Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity
- 70 Downloads
Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.
Key wordsRhizopus oryzae lipase (ROL) Yeast surface display Codon optimization Whole-cell biocatalyst
Unable to display preview. Download preview PDF.
- Bucarey, S.A., Noriega, J., Reyes, P., Tapia, C., Saenz, L., Zuniga, A., Tobar, J.A., 2009. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine, 27(42): 5781–5790. [doi:10.1016/j.vaccine.2009.07.061]PubMedCrossRefGoogle Scholar
- Esteban, L., Munio, M.D., Robles, A., Hita, E., Jimenez, M.J., Gonzalez, P.A., Camacho, B., Molina, E., 2009. Synthesis of 2-monoacylglycerols (2-MAG) by enzymatic alcoholysis of fish oils using different reactor types. Biochem. Eng. J., 44(2–3):271–279. [doi:10.1016/j.bej.2009.01.004]CrossRefGoogle Scholar
- Guo, Q., Zhang, W., Ma, L.L., Chen, Q.H., Chen, J.C., Zhang, H.B., Ruan, H., He, G.Q., 2010. A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 111 (1):41–51. [doi:10.1631/jzus.B0900185]CrossRefGoogle Scholar
- Kato, M., Fuchimoto, J., Tanino, T., Kondo, A., Fukuda, H., Ueda, M., 2007. Preparation of a whole-cell biocatalyst of mutated Candida antaretica lipase B (mCALB) by a yeast molecular display system and its practical properties. Appl. Microbiol. Biotechnol., 75(3):549–555. [doi:10.1007/s00253-006-0835-2]PubMedCrossRefGoogle Scholar
- Matsumoto, T., Takahashi, S., Kaieda, M., Ueda, M., Tanaka, A., Fukuda, H., Kondo, A., 2001. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production. Appl. Microbiol. Biotechnol., 57(4):515–520.PubMedCrossRefGoogle Scholar
- Murai, T., Ueda, M., Shibasaki, Y., Kamasawa, N., Osumi, M., Imanaka, T., Tanaka, A., 1999. Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface. Appl. Microbiol. Biotechnol., 51(1):65–70. [doi: 10.1007/s002530051364]PubMedCrossRefGoogle Scholar
- Prim, N., Blanco, A., Martinez, J., Pastor, F.I.J., Diaz, P., 2000. estA, a gene coding for a cell-bound esterase from Paenibacillus sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases. Res. Microbiol., 151(4):303–312. [doi:10.1016/S0923-2508(00)00150-9]PubMedCrossRefGoogle Scholar
- Tamalampudi, S., Talukder, M.R., Hama, S., Numata, T., Kondo, A., Fukuda, H., 2008. Enzymatic production of biodiesel from Jatropha oil: a comparative study of immobilized-whole cell and commercial lipases as a biocatalyst. Biochem. Eng. J., 39(1):185–189. [doi:10.1016/j.bej.2007.09.002]CrossRefGoogle Scholar