Biljana Dojnov

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.

Immobilization of cell wall invertase modified with glutaraldehyde for continuous production of invert sugar.

Yeast cell wall invertase (CWI) was modified with dimethyl suberimidate, glutaraldehyde, formaldehyde, and sodium periodate. Retained activity after modification was 45% for CWI modified with formaldehyde, 77% for CWI modified with sodium periodate, 80% for CWI modified with glutaraldehyde, and 115% for CWI modified with dimethyl suberimidate. Chemically modified and native CWIs showed significantly broad pH stability (pH 3-11), whereas after incubations at 50, 60, and 70 °C, CWI modified with glutaraldehyde showed the highest thermostability. Optimum pH for CWI modified with glutaraldehyde was between 4 and 5, whereas optimum temperature was at 60 °C. Comparison to CWI modified with glutaraldehyde after immobilization within alginate beads showed broader pH optimum (4.0-5.5) as well as broader temperature optimum (55-70 °C). Column bed reactor packed with the immobilized CWI modified with glutaraldehyde was successfully used for the 95% inversion of 60% (w/w) sucrose at the flow rate of 3 bed volumes per hour, pH 4.9, and 45 °C. A 1 month productivity of 3844 kg of inverted sugar/kg of the immobilisate was obtained.

Fast and reliable method for simultaneous zymographic detection of glucoamylase and α-amylase in fungal fermentation

Detection of α-amylase and glucoamylase in crude fermentation extracts using a single native electrophoresis gel and zymogram is described in this article. Proteins were printed on substrate gel and simultaneously onto a membrane in a three-sandwich gel. α-Amylase was detected on the substrate gel with copolymerized β-limit dextrins and iodine reagent. Glucoamylases were detected on the membrane using a coupled assay for glucose detection. Both amylases were detected in native gel using starch and iodine reagent. The described technique can be a helpful tool for monitoring and control of fermentation processes because fungal amylase producers almost always synthesize both amylases.

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.

Enhancement of amylase production by Aspergillus sp. using carbohydrates mixtures from triticale

With the purpose of finding a suitable available inducer in com- bination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced the production of an amylase cocktail (α-amylase and glucoamylase) in Aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and TLC analysis of the carbohydrate mixtures before and after fermentation. Glucoamylase production by A. niger was the highest in the presence of the extract obtained after auto- hydrolysis of starch from triticale (95.88 U mL -1 ). Carbohydrate mixtures from triticale induced the production of α-amylase in A. oryzae. More α-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. A 48-h induction was the most efficient using a triticale extract (101.35 U mL -1 ). Carbohydrates from triticale extracts could be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by Aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation.

Enhancement of amylase production using carbohydrates mixtures from triticale in Aspergillus sp

For the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale was used as inducers and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced production of amylase cocktail (α-amylase and glucoamylase) in Aspergillus niger , unlike induction with well-known inducers which induce only glucoamylase, showed by zymogram and TLC analysis of carbohydrates mixtures before and after fermentations. Glucoamylase production by A. niger was highest in the presence of extract obtained after autohydrolysis of starch from triticale (95.88 U/mL). Carbohydrate mixtures from triticale induced production of α-amylase in A. oryzae. More α-amylase isoforms were detected upon using complex carbohydrate mixture, compared to induction with maltose or starch. The 48 h induction was the most efficient by using triticale extract (101.35 U/mL). Carbohydrates from triticale extracts can be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as the inducer in amylase production by Aspergillus sp, such a way it could be used as sole substrate in fermentation.

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4‐methylumbelliferyl‐β‐d‐cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates.