Expression Of Cellobiose 2-epimerase Gene In Pichia Pastoris And Its Enzymatic Characterization

Lactulose is one kind of functional oligosaccharides, which has many kinds of characteristics, such as low calorific value, high safety, without Maillard reaction and so on. So lactulose is widely used in the pharmaceutical, food and health care products and animal feed industries. The role of traditional enzymatic synthesis for lactulose is to link fructose to galactose based on lactose by transglycosidation. But its conversion rate was low, and the current output can not meet the needs of industrial production. Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus(CsCE) can catalyze the single substrate lactose into lactulose, and was found to be the most efficient enzyme for the enzymatic synthesis of lactulose. Preliminarily, the research group expressed Cs CE in E.coli. And through random mutation screening, the mutant C4-C5 was gotten,which specific enzyme activity was 2.8 times more than that of the wild type CsCE.In this study, CsCE was firstly expressed in Pichia pastoris. To improve the production of CsCE, codon optimization, site directed mutagenesis and fermentation condition optimization method was used. After expressed in P. pastoris, the enzymatic properties of wide type CsCE(CsCEm) and mutant type CsCE(CsCEmt) were studied, and catalytic action for convering lactose to lactulose by the recombinant enzyme was preliminarily explored. The experimental results obtained were briefly described as follows:Firstly, according to the nucleotide sequence of CsCE and the codon preference of P. pastoris, the gene CsCEm was synthesized. The results of nucleotide sequence alignment showed that CsCEm had 78.09% homology with the wild type gene CsCE. Then the synthesized CsCEm gene was connected with the expression vector pPIC9 K and transformed into P. pastoris GS115 after sequencing.Then by G418-plate and microplate screening,the strain GS115/CsCEm4-19 was screened with the highest enzyme activity of the recombinant yeast. After optimization of fermentation conditions, the highest enzyme activity reached at 0.42 U/mL after induction for 144 h, and the concentration of supernatant protein was 204.1±5.67 mg/L. The enzyme activity was increased 3.5 times than that of the recombinant P. pastoris before optimization(0.12 U/m L).Secondly, based on codon optimization, five amino acid sites of R5 M, I52 V, A12 S, K328 I and F231 L were site-directed, and the mutant gene CsCEmt was connected with expression vector pPIC9 K. Then, the recombinant vector was transformed into P. pastoris GS115 after sequencing, and the GS115/CsCEm 7-14 was screened with highest enzyme activity of recombinant yeast transformed. After optimization of fermentation conditions, the highest enzyme activity reached at 2.40 U/mL after the induction for 144 h, which was the highest activity of Cs CE reported until now. And the concentration of supernatant protein was about 368.73±6.79 mg/L. Compared with the recombinant P. pastoris gene optimizated only, the enzyme activity of site-directed mutagenesis was 5.7 times higher than that(0.42 U/m L).Then, the wide type CsCE and the mutant were purified by ammonium sulfate salting and Q-Sepharose F.F anion column chromatography.And one apparent stripwhich showed on the molecular mass of 45 kDa by SDS-PAGE, which was similar to the theoretical value of 47.3 kDa. The final specific activity of purified CsCEm was increased from 2.43 U/mg to 5.6 U/mg, and the CsCEmt was increased from 5.52 U/mg to 14.75 U/mg. Compared with the wild type CsCE, the activity of mutant was 1.64 times higher than that.Finally, enzyme properties of the recombinant enzyme showed that the most suitable temperature range of the mutant was more extensive and the thermal stability was higher compared with the wild type CsCE. The Michaelis constant Km decreased from 120.27 mmol/L to 74.24 mmol/L and Kcat/Km increased, which indicated that the recombinant enzyme affinity for substrate lactose was enhanced after mutation. The result of metal ions effect for wide type CsCE and mutant showed that Zn2+、Cu2+、Ni2+、Co2+、Fe3 + had a strongly inhibitory effect, which residual enzyme activity was less than 5%. But the other metal ions and EDTA was not obvious for the recombinant enzyme activity. Under the condition of 500 g/L lactose solution, recombinant enzyme of 20 U/mL was added and reacted at 80℃. The conversion rate for lactulose based on lactose was 63.7%, and increased by 7.5% compared with the wild type CsCE. The mutant was more potential for lactulose synthesis in industrial application.