Part:BBa_K4093000

xynA

xynA

Usage and Biology

BBa_K4093000 is a coding sequence of from Bacillus subtilis. Xylanase is a kind of complex enzyme preparation specialized in degrading xylan in cereals.

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.

Construct design

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.

Experimental approach

Figure 2. electrophoregram of XynA.

The result of electrolysis can clearly show whether the XynA is successfully amplified. XynA was amplified by PCR. The length of XynA is 650 bp. By compared the marker and the place of DNA, the PCR of XynA is successful.

Proof of function

A．Protein expression and enzyme activity test of pMD19-T-xynA in E.coli BL21(DE3)

Figure 3. SDS PAGE result of BL21(DE3)/pMD19-xynA, Coomassie blue staining.

After the sample has been centrifuged and sonicated. we put it to SDS-PAGE(SDS-polyacrylamide gel electrophoresis) which could determine the level of protein expression. As seen in the gel map (Maker, culture solution, intracellular supernatant, intracellular precipitation), the second red line of Marker represents 25KDa and the target protein should be 23.28kDa(Fig. 5). The blue line in the culture solution was near 23 KDa, it indicates that our protein could be sucessfully expressed in E. coli.

Figure 4. DNS test results of E. coli/pMD19-xynA.

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. 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.

B. Secretory expression and enzyme activity test of pSIP403-PUS-xynA in L. reuteri

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 DNS method. This indicates that our PUS protein can help xynA protein to be secreted outside the cell.

Table 1. Xylanase activity (OD540 nm) was detected from broken supernatant by DNS method.

A standard curve for reducing sugar was prepared using glucose (Figure 5).

Figure 5. Standard curve for reducing sugar.

Table 2. Calculation of average unit enzyme activity.

Figure 6.

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 SppIP was induced for 8-12 hours. Is the best induction time.

Future plan

We aim at developing a probiotic (Lactobacillus) containing xylanase to produce feed additives and poultry beverages, which are believed to help poultry digest xylan and enhance their health. Maybe it is possible for us to produce a novel “mixed feed” that combines other feed additives to enhance more poultry and ruminants’ digestion. After in-depth research, we obtained a feasible plan of “mixed feed” and the details of this plan can be seen at our wiki page partnership.

References

6.https://wenku.baidu.com/view/bb1a6d76590216fc700abb68a98271fe910eafb9.html

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]