Development Of A Keratinase-recombinant Strain And Characterization And Efficacy Of The Recombinant Enzyme

Keratinases are metallo or serine proteinases which can degrade the insoluble structure forming keratin substrates and have immense potential application in the animal nutrition and feed industry. However, the industrial applications of keratinase are still not extensive because of its low level of expression. To increase its production, the keratinase (kerA) gene from Bacillus licheniformis S90was expressed in Escherichia coli BL21and Pichia pastoris X-33. In addition, the native gene was optimized by site-directed mutagenesis to improve its production and thermal stability, and then expressed in P. pastoris X-33. The biochemical properties of all recombinant keratinases were fully characterized.Experiment1:Cloning of the kerA gene from Bacillus licheniformisThe kerA gene was successfully amplified by PCR from B. licheniformis S90DNA. Sequence analysis revealed that the nucleotide sequence of this gene contains1047bp open reading frame (ORF) encoding349amino acids. As compared to the B. licheniformis PWD-1keratinase, there were only five amino acid mutations in the protein sequence of B. licheniformis S90keratinase (Arg-7vs.Glu-7, Asp-121vs. Gly-121, His-136vs. Asn-136, Glu-267vs. Gly-267, Ala-297vs.Val-297). However, both enzymes share the same triad of catalytic residues including Asp-32, His-63, and Ser-220.Experiment2:Expression of the kerA gene in E. coliThe gene kerA was cloned into two conventional vectors, pET30a and pET32a, and expressed in E. coli BL21. From SDS-PAGE analysis, the recombinant keratinases were45and55kDa. They had different optimal pH values (7.5and8.5) but the same optimum temperature of50℃. Both of them had improved thermal stability and kept approx.60%of its max activity after30min at60℃.Experiment3:Expression of the kerA gene in P. pastoris X-33The gene kerA was cloned into plasmid pPICZaA and expressed in P. pastoris X-33under the control of AOX1promoter. The highest keratinase activity produced by P. pastoris pPICZaA-kerAwt was195U/mL. The keratinase secreted by recombinant P. pastoris was named PK1. The molecular weight of PK1was approx.39kDa. The enzyme PK1was active at pH values from7-9and had optimum pH of7.5. It was stable at moderate temperature and had optimum temperature at50℃. The PK1is much more stable at optimum temperature (50℃), remained approx.50%of the maximum activity at this temperature for2h. However, it was rapidly inactivated at higher temperatures60-70℃for30min. When the substrate chicken feather (β-keratin) meal was used, the yield of enzymatic hydrolysis was significantly higher than the substrate keratin azure (a-keratin).Experiment4:Expression of the high-yield kerA gene in P. pastoris X-33The main kerA gene was optimized by two codon optimization strategies and expressed in P. pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The obtained recombinant keratinase was named PK2and PK3respectively. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in P. pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZaA-kerAoptil and pPICZaA-kerAopti2was324U/mL and293U/mL respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. And the secreted recombinant protein was determined by SDS-PAGE was approx.39kDa.Experiment5:Expression of the thermostable kerA gene in P. pastoris X-33Improvement of thermal stability of kerA was tried by engineering a de novo designed disulfide bridge. Disulfide design was performed firstly using Disulfide by DesignTM program. In the present study, only one disulfide bond (Gln320and Leu316) was selected, and the respective amino acids were substituted with Cys residues. The newly designed gene which was named kerAopti3expressed in Pichia pastoris. The highest keratinase activity produced by P. pastoris pPICZaA-kerAwt was24U/mL. The obtained recombinant keratinase was named PK4. The molecular weight of PK4was approx.39kDa. The PK4was optimally active at pH8.0and50℃. The PK4produced clearly improved the thermal stability. It was more stable than PK1at60-70℃.Experiment6:Production of recombinant keratinase by small-scale fermentation and its effect on growth performance and apparent crude protein digestibility in finisher pigsThe high density cultivation of Pichia pastoris pPICZaA-kerAopti1was performed in a30-L fermenter. The highest expression level of568U/mL was achieved after72h fermentation. Then, the recombinant keratinase was added to a feather meal dietary to detect its effect on growth performance and crude protein digestibility in finisher pigs. Sixty Duroc×Landrace×Yorkshire finisher pigs (58.1±1.1kg of initial body weight) were randomly allotted to five groups (n=12). The dietary groups include:(1) basal diet without feather meal and keratinase supplementation (CD);(2-5) feather meal dietary group (5%of feather meal inclusion) with keratinase supplementation at a final concentration of0U/kg (FD group)、3000U/kg (LFD group)、6000U/kg (MFD group)、9000U/kg (HFD group). The results indicated that the average daily body-weight gain (ADG) and the apparent digestibility of crude protein (ADCP) were significantly reduced for FD group compared to CD group (P<0.05), but the ADG and ADCP were increased by FD group keratinase supplementation. In addition, no significant effect of keratinase treatment on feed intake and efficiency.In summary, the broad the pH profile, substrate specificity and expression level make the recombinant keratinase from P. pastoris pPICZaA-kerAopti1a suitable applicant for various industrial applications.