We have previously introduced mutations into the aprN gene using site-directed mutagenesis to probe the importance of hydrogen bonds in the active site of the NK (Zheng et al., 2006), increase the oxidative stability of NK (Weng et al., 2009), and investigate the function of the propeptide of NK (Jia et al., 2010).
DNA family shuffling is a simple and efficient method for molecular-directed evolution by mimicking and accelerating the process of sexual recombination (Crameri et al., 1998). This approach involves the recombination of homologous sequences, which are the same gene from related species or related genes from a single species to create a library of chimeras. A library of chimeric subtilisins has been created by DNA family shuffling and the mutants have improved properties compared to the parental enzymes (Ness et al., 1999). NK belongs to the subtilisin family of serine protease, has the same conserved catalytic triad (D32, H64, S221) and substrate binding sites Lumacaftor purchase (S125, L126, G127) (Bryan,
2000). The homology of the encoding gene sequence between NK and subtilisin BPN′ (SB) from Bacillus amyloliquefaciens or NK and subtilisin Carlsberg (SC) from Selleck Navitoclax Bacillus licheniformis was 80% or 69%, respectively (Nakamura et al., 1992). Therefore, we introduced random mutagenesis in the aprN gene using the DNA family shuffling method. The three encoding genes were recombined and shuffled to establish chimeric gene libraries. Combined with a high-throughput plate-based screening method, mutants that had the desired properties were selected, purified Org 27569 and characterized. In the current study, we reported for the first time the application of directed evolution to improve the fibrinolytic activity of subtilisin NAT from Bacillus natto. Bacillus subtilis var. natto strain AS 1.107 (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China),
B. amyloliquefaciens strain CICC 20164 and B. licheniformis strain CICC 10092 (China Center of Industrial Culture Collection, Beijing, China) were used to isolate the genomic DNA. Escherichia coli BL21(DE3)pLysS and the plasmid pET-26b+ (Novagen) were used as the host-vector system for the cloning and expression of the gene encoding the enzymes. All of the enzymes for DNA manipulations were purchased from TaKaRa (Dalian). Thrombin and urokinase were purchased from the Chinese Medicine Testing Institute. Human fibrinogen and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (suc-AAPF-pNA) were purchased from Sigma (St. Louis, MO). The oligonucleotide primers and plasmids used in the current study are listed in Table 1. The gene encoding the precursor NK was amplified by PCR from genomic DNA of B. subtilis var. natto using the primers PNB and PNX. Similarly, the gene encoding the precursor SB from B. amyloliquefaciens (Vasantha et al., 1984) or Carlsberg from B. licheniformis (Jacobs et al., 1985) were also obtained by PCR using the two sets of primer PBB and PBX or PCB and PCX, respectively.