Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

Abstract

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per g of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.Proteolytic enzymes catalyze the hydrolytic cleavage of peptide bonds. These enzymes are present in all living organisms and are essential for cell growth and differentiation. Microorganisms produce a variety of intracellular and/or extracellular proteases. Intracellular microbial proteases are highly specific and are involved in several cellular and metabolic processes such as activation of inactive precursors, maintenance of the cellular protein pool, and sporulation. Extracellular proteases degrade proteins in cell-free environments. The resulting hydrolytic products (small peptides and amino acids) can be transported into the cells and utilized as carbon or nitrogen sources (13, 39). Especially, extracellular proteases are of industrial importance and are used as cleaning agents and food and feed additives.Proteases can be divided into various groups with respect to the functional group present at the active site and the pH profile of the activity. Microbial alkaline proteases, which are defined to be active in a neutral to alkaline pH range (13), possess either a serine center (serine proteases) or are of metal-type (metalloproteases). Extracellular...

Metagenomic Analyses: Past and Future Trends

“…Small-insert libraries can be employed for the identification of novel biocatalysts encoded by a single gene or a small operon, whereas large-insert libraries are required to recover large gene clusters, which code for complex pathways (18). Construction and screening of both types of metagenomic libraries have resulted in the identification of many novel biocatalysts, e.g., lipases/esterases (15,26,48,49), cellulases (25,47), chitinases (50), DNA polymerases (109), proteases (139), and antibiotics (95). To date, lipases/esterases are probably the biocatalysts which have been most frequently recovered from metagenomes.…”

“…Reasons for this include the reluctance of many microorganisms present in these samples to lyse by protocols that have been developed mainly for DNA extraction from mesophilic samples and the release of very stable nucleases upon cell lysis. Nevertheless, significant progress has been made, and various methods allowing the isolation of highquality DNA from a variety of environments, i.e., soil (45,87,134,139), marine picoplankton (117), contaminated subsurface sediments (1), groundwater (128), hot springs and mud holes in solfataric fields (94), surface water from rivers (145), glacier ice (109), Antarctic desert soil (48), and buffalo rumens (25), have been developed.…”

Bioprospecting in the Genomic Age

“…These two proteases are zinc-containing metalloproteases, many of which are involved in pathogenesis (Bitar, Cao, & Marquis, 2008;Chuang, Chang, & Chang, 1997;Häse & Finkelstein, 1993;Kim, Yang, & Cha, 2002;Miyoshi, Kawata, Tomochika, Shinoda, & Yamamoto, 2001). Screening for these proteases was possible as they were necessary to hydrolyze milk proteins, leading to detection of clear halos formed on the plates (Waschkowitz, Rockstroh, & Daniel, 2009). Other enzymatic functions identified using a similar detection methodology include phosphatase and lipolytic activities (Bornscheuer & Kazlauskas, 2006;Calamia & Manoil, 1990;Lämmle et al, 2007).…”

High throughput sequencing methods and analysis for microbiome research

“…A C C E P T E D M A N U S C R I P T (Hardeman and Sjoling, 2007, Pathak et al, 2009, Voget et al, 2006, Waschkowitz et al, 2009, marine picoplankton (Stein et al, 1996), saline soil (Purohit and Singh, 2009), contaminated subsurface sediments (Abulencia et al, 2006), groundwater (Uchiyama et al, 2005), hot springs and mud holes in solfataric fields (Rhee et al, 2005), Urania hypersaline basins (Ferrer et al, 2009), surface water from rivers (Wu and Sun, 2009), glacier ice (Simon et al, 2009), glacier soil (Yuhong et al, 2009), Antarctic and Arctic desert soil (Cieslinski et al, 2009, Heath et al, 2009, Jeon et al, 2009, and buffalo rumens (Duan et al, 2009).…”