Process optimization for simultaneous production of cellulase, xylanase and ligninase by Saccharomyces cerevisiae SCPW 17 under solid state fermentation using Box-Behnken experimental design.

Multienzyme complex has attracted increased attention in biofuel technology. They offer solutions to effective degradation of complex plant material into fermentable sugars. Microorganisms, especially bacteria and fungi, are well studied for their ability to produce enzymes complex unlike yeast. Yeast strain isolated from mushroom farm was studied for simultaneous production of cellulase, xylanase and ligninase enzymes using lignocellulose waste as substrates. A response surface methodology (RSM) involving Box-Behnken design (BBD) was used to investigate interaction between variables (moisture content, inoculum size, initial pH, incubation time) that affect enzyme production. Crude filtrate was partially purified and characterised. Yeast strain identified as Saccharomyces cerevisiae SCPW 17 was finally studied. Evaluation of lignocellulose waste for enzyme complex production revealed corn cob to be most effective substrate for cellulase, xylanase and ligninase production with enzyme activity of 17.63 ± 1.45 U/gds, 29.35 ± 1.67 U/gds and 150.75 ± 2.01 µmol/min respectively. Time course study showed maximum enzyme complex production was obtained by day 6 with cellulase activity of 12.5 U/gds, xylanase 48.3 U/gds and ligninase 90.8 µmol/min. Using RSM involving BBD, maximum enzyme activity was found to be 19.51 ± 0.32 U/gds, 56.86 ± 0.38 U/gds, 408.17 ± 1.04 µmol/min for cellulaase, xylanase and ligninase respectively. The developed models were highly significant at probability level of P = 0.0001 and multiple correlation co-efficient (R2) was 0.9563 for cellulase, 0.9532 for xylanase and 0.9780 for ligninase. Enzyme complex was stable at varying pH and temperature conditions. Saccharomyces cerevisiae (SCPW 17) studied produced enzyme complex which can be used for bioconversion of biomass to value-added chemicals.

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius.

Amylase capable of raw starch digestion presents a cheap and easier means of reducing sugar generation from various starch sources. Unfortunately, its potential for use in numerous industrial processes is hindered by poor stability. In this work, chemical modification by acylation using citraconic anhydride (CA) and maleic anhydride (MA) was used to stabilize the raw starch saccharifying amylase from A. carbonarius. The effect of the anhydrides on the pH and thermal stability of the free amylase was investigated. Enzyme kinetics and thermodynamic studies of the free and modified amylase were also carried out. Blue shifts in fluorescent spectra were observed after modification with both anhydrides. Citraconylation led to increased affinity of the enzyme for raw potato starch, unlike maleylation. The activation energy (kJ mol-1) for enzyme inactivation was increased by 94.8% after modification with CA while only 17.9% increase was noted after modification with MA. Acylation led to an increase in Gibb's free energy and enthalpy while a reduction in entropy was observed. At 80 °C the half-life (h) was 5.92, 11.18 and 14.74 for free, MA and CA enzyme samples, respectively. These findings have potential value in all industries interested in starch conversion to sugars.

Chemical modification with phthalic anhydride and chitosan: Viable options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius.

The raw starch digesting type of amylase (RSDA) presents greater opportunities for process efficiency at cheaper cost and shorter time compared to regular amylases. Chemical modification is a simple and rapid method toward their stabilization for a wider application. RSDA from Aspergillus carbonarius was modified with either phthalic anhydride (PA) or chitosan. Activity retention was 87.3% for PA-modified and 80.9% for chitosan-modified RSDA. Optimum pH shifted from 5 to 7 after PA-modification. Optimum temperature changed from 30°C (native) to 30-40°C and 60°C for PA-modified and chitosan-modified, respectively. Activation energy (kJmol-1) for hydrolysis was 13.5, 12.7, and 10.2 while the activation energy for thermal denaturation was 32.8, 80.3, 81.9 for free, PA-modified and chitosan-modified, respectively. The specificity constants (Vmax/Km) were 73.2 for PA-modified, 63.1 for chitosan-modified and 77.1 for native RSDA. The half-life (h) of the RSDA at 80°C was increased from 6.1 to 25.7 for the PA-modified and 138.6 for the chitosan derivative. Modification also led to increase in D value, activation enthalpy and Gibbs free energy of enzyme deactivation. Fluorescence spectra showed that center of spectral mass decreased for the PA-modified RSDA but increased for chitosan modified RSDA.

Structural and spectroscopic characterisation of a heme peroxidase from sorghum.

A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV-visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe(III)/Fe(II) reduction potential of -266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na(+) ion) and proximal (assigned as a Ca(2+)) sides of the heme, which is consistent with the Ca(2+)-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions.

Extracellular amylase production of a thermotolerant Fusarium sp: isolated from Eastern Nigerian soil

In this work, an α- amylase producing Fusarium sp. was isolated from the soil at 50 0C. Growth and enzyme production occurred at 30, 45 and 55 ºC. Soybean meal at 1 percent concentration, supplemented with 0.2 percent NH4Cl and 2.5 percent corn starch elicited the highest amylase yield. Optimum pH for the enzyme was pH 6.5 which retained over 60 percent of its activity after 24 h incubation at the pH range of 4.5-7.0. The enzyme showed high activity from 40-70 0C with optimal activity at 50 ºC and 78 percent activity was retained after incubation at 70 ºC for 30min. Catalytic function of the crude amylase was stimulated by Mg (136 percent), Ca (118 percent) and Zn (118 percent) at 2mM concentrations. The enzyme hydrolyzed cassava, potato and yam starches effectively.