286587 Production of the Recombinant Trichoderma Reesei Endogluconase Protein Cel7B by Using the Kluyveromyces Lactis Yeast
This research is about producing recombinant Trichoderma reesei endogluconase protein Cel7B by using Kluyveromyces lactis yeast as a host. Cel7B is one of the glycoside hydrolyses family proteins that are produced by T. reesei. Cel7B with other endoglucanases, exoglucanases, and β-glucosidases hydrolyze cellulose to glucose, which can then be fermented to fuels or other products.
Examining favorable fermentation conditions for enzyme production and improved activity were the research objectives. Production of protein on different types of media was examined, and measured the activities of the protein by using different tools or procedures. The first method was using different concentration of galactose as a carbon and energy source. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second method was to use different types of media: a complex media-yeast extract, peptone, galactose (YPGal); a minimal media-yeast nitrogen base (YNB); and a minimal media with supplement- yeast nitrogen base with casamino acid (YBC). The third method was using different types of reactor or fermenter: a small reactor (shake flask) and a big reactor (BioFlow 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production.
Different tools to determine the presence and activity of protein were used: for presence of protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed.
SDS-PAGE showed that the enzyme band was at 67,000 Da. CMC-DNS activity assay showed that protein activity was greatest with 1% sugar (Galactose) concentration. For the different type of media used in our fermentation, protein was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but protein was not produced and no activity was detected from yeast nitrogen base (YNB). That means the protein production requires the amino acid resources. The quantity of protein which was produced from the rich media (YPGal) over time of production was equal to the quantity of protein that was produced from the minimum media supplemented with casamino acids (YBC) over time with a concentration of 1% galactose.
See more of this Group/Topical: Sustainable Engineering Forum