Properties of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens RSB34, Isolated from Doenjang

Abstract

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as by 16S rRNA and gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity (83.23 mU/μl) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. was overexpressed in BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The and values of recombinant AprE34 were 0.131 ± 0.026 mM and 16.551 ± 0.316 μM/l/min, respectively, when measured using an artificial substrate, -succinyl-ala-ala-pro-phe--nitroanilide. was overexpressed in WB600 using pHY300PLK. transformants harboring pHYRSB34 (pHY300PLK with) showed higher fibrinolytic activity than RSB34.

Characterization of a Fibrinolytic Enzyme Secreted by Bacillus velezensis BS2 Isolated from Sea Squirt Jeotgal

“…Considering the results, AprEBS2 has moderate heat stability. The optimum temperature for AprEBS2 is similar to that for AprEs from other Bacillus strains such as B. subtilis HK176 [31] and B. amyloliquefaciens RSB34 [14], but lower than that for Bacillus sp. strain CK11-4 (70 o C) [32].…”

“…16S rRNA genes were amplified by using the following primers: bac-F (5'-CGGCGTGCCTAATACATGCAAG-3') and bac-R (5'-GGCATGCTG ATCCGCATTAC TA-3') [14]. A partial recA gene was amplified using primers: recA-F (5'-TGAGTGATCGTCAGG CAGCCTTAG-3') and recA-R (5'-CYTBRGATAAGAR TACCAW GMACCGC-3') [14]. Amplification was done using a thermocycler (MJ Mini Personal Thermal Cycler, BioRad, USA).…”

“…Aliquots were centrifuged at 4,000 ×g for 10 min at 4 o C, and the supernatant was filtered using a 0.45 μm filter (Sartorius Stedim, Germany). Filtered supernatant (FS) was assayed for fibrinolytic activity by fibrin plate method [14]. The size of lysis zones on fibrin plates were converted into plasmin units (U) by comparing them with those of plasmin with different units (Sigma, USA).…”

“…In this work, CH51-F and CH51-R primers were used to clone aprEBS2. The primer pairs were initially designed to clone a fibrinolytic gene (aprE51) from B. amyloliquefaciens CH51 [16], and later successfully used to clone aprE34 from B. amyloliquefaciens RSB34 and aprEBS15 from B. pumilus BS15 [14,26]. (Fig.…”

“…Calculated pI and molecular mass of mature enzyme were 6.65 and 27429.74 Da, respectively. Nucleotide sequence of aprEBS2 showed 99% identities to those of aprE genes from B. amyloliquefaciens CH51 (EU414203, 1,145/1,149) [16], B. pumilus BS15 (MF943247, 1,144/1,149) [26], and 98% identities to genes from B. amyloliquefaciens CB1 (KM086575, 1,133/1,149) [27], B. amyloliquefaciens RSB34 (KX098331, 1,132/1,149) [14], and B. amyloliquefaciens MJ5-41 (JF739176, 1,135/1,149) [28]. AprEBS2 showed high similarities with other fibrinolytic ) are conserved in AprEBS2 like other AprE enzymes [29].…”