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Genomic sequencing analysis and previous studies have shown that there are eight genes in Streptomyces coelicolor A3(2) encoding putative cellulases. One of these genes, sco6548, was cloned into the Streptomyces/Escherichia coli shuttle vector pUWL201PW. The recombinant protein was successfully overexpressed in S. lividans TK24 under the control of the strong ermE promoter. Sco6548 was 1740 bp in length, and encoded a 579-amino acid-, 60.8-kDa protein with strong hydrolyzing activity toward Avicel and filter paper, yielding cellobiose as the final product. SCO6548 showed optimal activity at 50°C and pH 5. The Km values of SCO6548 toward Avicel and filter paper were 15.38 and 16.1 mg/mL, respectively. The Vmax values toward Avicel and filter paper were 0.432 and 0.084 μM/min, respectively. EDTA did not affect cellulase activity; however, several divalent cations, including Co(2+), Cu(2+), Ni(2+) and Mn(2+) (at 10 mM) had severe inhibitory effects on enzyme activity. Our analysis showed that SCO6548 is a cellulose 1,4-β-cellobiosidase that hydrolyzes cellulose into cellobiose., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Random mutagenesis was used to improve the alkaline and thermal stability of the xylanase (XynA) from Thermomyces lanuginosus. Error-prone PCR reactions were carried out; the PCR products were cloned into Escherichia coli and a library of 960 clones was selected on xylan-containing agar plates. The crude filtrates of positive xylanase producers were screened at 80 degrees C and tested separately at pH 10 for alkaline tolerance. The native XynA lost 80% activity after 90 min at 80 degrees C and lost 70% activity at pH 10. Conversely, the most thermostable variant, G41, retained 75% activity after 90 min at 80 degrees C and the best alkali-stable variant, G53, retained 93% activity at pH 10. Sequence analysis revealed four amino acid substitutions in G41 and a single substitution in G53. These variants, therefore, have improved thermal and alkaline stability and are ideal candidates for DNA shuffling experiments to produce a robust xylanase for industrial application.

Lignin is a major determinant of the decomposition of plant materials in soils. Advances in transgenic technology have led to the possibility of modifying lignin to improve the pulping properties of plant materials for papermaking. Previous studies have shown that lignin modifications also affect the rate of plant material decay in soil. The aim of this work was to investigate short-term changes in soil microbial community structures when tobacco residues with reduced activity of enzymes in the monolignol pathway decompose. The residues from lignin-modified plants all decomposed faster than unmodified plant materials. The relative proportions of some of the structural groups of microbial phospholipid fatty acids were affected by genetic modifications, especially the proportion of double unsaturated chain fatty acids, indicative of fungi.

A gene that encodes a protein with azoreductase activity was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737. The enzyme, with a molecular weight of 18.7 kD, was heterologously expressed in Escherichia coli and its azoreductase activity was characterized. Furthermore, the reduction mechanism of azo dyes catalyzed by the azoreductase was studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.

Xylanases are used mainly in the pulp and paper industries for the pretreatment of Kraft pulp prior to bleaching to minimize use of chlorine, the conventional bleaching agent. This application has great potential as an environmentally safe method. Hydrolysis by xylanases of relocated and reprecipitated xylan on the surface of cellulose fibres formed during Kraft cooking facilitates the removal of lignin by increasing permeability to oxidising agents. Most of the xylanases reported in the literature contained significant cellulolytic activity, which make them less suitable for pulp and paper industries. The need for large quantities of xylanases which would be stable at higher temperatures and pH values and free of cellulase activity has necessitated a search for novel enzymes. We have isolated and characterised several xylanase-producing cultures, one of which (an alkalophilic Bacillus SSP-34) produced more than 100 IU ml(-1) of xylanase activity. The SSP-34 xylanases have optimum activity at 50 degrees C in a pH range 6-8, with only small amounts of cellulolytic activity (CMCase (0.4 IU ml(-1), pH 7), FPase (0.2 IU ml(-1), pH 7) and no activity at pH 9).