In the iGEM21_Beijing_United team last year, the gene of xylanase ccxynA was inserted into yeast genome by gene editing, so that yeast itself could produce these enzymes to further decompose non-starch polysaccharides in wheat starch-B. The yeast could directly use them to produce alcohol.
Aiming at the bottleneck problem of low efficiency and high cost of cellulase hydrolysis in the current market, we proposed a strategy of multi-enzyme synergistic degradation. Studies have shown that xylanase ccxynA has the characteristics of hydrolyzing hemicellulose and can cooperate with cellulase to promote the biotransformation of lignocellulose. We further added the xylanase xynA gene to the cloning recombinant vector so that it can be expressed simultaneously with the enzyme ccxynA to promote the degradation of xylosidase.
Compared to last year's team, we not only apply the gene ccxynA to feed additives but also put forward the idea of a multi-enzyme combination. We combine ccxynA with xynA to make a multi-enzyme mixture, which increases the rate of xylan degradation and can be put into production in large quantities, increasing the utilization rate of silage and cereal mixed feed and suitable varieties for livestock. For example, the preparation of bagasse or corn stalks into common feed for livestock has extremely high economic value.

β-xylosidase ccxynA and Xylanase xynA are skeleton degrading enzymes of heterogeneous xylan, which can degrade the main chain of xylan to produce xylooligosaccharides or xylose with different degrees of polymerization. β-xylosidases ccxynA is mainly distributed in GH3 and GH43 families. And β-xylosidases ccxynA degrades the low-polymerized xylan or xylobiose produced by enzymatic hydrolysis of xylanase, releases xylose from the non-reducing end, and attenuate the substrate inhibition of oligosaccharides. Strains that secrete xylanase can generally produce β-xylosidase and work together with xylanase. β-xylosidase ccxynA not only plays a key role in the complete degradation of xylooligosaccharides but also alleviates the inhibition of xylooligosaccharides on xylanase and cellulase.β-xylosidase ccxynA and Xylanase xynA are skeleton degrading enzymes of heterogeneous xylan, which can degrade the main chain of xylan to produce xylooligosaccharides or xylose with different degrees of polymerization. β-xylosidases ccxynA is mainly distributed in GH3 and GH43 families. And β-xylosidases ccxynA degrades the low-polymerized xylan or xylobiose produced by enzymatic hydrolysis of xylanase, releases xylose from the non-reducing end, and attenuate the substrate inhibition of oligosaccharides. Strains that secrete xylanase can generally produce β-xylosidase and work together with xylanase. β-xylosidase ccxynA not only plays a key role in the complete degradation of xylooligosaccharides but also alleviates the inhibition of xylooligosaccharides on xylanase and cellulase.

We amplify xynA by PCR, double-enzyme digestion, and inserted into the XbaI and BamHI site of pET28a (+) carrier, to obtain the plasmids pET28a-xynA. Then the pET28a-xynA transform into DH5α, the colony PCR identification results show that the construction of pET28a-xynA is successful (Figure 1).

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 2), and the plasmid pET28a-xynA was successfully constructed.We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 2), and the plasmid pET28a-xynA was successfully constructed.

The plasmid pET28a-xynA was extracted from DH5α, and transformed into BL21(DE3). The PCR identification results showed that the plasmid pET28a-xynA was successful (Figure 3).

Because the gene synthesis company delivered a tube of plasmid, we transformed the pET28a-ccxynA into E.coli BL21(DE3) for expressing proteins. The PCR identification results showed that the plasmid pET28a-ccxynA was successfully transformed into E.coli BL21(DE3).

In order to obtain the proteins (xynA and ccxynA), we transferred the recombinant plasmids into E.coli BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the protein we wanted.
At this point, we obtained the proteins solutions we wanted.

The molecular weights of xynA and ccxynA were 22.87 KD and 57.0KD; referring to the marker in Figure 5, we found the proteins (xynA and ccxynA) in lane S, indicating that they were successfully expressed in E. coli BL21 (DE3).

We can construct a model diagram between the activity of the enzyme solution ccxynA and xylan concentration. It can be used to predict the activity of the enzyme solution ccxynA.

Here, we establish differential equations:

Solved： y=aln(bx)+c；
Where a,b and c is the parameter.

With the increase of xylan concentration, the model showed a trend of increasing first and then stabilizing(Figure 6). We can use this model to predict the reaction rate and maximum activity of the enzyme.

Determination of reducing the sugar by DNS method. The absorbance OD540 value of the enzyme solutions xynA was measured after color reaction with DNS. The activity of the enzyme can be converted by the amount of sugar consumed and the working time.

The enzyme activity of xynA is 0.14U/mL. The results indicated that the proteins were successfully expressed, and the enzyme activity of xynA was active. The enzyme activity of xynA is 0.14U/mL. The results indicated that the proteins were successfully expressed, and the enzyme activity of xynA was active.

The results show the proteins ccxynA and xynA were successfully expressed in E. coli BL21(DE3). According to the different enzymes activity values of xynA and ccxynA, we also can adopt the strategy of multi-enzyme synergistic degradation to accelerate the degradation rate of cellulose or xylan in feed, which can be added to feed as a feed additive to improve the ability of animals or digest and utilize feed, promote animal appetite and improve the quality of animal husbandry products. It can solve the bottleneck problem of low efficiency and high cost of cellulase hydrolysis. In the future, we can prepare bagasse or corn stalk as a common feed for livestock, and improve the utilization rate of silage and grain mixed feed and the applicable varieties of livestock, to have high economic value.