Browsing by Subject "Lysobacter enzymogenes"

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    Herstellung und Charakterisierung einer rekombinanten, sequenzspezifischen Protease zur Generierung bioaktiver Peptide
    (2009) Hug, Thomas; Fischer, Lutz
    The aim of the present thesis was the production and biochemical characterization of a sequence specific microbial protease. This enzyme should be applied in food protein hydrolyzation in order to generate bioactive peptides. To determine the substrate specificity of serine protease PRT1 from Xanthomonas campestris pv. campestris this strain was cultivated in a shaking flusk. After dialysis of the culture broth and addition of 1,10-phenantroline for metalloprotease PRT2 inactivation an enzyme activity of 0,34 nkat Caseine/mL was detected. The conversion of several chromogenic peptide substrates revealed that PRT1 does not offer a clear substrate specificity. The lysyl endopeptidase LysC from Lysobacter enzymogenes ssp. enzymogenes was obtained by cultivation of the wild type strain (ATCC 27796). In a bioreactor (1 L scale) a maximum protease activity of 0,084 nkat Tos-Gly-Pro-Lys-pNA/mL in the culture broth was detected after 45 h. The LysC gene was amplified by PCR using genomic template DNA and was cloned into the E. coli expression vector pET20b(+), leading to no detectable recombinant protease when expressed in E. coli BL21(DE3). Thus for the heterologous expression a synthetic gene construct was applied which was formerly described in literature. It contained a short N-terminal pro-sequence (MGSK) and a codon usage adapted to E. coli. The bioreactor cultivation (5 L scale) of E. coli BL21(DE3) pET20b-MGSK-LysC led to LysC inclusion bodies. The solubilization of the inclusion bodies and the following enzyme renaturation using L-arginine as an unspecific folding additive resulted in a maximum protease activity of 0,06 nkat Tos-Gly-Pro-Lys-pNA/L Culture 5h after IPTG induction. To increase the yield of recombinant protease activity the influence of the LysC propeptides on the in vitro renaturation of the protease was investigated. For this purpose both pro-peptide DNAs were sequenced, cloned and heterologously expressed in E. coli BL21(DE3). The addition of C-terminal and N-terminal propeptide to the LysC renaturation led to a maximum of 27fold (1.56 Μkat Tos-Gly-Pro-Lys-pNA/L Culture) LysC activity increasion compared to the L-arginine renaturation. As an alternative food-grade expression system the in the literature already established system of Lactobacillus plantarum NC8, L. sakei Lb790 and the E. coli-Lactobacillus shuttle vector pSIP409 was tested. However, the shaking flusk cultivations of neither L. plantarum NC8 pSIP409-MGSK-LysC nor L. sakei Lb790 pSIP409-MGSK-LysC led to detectable recombinant lysyl endopeptidase. As a possible reason therefor the different codon usage of E. coli and Lactobacillus was assumed. So expression experiments were performed using point mutated variants of the beta-galctosidase gene from Kluyveromyces lactis. It could be shown that the exchange of the serine codons tca/agt and tcc had a significant effect on the resulting enzyme activity. The exchange of three codons led to a decreation of beta-galactosidase activity of 38 %. The characterization of the recombinant lysyl endopeptidase LysC confirmed the high substrate specificity for lysine residues at P1 position. The pH and temperature optimum was 8.5 and 45°C, respectively. At 4°C and pH 9 the enzyme was stable for at least 20.5 h, whereas at 45°C only 40 % residual activity were detected after 1 h. An inhibiting effect on LysC was demonstrated for Ba2+, NH+ and PMSF. Hydrolysis of bovine caseine by LysC for generated the ACE inhibiting peptides EMPFPK, FALPQYLK, NMAINPSK and ALNEINQFYQK as well as the antioxidative VLPVPQK, which all were unambiguously identified by LC-ESI-MS/MS. Performing appropriate in vitro assays, the radical scavenging acticvity (IC50 = 4,85 Μg/mL), lipoxygenase inhibition (IC50 = 23,6 Μg/mL) and ACE inhibition (IC50 = 2,78 Μg/mL) of the caseine hydrolysate were quantified.