Heterologous expression and structure-function relationship of low-temperature and alkaline active protease from Acinetobacter sp. IHB B 5011(MN12).

Abstract

The gene encoding protease from Acinetobacter sp. IHB B 5011(MN12) was cloned and expressed in Escherichia coli BL21(DE3). The nucleotide sequence revealed 1323 bp ORF encoding 441 amino acids protein with molecular weight 47.2 kDa. The phylogenetic analysis showed clustering of Alp protease with subtilisin-like serine proteases of S8 family. The amino acid sequence was comprised of N-terminal signal peptide 1–21 amino acids, pre-peptide 22–143 amino acids, peptidase S8 domain 144–434 amino acids, and pro-peptide 435–441 amino acids at C-terminus. Three constructs with signal peptide pET-Alp, without signal peptide pET-Alp1 and peptidase S8 domain pET-Alp2 were prepared for expression in E. coli BL21(DE3). The recombinant proteins Alp1 and Alp2 expressed as inclusion bodies showed∼50 kDa and∼40 kDa bands, respectively. The pre-propeptide∼11 kDa removed from Alp1 resulted in mature protein of∼35 kDa with 1738 U mg−1 specific activity. The recombinant protease was optimally active at 40 ◦C and pH 9, and stable over 10–70 ◦C and 6–12 pH. The activity at low-temperature and alkaline pH was supported by high R/(R + K) ratio, more glycine, less proline, negatively charged amino acids, less salt bridges and longer loops. These properties suggested the suitability of Alp as additive in the laundry.