Zoran Vujčić

Immobilization of invertase on a new type of macroporous glycidyl methacrylate

Invertase was immobilized via its carbohydrate moiety. The immobilized enzyme has a specific activity of 5500 IU g−1, with 45% activity yield on immobilization. In a packed bed reactor, 90% 2.5 M sucrose was converted at a flow rate of 4 bed volumes h−1. The obtained specific productivity at 40 °C of 3 kg l−1 h−1 is the best one so far. Long-term stability was 290 days in 2.5 M sucrose at 40 °C and at a flow rate of 3 bed volumes h−1.

Bacillus amyloliquefaciens laccase--from soil bacteria to recombinant enzyme for wastewater decolorization.

One hundred wild type strains of Bacillus sp. were isolated from industrial and agricultural soil across Serbia and screened for laccase activity. Three strains showed high laccase activity temperature optimum of 65 and 80 °C towards ABTS. A new laccase gene from the strain with highest temperature optimum, namely Bacillus amyloliquefaciens 12B was cloned and expressed in Escherichia coli. Recombinant laccase degraded dye Reactive blue 52 at pH 7.0 and pH 4.0 and at elevated temperature, while fungal laccases was unable to act on this substrate at pH higher than 4.0 and was quickly inactivated at temperatures higher than 45 °C. Degradation of dye was monitored by HPLC-DAD and resulting precipitate was analyzed by FTIR spectroscopy. Single product peak without chromophore was detected in solution, while water insoluble aggregate, presumably dye polymer is formed retaining blue color.

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.

Efficient stabilization of Saccharomyces cerevisiae external invertase by immobilisation on modified beidellite nanoclays.

The external invertase isoform 1 (EINV1) was immobilised on eight differently modified beidellite nanoclays. Modifications were composed of organo-modification with different amounts of surfactant - hexadecyl trimethylammonium cation (HDTMA), pillaring with Al/Fe containing polyhydroxy cations and acid modification of Na-enriched and pillared clays. The modified nanoclays were characterised by XRD, N2-physisorption, SEM and FT-IR spectroscopy. The amount of bound enzyme activity was significantly influenced by the modification of beidellite ranging from 50 to remarkable 2200U/g. Biochemical characterization was performed for five modified nanoclays showing the highest enzyme activity after invertase immobilisation. The investigation demonstrated that after immobilisation the structure and the catalytic properties of invertase were preserved, while Km values were slightly increased from 26 to 37mM. immobilisation significantly improved thermal and storage stability of EINV1. Results indicate that beidellite nanoclays obtained by low cost modifications can be applied as a suitable support for the immobilisation of invertase. The immobilizate can be efficiently engaged in sucrose hydrolysis in batch reactor.

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.

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.

Activity and stability of soluble and immobilized α-glucosidase from baker's yeast in cosolvent systems

The activity of α-glucosidase from bakers yeast was determined in various concentrations of dioxan, tetrahydrofuran, tert-butanol, dimethylformamide, methanol and dimethylsulfoxide (DMSO). Higher activities were observed with sucrose than with nitrophenylglucoside as substrate in cosolvent mixtures. In 30% (v/v) DMSO, 25% of the activity obtained in pure water was detected, and in 30% (v/v) methanol 12.5% of the activity in pure water was detected, while in other cosolvents there was almost no activity under these conditions. α-glucosidase was immobilized onto a macroporous copolymer of ethylene glycol dimethacrylate and glycidyl methacrylate, poly(GMA-co-EGDMA), by the glutaraldehyde method. By immobilization, the half-life of the enzyme in 35% (v/v) methanol was increased from 6 to 60 min and from 4 to 15 min in 45% (v/v) DMSO. The activity of the immobilized enzyme in 30% (v/v) DMSO and 30% (v/v) methanol was 22% and 18% of the activity in pure water, respectively.

Raw starch degrading α-amylases: an unsolved riddle

Abstract Starch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

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.