30% residual activity was reported for the PepN from LHE-511 after 2 h of incubation at 50C [10]. The kinetic parameters of PepN from ATCC 12046 were decided using nine commercially available chromogenic peptides. [5], [11], [12] or other LAB exopeptidases been reported. Product inhibition for endopeptidases is usually a known problem that occurs during food Desvenlafaxine succinate hydrate protein hydrolyses [13], [14]. We report the production of recombinant CAPZA1 PepN and PepX from ATCC 12046 was Desvenlafaxine succinate hydrate cultivated in de Man, Rogosa and Sharpe (MRS) medium [15] with constant shaking at 37C. DH5 (Invitrogen, Carlsbad, USA) and BL21(DE3) (Novagen, Madison, USA) strains were used as hosts for plasmid maintenance and T7 expression work, respectively. Standard protocols were used for the preparation and transformation of qualified cells with plasmid DNA via heat shock [16]. Cells were cultivated in Luria Bertani (LB) medium supplemented with the appropriate antibiotic (100 g mL?1 ampicillin) and agar (15 g L?1) for agar plates. All cultures were produced with continuous shaking at 37C unless otherwise stated. Cloning, Construction of Expression Desvenlafaxine succinate hydrate Vectors and Sequencing of and ATCC 12046 was extracted using an identical method as previously described [17]. Polymerase chain reaction (PCR) was performed using HotStar HiFidelity polymerase (Qiagen), according to the manufacturers instructions. The primers gene based on the nucleotide sequence of from CNRZ 32 (EMBL: “type”:”entrez-protein”,”attrs”:”text”:”AAB50275″,”term_id”:”984347″,”term_text”:”AAB50275″AAB50275) that is available in the UniProt database (UniProt ID: “type”:”entrez-protein”,”attrs”:”text”:”Q59485″,”term_id”:”34222706″,”term_text”:”Q59485″Q59485). The PCR product (approx. Desvenlafaxine succinate hydrate 2,400 bp) of (2,379 bp) was cloned into the pJET1.2 vector (Fermentas), according to the manufacturers instructions. Similarly, the gene was amplified with the primers gene (2,532 bp) based on the nucleotide sequence of the gene from (EMBL: “type”:”entrez-protein”,”attrs”:”text”:”CBK51574″,”term_id”:”291048136″,”term_text”:”CBK51574″CBK51574) that is available in the UniProt database (UniProt ID: “type”:”entrez-protein”,”attrs”:”text”:”Q10730″,”term_id”:”1703285″,”term_text”:”Q10730″Q10730), resulting in an approx. 2,500 bp PCR product. The PCR products of (pJET1.2 as template) and (genomic DNA as template) were purified (QIAquick Gel Extraction Kit; Qiagen) after electrophoresis through an agarose gel (0.8%). The construction of the expression vectors pET-20b(+)_and pET-20b(+)_BL21(DE3) Transformed BL21(DE3) strains were grown in 2 YT medium that contained glucose (10 g L?1) supplemented with ampicillin (100 g mL?1). Precultures were incubated at 37C on a rotary shaker. The first precultures were cultivated for 18 h and the second precultures for 13 h. The main cultures (800 mL) were grown in a bioreactor parallel system (Multifors), following the analytical methods previously described [17], with some modifications. The stirrer velocity varied between 500 and 1000 rpm. The heat was maintained at 30C until the OD600 reached a value of 5 to minimize the formation of inclusion bodies, and protein expression was induced by the addition of 0.5 mM IPTG. During the cultivations, samples were removed at various time points, and the enzymatic activity was decided from the cell-free extract after cell disruption [17]. The cultures were harvested after 23 h of cultivation, as previously described [17]. Automated Purification of PepX and PepN Both PepX and PepN were individually purified using Ni2+ immobilized metal affinity chromatography (IMAC) and subsequently desalted via two HiPrep? 26/10 columns using an automated operating procedure, as previously reported [17], [18]. Cell suspensions of 15% (w/v) were prepared in Desvenlafaxine succinate hydrate 50 mM Na2HPO4/KH2PO4 buffer (pH 6.5) containing 500 mM NaCl and 20 mM imidazole (PepX) or 10 mM imidazole (PepN). Both enzymes were eluted by increasing the imidazole concentration to 500 mM in an identical buffer. Subsequently, the enzymes were desalted in 50 mM Na2HPO4/KH2PO4 buffer (pH 6.5). Polyacrylamide Gel Electrophoresis (PAGE) The samples, following cell disruption.
30% residual activity was reported for the PepN from LHE-511 after 2 h of incubation at 50C [10]
