associated with trypsin‐like proteinase, rendering an improvement in surimi gelation set at 37–40 °C. Copyright © 2009 Society of Chemical Industry     

Characteristics of the proteins of the tubers of winged bean (Psophocarpus tetragonolobus (L.) DC)

The crude protein content (amino acid Nx6.25) of six varieties of Psophocarpus tetragonolobus tuber varied from 12.7 to 16.9%. Free amino acids and low molecular weight peptides account for about 40% of this crude protein value. The non-dialysable protein content of winged bean tuber is therefore less than previously assumed, but is still relatively high for a tuber crop. The amino acid contents of the tuber varieties were similar and nutritionally acceptable, except for the sulphur containing amino acids. Fresh tubers showed trypsin and chymotrypsin inhibitory activity and haemagglutinin activity. The inhibitor levels showed little variation but differences in the levels of haemagglutinin activity were found. On SDS-polyacrylamide gels the tuber proteins revealed a simple subunit pattern of two major polypeptides of M_r 30000 and 20000. Fractionation of the tuber proteins showed that the inhibitors and haemagglutinins comprise about 35% of the tuber protein. In addition, three acidic protein (about 12% of tuber protein), three basic proteins (ca. 34% of tuber protein) and a minor component (M_r～10000) with a high cystine content (11 residue %) were identified.     

Properties of Trypsin from the Pyloric Ceca of Atlantic Cod (Gadus morhua)

Trypsin (EC 3.4.21.4) was isolated from the pyloric ceca of Atlantic cod and purified to homogeneity by affinity chromatography. The enzyme catalyzed the hydrolysis of benzoyl arginine p-nitroanilide (BAPA, pH 8.2 and 25°C) such that V_max was 250 BAPA units per micromole trypsin and K_m was 1.48 mM. For the hydrolysis of tosyl arginine methyl ester (TAME, pH 8.1 and 25°C), V_max was 18.2 × 10³ TAME units/micromole trypsin, and K_m 0.22 mM. The pH and temperature optima with BAPA substrate were 7.5 and 40°C, respectively. Atlantic cod trypsin was most active and stable at alkaline pH. The enzyme was heat labile, losing more than 50% of its activity after incubation at 50°C for 30 min. Amino acid analysis of Atlantic cod trypsin revealed that the enzyme was rich in residues such as serine, glycine, glutamate and aspartate, but poor in basic amino acid residues compared to trypsins from warm blooded animals.     

Isolation and characterisation of invertase inhibitor from sweet potato storage roots

BACKGROUND: Plant invertases play important roles in sucrose metabolism. Cell wall invertase has been reported to participate in phloem loading and unloading. Soluble invertases are involved in hexose level regulation in mature tissues and in utilisation of stored sucrose within vacuoles. Invertase inhibitory proteins have been described as one of the possible components for invertase activity regulation in some plant species. RESULTS: In this work an invertase inhibitor (ITI) coding sequence was cloned by differential display from sweet potato (SP) storage roots. SPITI codes for a protein of 192 amino acids with a predicted molecular mass of 20 624 Da containing a 20‐amino‐acid signal peptide and four cysteines. Computer analysis of the deduced amino acid sequences of the conserved domain revealed that the protein belonged to the plant invertase/pectin methylesterase inhibitor. Both the corresponding mRNA and protein levels were found to be highest in storage roots, followed by veins. Recombinant SPITI protein from the storage root cDNA clone overproduced in Escherichia coli (M15) was purified by affinity chromatography. This protein effectively inhibited the invertase activity in a dose‐dependent manner. The results presented in the Lineweaver‐Burk plots indicated that the invertase inhibitor displayed a mode of competitive inhibition towards the invertase tested, with a K_i of 3.82 × 10^?6 mol L^?1. CONCLUSION: These results suggested that SPITI is a novel member of the ITI family in plants. SPITI genes of sweet potato storage roots display differential gene expression patterns, which may be associated with sucrose metabolism to cope with particular developmental requirements. Copyright © 2008 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF TWO DOUBLE-HEADED TRITICALE ISOINHIBITORS OF ENDOGENOUS ALPHA-AMYLASE AND SUBTITLISIN1

Two double-headed isoinhibitors of endogenous alpha-amylase and subtilisin have been purified from triticale by immobilized copper affinity chromatography followed by S-Sepharose Fast Flow ion exchange chromatography and preparative isoelectric focusing on a sucrose density gradient ampholyte column. The inhibitors had isoelectric points at pH 7.25 and 6.95, and were present in triticale grains in approximately equal amounts. Both proteins, designated inhibitor 7.25 and inhibitor 6.95, have been purified to homogeneity as assessed by nondenaturing polyacrylamide gel electrophoresis (PAGE) at pH 8.3. They also showed approximately 90% purity as determined by size exclusion limit FPLC. Their properties were similar to the endogenous alpha-amylase/subtilisin inhibitors found in wheat and barley, since they were active against cereal alpha-amylases and subtilisin and they were inactive against salivary and bacterial alpha-amylases as well as against trypsin. Apparent MW of both inhibitors was 20 kilodaltons as estimated from sodium dodecyl sulfate-PAGE under reducing conditions. No carbohydrate was detected in isoinhibitor preparations. The triticale alpha-amylase-triticale inhibitor systems studied revealed a mixed type of inhibition with apparent dissociation constant (Ki) values of 6.2 and 5.8 nM for the inhibitor 7.25 and inhibitor 6.95, respectively.     

PURIFICATION AND CHARACTERIZATION OF CHYMOTRYPSIN FROM PENAEUS VANNAMEI (CRUSTACEA: DECAPODA)

The purification and characterization of a chymotrypsin from the hepatopan-creas of the white shrimp Penaeus vannamei is described. Only one chymotrypsin was detected in contrast to other shrimp that have two major forms. P. vannamei chymotrypsin has a molecular mass of 33.2 kDa and a pI of 3.1. The molecular mass is high relative to other penaeid chymotrypsins. The proteinase is acid labile and exhibits optimum activity at pH 8. The enzyme is thermostable both at 25 and 37C. It is a serine proteinase. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor blocked the activity of the enzyme, and it was not affected by chymotrypsin inhibitors such as tosyl-PheCH₂Cl or benzyloxycarbonyl-Phe-CH₂Cl. Protein profiles of the hepatopancreas from two populations varied     

PURIFICATION AND PARTIAL CHARACTERIZATION OF WHITE KIDNEY BEAN (PHASEOLUS VULGARIS)β-AMYLASE INHIBITORS FROM TWO EXPERIMENTAL CULTIVARS

β-Amylase inhibitors WKB 858B and WKB 858B were purified to homogeneity from different cultivars of white kidney beans by extraction from the ground beans and by sequential heat treatment, ethanol fractionation, DEAE-cellulose chromatography, Sephadex G-75 gel chromatography and CM-cellulose chromatography. The inhibitors were homogeneous by 7.5% polyacrylamide gel electrophoresis; no isoinhibitors were found. Inhibitors WKB 858A and WKB 858B had isoelectric points of 5.0 and 4.65, respectively, and molecular weights of 42,000 and 20,000, respectively, by FPLC Superose 12 gel filtration chromatography. Inhibitor WKB 858A had molecular weights of 40,000 and 38,000 by Sephadex G-75 gel filtration chromatography and by native gel electrophoresis, respectively. Inhibitor WKB 858A contained 11.0% carbohydrate, N-linked to asparagine residues, with a composition of 1 fucose, 1 xylose, 4 galactose, 8 N-acetylglycosamine and 13 mannose residues per mol of inhibitor. Amino acid analysis of Inhibitor WKB 858A gave a high content of Asx, Glx, Ser, Thr and Val (combined total of 60% molar ratio) and low content of sulfur amino acids (0.8% molar ratio of Met and no 1/2 cystine). No-SH groups were found. The amino acid composition was similar to that of eight other a-amylase inhibitors from beans. Inhibitor WKB 858A formed a 1:1 stoichiometric complex with porcine pancreatic a-amylase with a Ki of 1.0 × 10^?11 M at pH 5.4 and 30C; it had no trypsin inhibitory activity. At pH 6.90 and 30C, the rate of complex formation between Inhibitor WKB 858A and porcine pancreatic β-amylase was 2.76 times faster at 1.385 vs 0.035 ionic strength (with Na₂SO₄), indicating hydrophobic bonds are most important in complex formation.