Trypsin-chymotrypsin Inhibitor Research Articles

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legumecropproductivities. Protease inhibitors affectprotein degradation and reducethe availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases andthe growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmataliswas delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage whereinA. gemmatalis fedvoraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline informationabout the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Extraction, partial purification and characterization of alkaline protease from rainbow trout (Oncorhynchus Mykiss) viscera

In this study, crude alkaline proteases were recovered from rainbow trout (Oncorhynchus mykiss) viscera and partially purified by use of different saturation of ammonium sulfate. The enzyme exhibited highest yield, purity and activity when precipitated at a saturation of 40–60% compared to other ranges of saturation. Molecular weight for extracted protease was between 8-24 kDa. The protease had caseinolytic activity over a wide range of temperatures (30-55 °C) and pH (4-12). Soybean trypsin inhibitor and trypsin-chymotrypsin inhibitor strongly inhibited the enzyme activity but it was stable in the presence of surfactant, oxidizing reagents and organic solvents. The proteases had serine protease activity but no collagenase activity was detected. The current study showed that partially purified protease from the digestive tract of rainbow trout could be applicable in food and detergent industry because of its good activity over a wide temperature and pH range and its good thermal stability.

First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin-trypsin inhibitor purified from Lonchocarpus sericeus seeds.

Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin-trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases. LsCTI showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mass determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (Ki ) for chymotrypsin (8.24 x 10-8 m). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50-percent enzyme inhibition (IC50 ) of LsCTI was 4.7 x 10-7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL-1 , but caused a high larval mortality rate (77%) and delayed development (37%). LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development. © 2017 Society of Chemical Industry.

Assessment of soybean inhibitor as a biopesticide against melon fruit fly, Bactrocera cucurbitae (Coquillett)

In the current study, the soybean trypsin–chymotrypsin inhibitor (Bowman–Birk Inhibitor, SBBI) was tested against Bactrocera cucurbitae (Coquillett), a major pest of cucurbit crops. Bioassays conducted using different concentrations (12.5, 25, 50, 100 and 200 ppm) revealed a detrimental effect of the inhibitor on the growth and development of the second instar larvae of the melon fruit fly. SBBI prolonged the larval and total development period and reduced the percentage pupation and emergence. Enzymatic assays of proteases conducted at three time intervals using the LC40 (59 ppm) concentration of SBBI showed an inhibitory effect on trypsin activity, whereas an increase was observed in the activity of chymotrypsin, elastase and leucine aminopeptidase. Among the enzymes involved in detoxification, antioxidant and general metabolism, an increase was observed in the activity of catalases, and acid and alkaline phosphatases at most treatment intervals. The activity of esterases was induced only with prolonged treatment whereas that of glutathione S-transferases was suppressed in larvae treated with SBBI. The findings revealed the potential of SBBI to disrupt the growth of the melon fruit fly.

Purification and antiparasitic activity of a few legume serine proteinase inhibitors: Effect on erythrocyte invasion, schizont rupture and proteolytic processing of the Plasmodium falciparum AMA1 protein

Abstract Legume proteinase inhibitors (PI/s) abrogate proteolytic activity of proteins and cause adverse effects on growth. In this study, two legume PIs – Archidendron ellipticum Trypsin Inhibitor (AeTI) and Derris trifoliata Trypsin Chymotrypsin Inhibitor (DtTCI) were purified and used along with standard, Soybean Trypsin Inhibitor (STI), either singly or in combination, as antiplasmodial agents against two Plasmodium forms (Pf 3D7/Pf FCR3). Both AeTI/DtTCI were purified to homogeneity by HPLC and showed over 98% trypsin/chymotrypsin inhibition, respectively. DtTCI severely arrested growth of both the Plasmodium forms (IC50 of 9.59 μM and 16.86 μM, respectively). In addition, combination of DtTCI/AeTI had synergistic effect against both Pf 3D7 (FIC – 0.19) and Pf FCR3 (FIC − 0.23). Combinations of DtTCI/STI and AeTI/STI showed additive effects for the parasite forms. Time-course studies indicated DtTCI and combination of DtTCI/AeTI, to be potent inhibitor of schizont rupture. Antiproteolytic action of the PIs on Pf Apical Merozoite Antigen 1 (AMA1) protein revealed that none of the inhibitors (singly/combinations) affected the primary proteolysis of PfAMA1 protein. Notably however, the normal secondary proteolysis of PfAMA1 was abrogated by both DtTCI (singly/combinations) and AeTI. Incidentally, the proteolytic processing of PfAMA1 remained unaffected following STI treatment.

Rational tailoring of substrate and inhibitor affinity via ATRP polymer-based protein engineering.

Atom transfer radical polymerization (ATRP)-based protein engineering of chymotrypsin with a cationic polymer was used to tune the substrate specificity and inhibitor binding. Poly(quaternary ammonium) was grown from the surface of the enzyme using ATRP after covalent attachment of a protein reactive, water-soluble ATRP-initiator. This "grafting from" conjugation approach generated a high density of cationic ammonium ions around the biocatalytic core. Modification increased the surface area of the protein over 40-fold, and the density of modification on the protein surface was approximately one chain per 4 nm(2). After modification, bioactivity was increased at low pH relative to the activity of the native enzyme. In addition, the affinity of the enzyme for a peptide substrate was increased over a wide pH range. The massively cationic chymotrypsin, which included up to 2000 additional positive charges per molecule of enzyme, was also more stable at extremes of temperature and pH. Most interestingly, we were able to rationally control the binding of two oppositely charged polypeptide protease inhibitors, aprotinin and the Bowman-Birk trypsin-chymotrypsin inhibitor from Glycine max, to the cationic derivative of chymotrypsin. This study expands upon our efforts to use polymer-based protein engineering to predictably engineer enzyme properties without the need for molecular biology.

Endonuclease com incompatibilidade heteroduplex para detectar mutação e variações genéticas de inibidores da tripsina em soja

O objetivo deste trabalho foi avaliar a variação genética do inibidor de tripsina em variedades cultivadas (Glycine max) e silvestres (Glycine soja) de soja. Foram avaliadas as variações genéticas do inibidor de tripsina Kunitz, representado pela proteína 21-kDa (KTI), e do inibidor de tripsina-quimotripsina Bowman-Birk (BBI), em variedades de soja cultivadas (G. max) e selvagens (G. soja). Ensaios de clivagem foram feitos com endonuclease de incompatibilidade heteroduplex, para a detectar mutações no gene de KTI, com uma única nuclease específica de cadeia simples, obtida a partir de extractos de aipo (CEL I). As variedades de soja estudadas apresentaram baixo nível de variação genética em KTI e BBI. A análise por PCR -RFLP dividiu o BBI-A em A1 e A2 e mostrou que o Tib do KTI é o tipo dominante. A digestão com enzimas de restrição não foi capaz de detectar diferenças entre os tipos de ti-null e outros alelos Ti, enquanto o ensaio com endonucleases com incompatibilidade heteroduplex com CEL I pôde detectar o tipo ti-null. O método de digestão com CEL I fornece uma ferramenta genética simples e útil para a análise de SNP. O método apresentado pode ser utilizado como ferramenta para a triagem rápida e útil de genótipos desejáveis em futuros programas de melhoramento de soja.

Analysis of sequential accumulation of individual pigeonpea protease inhibitors during seed development

Seeds of pigeonpea are known to accumulate protease inhibitors (PIs), belonging to the Bowman-Burk inhibitor family. PIs are important for natural defense against phytophagous insect pests. Most insects attack crops at the early stages of seed development. Accumulation patterns of individual PIs and their relationship with each other were studied in developing seeds of 76 pigeonpea lines derived from BDN2 cultivar by ethyl methane sulfonate induced chemical mutagenesis. PIs extracted from these lines, collected between 10 and 40 days after flowering (DAF) and from mature seeds of BDN2 cultivar, were detected by using gel X-ray film contact print method. A total of 9 trypsin–chymotrypsin inhibitors were detected in mature seeds. All the nine PIs were capable of inhibiting proteases. Appearance of detectable levels of individual PIs started around 10 DAF. The PI-3 appeared early and was the most stable. It was accumulated in all the studied lines and was also detected in 84 % of the samples collected 10 DAF. In particular, 14 DAF, the individual PIs were accumulated and accumulation sequence was observed (PI-3, PI-2, PI-5, PI-7, PI-6, PI-4, PI-8, PI-9 and PI-1). Accumulation continued up to 40 DAF when seeds started hardening. Chemical mutagenesis could not produce any variation in the profile of individual PIs in the 76 studied lines in mature seeds. The process of accumulation of inhibitors is sturdy and mutagenesis fails to alter it. The robust mechanism is responsible for early appearance of PI-3. Early accumulated PIs in this study need further exploration for strengthening natural defense of pigeonpea against pests.

Isolation of a thermostable trypsin inhibitor with exploitable potential.

A novel trypsin inhibitor with considerable thermal and pH stability, designated Glytine, was isolated from seeds of the Chinese black soybean Glycine max (L.) Merr. The purification procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, gel filtration chromatography on Sephacryl S-200HR, and gel filtration chromatography on POROS HS-20. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating close homology with the sequences of leguminous trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by SDS-PAGE and isoelectric focusing to be 19.9 kDa and 6.2, respectively. Trypsin could be completely inhibited by Glytine when the weight ratio was 1.5. The inhibitory activity of Glytine was unaffected by exposure to temperatures up to 100 °C, or within the pH range 2–12. Besides trypsin–chymotrypsin inhibition activity, Glytine demonstrated other biological activities including antiproliferative activity against tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. In addition, the inhibitor showed antifungal activity against Pythium aphanidermatum, Fusarium oxysporum, Alternaria alternata (Fr.) Keiss, Fusarium solani, and Botrytis cinerea. This study extended research on leguminous trypsin–chymotrypsin inhibitor and suggested exploitable potential.

A potential human hepatocellular carcinoma inhibitor from Bauhinia purpurea L. seeds: from purification to mechanism exploration

A 20-kDa Kunitz-type trypsin-chymotrypsin inhibitor, Bauhinia purpurea trypsin inhibitor (BPLTI), has been isolated from the seeds of B. purpurea L. by using liquid chromatography procedures that involved ion exchange chromatography on Sp-Sepharose and Mono S and gel filtration on Superdex 75. BPLTI demonstrated protease inhibitory activities of 7226 BAEE units/mg and 65 BTEE units/mg toward trypsin and α-chymotrypsin, respectively. BPLTI was relatively thermal (0-60°C) and pH (3-10) stable and its activity could be decreased by dithiothreitol treatment. BPLTI exhibited a wide spectrum of anti-proliferative and pro-apoptotic activities especially on human hepatocellular carcinoma Hep G2 cells. However, it was devoid of a significant antiproliferative effect on immortal human hepatic WRL 68 cells. We show here that BPLTI stimulates apoptosis in Hep G2 cells, including (1) evoking DNA damage including the production of chromatin condensation and apoptotic bodies; (2) induction of cell apoptosis/necrosis; (3) mitochondrial membrane depolarization; and (4) increasing the production of cytokines. Taken together, our findings show for the first time that purified protease inhibitor from B. purpurea L. seeds is a promising candidate for the treatment of human hepatocellular carcinoma.

Site-Directed Mutagenesis to Identify Key Residues in Structure-Function Relationship of Winged Bean Chymotrypsin-Trypsin Inhibitor and 3-D Structure Prediction

Winged bean chymotrypsin trypsin inhibitor (WbCTI) is a Kunitz type serine protease inhibitor that inhibits both trypsin and chymotrypsin at 1:1 molar ratio. Site-directed mutagenesis study was employed to generate two mutants of WbCTI, with an aim to explore its dual inhibitory properties against the proteases. The mutants were expressed in Escherichia coli and, were purified to homogeneity using a single step immunoaffinity column. The two mutants, each containing a single mutation at the amino acid sequence positions of 63 and 64, were named as L63A and R64A, respectively. Purified L63A-WbCTI exhibited anti-trypsin activity with no anti-chymotrypsin activity whereas R64A-WbCTI could inhibit chymotrypsin but not trypsin. To investigate the binding interactions between the mutated forms of WbCTI with the putative proteases, binding studies were carried out using gel filtration chromatography which further confirmed the formation of enzyme-inhibitor complexes. Finally, 3D model structure of WbCTI was designed using computer simulations which further emphasize the roles of L63 and R64 residues for dual inhibitory characteristics of WbCTI.

Growth and development of melon fruit fly larvae under the influence of soybean trypsin inhibitors

Protease inhibitors (PIs) retard growth and development and cause mortality in a range of insect pests belonging to various orders. Some transgenic plants expressing PI genes have also been effective against insect pest attack. The effects of two purified soybean PIs, i.e. trypsin inhibitor (Kunitz type; SBTI) and trypsin-chymotrypsin inhibitor (Bowman-Birk type; SBBI), were investigated on first and third instar larvae of the melon fruit fly Bactrocera Cucurbitae (Coquillett) in laboratory feeding bioassays. The larval period of both the first and third instar larvae was prolonged when the larvae were treated with various concentrations of SBTI and SBBI. The total development period prolonged significantly in both the instars of larvae treated with SBTI, but in SBBI-treated larvae, the development period was delayed significantly only in the third instar larvae. A decrease in percentage pupation and adult emergence was observed in the first and third instar larvae treated with different concentrations of both SBTI and SBBI. The present studies showed that SBTI and SBBI have a potential in inhibiting the growth and development of melon fruit fly, but further detailed and molecular level studies are required before their use in transgenics.

A leguminous trypsin-chymotrypsin inhibitor Limenin with antifungal activity from Phaseolus limensis.

A trypsin-chymotrypsin inhibitor, designated Limenin, with both antifungal and antibacterial activity, and exhibiting a molecular mass of 18.0 kDa in SDS–polyacrylamide gel electrophoresis, was isolated from the large lima bean (Phaseolus limensis) legumes by a combination of extraction, ammonium sulfate precipitation, ion exchange chromatography on SP-Toyopearl and high performance liquid chromatography (HPLC) on Mono S. The isoelectric point was estimated to be 7.6 by isoelectric focusing. The 15 N-terminal amino acid sequences were determined to be DFVIDNEGNPLENGG, demonstrating some resemblance to those other protease inhibitors and inhibitor precursors from leguminous plants. It exerted potent antifungal action toward Botrytis cinerea, Alternaria alternata(Fr.) Keissl, and Pythium aphanidermatum. It showed antiproliferative activity toward tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. However, it had no effect on bacteria Staphylococcus aureus and Salmonella.

Inhibition of proteolysis in luminal extracts from the intestine of the brushtail possum.

The proteolytic activity of luminal extracts from five regions (duodenum, jejunum, ileum, caecum and colon) of the brushtail possum intestine towards bovine serum albumin (BSA) and human luteinizing hormone releasing hormone (LHRH) was investigated. There were no significant differences in degradation rates between fresh and previously frozen extracts from any region of the possum intestine. The inhibition of degradation of BSA by luminal extracts from two regions (jejunum and ileum) and of LHRH from four regions (jejunum, ileum, caecum and colon) was evaluated. Soybean trypsin-chymotrypsin inhibitor (SBTI), sodium deoxycholate, Carbopol 934P, bacitracin and bestatin significantly inhibited the degradation of both LHRH and BSA (P < 0.05). SBTI almost totally inhibited the proteolysis of BSA and the peptidolysis of LHRH in extracts from the small intestine. This finding suggests that serine proteases such as chymotrypsin are responsible for the protein and peptide degradation in luminal extracts. It is concluded that including serine protease inhibitors in a formulation may enhance oral delivery of bioactive peptides and proteins to possums.

Genomic and cDNA Cloning, Expression, Purification, and Characterization of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds

A 516-bp winged bean chymotrypsin-trypsin inhibitor (WbCTI) gene was amplified from genomic DNA and cDNA isolated from winged bean using a pair of degenerate primers designed on the basis of the amino acid sequences of WbCTI. The amplified PCR products were cloned and sequenced to confirm their authenticity. DNA sequence analysis of the genomic and cDNA clones of WbCTI revealed the same nucleotide sequence in the coding region and showed WbCTI to be an intron less gene. WbCTI was subcloned into pTrc99A and expressed in Escherichia coli to yield a recombinant protein (rWbCTI). rWbCTI was purified by a rapid and single step immunoaffinity chromatography method, with an overall yield of 1.1 mg/g of wet cells. The homogeneity of the purified protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the presence of a single protein band. Functionally rWbCTI is indistinguishable from WbCTI, since both inhibit bovine trypsin and chymotrypsin in a 1:1 molar ratio. FPLC binding studies also confirmed that rWbCTI binds the proteases in a 1:1 molar ratio.

Trypsin-Chymotrypsin Inhibitors from Vigna mungo Seeds

Three trypsin-chymotrypsin inhibitors were isolated from seeds of the black gram (Vigna mungo) with a procedure that entailed cation exchange chromatography on SP-Sepharose, anion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. Two of the trypsin-chymotrypsin inhibitors were adsorbed on the first four types of chromatographic media. All three inhibitors have a molecular mass of 16 kDa as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitors was attenuated in the presence of the reducing agent dithiothreitol. The remaining inhibitor was unadsorbed on SP-Sepharose but adsorbed on Q-Sepharose, Mono Q and Mono S. The protease inhibitors did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cells or antifungal action toward Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. Two of the inhibitors slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 in the millimolar range.

Purification of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds using Single Step Immunoaffinity Column

Problem statement: Winged bean is a rich source of protease inhibitors. The seeds of winged bean contain several serine protease inhibitors of which winged bean chymotrypsin-trypsin inhibitor (WbCTI) is of significant importance due to its dual inhibition of both chymotrypsin and trypsin. The purification of WbCTI has always been difficult and a time taking process that involved large number of steps and various purification columns. Approach: This study was focused on the single step method of purification of WbCTI to homogeneity from winged bean seeds using immunoaffinity column chromatography. Results: WbCTI was purified to homogeneity using anti-WbCTI antibody column in a single step which gave a single band on SDS-PAGE analysis and with almost one and half times more yield of purified protein than obtained from previously mentioned purification methods. Conclusion: This method using immunoaffinity column is high yielding and time saving process of purification.

Kunitz‐type protease inhibitors group B from Solanum palustre

Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.

Digestive Endo-Proteases from the Midgut Glands of the Indian White Shrimp, &lt;i&gt;Penaeus Indicus&lt;/i&gt; (Decapoda: Penaeidae) from Kenya

In order to provide information on the digestive capacity of marine crustacea of mariculture potential in Kenya with a view to aiding the development of suitable feeds to support the industry, a biochemical survey was made of enzymes of extracellular digestion in the Indian White shrimp, Penaeus indicus. Results showed midgut gland endo-proteases in wild adult shrimp from the Kenya coast to have optima between pH 7.2 and 8.5 (Trypsin pH 7.5-8.0, Chymotrypsin pH 7.2-7.8, Elastase pH 6.8-8.5) with maximum specific activities of 101-408, 37-516, 70-90 Units mg protein-1 min-1 for trypsin, chymotrypsin and elastase respectively. There was no pepsin. The North Sea Norway lobster, Nephrops norvegicus, was investigated to a lesser extent and found to have much lower trypsin activity than the shrimp and no chymotrypsin. In addition to the cited serine endo-proteases, significant activity in the shrimp was thought to originate from non-serine proteases. This situation may differ from other shrimps in which serine endo-protease activity, especially trypsin, is dominant. Diphenylcarbamyl chloride (DPCC) and 2-Nitro-4-Carboxyphenyl N,N-Diphenylcarbamate (NCDC) inhibited chymotrypsin but not trypsin, Soybean Trypsin Inhibitor (SBTI), Bowman-Birk Chymotrypsin-Trypsin Inhibitor (BBSTCI), N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK), 4-(2-Aminoethyl)-Benzenesulfonylfluoride Hydrochloride (AEBSF) and N-Tosyl-LLysine Chloromethyl Ketone/1-Chloro-3-Tosylamido-7-Amino-L-2-Heptanone Hydrochloride (TLCK) inhibited both, while Phenyl Methanesulfonyl Fluoride/ Phenylmethyl Sulfonyl Fluoride/ &#945;-Toluenesulfonyl Fluoride (PMSF) and Ovomucoid Trypsin Inhibitor (Ovomucoid/OTI) precipitated shrimp homogenate. The effect of the former was inferred from the action of AEBSF which together with TLCK inhibited shrimp trypsin more than chymotrypsin. In contrast, TPCK inhibited shrimp chymotrypsin more than trypsin. These results indicate that relying on imported commercial feeds, usually developed for other species or strains of farmed shrimp in other parts of the world, may not only be too uneconomical but may not provide adequate nutrition to local animals if not efficiently digested. There is, therefore, greater need and urgency to establish detailed enzymic profiles and digestive capacities of locally cultured fin and shellfish. Such studies should parallel those prospecting for suitable feed ingredients while developing local capacity for feed technology.br> West Indian Ocean Journal of Marine Science Vol. 4 (1) 2005: pp. 109-121

Isolation and Characterization of a Trypsin-Chymotrypsin Inhibitor from the Seeds of Green Lentil (Lens culinaris)

A Bowman-Birk type trypsin-chymotrypsin inhibitor was isolated from seeds of the legume green lentil (Lens culinaris) by means of affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. The trypsin-chymotrypsin inhibitor was bound on the first three types of chromatographic media. It appeared as a single 16-kDa peak in gel filtration and a single 16-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitor was sensitive to the reducing agent dithiothreitol. It was completely abrogated after treatment with 10 mM dithiothreitol for 20 minutes. The protease inhibitor did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cell lines. There was no suppressive action on several fungal species including Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 of 30 mM.