Paecilomyces variotii xylanase production, purification and characterization with antioxidant xylo-oligosaccharides production.

Paecilomyces variotii xylanase was, produced in stirred tank bioreactor with yield of 760 U/mL and purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.29-fold purification with 34.47% activity recovery. The enzyme purity was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirming its monomeric nature as single band at 32 KDa. Zymography showed xylan hydrolysis activity at the same band. The purified enzyme had optimum activity at 60 °C and pH 5.0. The pH stability range was 5-9 and the temperature stability was up 70 °C. Fe2+and Fe3+ exhibited inhibition of xylanase enzyme while Cu2+, Ca2+, Mg2+ and Mn2+ stimulated its activity. Mercaptoethanol stimulated its activity; however, Na2-EDTA and SDS inhibited its activity. The purified xylanase could hydrolyze beechwood xylan but not carboxymethyl cellulose (CMC), avicel or soluble starch. Paecilomyces variotii xylanase Km and Vmax for beechwood were determined to be 3.33 mg/mL and 5555 U/mg, respectively. The produced xylanase enzyme applied on beech xylan resulted in different types of XOS. The antioxidant activity of xylo-oligosaccharides increased from 15.22 to 70.57% when the extract concentration was increased from 0.1 to 1.5 mg/mL. The enzyme characteristics and kinetic parameters indicated its high efficiency in the hydrolysis of xylan and its potential effectiveness in lignocellulosic hydrolysis and other industrial application. It also suggests the potential of xylanase enzyme for production of XOS from biomass which are useful in food and pharmaceutical industries.

Catalytic and thermodynamic properties of ß-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis.

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of ß-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of ß-glucosidase produced by L. corymbifera was 39 U/g dry substrate (or 3.9 U/mL), and that by B. spectabilis was 77 U/g (or 7.7 U/mL). The optimum pH and temperature were 4.5 and 55 °C and 4.0 and 50 °C for the enzyme from L. corymbifera and B. spectabilis, respectively. ß-Glucosidase produced by L. corymbifera was stable at pH 4.0-7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0-6.0. Regarding the thermostability, ß-glucosidase produced by B. spectabilis remained stable for 1 h at 50 °C, and that from L. corymbifera was active for 1 h at 45 °C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (Ea), and half-life t(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.

Optimization of production and reaction conditions of polygalacturonase from Byssochlamys fulva.

In the present study, the optimization of production and reaction conditions of polygalacturonase produced by a fungus Byssochlamys fulva MTCC 505 was achieved. The production of polygalacturonase with a considerable activity of 1.28 IU/ml was found when the culture was shaken at 30°C for 5 days in 100 ml of medium containing (w/v) 10 g/l pectin, 2 g/l NaNO3, 1 g/l KH2PO4, 0.5 g/l KCl, 0.5 g/l MgSO4. 7H2O, 0.001 g/l FeSO4. 7H2O, 0.001 g/l CaCl2. The best carbon and nitrogen source for this enzyme were pectin (1%) and Ca(NO3)2 (0.1%), respectively. The enzyme gave maximum activity at incubation time of 72 h, temperature of 30°C and pH 4.5. During the optimization of reaction conditions, the enzyme showed maximum activity in sodium citrate buffer (50 mM) of pH 5.5 at 50°C reaction temperature for 15 minutes of incubation. The enzyme showed greater affinity for polygalacturonic acid as substrate (0.5%). Km and Vmax values were 0.15 mg/ml and 4.58 µmol/ml/min. The effect of various phenolics, thiols, protein inhibitors and metal ions on the enzyme activity was investigated. The enzyme was quite stable at 4°C and 30°C. At 40°C the half life of the enzyme was 6 h and at 60°C it was 2 h.

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae.

Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298bp polyketide synthase gene "aomsas" has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40-56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant "aoDeltamsas" of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone, but also 6-methylsalicylic acid. The genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aoDeltamsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone.