Demonstration
Major achievements:
(1) We screened out six signal peptides, and finally selected the signal peptide FLO10- α pro, which has the highest expression level, and combined it with our genes that degrade lignin and pectin to form a system for extracting cellulose from banana stalk.
(2) We fermented banana stalk in a fermentation device and obtained preliminary results in preparation for the next step of industrialization.
(2) We fermented banana stalk in a fermentation device and obtained preliminary results in preparation for the next step of industrialization.
signal peptide selection
We constructed the pathway, linked the six signal peptides to SLAC,VP and pelA, and electro transferred them into Pichia pastoris for expression. Then we first broke up the cells and verified the protein content using protein glue (figure).
Then we induced the expression of Mut+ phenotypic recombinant yeast. In SLAC, we induced the expression by adding methanol to BMM medium, and then the fluorescence intensity was determined by DNS spectrophotometry. The optimal signal peptide FLO10- α pro was screened out.
As for pelA, We failed of determination by DNS spectrophotometry at the beginning, then we changed to titration for the determination of Pectinase activity, and finally obtained the results
Then we induced the expression of Mut+ phenotypic recombinant yeast. In SLAC, we induced the expression by adding methanol to BMM medium, and then the fluorescence intensity was determined by DNS spectrophotometry. The optimal signal peptide FLO10- α pro was screened out.
As for pelA, We failed of determination by DNS spectrophotometry at the beginning, then we changed to titration for the determination of Pectinase activity, and finally obtained the results
Banana stalk degumming test.
The untreated banana stalk is dark brown, bundled with fiber, and there are a large number of sand-like and sticky substances in the middle of the fiber bundle, which make the fiber difficult to peel off and can not be used. There are also a large number of structures distributed perpendicular to the next fiber bundles in the middle of each of them, which are used to connect the adjacent fiber bundles and connect them into a network. On the other hand, the banana stalk treated by us not only lost the connection structure between the fiber bundles, but also almost all the viscous particles filled between the fiber bundles were degraded. The fiber is transparent and white and can be separated.
We could clearly see that the fiber without degumming are clustered together and difficult to separate. (Fig.1). And the degummed fiber are loose. (Fig.2). At a higher magnification, we could see that there are many impurities on the fiber without degumming, including pectin, lignin, etc. They are attached to the fiber, as shown in figure 3 (the brown part is impurities). These impurities make the fiber look very dirty and they greatly affect the properties of the fiber. It can be seen more clearly under a transmission electron microscope (Fig.5, 6), with all the impurities clustered.
After biological degumming, we can clearly see that almost all the impurities attached to the fiber are removed, and the whole fiber is clean and transparent under the optical microscope (Fig. 4). This is also confirmed by transmission electron microscopy (Fig.7, 8). This shows that our engineering yeast can secrete enzymes to degrade lignin and pectin, and can be used on a large scale.
Figure7: A pilot test of banana stalk degumming with Banamax
We could clearly see that the fiber without degumming are clustered together and difficult to separate. (Fig.1). And the degummed fiber are loose. (Fig.2). At a higher magnification, we could see that there are many impurities on the fiber without degumming, including pectin, lignin, etc. They are attached to the fiber, as shown in figure 3 (the brown part is impurities). These impurities make the fiber look very dirty and they greatly affect the properties of the fiber. It can be seen more clearly under a transmission electron microscope (Fig.5, 6), with all the impurities clustered.
After biological degumming, we can clearly see that almost all the impurities attached to the fiber are removed, and the whole fiber is clean and transparent under the optical microscope (Fig. 4). This is also confirmed by transmission electron microscopy (Fig.7, 8). This shows that our engineering yeast can secrete enzymes to degrade lignin and pectin, and can be used on a large scale.
Lignin degradation ration
Lignin is one of the most abundant impurities in fiber, and the degradation of lignin is difficult. Therefore, we use the relevant enzymes to help us degrade lignin. We determined the degradation rate of lignin after pretreatment, chemical treatment and chemical and enzyme treatment under the same conditions.
We burned the fiber and measured the lignin content of ash. The experimental result show that the combination of chemistry and enzyme can better degrade lignin. Based on this result, we can believe that the enzyme-treated fiber contains less lignin.
We burned the fiber and measured the lignin content of ash. The experimental result show that the combination of chemistry and enzyme can better degrade lignin. Based on this result, we can believe that the enzyme-treated fiber contains less lignin.
