Expression and distribution of cellulase, amylase and peptidase isoforms along the midgut of Morimus funereus L. (Coleoptera: Cerambycidae) larvae is dependent on nutrient substrate composition.

The influence of diet composition--two substrates, wheat bran and sawdust--on isoform expression of digestive enzymes (cellulase, amylase and peptidase) in the midgut of Morimus funereus larvae was examined. Their impact on larval development was demonstrated by measuring the increase of larval weight during development and by analysis of digestive enzymes zymographic profiles, where the expression of cellulase isoforms from M. funereus larvae midgut has been examined for the first time in this study. Larvae reared on wheat bran had higher body weight between day 60 and day 100 than larvae reared on sawdust; however, both groups achieved similar body weight after day 110. Wheat bran as substrate induced different cellulase and amylase isoforms. Oak sawdust in substrate acted as inducer of peptidases. The highest cellulase activity and the greatest isoform variability were detected in the midgut extracts of larvae reared on wheat bran. From our results it can be assumed that M. funereus endocellulase, amylase and peptidase are secreted in the anterior midgut, and their concentration gradually decreases towards the hindgut.

Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae.

Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5-9.5) and temperature optimum of 45 degrees C with the K(M) ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K(I) value of 0.012 mM, Ic(50) value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer.

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae.

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.

Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae.

Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.

Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae.

Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0-9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K(m) for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 degrees C, the enzyme was active over a broad temperature range from 2 to 60 degrees C. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity.

Metabolic response of cerambycid beetle (Morimus funereus) larvae to starvation and food quality.

The response of xylophagous Morimus funereus larvae to a direct change of diet demonstrated that the larvae from nutrient-poor substrates, e.g. oak, are very sensitive to such a change. Depending on dietary protein quality and quantity, an increase of proteolytic activity, i.e. an intensified protein metabolism accompanied by changes in body mass gain, was observed. At the same time, amylolytic activity was usually decreased. In the larvae reared on Robert's diet, sensitivity to the switch in diet was lower at the level of proteolytic enzymes that remained at the control level, while amylolytic activity was elevated. If the switch to a new diet was preceded by 7-day-starvation that disturbed nutritional homeostasis, the response of the larvae was similar to that recorded upon a direct switch only after short-term feeding (24 h) upon starvation. Differences in the response to changes in the diet of the larvae from nature, those reared under laboratory conditions and those of different physiological status could be ascribed to plasticity in the expression of the genes coding for proteases and their isoenzymes, as well as to the multi-functionality of some neurosecretory neurons, synthetic products that participate in the regulation of digestive enzyme activities.