Midgut proteinases of the cockroachNauphoeta cinerea

The study of properties of proteolytic enzymes in midgut of imago of the cockroachNauphoeta cinerea Oliv. Has been carried out. It is shown that the total proteolytic activity of digestive proteases, measured with azocasein as substrate, is maximal at pH 11.5 both in the anterior and in the posterior parts of the midgut. The predominant part of this activity (67%) was present in the posterior part. Fractionation of preparation from the posterior part on a column with Sephadex G-50 and subsequent analysis of the activity in the obtained fractions using specificp-nitroanilide substrates and effects of activators and inhibitors of active center have allowed revealing three types of activity of serine proteinases and one cysteine proteinase. No activity of aspartic and metalloproteinases were detected. Among serine proteinases, one trypsin-like, one unusual SHdependent serine, one chymotrypsin-like, and not less than two enzymes hydrolyzing specific substrate of subtilisin were established. The fractionation of the preparation from the anterior part has allowed revealing only three proteinases that were similar by their properties to cysteine, SHdependent serine, and chymotrypsin-like ones in the posterior part of midgut. Their activity was lower in the anterior, than in the posterior part of the midgut. The probable causes of the low proteolytic activity in the anterior part of the midgut are discussed.     

Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

Increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to Tribolium castaneum larvae, and the properties of digestive proteinases were compared in vitro. Cysteine proteinases were the major digestive proteinase class in control larvae, and serine proteinase activity was minor. Dietary serine proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine proteinase inhibitors, there was a dramatic shift from primarily cysteine proteinases to serine proteinases in the proteinase profile of the midgut. Moreover, a combination of cysteine and serine proteinase inhibitors in the diet prevented this shift from cysteine proteinase-based digestion to serine proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.     

Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.     

Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum)

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6–6.0 in the anterior to 7.0–7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p‐nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin‐, chymotrypsin‐, and elastase‐like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases. © 2009 Wiley Periodicals, Inc.     

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.     

Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.     

Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.     

Study of Inhibitors of Proteinases in the Midgut Anterior Part of the Cockroach Nauphoeta cinerea

The study of proteinase inhibitors in the midgut of the omnivorous cockroach Nauphoeta cinerea was carried out under conditions excluding their food origin. One trypsin inhibitor of molecular mass of 8.0 kDa and three subtilisin inhibitors of molecular masses of 13.0, 8.0, and 4.5 kDa were found in the protein preparations, using Sephadex G-50 fractionation. 94% of the activity of the both inhibitor types were located in the anterior midgut part. Using a high performance liquid chromatography on Mono Q column, the preparation of trypsin inhibitor was purified 120 times. Its isoelectric point was to 4.3. The inhibitor lost a part of its activity both under acidic and, especially, under alkaline conditions and was completely inactivated at pH 10. The studied inhibitors inhibited effectively activities of trypsin-like and subtilisin-like proteinases from the cockroach posterior midgut part. The possible physiological role of the proteinase inhibitors and, particularly, their participation in regulation of digestion in the midgut of N. cinerea are discussed.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

Cysteine proteinases substitute for serine proteinases in the midgut glands of Crangon crangon and Crangon allmani (Decapoda: Caridea)

The utilization of dietary proteins in crustaceans is facilitated by a set of peptide hydrolases which are often dominated by “trypsin-like” serine proteinases. As expected, the North Sea shrimps Crangon crangon and Crangon allmani showed in their midgut glands high proteolytic activities. However, the majority of animals lacked trypsin and chymotrypsin. Conversely, a minority of about 10% of the animals had elevated trypsin activities. The appearance of trypsin was neither related to the mode of feeding nor to the nutritive state of the animals. When present, trypsin was expressed in both species as a single isoform of apparently 20 kDa. The lack of serine proteinases was also confirmed by inhibitor assays. AEBSF, a serine proteinase inhibitor, slightly reduced total proteinase activity by less than 10%. In contrast E 64, a cysteine proteinase inhibitor, caused a reduction of more than 70% of total proteinase activity, indicating that a substantial share of proteolytic activity is caused by cysteine proteinases. Cathepsin L-like proteinases were identified as major cysteine proteinases.A comparison with the eucarid crustaceans Pandalus montagui, Pagurus bernhardus, Cancer pagurus and Euphausia superba showed a similar high level of total proteinase activity in all species. Trypsin, however, varied significantly between species showing lowest activities in Caridea and the highest activity in E. superba. E 64 suppressed total proteinase activity by more than 70% in Crangon species but not in C. pagurus and E. superba. In contrast, the serine proteinase inhibitor AEBSF had only little effect in Caridea but was most effective in P. bernhardus, C. pagurus and E. superba. The results may indicate different traits of food utilization strategies in some eucarid crustaceans. Caridea may express predominantly cysteine proteinase, while in Anomura, Brachyura and Euphausiacea, serine proteinases may prevail.     

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae)

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.     

Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

An hypothesis of the mechanism controlling proteolytic digestive enzyme production levels in Stomoxys calcitrans

Flies fed a human blood meal and sacrificed 9 h later were assayed to give information on unfed fly weight, meal weight, total midgut protein, total midgut proteolytic activity, anterior midgut protein, anterior midgut proteolytic activity, posterior midgut protein, and posterior midgut proteolytic activity; correlation coefficients were calculated for all pairings of these parameters. Posterior midgut protein showed a positive correlation with posterior midgut proteolytic activity and on this evidence it is concluded that proteolytic digestive enzyme secretion in the midgut of Stomoxys calcitrans is controlled by a secretogogue mechanism.It is proposed that the only direct stimulus the food supplies in the control of digestive enzyme production is that for digestive enzyme release from the production cells. It is also proposed that the basis of the secretogogue mechanism is that digestive enzymes are produced in direct proportion to the quantities of amino-acids available for their synthesis and that this is a consequence of the quantities of amino acids released from the food during digestion.     

Digestive proteinases of yellow mealworm (<Emphasis Type="Italic">Tenebrio molitor</Emphasis>) larvae: Purification and characterization of a trypsin-like proteinase

A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55 degrees C, pH optimum at 8.5, and K(m) value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein. The BGPF1 proteinase completely hydrolysed only beta-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.