Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris

Abstract

The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/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. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.

Figure 1. Comparative the target gene copy numbers of P. pastori pPICZα-kerAwt, pPICZα-kerAopti1 and pPICZα-kerAopti2.

The values represent means ± SD (n = 3). Results showed no significant difference in gene copy numbers of every positive transformant (P>0.05).

Figure 2. Comparative the target gene relative mRNA levels of P. pastori pPICZα-kerAwt, pPICZα-kerAopti1 and pPICZα-kerAopti2.

The values represent means ± SD (n = 3). Results showed no significant difference in target gene relative mRNA levels of every positive transformant (P>0.05).

Figure 3. SDS-PAGE analysis.

Lane M: protein MW markers (14–94 kDa); Lane 1: the purified keratinase of P. pastoris pPICZα-kerAwt; Lane 2: the purified keratinase of P. pastoris pPICZα-kerAopti1; Lane 3: the purified keratinase of P. pastoris pPICZα-kerAopti2.

Figure 4. The glycoprotein staining and deglycosylation of purified recombinant keratinase in the SDS-PAGE.

A: The glycoprotein staining experiment. Lane 1: BSA; Lane2: purified glycosylated keratinase. B: The deglycosylation of purified recombinant keratinase. Lane M: protein MW markers (14–94 kDa); Lane 1: deglycosylated keratinase.

Figure 5. Recombinant keratinases production by P. pastoris X-33.

A: Kinetics of keratinase production by P. pastoris pPICZα-kerAwt, pPICZα-kerAopti1 and pPICZα-kerAopti2. B: Comparative expression of kerAwt, kerAopti1 and kerAopti2 in P. pastoris at 96 h. The values represent means ± standard deviations (SD) of six independent positive transformants. Every transformant was examined in four independent experiments in quadruplicate. ^a,bMean values were significantly different (P<0.05) when labeled with unlike letters.

Figure 6. Comparative biomass concentration of P. pastori pPICZα-kerAwt, pPICZα-kerAopti1 and pPICZα-kerAopti2 at 96 h.

The values represent means ± SD (n = 3). Results showed there was no significant difference in biomass concentration of every positive transformant (P>0.05).

Figure 7. The keratin azure degradation by recombinant keratinase at 50°C after 1 h (1, control without keratinase; 2, keratinase treatment).

Figure 8. Enzymatic properties of the purified keratinase.

A: The optimum pH of keratinase; B: The optimum temperature of keratinase; C: The keratinolytic temperature stability; D: The keratinolytic thermostability. The maximum value was taken as 100%.

Figure 9. Enzymatic hydrolysis of different keratin.

The values represent means ± standard. ^a,bMean values were significantly different (P<0.05) when labeled with unlike letters.