Characterization and partial purification of the digestive proteases of the black field cricket,Teleogryllus commodus (Walker): Elastase is a major component

The major proteases of the black field cricket, Telleogryllus commodus, digestive system have been identified, partially purified and characterized. Classification of proteases into different classes of endo- and exopeptidases was made on the ability to hydrolyse specific synthetic substrates, pH optima and their interaction with a range of specific chemical and proteinaceous inhibitors. The major activities detected were trypsin, elastase, an uncharacterized proteinase (proteinase Tc), leucine aminopeptidase and carboxypeptidases A and B. Chymotrypsin activity was very low and neither cysteine endopeptidase nor metalloendopepitidase activities were found. Elastase is a newly discovered protease activity for insects.Trypsin, elastase and proteinase Tc have molecular weights of 24,300, 19,500 and 23,600, respectively; show alkaline pH optima and chemical inhibition indicative of serine endopeptidases; and interact most strongly with their characteristic class of proteinaceous inhibitors. Elastase and proteinase Tc are inhibited by a very similar spectrum of specific inhibitors, but the latter lacks activity against all specific synthetic substrates tested. Leucine aminopeptidase and carboxypeptidase A have molecular weights of 94,000 and 39,700, respectively, and show optimum activity at pH 8 and pH 9, respectively.The equilibrium dissociation constants for trypsin, elastase and proteinase Tc with 25 serine proteinase inhibitors were measured. Values spanning a 1000-fold range were obtained in each case.     

Biochemical characterization of digestive proteases and carbohydrases of the carob moth,Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.     

Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications

We characterized trypsin‐ and chymotrypsin‐like serine alkaline proteases from cotton bollworm, Helicoverpa armigera, for their probable potential application as additives in various bio‐formulations. Purification was achieved by using hydroxylapatite, DEAE sephadex and CM sephadex columns, which resulted in increased enzyme activity by 13.76‐ and 14.05‐fold for trypsin and chymotrypsin, respectively. Michaelis–Menten constants (K_m) for substrates of trypsin and chymotrypsin, BApNA and SAAPFpNA, were found to be 1.25 and 0.085 mM, correspondingly. Fluorescent zymogram analysis indicated the presence of five trypsin bands with molecular masses of ～21, 25, 38, 40, and 66 kDa and two chymotrypsin bands with molecular masses of ～29 and 34 kDa in SDS‐PAGE. The optimum pH was 10.0 and optimum temperature was 50°C for proteolytic activity for the purified proteases. The proteases were inhibited by synthetic inhibitors such as PMSF, aprotonin, leupeptin, pefabloc, and antipain. TLCK and TPCK inhibited about 94 and 90% of trypsin and chymotrypsin activity, respectively, while EDTA, EGTA, E64, pepstatin, idoacetamide, and bestatin did not affect the enzymes. The purified enzymes exhibited high stability and compatibility with metal ions; oxidizing, reducing, and bleaching agents; organic solvents; and commercial detergents. Short life cycles, voracious feeding behavior, and production of multiple forms of proteases in the midgut with rapid catalytic activity and chemostability can serve H. armigera as an excellent alternative source of industrially important proteases for use as additives in stain removers, detergents, and other bio‐formulations. Identification of enzymes with essential industrial properties from insect species could be a bioresource.     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.     

Purification and properties of three intracellular proteinases from Candida albicans

Three intracellular proteinases termed A, B and C were purified to homogeneity from the unicellular form of the yeast Candida albicans. Enzyme A is an aspartic proteinase that acts on a variety of proteins. Its optimal pH is around 5 and it is displaced to 6.5 by KSCN. It is not significantly inhibited by PMSF, TLCK (Tos-Lys-CHCl₂) or soybean trypsin inhibitor but it is inhibited by pepstatin. Its molecular weight is 60 000. Enzyme B is a dipeptidase that acts on esters or on dipeptides without blocks in either the carboxyl or amino ends. Its pH optimum is around 7.5 and the molecular weight is 57 000. It is inhibited by PMSF, TLCK and DANME (N₂Ac-Nle-OMe). Proteinase C is an aminopeptidase with an optimum pH around 8. Its molecular weight was 67 000 when determined by SDS gel electrophoresis and 243 000 when determined by gel filtration. It is active towards dipeptides in which at least one amino acid is apolar and is not active when the N-terminal amino acid is blocked. It is inhibited by EDTA or o-phenanthroline and activated by several divalent cations.     

Consumption of food and spatial organization of digestion in the cassava hornworm, Erinnyis ello

Fifth-instar Erinnyis ello larvae eat 2.1 times their own weight per day of Euphorbia pulcherrima leaves, with a coefficient of digestibility of 45% and an efficiency of food conversion into tissue of 25%. The food takes about 150 min to go through the gut. Midgut contents have a pH of 9.3–9.8, depending on the region. Cellulase is absent from the gut in E. ello. Significant gut hydrolase activities are found only in midgut. Amylase and trypsin occur in the midgut tissue and contents and in regurgitated material, whereas aminopeptidase, α-glucosidase, β-glucosidase and trehalase are found in major amounts in the midgut tissue, in minor amounts in the midgut contents and are absent from regurgitated material. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase and trypsin and is largely completed in the ectoperitrophic space through the catalytic action of several oligomer and dimer hydrolases. Involvement of a membrane-bound aminopeptidase in the terminal digestion of oligopeptides cannot, at present, be excluded. The finding that less than 7% of the total amylase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal for the existence of some mechanism by which those enzymes are recovered from the undigested food before it is excreted.     

Digestive enzymes associated with the glycocalyx,microvillar membranes and secretory vesicles from midgut cells of Tenebrio molitor larvae

Acetylglucosaminidase, amylase, cellobiase and maltase are more active in anterior midgut cells, whereas aminopeptidase, carboxypeptidase and trypsin are more active in posterior midgut cells of Tenebrio molitor larvae. Differential centrifugation of midgut homogenates prepared in saline (or mannitol) isotonic buffered solutions revealed that aminopeptidase is associated with membranes, which occur in subcellular fractions displaying many microvilli. Carboxypeptidase, trypsin and the carbohydrases are mostly found in the soluble fraction, although significant amounts sediment together with cell vesicles. Data on differential calcium precipitation of midgut homogenates and on partial ultrasound disruption of midgut tissue suggest that aminopeptidase is a microvillar enzyme and that the digestive enzymes recovered in the soluble fraction of cells are loosely bound to the cell glycocalyx. About 5% of the non-absorbable dye amaranth fed to T. molitor larvae remains in the midgut tissue after rinsing. Most dye was recovered in the soluble fraction of midgut cells. This provided further support for the hypothesis that the digestive enzymes found in the soluble fraction are actually extracellular and that the true intracellular enzymes are those associated with cell vesicles. The results suggest that the carbohydrases are secreted by exocytosis from the anterior midgut and carboxypeptidase and trypsin from the posterior midgut.     

Characterization and distribution of digestive proteases of the stalk corn borer,Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae)

Larval midgut extracts from the noctuid Sesamia nonagrioides Lef. were assayed for protease activity. Total proteolytic activity, as measured by azocasein hydrolysis, showed a pH optimum in the range 10.0 to 11.5, suggesting a digestive system based largely on serine-like proteases. The ability of midgut extracts to hydrolyze specific synthetic substrates, the elucidation of the pH at which maximal hydrolysis occurs, and their sensitivity to protease inhibitors confirmed the presence of the serine endoproteases: trypsin, chymotrypsin, and elastase; and the exopeptidases: carboxypeptidase A, carboxypeptidase B, and leucine aminopeptidase. The distribution of these digestive proteases along the gut sections and among the different midgut regions was examined. All types of endoproteases and exopeptidases were mainly located in the midgut, with less than 5% of the activity in the foregut and hindgut. When the two halves of the midgut were compared, all proteolytic activities were higher in the anterior portion of the midgut. Trypsin, chymotrypsin, elastase, and carboxypeptidase B activities were mainly located in the endoperitrophic space of the midgut, with some activity in the ectoperitrophic space, whereas aminopeptidase and carboxypeptidase A activities were preferentially located in the midgut epithelium. © 1996 Wiley-Liss, Inc.     

Spatial organization of digestion,secretory mechanisms and digestive enzyme properties in Pheropsophus aequinoctialis (Coleoptera: Carabidae)

Aminopeptidase (soluble form M_r 110,000), carboxypeptidase A (soluble form M_r 47,000), maltase (a dimer composed of two identical M_r 60,000 subunits) and trypsin (two charge isomers with M_r 34,000) are found in major amounts in the crop and midgut tissue, whereas amylase (a trimer of three identical M_r 18,000 subunits) and cellobiase (a trimer of three identical M_r 27,000 subunits) occur mainly in the crop and midgut contents. Subcellular fractions of midgut cells were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation. The results suggest that part of the aminopeptidase and carboxypeptidase A activity is bound to microvilli, that major amounts of trypsin and maltase are trapped in the cell glycocalyx and finally that soluble aminopeptidase, amylase and cellobiase occur in intracellular vesicles. The data support the hypothesis that most protein and carbohydrate digestion takes place in the crop under the action of enzymes passed forward from the midgut, after being secreted by exocytosis. Nevertheless, part of the intermediate and final digestion occurs at the surface of the midgut cells. The peculiar features of the digestion of P. aequinoctialis beetles, including their partly fluid peritrophic membranes, are thought to be derived from putative Coleoptera ancestors.     

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of ～ 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50–60 °C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca^+ 2, Mn^+ 2 and Mg^+ 2 somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.     

Isolation and partial characterization of two alkaline proteases of the greater wax moth Galleria mellonella (L.)

Two alkaline proteases were isolated from whole-body extracts of Galleria mellonella larvae. The two proteases were separated by cation-exchange chromatography on CM-Sepharose CL6B and further purified by gel filtration on Ultrogel ACA 54. The optimal pH of activity using Azocoll as substrate was 10.5 for protease P-1 and 11.2 for protease P-2. The molecular weights of the two enzymes determined by gel filtration were respectively 12,500 and 10,500. Protease P-1 was inhibited by soybean trypsin inhibitor, TPCK, TLCK and activated by non-ionic detergents. Protease P-2 was inhibited by soybean trypsin inhibitor, 4-aminobenzamidine, ovomucoid and activated by dithiothreitol. Both enzymes were partially inhibited by PMSF.Distribution studies suggested that the two proteases were digestive enzymes.     

Ultrastructural and biochemical aspects of digestion in the imagoes of the flyRhynchosciara americana

The midgut of adultRhynchosciara americana Wiedemann (Diptera: Sciaridae) displays, in contrast to the midguts of other adult Diptera, two caeca connected to a ventriculus. All midgut cells exhibit long apical microvilli, and narrow and ramified basal channels with openings to the underlying space. These morphological features are thought to be involved in the absorption of nutrients from food. Enzymatic assays inR. americana adults revealed that amylase occurs in salivary glands and midgut, whereas aminopeptidase, α-glucosidases and trypsin occur only in the midgut, mainly in the ventriculus. There is a soluble (Mr 105000) and a membrane-bound aminopeptidase (solubilized form, Mr 110000). Soluble α-glucosidase inactivates easily and could not be characterized, whereas membrane-bound α-glucosidases were resolved after solubilization into three molecular species (Mr 186000, 105000 and 84000) with different substrate specificities. The activities of trypsin (pH optimum 9.0), which was inhibited completely by soybean trypsin inhibitor, and of amylase (pH optimum 5.5), were not sufficiently high to be further characterized. The data support the assertion thatR. americana adults are able, to a limited extent, to digest and absorb starch and proteins, in addition to nectar sugars. The results, supported by published data, suggest that there is an inverse correlation between the digestive enzyme activities and midgut absorptive surface in insects which has nectar as a major food.     

Proteinases in molting fluid of the tobacco hornworm,Manduca sexta

Manduca sexta pharate pupal molting fluid contains more than 10 proteolytic enzymes that differ in relative mobility during electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and gelatin. The major gelatin digesting enzyme was an endoprotease with an apparent molecular weight of 100 kDa. Gel filtration on a Sephacryl S-300 column resolved another endoprotease of similar size that digests azocoll and [³H]casein. In addition we found an aminopeptidase-like enzyme (MW_app 500 kDa) and at least three carboxypeptidase-like enzymes (MW_app 10–60 kDa). Use of pseudosubstrates and inhibitors suggested the presence of both trypsin-like and chymotrypsin-like enzymes with the former activity approx. 10-fold greater than the latter. However, none of the proteolytic enzymes were substantially inhibited by diisopropylphosphorofluoridate or phenylmethylsulfonyl fluoride which are poteint inhibitors of trypsin and chymotrypsin. No carboxyl or sulfhydryl proteases were detected. The enzymes were most active in the neutral to alkaline pH range, but they were relatively unstable during storage which precluded their purification to homogeneity. Proteolysis of Manduca cuticular protein appears to involve a rather complex and unique mixture of endo- and exo-cleaving proteolytic enzymes.     

Comparison of phospho- and dephospho-ATP citrate lyase

The dephospho- form of rat liver citrate lyase has been prepared by treating purified [³²P]-ATP citrate lyase with a partially purified phosphatase. A comparison of the properties of the phospho- and dephosphoenzyme has been performed. The pH optima were the same for both forms of the enzyme in four different buffer systems although the optimum values varied identically for both enzyme forms with the buffer. Both the phospho- and dephosphoenzymes show the same kinetic properties except for the K_m observed for ATP in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system where it was 54 μm for the phosphoenzyme and 292 μm for the dephosphoenzyme. The present study also indicates that both enzymes are cleaved by trypsin and lysosomal proteases in a similar manner. Both forms of the enzyme tend to associate with mitochondria to the same extent and both enzymes have identical temperature stability curves.     

The proteases in the gut of the locust, Locusta migratoria

The intestinal fluid of Locusta migratoria was purified by ionexchange chromatography on a DEAE-cellulose column. Four fractions (P_I–P_IV) with endopeptidase activity have been obtained and characterized in further studies. All proteolytic fractions were found to react with PMSF. Therefore, they seem to be typical serine proteases. Two of them, P_I and P_IV, resemble bovine trypsin and bovine chymotrypsin, respectively. These proteases hydrolyse the B-chain of oxidized insulin and the synthetic substrates BTEE,² APNE and BAEE, BANA with a specificity very similar to the bovine enzymes. Moreover, they show similar inhibition characteristics and pH activity profiles. Their molecular weights were found to be 17,000 and 18,200, respectively, according to gel filtration. Fraction P_III did not hydrolyse any of the applied synthetic substrates, P_II was active only with GluPNA. The pH optima of these enzymes lay near neutrality. Their molecular weights were found to be 27,000 and 32,000, respectively. Probably they belong to a type of proteases hitherto scarcely described and not to be found in vertebrates.     

Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.     

Midgut amylase,lysozyme, aminopeptidase,and trehalase from larvae and adults of Musca domestica

Based on polyacrylamide gel electrophoresis, density-gradient ultracentrifugation and thermal inactivation, there is only one major molecular species of each of the following larval enzymes (soluble in water or solubilized in Triton X-100): membrane-bound aminopeptidase (pH optimum 8.5; K_m 0.21 mM L-leucine p-nitroanilide; M_r 322,000), amylase (pH optimum 6.5; K_m 0.14% starch; M_r 66,000), lysozyme (pH optimum 3.5; K_m 0.3 mg/ml; Mr 24,000); and membrane-bound trehalase (pH optimum 5.0; K_m 1.09 mM trehalose; M_r 94,000). Except for lysozyme, the properties of adult digestive enzymes are different from those described for larval enzymes. Larval aminopeptidase and trehalase were purified by electrophoresis and larval lysozyme (contaminated with amylase) by density-gradient ultracentrifugation, and were used to raise antibodies in a rabbit. Antibodies raised against larval aminopeptidase, trehalase, and amylase did not recognize the imaginal enzymes, whereas those against larval lysozyme recognize imaginal lysozyme. The data suggest that the genes coding for digestive enzymes (except for lysozyme) are different in larvae and imagoes.     

Digestive proteolytic activity in the pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators, respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m₁ and m₃, respectively, and maximum chymotrypsin and trypsin activities were observed in m₃. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.