Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae.
ABSTRACT 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.
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ABSTRACT: A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.Archives of Insect Biochemistry and Physiology 12/2013; · 1.52 Impact Factor
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ABSTRACT: Captive breeding has been suggested as a method of conservation for many vertebrates, and is increasingly being proposed as a strategy for invertebrates. In this study, the growth, development and fertility of adults of the vulnerable cerambycid Morimus funereus reared in captivity are examined. Two oviposition cycles; from May to September and from January to March were studied and larvae from wild adults and from the progeny of captive adults (second generation larvae) were examined. Five to 12 instars were observed during larval development. Larval development was completed in 218 days (average) for the progeny of wild adults with an average mortality rate of 10.3% and in 226 days (average) for larvae from captive adults with mortality rate of 34.9%. First generation larval body weights were disparate during development, while second generation larvae had similar weights with no significant differences. In this study we have tested the potential of captive breaded M. funereus larvae as a model for investigation of digestive enzymes. Amylase from the midgut of larvae reared under laboratory conditions showed twofold higher specific activities with a decreased number of isoforms expressed, as compared to the enzyme from field-collected larvae. Captive breeding of M. funereus can be used in the future as a part of an effective conservation strategy for this rare insect species.Journal of Insect Conservation 01/2012; 16:239-247. · 1.80 Impact Factor
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ABSTRACT: 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.Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 02/2013; 164(4):259-267. · 1.61 Impact Factor