Properties of post-proline cleaving enzymes from <Emphasis Type="Italic">Tenebrio molitor</Emphasis>

Two post-proline cleaving peptidases PPCP1 and PPCP2 with molecular masses of 101 and 63 kDa, respectively, hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the larval midgut of the yellow mealworm Tenebrio molitor and characterized. PPCP1 was active only in acidic media, with a maximum at pH 5.6, whereas PPCP2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PPCP1 and PPCP2 were shown to belong to serine peptidases. The data obtained indicate that a Cys residue is located close to the PPCP2 substrate binding site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, completely inhibited PPCP2 and partially PPCP1. The substrate specificities of the isolated enzymes were studied. Z-Ala-Ala-Pro-pNA was the best substrate for PPCP1, and Z-Ala-Pro-pNA, for PPCP2. The combination of the properties allows characterization of PPCP2 as a proplyl oligopeptidase.     

Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests

The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine.     

Dipeptidyl peptidase 4 – An important digestive peptidase in Tenebrio molitor larvae

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0–9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor.     

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.     

Sunn pest,Eurygaster integriceps Putton (Hemiptera: Scutelleridae), digestive α-amylase, α-glucosidase and β-glucosidase

Morphology, pH and carbohydrate hydrolyzing enzyme activities of the Sunn pest gut were investigated in this study. The Sunn pest midgut is separated into the first ventriculus (V1), the second ventriculus (V2), the third ventriculus (V3) and the fourth ventriculus (V4). The first three regions of the midgut were acidic (pH 5.0–5.2), while the fourth region of the midgut and rectum was moderately acidic (pH 6.2–6.4 and pH 6.5–6.8, respectively). Activity of α-amylase was highest at pH 6 to 7, which correlates with the pH of the midgut. The optimum pH for α-glucosidase and β-glucosidase is 4 to 6 and 5 to 6, respectively. Different gut regions had different carbohydrate hydrolyzing enzyme activities. Carbohydrate hydrolyzing enzyme activities in V2 and V4 were the same, but activities in V1 were slightly higher than in V2 and V4 and lower than in V3. Levels of α- and β-glucosidase activities were similar in various midgut sections. However, the V3 had the highest activity followed by V4, V2, V1, respectively.     

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.     

Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline-Containing Neuropeptides

From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.     

Resolution of the phosphoinositide-specific phospholipase C isolated from porcine lymphocytes into multiple species. Partial purification of two isoenzymes.

Phospholipase C isolated from porcine mesenteric lymph node lymphocytes was distributed between the soluble and particulate fractions. Enzyme activity was found predominantly in the soluble fraction with optimal activity at pH 5.5. Gel filtration chromatography of the soluble phospholipase C revealed that it was composed of multiple species of enzyme activity. The activity associated with the particulate fraction had optimal activity at pH 7.0, as also did one of the species of soluble phospholipase C. Cellulose phosphate chromatography resolved the major soluble form into two species designated PLC-A and PLC-B. Both phenyl-Sepharose chromatography and hydroxyapatite chromatography purified these species still further. PLC-A and PLC-B demonstrated similar activities against phosphatidylinositol with a pH optimum near 5.5. The phospholipase C activities were abolished against this substrate by the addition of 1 mM-EDTA. When assayed in the presence of Ca2+-EDTA buffers providing a range of Ca2+ free concentrations, both enzymes exhibited optimal activity near 10(-3) M free Ca2+, but PLC-B was inhibited above this concentration more than PLC-A. PLC-B exhibited markedly lower activity against phosphatidylinositol 4,5-bisphosphate, suspended as liposomes of the pure phospholipid, than did PLC-A.     

Digestive enzymes in larvae of the leaf cutting ant, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini)

The digestive physiology and biochemistry of larvae of the leaf-cutting ant Acromyrmex subterraneus were investigated here. The activity of digestive enzymes was evaluated in the labial glands, midgut epithelium (soluble and particulate fractions), and in the lumen contents, separated into endo and ectoperitrophic regions. Enzymes with high levels of activity were partially characterised using chromatography and electrophoresis techniques. Microscope observations were carried out and the anatomy of the larval digestive tract was described here for the first time. Larvae fed with pH indicator solutions showed the anterior portion of the midgut to be acidic and the posterior portion neutral to alkaline, indicating that the pH of the different regions of the midgut could optimise certain enzyme activities, whilst inhibiting others. The flow rate of the intestinal contents was also evaluated in larvae fed with a dye solution. The slow flow rate is probably due to closure of the rear end of the larval midgut. No compartmentalisation of digestive enzymes acting on oligosaccharides and disaccharides in the ectoperitrophic space and on polysaccharides in the endoperitrophic space was observed here, which could also be related to the closure of the midgut. The digestive physiology of these larvae is therefore similar to ancestral Holometabola, a paradox when considering the highly evolved nature of these insects. The larval midgut demonstrated a large diversity of enzyme activities with high levels of alpha-amylase, alpha-mannosidase, chitinase, alpha-glucosidase, beta-glucosidase and proteinase. High levels of chitinase and amylase activities were detected in the labial glands of larvae. The enzyme profile reflected the necessity of the larvae to degrade the fungal substrate, their sole diet, and a probable source of some of the digestive enzymes detected here. When compared to adults, the larvae had a greater diversity and higher levels of enzyme activity, highlighting their importance as the "digestive caste" of the colony.     

Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive α-amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for α-amylase against starch at acidic pH (pH 5.0). α-Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. α-Amylase activity was significantly decreased in the presence of Ca²⁺, Mg²⁺ and sodium dodecylsulfate. On the contrary, K⁺ and Na⁺ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of α-amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for α-amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.     

Hydrolysis of sphingomyelin and phosphatidylcholine by midgut homogenates of the stable fly

Qualitative and quantitative analyses were made to characterize the enzymatic degradation of sphingomyelin and phosphatidylcholine by midgut homogenates of the adult stable fly, Stomoxys calcitrans (L.). The results indicated that sphingomyelin was hydrolyzed by an enzyme with sphingomyelinase-like properties, and that phosphatidylcholine was hydrolyzed by an enzyme with properties similar to phospholipase C. The optimum pH for the sphingomyelinase was 7.6, and the rate of hydrolysis of sphingomyelin at that pH was linear from 1 to 4 nmol of substrate and 5 to 25 micrograms of enzyme preparation. Dialysis of the homogenates against Tris-HCl and imidazole buffers resulted in a decrease of sphingomyelinase activity by 59% and 98%, respectively, and the original activity was not restored with the addition of Ca++, Mg++, or Mn++.     

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.     

Properties of post-proline cleaving enzymes from Tenebrio molitor

Two post-proline cleaving enzymes PRE1 and PRE2 with molecular masses of 101 and 62 kDa, respectively, capable of hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the midgut of the flour beetle Tenebrio molitor and characterized. PRE1 is active only in acidic media, with a maximum at pH 5.6, whereas PRE2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PRE1 and PRE2 were shown to belong to serine peptidases. Some data indicate that a Cys residue is located close to the PRE2 active site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, inhibits completely PRE2 and partially PRE1. The substrate specificities of the isolated enzymes were studied. It was shown that Z-AlaAla-Pro-pNA was the best substrate for PRE1, and Z-AlaPro-pNA, for PRE2. The combination of the studied properties allowed characterization of PRE2 as a prolyl oligopeptidase.     

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 °C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k_cat/K_m = 37.53 mM S⁻¹), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K_i = 0.196 nM), indicating that this protease is a potential target for pest control.     

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.     

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.     

Buffer, pH, and ionic strength effects on the (Na+ + K+)-ATPase

A dog kidney (Na+ + K+)-ATPase preparation also catalyzes K+-independent and K+-activated phosphatase reactions with p-nitrophenyl phosphate as substrate. K+-independent activity increases with declining pH over the range 7.5 to 5.8, whereas the other two activities decrease. The increased K+-independent activity is similar with imidazole, histidine, and several Good buffers, and is thus attributable to free H+, probably by affecting enzyme conformations rather than by changing affinity for Mg2+ or substrate or by H+ occupying specific K+-sites. The decrease in K+-phosphatase and (Na+ + K+)-ATPase activities with pH also occurs similarly with those buffers, and is not due to changes in apparent affinity for substrate or for cation activators. However, the Good buffers Pipes and ADA inhibit the K+-independent phosphatase reaction strongly, the K+-activated reaction moderately, and the (Na+ + K+)-ATPase reaction little; both contain two acidic groups, unlike the other buffers tested. Inhibition of the phosphatase reaction by Pipes is associated with a decreased apparent affinity for K+ and an increased sensitivity to inhibition by Na+ and ADP, consistent with Pipes hindering conformational transitions to the E2 enzyme forms required for phosphatase hydrolytic activity.     

Ionic liquids as alternative co-solvents for laccase: study of enzyme activity and stability

The activity and stability of commercial laccase (DeniLite base) in three different water soluble ionic liquids (ILs) (1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate, [emim][[MDEGSO4], 1-ethyl-3-methylimidazolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazolium methanesulfonate, [emim][MeSO3]) have been studied and compared to that in two organic solvents (acetonitrile and dimethyl sulfoxide). Initial enzyme activities were similar among the ILs if the same conditions were used. A high reduction on initial enzyme activity was found with acidic pH (5.0). The effect of pH and solvent concentration on enzyme stability were investigated in more detail for 1 week. The enzyme maintained a high stability at pH 9.0 for all ILs tested. [emim][MDEGSO4] was the most promising IL for laccase with an activity loss of about 10% after 7 days of incubation. The kinetic studies in the presence of ABTS as substrate allowed to calculate the Michaelis- Menten parameters. Good agreement was found between experimental data and calculated values using the Michaelis-Menten mechanism, with a total average relative deviation of 2.1%.     

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF PROTEASES IN LARVAL PREPARATIONS OF THE CEREAL LEAF BEETLE (Oulema melanopus,CHRYSOMELIDAE, COLEOPTERA)

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole‐larval preparations, permitting studies of the very small whole‐larval preparations. Protein preparations were analyzed using FITC–casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0–4.0). Cysteine protease activity increased at acidic pH and in the presence of β‐mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe²⁺, Ca²⁺, Mg²⁺, Zn²⁺, and K⁺ (10 mM). Fe²⁺ and Zn²⁺ significantly decreased enzyme activity at all pH values, Ca²⁺ and Mg²⁺ at pH 6.2 and Mg²⁺ at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, N_α‐tosyl‐l‐lysine chloromethyl ketone hydrochloride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone, trans‐epoxysuccinyl‐l‐leucylamido‐(4‐guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin‐like) proteases and metalloproteases act in cereal leaf beetle digestion.