A novel carboxyl-terminal protease derived from Paenibacillus lautusCHN26 exhibiting high activities at multiple sites of substrates

Abstract

BackgroundCarboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), was derived from P. lautus CHN26, a Gram-positive bacterium isolated by functional screening. Recombinant protein was obtained from protein over-expression in Escherichia coli and the biochemical properties of the enzyme were investigated.ResultsScreening of environmental sediment samples with a skim milk-containing medium led to the isolation of a P. lautus CHN26 strain that exhibited a high proteolytic activity. A gene encoding a carboxyl-terminal protease (ctpAp) was cloned from the isolate and characterized. The deduced mature protein contains 466 aa with a calculated molecular mass of 51.94 kDa, displaying 29-38% amino acid sequence identity to characterized bacterial CtpA enzymes. CtpAp contains an unusual catalytic dyad (Ser309-Lys334) and a PDZ substrate-binding motif, characteristic for carboxyl-terminal proteases. CtpAp was expressed as a recombinant protein and characterized. The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites. Furthermore, CtpAp exhibited a high activity at room temperature and strong tolerance to conventional protease inhibitors, demonstrating that CtpAp is a novel endopeptidase.ConclusionsOur work on CtpA represents the first investigation of a member of Family II CtpA enzymes. The gene was derived from a newly isolated P. lautus CHN26 strain exhibiting a high protease activity in the skim milk assay. We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications.

Structural and biochemical properties of a novel pullulanase of Paenibacillus lautus DSM 3035

“…The ExPASy compute pI/Mw program algorithm estimated the molecular weight and the pI value of Pul PL was 87.9 kDa and 5.16, respectively. Although Paenibacillus species are well known for their ability to secrete kinds of useful extracellular enzymes , there are few reports of Paenibacillus pullulanase, and no report of P. lautus pullulanase including wild strain and recombinant strain. There was only one pullulanase gene report from Paenibacillus spp.…”

“…Although many pullulanase genes from Bacillus spp. have been cloned and sequenced, pullulanase in Paenibacillus is underreported . Castro et al .…”

Conversion of Shrimp Head Waste for Production of a Thermotolerant, Detergent-Stable, Alkaline Protease by Paenibacillus sp.

“…As such, TKU047 was simply identified as Paenibacillus sp. The biosynthesis of proteases by Paenibacillus have rarely been reported [52][53][54], especially on fishery processing by-products [13], therefore the current study shows promise in seeking to discover a novel protease.…”

“…Compared to other reports, TKU047 showed a higher optimal temperature than P. tezpurensis sp. nov. AS-S24-II protease (45-50 • C) [52] or P. lautus protease (20-30 • C) [54], and could be comparable with thermostable proteases from Bacillus strains [45]. A higher optimal temperature and thermostability are important factors for industrial applications; therefore, Paenibacillus sp.…”

“…TKU042 protease (35 kDa) [13], but different from other Paenibacillus proteases, such as P. tezpurensis sp. nov. AS-S24-II (43 kDa) [52], P. larvae strain 44 (59 kDa) [53], and P. lautus CHN26 (52 kDa) [54]. The purification of Paenibacillus sp.…”