Characterization of a new oxidant-stable serine protease isolated by functional metagenomics

A novel serine protease gene, SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent (Tween 20, Tween 80, or Triton X-100). Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H(2)O(2). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2193-1801-2-410) contains supplementary material, which is available to authorized users

Which properties of cutinases are important for applications?

Cutinases (EC 3.1.1.74) are extracellular enzymes that belong to a/ß hydrolases. They are serine esterases with the classical Ser-His-Asp triad similar to several lipases and serine proteases. In nature, cutinases catalyse the hydrolysis of the polyesters of the cuticle and the suberin layers, which protect plant surfaces. Cutinase production is typical for plant pathogenic fungi, but also, bacterial cutinases and cutinases from plant pollen have been discovered. Cutinases are promiscuous esterases catalysing reactions with a wide range of different substrates, such as short-chain soluble esters, water-insoluble medium and long-chain triacylglycerols, polyesters and waxes. In the current work, an overview is given on suggested applications of cutinases in the textile industry, in laundry detergents, in processing of biomass and food, in biocatalysis and in detoxification of environmental pollutants. The applications are discussed from the point of view of cutinase properties - which properties of cutinases are already advantageous and which would be desired. In addition, improvements that have been made on cutinase performance by protein and reaction engineering are reviewed

Occurrence of lignin degradation genotypes and phenotypes among prokaryotes

A number of prokaryotes actively contribute to lignin degradation in nature and their activity could be of interest for many applications including the production of biogas/biofuel from lignocellulosic biomass and biopulping. This review compares the reliability and efficiency of the culture-dependent screening methods currently used for the isolation of ligninolytic prokaryotes. Isolated prokaryotes exhibiting lignin-degrading potential are presented according to their phylogenetic groups. With the development of bioinformatics, culture-independent techniques are emerging that allow larger-scale data mining for ligninolytic prokaryotic functions but today, these techniques still have some limits. In this work, two phylogenetic affiliations of isolated prokaryotes exhibiting ligninolytic potential and laccase-encoding prokaryotes were determined on the basis of 16S rDNA sequences, providing a comparative view of results obtained by the two types of screening techniques. The combination of laboratory culture and bioinformatics approaches is a promising way to explore lignin-degrading prokaryotes