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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
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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. · 1.69 Impact Factor
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ABSTRACT: Highly efficient raw starch digesting α-amylase was produced after 24 h of batch fermentation of Bacillus licheniformis ATCC 9945a in laboratory bioreactor at 37 °C. The enzyme was purified by gel filtration chromatographies with 6-fold increase of specific activity and 38% recovery and showed a molecular mass of 31 kDa by SDS-PAGE. The purified enzyme had an optimum pH of 6.5 and optimum temperature of 90 °C. The purified α-amylase in the presence of CaCl2 retained 55% of its activity after 6 h of incubation at 70 °C. Co2+, Ni2+ and Ca2+ slightly stimulated, while Hg2+ completely inhibited α-amylase activity. Hydrolysis rates of raw triticale, wheat, potato, horseradish and corn starches, at 1% concentration were 63, 60, 59, 52 and 37%, respectively, in a period of 4 h. The properties of the purified enzyme proved its high efficacy for digesting diverse raw starches below gelatinization temperature and, hence, its potential commercial value to use as an industrial enzyme.
Biochemical Engineering Journal 01/2011; 53(2):203-209. · 2.64 Impact Factor
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ABSTRACT: Cell growth and the level of α-amylase in response to the carbon and
nitrogen sources used for the growth of the strain Bacillus subtilis IP 5832 were
examined. Based on the amylase productivity level in shake flask cultures after
24 hours of growth, the growth medium containing starch and peptone was
selected as the best medium. Amylase production was greatly reduced when
glutamate or citrate as sources of carbon were used. Experiments performed at
different initial concentrations of starch showed that although the strain grew
well with all the starch concentration used, 0.5 % starch was necessary for
maximum α-amylase production, inducing 1.55 IU mL
-1
of amylase to be secreted after 8 h of cultivation in shaking flasks. During the batch fermentation of
B. subtilis IP 5832 strain in 2 L laboratory fermenter, a 60 % higher activity
(2.5 IU mL
-1
) was obtained. The production of the enzyme was directly related
to the growth of the strain. Maximum enzyme activity was obtained at the beginning of the stationary growth phase.
Journal of the Serbian Chemical Society 01/2011; 76(7):965-972. · 0.88 Impact Factor
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ABSTRACT: α-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL) and reared in the laboratory (ADL) were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF). All C. cerdo amylase isoforms were acidic proteins (pI < 3.5). Seven amylase isoforms (ACC 1-7) from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa. e, 62 (3), 575-583, 2010
Archives of Biological Sciences 01/2010; 62(3):575-583. · 0.36 Impact Factor
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ABSTRACT: Using soluble starch as a substrate five isoforms of α-amylase were identified in a crude extract of Morimus funereus larvae. The main α-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 °C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 °C. AMF-3 exhibited a high affinity towards soluble starch with a Km value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl2, while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by α-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.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology.
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ABSTRACT: Yeast cell wall invertase (CWI) was immobilised within 10% gelatin hydrogel. The result of entrapment was the complete immobilisation of all the added CWI. The activity of immobilised biocatalyst was 93 ± 3 U/g, with activity yield of 30%. The optimum pH was in range of 4.5–5.0 for free and immobilised biocatalyst. The optimum temperature was 60 °C for both free and gelatin immobilised CWI. Immobilised CWI was more stable than free CWI above optimum activity temperatures. The Km of free and immobilised CWI was 35.10 ± 2.99 mM and 71.45 ± 3.23 mM, respectively. The Vmax values were estimated as 8.23 ± 0.24 mM/min and 0.121 ± 0.002 mM/min, respectively. Immobilised CWI was tested in a batch reactor using 50% sucrose (w/v). After 70 consecutive cycles gelatin immobilised CWI retained 75% of its original activity.Research highlights► The first successful entrapment of CWI within gelatin hydrogel has been reported. ► The immobilisation was complete without loss or leakage of enzyme. ► Obtained biocatalyst is cheap and available in large amount. ► Very stable biocatalyst even at high temperatures and with long shelf-life. ► Obtained biocatalyst can be easily separated from the reaction mixture.
Food Chemistry. 126(1):236-240.
