Expression of the inulinase gene from the marine-derived Pichia guilliermondii in Saccharomyces sp. W0 and ethanol production from inulin.
Bottom Line: It has been confirmed that Saccharomyces sp.It was found that 15.2 U of inulinase activity per one gram of inulin was suitable for inulin hydrolysis and ethanol production by the transformant Inu-66.During the 2 l fermentation, 14.9% (v/v) of ethanol was produced from inulin and 99.5% of the added inulin was converted into ethanol, CO₂ and cell mass.
Affiliation: Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China.Show MeSH
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Mentions: The INU1 gene encoding the extracellular inulinase was amplified from the genomic DNA of P. guilliermondii strain 1 by PCR and inserted into the expression vector YCPlac33 PGK/CYC1. The resulting plasmid was named YCPlac33‐INU1. The construction of the expression vector carrying the inulinase gene is shown in Fig. 1. From this figure, it can be seen that the recombinant plasmid contains the constitutive promoter PGK and the effective terminator CYC1. Therefore, the cloned inulinase gene can be expressed constitutively in S. cerevisiae and the gene expression can be effectively terminated. The expression vector also contains CEN4 sequence so that the vector in the host can be distributed in each daughter cell during the vegetable growth. The recombinant plasmid was transformed into the uracil mutant Saccharomyces sp. W101. After determination of extracellular inulinase activity from over 100 positive transformants, the results in Table 1 indicate that the inulinase activity was in the range of 17 and 33 U ml−1, while no inulinase activity of Saccharomyces sp. W0 was detected. The results in Table 1 also show that the inulinase activity produced by the transformant Inu‐66 was the highest (33.67 ± 0.42 U ml−1). Therefore, it was used in the subsequent investigation. The results in Fig. 2 confirm that the cloned INU1 gene was amplified from the genomic DNA of the transformant Inu‐66 by PCR and no such PCR products were obtained from the genomic DNA of Saccharomyces sp. W0. This means that the transformant Inu‐66 indeed carried the cloned INU1 gene.
Affiliation: Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China.