Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5516133 | Protein Expression and Purification | 2017 | 7 Pages |
â¢Two thermophilic β-mannanases (ManA and ManB) were successfully expressed in Yarrowia lipolytica using vector pINA1296I.â¢The DNA sequences of these two genes were modified according to Yarrowia lipolytica codon usage preferences.â¢Multi-copy expression strategy could increase the yield of the two recombinant thermophilic β-mannanases.
Two thermophilic β-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pHâ¼5 and â¼70 °C, and for manB activity were pHâ¼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications.