Part:BBa_K4277002

CcxynA

CcxynA

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.

Improvement of existing data

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.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 458
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1242
  Illegal AgeI site found at 835
  
• 1000
  COMPATIBLE WITH RFC[1000]