Maria Costa-Ferreira

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

Abstract A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml−1 was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l−1 h−1, and in reactor, 104,000 U l−1 h−1 was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l−1 h−1, compared to shake-flask fermentations, 12,000 U l−1 h−1. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.

Enzymatic properties of a neutral endo-1,3(4)-β-xylanase Xyl II from Bacillus subtilis

A Bacillus sp. CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min. It produces at least two extracellular xylanases, Xyl I and Xyl II. The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile. The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3. The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa. Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography. The enzyme showed an optimum pH and temperature of 6.0 at 60 degrees C. Xyl II was stable at 40 degrees C for 180 min at pH 6.0. The specificity of Xyl II for different substrates was evaluated. Xyl II presents a higher affinity towards OSX, with a K(m) of 1.56 g l(-1) and showed the ability to hydrolyse laminarin, with a K(m) of 1.02 g l(-1). Xylotetraose is the main product of xylan degradation. The Xyl II ability for binding to cellulose and/or xylan was also studied.

Decolourisation of reactive textile dyes by Irpex lacteus and lignin modifying enzymes

Abstract Irpex lacteus decolourised the textile reactive dyes Reactive Blue 19 (RBBR) and Reactive Black 5. Less than 10% RBBR remained following 2 days of cultivation with the fungus whereas Reactive Black 5 was decolourised slower, requiring 10 days for about 90% decolourisation. No lignin peroxidase activity was detected in the extracellular medium of I. lacteus . Low levels of Mn peroxidase and laccase activity were detected but only after most of the decolourisation had taken place. Addition of dyes to grown cultures containing these enzymes did not enhance the decolourisation rate. It is suggested that enzymic systems or processes, other than the lignin modifying enzymes, may be responsible for dye decolourisation.

Specificity of phenolic disazo dyes in relation to transformation by laccase

Four novel disazo dyes were synthesized and characterized using FTIR and NMR spectroscopy. These water-soluble dyes had a hydroxyl group in the para position of the phenolic ring in relation to the azo bond, whereas the other substituents on the phenolic ring and/or between the two azo bonds varied. The transformation of the dyes was studied using purified fungal laccase. The rank order in which the dyes were transformed was IV > III ≫ I. Dye II was not a laccase substrate. As both dye I and II had a carboxylic substituent on the phenolic ring but the ring structures between the two azo linkages were different, we suggest that the latter also influenced laccase catalysis. Both of the redox mediators tested, violuric acid and N-hydroxybenzotriazole, enhanced the laccase reaction with violuric acid, being generally more effective. The results show that the chemical structure of both the phenolic ring as well as the ring distal to the phenolic moiety affect the reaction kinetics.

On the relationship between cellobiose dehydrogenase and cellobiose:quinone oxidoreductase under conditions where [14C]DHP is mineralized by whole cultures of Phanerochate chrysosporium

Phanerochaete chrysosporium ME-446 cultivated in celluose-based media degraded synthetic lignin to about the same extent in high-nitrogen (24 mM N as asparagine) and low-nitrogen (2.4 mM) media when Tween 80 was employed at 0.15%, and 0.075%, respectively. Under these specified conditions of high 14C-DHP itmineralization which occurred after 6 days of cultivation, the amount of lignin peroxidase (113 U l−1) in high-N media was much higher than that in low N (17 U l−1 LiP). In contrast, the Mn-dependent peroxidase activity was not affected by the N concentration. Likewise, the total (extracellular plus bound) cellobiose:quinone oxidoreductase activity was of the same order (about 15 to 25 U l−1) in both high- and low-N/cellulose-based media. These data suggest that the amount of cellobiose:quinone oxidoreductase/cellobiose dehydrogenase formed is critical for optimal ligninolytic activity and that too high levels of these enzymes may be inhibitory. The use of the cellulose-based media has made it possible to observe a wide spectrum of enzymatic activities, including protease activity. The relationship among these enzymes is analyzed in the light of the synthetic lignin degradation.

Pilot-scale Enzymatic Decolorization of Industrial Dyeing Process Wastewater

Manuela Lageiro and Maria Costa-Ferreira1 Bioengineering and Bioprocessing Unit, Department of Biotechnology, National Institute for Industrial Engineering and Technology (INETI), Portugal Abstract An enzymatic cocktail was used for the decolorization of process wastewater, containing mainly three reactive azo dyes, from a textile dyeing and printing company. Water sampled from different processing streams and combinations thereof was decolorized to different extents. The decolorization was greatest for C.I. Reactive Black 5, followed by C.I. Reactive Red 158, whereas C.I. Reactive Yellow 27 was the least decolorized. As wastewater from the printing process was inhibitory, the 1 mistirred tank type reactor prototype was installed near the outlet prior to the discharge of wastewater from the printing process. Temperature and pH control systems were installed to ensure optimal enzymatic decolorization, this being about 45 ° C and pH 6.4. The average temperature observed at the reactor was 42.1 ° C, which indicated the need for improved temperature control. Laboratory scale tests were done to assess the reuseability of the biotreated wastewater for the washing of dyed cotton fabrics.

Glucose utilization by lysine-producing fluoroacetate-sensitive mutants ofCorynebacterium glutamicum

A fluoroacetate-sensitive mutant was isolated fromCorynebacterium glutamicum, ATCC 21513, following mutagenesis with NTG. Batch fermentations show that in terms of growth kinetics, glucose utilization, and lysine formation, there are significant differences between the mutant and the parent. The mutant’s specific growth rate (0.22/h) is lower than that for the parent (0.34/h). Also, the yield expressed as lysine/glucose consumed does not alter as a function of the glucose concentration for the mutant, and is about 0.22, whereas for the parent, this coefficient decreases with increasing glucose concentration. The maximum specific rate of lysine production for the mutant is 1.3 g/L/h that is about two-fold higher than that for the parent.

Amino acid accumulation by an analogue sensitive mutant ofCorynebacterium glutamicum

SummaryA fluoroacetate/fluoropyruvate-sensitive mutant was derived from the parent strainCorynebacterium glutamicum ATCC 21513. Accumulation of various amino acids in the fermentation broth using the two strains was compared. The FA/FP-sensitive mutant accumulated about 26.5 g/L L-lysine and 2.2 g/L aspartic acid which was about 3-fold and 10-fold respectively, more than the amount produced by the parent strain.

An Overview on the Use of Microbial and Enzymatic Systems for Dye Biotransformation

Abstract Dyes are of primary importance to textile manufacturing. These are xenobiotic compounds that are very recalcitrant against biodegradative processes. Nevertheless, in recent years, the knowledge base for using biocatalysts as remediation agents has increased substantially. In particular, various lignolytic fungi have been shown to transform several dyes to non-coloured products. The involvement of lignolytic enzymes, lignin peroxidase, Mn-peroxidase, and laccase, is described in relation to dye biotransformation. In the case of bacterial systems, azo dye transformation begins in most cases by a reductive cleavage leading to the formation of colourless amines. Some of the issues that remain to be addressed before biological systems, either whole cells or enzymes thereof, can be used for the industrial remediation of coloured wastewater from the textile dyeing process are highlighted.

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the kLa increased with the gas fractional hold-up (εG) and the aeration rate. A linear relationship between εG and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher kLa and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets. Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns. Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.