Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library

Abstract

It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42°C and pH 9.0; Ca 2؉ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making ␣-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, ␣-amylase, and 4-␣-glucanotransferase.

Soil eukaryotic functional diversity, a metatranscriptomic approach

“…In this respect, this approach developed for eukaryotic micro-organisms has the same potential as the traditional metagenomic approach for the cloning of single genes, coding enzymes of interest, from the environment (for example, Voget et al, 2003;Yun et al, 2004). In the context of natural product discovery from the environment the metatranscriptomic approach is however limited to the independent cloning of single genes and cannot be used to recover, at once, entire biosynthetic pathways despite the fact that genes coding for the synthesis of secondary metabolites often cluster in fungal genomes (Keller and Hohn, 1997).…”

“…Such libraries have been generated for different purposes that encompass basic and applied research. The metagenome is considered as a treasure-trove for new enzymes (see, for example, Voget et al, 2003;Yun et al, 2004) and bioactive compounds whose biosynthetic pathways can be coded by full-length genes, operons or gene clusters present on single, long DNA inserts of the libraries (see, for example, Gillespie et al, 2002;Courtois et al, 2003;Schirmer et al, 2005). Analysis of metagenomic libraries also offers the opportunity to get insights into genome organization and gene content of new bacterial species belonging to phyla with no cultivable representative (see, for example, Tyson et al, 2004;Nesbø et al, 2005;García Martín et al, 2006;Strous et al, 2006).…”

From bacterial genomics to metagenomics: concept, tools and recent advances

“…The quality of DNA in terms of size, amount and presence of contaminants should be considered before the library is made as poor quality DNA can lead to low coverage of environmental genomes in the library [80]. Extracted DNA is purifi ed if required and cloned in suitable vectors, such as plasmid [84,85], cosmid [86], fosmid [63,87] or BAC [88,89]. As small insert libraries do not permit detection of large gene clusters and operons, large insert libraries in cosmid/ fosmid/ BAC vectors are preferred [80].…”

HSV (Herpes Simplex Virus)

“…Les micro-organismes saprophytes sont de puissants recycleurs de la matière organique et ils disposent d'une panoplie biochimique qui intéresse fortement la communauté scientifique (Yamagushi et Yokoe, 2000 ;Sanchez et al, 2004 ;Yun et al, 2004). La mise en évidence de micro-organismes dépolluants ou recycleurs représente en effet une grande partie de l'activité de recherche des équipes spécialisées dans les microflores du sol.…”