Part:BBa_K1175005

endo-1,4-beta-xylanase xynA from Bacillus Subtilis Subtilis 168

The endo-1,4-beta-xylanase gene xynA cleaves xylan polysaccharide chains to form shorter xylan chains. This gene has been isolated from the bacterium Bacillus subtilis subtilis 168.

Usage and Biology

Xylan is a molecule similar to cellulose, and after cellulose the most abundant biomass material on earth. It is a major structural component of plant cell walls. Furthermore, xylan crosslinks with cellulose and other cell wall components, inhibiting access of cellulases (1). Xylose is the sugar monomer of xylan as glucose is to cellulose. Xylose cannot be used in the human body as a source of energy. Endo-1,4-beta-xylanase (xynA) breaks the xylan chains into shorter chains, and may be stearically hindered by side chains (2).

A beta-xylanase such as Endo-1,4-beta xylanase may be used to degrade xylan to facilitate cellulase activity. Another use may be in conjunction with an exo-xylanase to efficiently break down xylan into xylose monomers (a pentose sugar).

Enzymatic Activity of Gene Product

As stated above, the function of the gene product is xylan degredation. The enzyme's catabolic activity results from endohydrolysis of 1,4-beta-D-xylosidic linkages in xylan molecules (4).

(1) http://newscenter.lbl.gov/feature-stories/2012/11/12/a-better-route-to-xylan/ (2) http://www.nutrex.be/sites/default/files/wysiwyg-upload/nutrase-xyla-nsp-enzyme.pdf (3) http://subtiwiki.uni-goettingen.de/wiki/index.php/XynA (4) http://www.uniprot.org/uniprot/P18429

xynA The endo-1,4-beta-xylanase xynA is a globular protein that has two residues of interest the nucleophile and acid-base cleavage sites at the E residues 78 and 172 highlighted in red.
xynA Enzyme Activity
This graph depicts the inhibition of the gene product of XynA found in Bacillus Subtillis Subtillis 168 (BsX) in comparison to the inhibition of the XynA found in Aspergillus Niger (AsX). Sorensen and Sibbensen were observing the inhibitory effects of the TAXI (Triticum Aestivum Xylanase Inhibitor) , specific to Glycoside hydrolase family 11 (GH 11), and XIP (xylanase inhibitor protein), specific to fungal GH 11 but not bacterial GH 11. which XynA is a member. Inhibition was tested with either pure XIP (BsX-XIP and AnX-XIP) or both XIP and TAXI ( BsX-Inhibitor Prep and AnX-Inhibitor Prep. It is evident from this graph that BsX is not effected by XIP but is strongly inhibited by TAXI, causing a decrease in residual xylanase activity by approximately 80%.	This graph depicts the effect of pH on the interaction between the BsX xylanase and the inhibitor TAXI at a 1:5 concentration. The pH profile of the of the inhibition resembles the ph profile of the enzyme, indicating that TAXI is a competitive inhibitor for the BsX Xylanase. This graph also depicts the optimal pH for the XynA enzyme from Bacillus Subtillis Subtillis 168 to be around 5.5.

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Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 85
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 489
  Illegal SapI.rc site found at 543
  

Improvement

The iGEM Team Heidelberg 2014 improved this part by removing the first 84 basepairs and adding a Ribosome Binding Site thus making the gene product (xylanase) retainable in the cytoplasm in E. coli.

The first 84 bases of the gene code for a signal peptide 1, 2 that is important for the secretion of the gene product into the extracellular medium in B. subtilis. This signal peptide was shown to be recognized and functional in E. coli as well 1 . After secretion, the signal peptide is removed and the mature protein, therefore, consists of the remaining 185 amino acids. In some applications, it is desirable to prevent secretion of the protein into the extracellular medium (for instance, for our own purposes of comparing the activity of linear and circular xylanase, we had to remove the signal peptide since it would not be exposed in the circular protein and the two proteins needed to be identical in their amino acid composition and localization). By eliminating the signal peptide and adding a Ribosome Binding Site (RBS) in our new part BBa_K1362020 we provide a new option for future users of xylanase.

Characterization

Activity after Heatshock assay for 30 minutes of linear Xylanase

Activity after Heatshock assay for 30 minutes of circular Xylanase

Fig. 3: Activity after Heatshock assay for 5 minutes of linear and circular Xylanase

We determined the activity of the linear and the circular Xylanase after Heatshock at different temperatures. Firstly the Xylanases were incubated at 37 °C, 50 °C, 56 °C, 60 °C and 66 °C. After 30 minutes substrate (EnzChek® Ultra Xylanase Assay Kit) was added and the fluorescence was measured for 150 minutes in a platereader at 37 °C. The results indicate a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.

For further characterization and as another control the 2 constructs were again expressed and tested in a shorter heatshock assay (5 minutes at 63 °C). The results in Fig. 3 show an increased heatstability of the circular xylanase compared to the linear after heatshock, which shows no more activtiy.

References

1. Is Helianti, Niknik Nurhayati, Maria Ulfah, Budiasih Wahyuntari, and Siswa Setyahadi, “Constitutive High Level Expression of an Endoxylanase Gene from the Newly Isolated Bacillus subtilis AQ1 in Escherichia coli,” Journal of Biomedicine and Biotechnology, vol. 2010, Article ID 980567, 12 pages, 2010. doi:10.1155/2010/980567

2. http://www.uniprot.org/uniprot/P18429

Characterization by 2021iGEM_Shanghai_City_United

Improvement of an existing part

According to the registry, the part BBa_K1175005 designed by group iGEM13_WLC-Milwaukee, described a endo-1,4-beta-xylanase gene xynA. And there was xylanase enzyme activity data in support of its function. In 2014, iGEM team Heidelberg improved this part by removing the first 84 basepairs and adding a Ribosome Binding Site thus making the gene product (xylanase) retainable in the cytoplasm in E. coli. They characterize their part BBa_K1362020 in respect to its thermal stability by conducted a Heatshock Assay. Their results indicated a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.

In 2015, iGEM team TU_Darmstad further improved the initial part BBa_K1175005 by adding a linker-ligand sequence to fuse the protein to an in vitro scaffold for easy purification and increasing the degradation efficiency of xylan (BBa_K1602035). However, they just exemplarily the overexpression of degradation enzyme Acetyl Esterase fused to a linker binding the SH3 domain of the protein scaffold without enzymes activity.

Compared to the above parts BBa_K1175005, BBa_K1362020 and BBa_K1602035, we improved the sequence of xynA and added a coding sequence of lactic acid bacteria, which can help xylanase to be secreted outside the cell.

Figure 6. The blast results about the DNA sequence of our new part BBa_K4093005 and the old parts BBa_K1175005, BBa_K1362020 and BBa_K1602035.

Besides, our results show that broken supernatant presents an obvious enzyme activity than that of blank control indicate recombinant protein secret outside cell successfully.

In addition, we will establish a business development department aiming at an edible "drink" that can produce xylanase for monogastric animals. This feed additives or liquid beverage and can be sold to poultry breeders.

PUS-xyn A

Profile

Name: PUS-xynA

Base Pairs: 936bp

Origin: E. coli , Bacillus subtilis ,synthetic

Properties: Produce recombinant xylanase with secreted protein PUS

Usage and Biology

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

Figure 1. Design diagram.

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. The profiles of every basic part are as follows:

BBa_K4093000

Name: xynA

Base Pairs: 642bp

Origin: Bacillus subtilis

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.

BBa_K4093001

Name: PUS

Base Pairs: 294bp

Origin: lactic acid bacteria

Properties: Secreted protein

Usage and Biology

BBa_K4093001 is a coding sequence of lactic acid bacteria, which can help protein to be secreted outside the cell.

Experimental approach

Figure 2. The workflow of constructing pSIP403-PUS-xynA.

Proof of function

A. Secretory expression of pSIP403-PUS-xynA in L. reuteri.

At this time, start induction. The recombinant strain is added with 25 ng/mL SppIP to induce expression. After shaking at 37°C and 220r/ml for 3 hours, samples are collected at different time points to establish a function model of time and secreted expression;

Take 20ml of the recombinant strain after induction culture, of which 1ml is used to detect the OD600 of the sample with an ultraviolet spectrophotometer.

Figure 3.

B．DNS enzyme activity detection 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 7).

Figure 4. Standard curve for reducing sugar.

Table 2. Calculation of average unit enzyme activity.

Figure 5.

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.