1.1 Materials and reagents
A.niger CGMCC 3.7193 was stored in the China Common Microbe Culture Collection Management Center (CGMCC); E. coli JM109, P. pastoris GS115 and the pPIC9K plasmid were obtained and stored in our laboratory. A. niger and E. coli were cultured on PDA medium and LB medium, respectively. P. pastoris was cultured in YPD, MD, BMGY and BMMY media. The preparation and culture methods were performed according to the Pichia Expression Kit (Version M) provided by Invitrogen. The PCR primers for CapA were F: 5'- GTAGTCCTCCAGCCAGAGGAACCATC-3', and R: 5'-TGCTCTAGATCACTCAGTAAAC GATGCCCCG-3'; the restriction enzyme sites are underlined, and these primers were generated by Sangon Biotech Co., Ltd. (Shanghai). Restriction endonuclease, Pyrobest DNA polymerase and T4 DNA ligase were purchased from the TaKaRa company; the Plasmid Extraction Kit and DNA Purification and Recovery Kit were from Beijing Zoman Biotechnology Co., Ltd; the RNAqueous™-Micro Total RNA Isolation Kit and SuperScript III First-Strand Synthesis System were from Invitrogen; and the following chemical substrates were synthesized by Ontores Biotech Co: CBZ-ala-Arg, CBZ-Pro-Gly, CBZ-Ala-Lys, CBZ-Gly-Ala, CBZ-Ala-Glu and CBZ-Phe-Leu.
1.2.1 Gene cloning and recombinant P. patoris construction
Total RNA was extracted from A. niger, and the first-strand cDNA was synthesized by reverse transcription. The first-strand cDNA was used as a template for PCR amplification with the synthesized primer, and the subsequent PCR product purification, enzyme digestion, ligation, E. coli JM109 transformation, and the positive clone screening was performed according to routine laboratory methods (Zhu and Wang 1994). The recombination plasmid linearization and electric transformation of P. pichia GS115 were conducted, and the recombinant P. pastoris GS115 (pPIC-capA) were screened, according to the methods provided by the Pichia Expression Kit.
1.2.2 Induction of expression and preliminary purification of recombinant enzyme
Recombinant P. pastoris GS115 (pPIC-CapA) was purified on YPD plates. Single colonies were selected and inoculated into YPD liquid medium (25 mL) and cultured at 30°C and 200 r/min for 18 h to 20 h. Then, 1% of the inoculation was transferred into BMGY medium (25 mL). The bacteria were cultured at 30°C and 200 r/min for 16 h-18 h until the cells reached the logarithmic stage of growth (OD600=2.0-6.0). Then, the bacteria were centrifuged at 8,000 r/min for 5 min for collection, and the bacteria were suspended in the proper volume of BMMY medium and cultured until the OD600 value reached 1.0. For the induction, the bacteria were cultured at 30°C and 200 r/min. Methanol was added every 24 h until the final concentration reached 0.5%. The fermentation was maintained for 120 h and then ended. The supernatant of the fermentation broth was centrifuged at 4°C and 8,000 r/min to collect the crude enzyme broth. The crude enzyme solution was precipitated by 30%-70% ammonium sulfate and dialyzed by a dialysis bag with a molecular weight cut-off of 50 kDa to achieve a preliminary purification.
1.2.3 Determination of recombinant carboxypeptidase activity
Carboxypeptidase activity was determined by referring to the method described by Morita et al (2009), and a simple optimization was performed. Using CBZ-Phe-Leu as the substrate, a 1 mmol/L substrate solution was prepared with a pH 6.0 disodium hydrogen phosphate and citric acid buffer solution (0.1 mol/L). A total of 450 μl substrate and 50 μl of the suitably diluted enzyme solution were mixed and reacted at 37°C for 60 min. A 500-μl aliquot of a 0.5% indanone solution was immediately added; the mixture was heated in a water bath at 100°C for 15 min, and then it was cooled by tap water for 5 min. The absorbance value of A570 was determined by spectrophotometry (SP-2012UV spectrophotometer: Shanghai Spectral Instrument Co., Ltd.). Standard tyrosine solutions of different concentrations were prepared and reacted with ninhydrin under the same conditions. The standard curve was generated.
The definition of the enzyme activity is as follows: the unit of enzyme activity (U) is the amount of enzyme that hydrolyzes the substrate to generate 1 μg tyrosine at 37°C for 1 min.
1.2.4 Analysis of the enzymatic properties of recombinant carboxypeptidase
188.8.131.52 Determination of optimum temperature and temperature stability
The activity values of carboxypeptidase were determined at 30°C-70°C and pH 6.0, and the optimal reaction temperature of the enzyme was determined.
The enzyme solution was incubated at 30°C-70°C for 0.5, 1, 1.5 and 2 h. The enzyme activity was measured according to the method described in section 1.2.3. The enzyme solution without heat treatment was used as a control (the enzyme activity was 100%) to calculate the relative enzyme activity and investigate the thermal stability of carboxypeptidase.
184.108.40.206 Determination of the optimum pH and pH stability
The activity values of carboxypeptidase at pH 4.0-8.0 and 45°C were measured to determine the optimal pH of the enzyme.
The enzyme solution was incubated in a pH 4.0-8.0 buffer solution for 1 h (45°C), and the enzyme activity was determined according to the method described in section 1.2.3. The maximum value of the enzyme activity was 100%, and the pH stability of carboxypeptidase was investigated. The buffer used was the 0.1 mol/L disodium hydrogen phosphate-citric acid buffer (pH 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, and 8.0).
220.127.116.11 Influence of metal ions and chemical reagents on enzyme activity
Metal ions and chemical reagents at final concentrations of 1 mmol/L were added to the carboxypeptidase and substrate reaction systems. The enzyme activity was determined according to the method described in section 1.2.3. The enzyme activity in the system without metal ions and chemical reagents was 100%, and the relative enzyme activity in the presence of the metal ions and chemical reagents was calculated.
18.104.22.168 Substrate specificity analysis
Carboxypeptidase was reacted with 1 mmol/L CBZ-Ala-Arg, CBZ-Pro-Gly, CBZ-Ala-Lys, CBZ-Gly-Ala, CBZ-Ala-Glu or CBZ-Phe-Leu, and the enzyme activity was determined according to the method described in section 1.2.3. When CBZ-Phe-Leu was used as the substrate, the enzyme activity was 100% in the assay. The relative enzyme activity value of other substrates reacting with carboxypeptidase was calculated.
1.2.5 Bioinformatics analysis
NCBI (https://www.ncbi.nlm.nih.gov/genbank/) was used to identify the serine carboxypeptidase amino acid sequences from A. niger and other sources; Clustal X2 and Biodit were used for sequence alignment analysis. The phylogenetic relationship was analyzed with Mega 5.0 by the neighbor-joining method (Tamura et al. 2011).