With the continuous expansion of the animal husbandry production scale, the demand for high-quality feed is also increasing greatly, which makes the development of animal production face severe challenges of supply and demand of feed resources. Therefore, it is urgent to find a way for improving feed efficiency. Cereal energy feed has high potential nutritional value as feed for poultry. However, grain feed contains high soluble non-starch polysaccharides, which hinders the utilization and absorption of poultry. Silage contains a large amount of cellulose, which is the main structural component of the plant cell wall. It is usually combined with hemicellulose, pectin, and lignin, and is difficult to digest and absorb. To solve this bottleneck problem, cellulase hydrolysis has low efficiency and high cost. We adopt the strategy of multi-enzyme synergistic degradation, which can promote the bioconversion of lignocellulose with cellulase. We further added the xylanase xyl3A gene to the vector, so that it can be combined with the cellulase PKC-01 and simultaneously express the β-xylosidase to improve the utilization rate of feed and the applicable varieties of livestock.

Due to the T7 promoter and T7 RNA polymerase having strong ability in translation and usually being used as protein expression, we choose pET28a-vector and E.coli BL21(DE3), to express our target protein xyl3A. Moreover, we inserted the DNA sequences of xyl3A into the NheI and HindIII sites of the pET-28a vector (Figure 1.) and transferred the plasmid into E.coli BL21(DE3) for protein expression.

In order to build our plasmids, we let the company synthesize the xyl3A integrate it into the pET28a vector. We transform the plasmid pET28a-xyl3A into E.coli DH5α. Figure 2 showed that the plasmid pET28a-xyl3A was successfully transform.

We send the plasmid pET28a-xyl3A to biological company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 3.)

Next, the plasmid pET28a- xyl3A was extracted from E.coil DH5α, then transformed into E.coil BL21(DE3). The PCR identification results showed that the transformation of plasmid pET28a-xyl3A was successful (Figure 2).

We 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 xyl3A. Next, we used nickel column purification to purify the protein xyl3A we wanted.

Determination of reducing the sugar by DNS method：The absorbance OD540 value of the purified enzyme solutions (xyl3A) 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 xyl3A is 0.3U/mL. Figure 5 indicated that protein xyl3A was successfully expressed, and the enzyme activity of xyl3A was active. It indicated that the xyl3A has the function of decomposing xylan.

Our results showed that the pET28a-xyl3A plasmid was successfully constructed, and the proteins xyl3A were successfully expressed in E.coli BL21 ( DE3). Meanwhile， the results indicated that the xyl3A has the function of decomposing xylan. We can add an appropriate amount of xyl3A enzyme to feed to improve the efficiency of xylan decomposition. We 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 put into large-scale use in animal husbandry, such as farms, and feed processing plants.