New robust subtilisins from halotolerant and halophilic Bacillaceae

Abstract

The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465T and Alkalibacillus haloalkaliphilus DSM 5271T and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976T served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0–12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications. Key points • Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins. • Four new subtilisins were biochemically characterised in detail. • The four proteases show potential for future biotechnological applications.

Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicusDSM 15822T

“…The comparison of the mature SPFA amino acid sequence to the well‐characterised true subtilisins Carlsberg [ 34 ], BPN’ [ 35 ] and subtilisin DY [ 36 ] reveals a sequence identity of 61.8%, 61.3% and 71.9%, respectively. When comparing SPFA amino acid sequence to our previously reported true subtilisins from halophilic and halotolerant background a sequence identity of 64.2% (SPMI), 65.4% (SPPM) and 71.7% (SPLA) was reached [ 27 ]. A more distant relationship exists to the high‐alkaline subtilisin Savinase from Lederbergia lenta (formerly Bacillus lentus ; 52.2%) [ 37 ] and our previously reported high‐alkaline subtilisin SPAO from Halalkalibacter okhensis K10‐101 T (47.8%) [ 24 ], as well as the PIS SPAH (48.1%) from Alkalibacillus haloalkaliphilus DSM 5271 T [ 27 ].…”

“…When comparing SPFA amino acid sequence to our previously reported true subtilisins from halophilic and halotolerant background a sequence identity of 64.2% (SPMI), 65.4% (SPPM) and 71.7% (SPLA) was reached [ 27 ]. A more distant relationship exists to the high‐alkaline subtilisin Savinase from Lederbergia lenta (formerly Bacillus lentus ; 52.2%) [ 37 ] and our previously reported high‐alkaline subtilisin SPAO from Halalkalibacter okhensis K10‐101 T (47.8%) [ 24 ], as well as the PIS SPAH (48.1%) from Alkalibacillus haloalkaliphilus DSM 5271 T [ 27 ]. The comparison of the amino acid sequences in the MSA confirms that SPFA belongs to the group of true subtilisins.…”

“…[ 43 ] reported on the high‐alkaline subtilisin aprM from Halalkalibacterium halodurans C‐125 that also displays a high activity (> 70%) between pH 7.0 and 12.0 but with a clear optimum at pH 12.0. We also recently reported on three true subtilisins and one PIS derived from halotolerant and halophilic Bacillaceae that exhibited good activity even at a pH of 12.0, probably due to the salt adaptation through a charged molecular surface that facilitates the adaptation to high pH [ 27 ]. This charged molecular surface can also be observed in SPFA.…”

“…The oligonucleotides for PCR were obtained from Eurofins Genomics GmbH (Ebersberg, Germany; Table S1 ). Cloning and transformation were performed as previously described [ 27 ]. Bioinformatic analysis of the amino acid sequence, homology modelling and multiple sequence alignment (MSA) as previously reported [ 27 ].…”

“…Cloning and transformation were performed as previously described [ 27 ]. Bioinformatic analysis of the amino acid sequence, homology modelling and multiple sequence alignment (MSA) as previously reported [ 27 ].…”