Team:Shanghai City United/Engineering
Engineering
Background
Feed grains are whole grains such as corn, wheat and barley used in the feeding of
livestock and poultry. At present, corn is the main fodder in the feed industry. Due to the increasing
shortage of raw materials and the rising price, the development of the feed industry has been greatly
limited. One of the important measures to alleviate the shortages of corn is to fully develop wheat, grain,
and bran, which are abundant in China, to replace corn. However, the cell walls of cereals such as wheat,
cereal, and bran contain anti-nutritional factors such as arabinoxylan and non-starch polysaccharide (NSP),
which will affect the digestibility of nutrients in single-stomach animals and the absorption of nutrients
in poultry. Arabinoxylan is a polysaccharide found in rice bran (hemicellulose B) edible fiber. Therefore,
we decide to aim at adding xylanase to the feed to degrade arabinoxylan, so as to improve the feed
absorption efficiency.
Design
Our project is to design an edible "drink" for monogastric animals, mainly poultry
through biosynthesis technology. The core product will be a probiotic that can produce xylanase. In this
project, we designed to construct a plasmid expressing xylanase gene, then we transformed them into
Escherichia coli and Lactobacillus reuteri for further performance analysis.
Compared to traditional feed, our drink contains the probiotics that could help
poultry digest xylan, thus
increasing feed efficiency. In this way, not only can the time and economic cost of feeding be saved, but
also the gastrointestinal tract of the animal can be protected and thus the disease rate can be reduced.
Build
Plasmids pMD19-T-xynA and pSIP403-PUS-xynA were constructed to produce
recombinant
xylanase in E. coli and L. reuteri, respectively (Figure 1).
Figure 1. Schematic maps of pMD19-T-xynA and pSIP403-PUS-xynA.
Test
pMD19-T-xynA was transformed into E. coli BL21(DE3). After the sample has been
centrifuged and sonicated.
The level of protein expression was determined using SDS-PAGE. As seen in the gel (Maker, culture solution,
intracellular supernatant, intracellular precipitation), the second red line of Marker represents 25KDa and
the target protein should be 23.28kDa (Figure 2). The blue line in the culture solution was near 23 KDa, it
indicates that our protein could be successfully expressed in E. coli (Figure 2).
Figure 2. SDS-PAGE result of BL21(DE3)/pMD19-xynA.
It could be seen by naked eyes that in the seven bottles of solution, the culture
medium solution showed
red after DNS reaction, which indicated that we had xylanase with well enzyme activity to degrade xylan and
this result is consistent with the SDS-PAGE result.
Figure 3. The xylanase activity was tested by DNS method. The enzyme activity test
results showed groups
from the left to the right, respectively: blank, culture medium supernatant * 2, intracellular supernatant *
2, and intracellular precipitation * 2.
Plasmid pSIP403-PUS-xynA transformed into L. reuteri to for secretory expression of
xynA in L. reuteri. As
shown in Table 1, significant xylanase activity was detected from broken supernatant by DNA method.
Table 1. Xylanase activity (OD540 nm) was detected from broken supernatant by DNA
method.
A standard curve for reducing sugar was prepared using glucose (Figure 4).
Figure 4. Standard curve for reducing sugar.
The DNS color method was used to detect the unit enzyme activity of two parallel
group samples at different induction time points, and the average unit enzyme activity was calculated. The
experimental data showed that the enzyme activity was maximum when 25 ng/mL inducer was added for 8-12 hours
(Table 2).
Table 2. Calculation of average unit enzyme activity.
