Difference between revisions of "Part:BBa K4093000"

Latest revision as of 14:14, 12 October 2022

xynA

xynA

Profile

Name: xynA

Base Pairs: 642bp

Origin: Bacillus subtilis, synthetic

Properties: endo-1,4-beta-xylanase

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

1.Beasley S S,Takala T M， Reunanen J， Apajalahti J， Saris P E.2004.Characterization and electrotransformation of Lactobacillus crispatus isolated from chicken crop and intestine. Poult Science，83(1)：45-48;

2.De Vos W M.1999.Gene expression systems for lactic acid bacteria. Current Opinion in Microbiology，2(3)：289-295;

3.Silversides F G， Scott T A， Korver D R，Afsharmanesh M，Hruby M.2006. A study on the interaction of xylanase and phytase enzymes in wheat-based diets fed to commercial white and brown egg laying hens.Poultry Science，85(2)：297-305;

4.崔罗生，祝茂生，徐顺清，朱辉，梁运祥，张忠明.2009.黑曲霉木聚糖酶基因(xyn A) 在大肠杆菌中的表达及酶学分析.华中农业大学学报，28(1)：48-53；

5.李慧.2010. 蛋白酶和木聚糖酶对肉鸡生长性能，消化机能及血液指标的影响.[研究生学位论文].杨凌：西北农林科技大学；

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

Improvement by 2022 Shanghai_United

In the part BBa_K4093000 of iGEM21_Shanghai_City_United, xynA gene inserted into the two vectors pMD19-T-xynA and pSIP403-PUS-xynA in order to produce xylanase in E. coli and L. reuteri. The functional result in E.coli showed the xylanase only expressed in the precipitation, indicating the vector is not suitable for xylanase expression. Thus, we employed the pET28a to express the xylanase in E. coli BL21(DE3). We also performed protein purification and analyzed the yield by SDS-page. Our results showed the proteins xylanase successfully expressed in E. coli BL21(DE3). Moreover, we further determined the enzymatic activity.

Both xynA and ccxynA is capable to hydrolysis of xylan, we analyzed the amino acid sequence of xynA and ccxynA though NCBI blast. The result showed that the identities of two enzymes is 43% in figure 5.

Figure 5. Amino acid sequence alignment of xynA and ccxynA.

CcxynA

Characterization by Shanghai_United

BBa_K4277002

Name: CcxynA

Length: 1563 bp

Origin: Clostridium fibrinophilus, sythesis

Properties: hydrolysis of xylan

Usage and Biology

The acetylxylan esterase (CcXynA) belongs to the hydrolases family which catalyzes the deacetylation of xylans and xylooligosaccharides. The enzyme plays an important role in the hydrolysis of xylan, since the acetyxylan interferes with the approach of enzymes that cleave the cleave the backbone. Thus, the enzyme catalyzation of CcXynA is necessary for digesting xylan sufficiently.

Construct design

In order to obtain the enzyme CcXynA in vivo, we designed the plasmid pET28a-ccxynA. This plasmid was synthesized in gene company, the plasmid map is shown as Figure 1.

Figure 1. The map of pET28a-ccxynA.

Experimental approach

1.1 The colony PCR of pET28a-ccxynA in competent cells DH5α

Due to the gene synthesis company delivered a plasmid pET28a-ccxynA, we directly transformed the pET28a-ccxynA into E.coli BL21(DE3) for protein expression. The colony PCR results showed that lane 1 and 3 has the correct band, indicating plasmid pET28a-ccxynA was successfully transformed into E.coli BL21(DE3).

Figure 2.Colony PCR result of pET28a-ccxynA M:2000 kd marker; Lane 1-3. pET28a-xynA (DH5α).

Functional assay

2.1 Protein expression of ccxynA

The colony containing plasmid pET28a-ccxynA was inoculated in LB medium and the protein expressed E.coli BL21(DE3). The cells enlarged in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and cultivation, the cells were collected and lysed by ultrasonication to release the intracellular proteins. Next, we used nickel resin to purify the protein we wanted.

According to SDS-PAGE, we obtained the protein ccxynA.

Figure 3. SDS-page M: maker; S: supernatant; P: precipitant; E: elution.

The molecular weight of ccxynA is 57.0KD, referring to the marker in Figure 3. SDS-page showed the protein ccxynA is in supernatant after ultrasonication and centrifugation, indicating that they were successfully expressed in E. coli BL21 (DE3).

2.2 Determination of ccxynA enzymatic activity

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

Here, we establish the relationship using differential equation:

Solved: y=aln(bx)+c Where a, b and c is the parameter. according to the equation, we obtained the experiment data, shown in Table 1.

Figure 4. Model of ccxynA activity and xylan solution concentration.

With the increase of xylan concentration, the result showed an increasing trend first and then stabilizing (Figure 4). We can use this model to predict the reaction rate and maximum activity of the enzyme.

Future Plan

Based on the previous result, we also want to develop the strategy of multi-enzyme synergistic degradation to accelerate the degradation rate of xylan in feed. For instant, xynA and ccxynA can be added simultaneously 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.

Sequence and Features