Abstract

A sequence-based polymerase chain reaction (PCR) was employed to screen fibrinolytic enzymes from soil metagenomes. A basic alignment search tool (BLAST) sequence homology analysis of 15 positive amplicons indicated a high degree of nucleotide sequence identity (>98%) to a fibrinolytic enzyme, nattokinase in Bacillus sp. Among the positive clones, KSL79_FE was selected for further characterization. Sequence analysis showed that its open reading frame (ORF) consisted of 1,146 nucleotides encoding 375 amino acids, of which two differed from the nattokinase (T268S and V298A). To overexpress the fibrinolytic enzyme, we transformed the plasmid pET28a/KSL79_FE into E. coli BL21 Codon (+) cells, leading to yield optimal expression by using a 9-h induction with 30 uM isopropyl thio-β-D-galactoside (IPTG) at an OD₆₀₀ of 0.5. The resulting KSL79_FE enzyme exhibited caseinolytic and fibrinolytic activities similar to those of nattokinase. In contrast to the nattokinase which showed the optimal conditions for proteolytic activity at 37°C and pH 8.0, KSL79_FE enzyme displayed maximal proteolytic activity at 50°C and pH 9.0. In addition, the enzyme activity of KSL79_FE was inhibited by Zn⁺² ions, but not by Cu⁺² ions, not similar to nattokinase. The two residues varied from amino acid sequence of nattokinase which might change the biochemical properties and optimal enzyme activity of KSL79_FE.

Key words: Nattokinase, proteolytic activity, metagenome, fibrinolytic activity, cloning and expression.

Abbreviation

Abbreviations: tPA, Plasminogen activator; PCR, polymerase chain reaction; FE, fibrinolytic enzyme gene; IPTG, isopropyl thio-β-D-galactoside; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; IgG, immunoglobulin G; TCA, trichloroacetic acid; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis (2-aminoethyl ether) tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; DTT, DL-dithiothreitol; BLAST, Basic Alignment Search Tool; ORF, open reading frame; NA, p-nitroaniline; DGGE, denaturing gradient gel electrophoresis.