Difference between revisions of "Part:BBa K2617000"

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<partinfo>BBa_K2617000 short</partinfo>
 
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Xyn10D-fae1A is a bifunctional enzyme, which has the activity of ferulic esterase and xylanase. Feruloyl esterase can hydrolyze ferulic acid ester groups, which are responsible for attaching in complex cellular cell wall structures. Xylanase can hydrolyze plant cell wall component xylan. It can cut the β-1,4 glycosidic bond between the xylose residues in the xylan backbone. This part is responsible for pretreatment of straw.
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Xyn10D-fae1A is a bifunctional enzyme, which has the activity of ferulic esterase and xylanase. Feruloyl esterase can hydrolyze ferulic acid ester groups, which are responsible for attaching in complex cellular cell wall structures. Xylanase can hydrolyze plant cell wall component xylan. This part is responsible for pretreatment of straw.
  
 
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===Molecular weight===
 
===Molecular weight===
  
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This halohydrin dehalogenase gene codes for a protein of 726 amino acids with a molecular mass of 82.3 kDa.
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This gene codes for a protein of 726 amino acids with a molecular mass of 82.3 kDa.
  
 
===Detection of ferulic acid===
 
===Detection of ferulic acid===
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To detect the expression of piGEM2018-Module001, we will convert the piGEM2018-Module001 strain BL21(DE3) to co-culture with straw culture medium. The supernatant was collected 12 hours later and then detected ferulic acid by gas chromatography. Gas chromatographic procedure for 80 ℃ to 130 ℃, rising per minute 10 ℃; 130 ℃ to 190 ℃, rising per minute 2 ℃; 190 ℃ to 230 ℃, rising per minute 10 ℃. The melting point of ferulic acid is 169-173 ℃ and the boiling point is 372.3 ℃ at one atmospheric pressure. In the case of ferulic acid in gas chromatography with nitrogen as carrier gas, ferulic acid will undergo the following reaction and decompose into 9 carbon compounds.  
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To detect the expression of Xyn10D-Fae1A gene(piGEM2018-Module001), we converted carrying Xyn10D-Fae1A gene to co-culture with straw culture medium. Gas chromatography was used to detect ferulic acid [1]. The ferulic acid production was monitored by periodically taking samples from the fermentation liquid of E. coli BL21(DE3) carrying Xyn10D-Fae1A gene.
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[[File:T--UESTC-China--ferulic acid decomposition products.png|500px|thumb|center|'''Fig. 1''' Ferulic acid decomposition equation.]]
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GC results showed that the peak of ferulic acid was appeared after fermentation for 24h with the strain carrying Xyn10D-Fae1A gene, while no ferulic acid was detected in the sample of E. coli BL21(DE3) without vector (Fig. 1).  
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[[File:T--UESTC-China--detection of ferulic acid.png|500px|thumb|center|'''Fig. 1''' The production of ferulic acid determined using GC. The supernatants of fermented liquid of E. coli BL21(DE3) with or without Xyn10D-Fae1A gene were used for the determination of ferulic acid, respectively. Samples were taken from reaction mixtureat 0h, 24h.]]
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The peak time of ferulic acid decomposition products was about 22-23min. The internal standard is carvacrol, and its peak time is after ferulic acid decomposition products. We compared them through four groups of experiments: only medium group, straw plus medium group, BL21(DE3) original strain group, and transformed piGEM2018-Module001 group. The following four graphs were obtained, and ferulic acid was generated by comparison.
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The ferulic acid peak was further analyzed by using GC-MS (Fig. 2).
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[[File:T--UESTC-China--detection of ferulic acid.png|500px|thumb|center|'''Fig. 1''' Quantitative detection of ferulic acid.]]
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[[File:T--UESTC-China--GC-MS.png|500px|thumb|center|'''Fig. 2''' Mass spectrums of ferulic acid derivative. Sample was the supernatants of E. coli BL21(DE3) carrying Xyn10D-Fae1A gene after fermentation for 24h.]]
  
 
===Enzyme activity of xylanase===
 
===Enzyme activity of xylanase===
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Xylanase can decompose xylan to xylose. Comparing with wild type,our E. coli carrying Xyn10D-Fae1A gene take effect [2] (Fig. 3).
  
  
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[[File:T--UESTC-China-standard curve of xylose .png|500px|thumb|center|'''Fig. 2''' Standard Curve of Xylose.]]
 
  
 
[[File:T--UESTC-China-xylanase activity.png|500px|thumb|center|'''Fig. 3''' Xylanase Activity on Xylan. Data were measured inphosphate buffer solution at pH 6.0 and 40℃.]]
 
[[File:T--UESTC-China-xylanase activity.png|500px|thumb|center|'''Fig. 3''' Xylanase Activity on Xylan. Data were measured inphosphate buffer solution at pH 6.0 and 40℃.]]
  
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===Enzyme activity of xylanase===
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===Enzyme activity of ferulic acid esterase===
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Ferulic acid esterase can decompose ferulic acid p-nitrophenol ester to produce p-nitrophenol and ferulic acid.  
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Ferulic acid esterase can decompose 4-nitrophenyl trans-ferulate to produce p-nitrophenol and ferulic acid. We used crude extract fraction of strain to detect enzyme activity [3].
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The strain BL21 (DE3) transformed with piGEM2018-Module001 was cultivated overnight and centrifuged to obtain the supernatant. The remaining bacteria were broken and broken products were obtained. DSMO solution of ferulic acid p-nitrophenol ester was added to phosphate buffer solution of 630 L pH=6.4 with a concentration of 400 L of 10 mmol/L. Heat preservation 5min at 40℃ and add 0.2ml sample solution. The initial OD values were measured before incubation. Gently mix and incubate 4h at 40 C, determine the final OD value and compare the difference.
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[[File:T--UESTC-China--Enzyme Activity of Ferulic acid esterase.png|500px|thumb|center|'''Fig. 4''' Enzyme Activity of Ferulic acid esterase. Data were measured in phosphate buffer solution at pH 6.4 and 40℃.]]
 
[[File:T--UESTC-China--Enzyme Activity of Ferulic acid esterase.png|500px|thumb|center|'''Fig. 4''' Enzyme Activity of Ferulic acid esterase. Data were measured in phosphate buffer solution at pH 6.4 and 40℃.]]
  
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The results (Fig. 4) showed that the ferulic acid enzyme activity of our strain was much higher than that of the wild type.
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===References===
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[1] Qiu-Yi, L. I., Gan, G. P., Wang, G. Z., & Liu, Y. W. (2007). Determination of ferulic acid in ligusticum chuanxiong hort. oil by gc. Lishizhen Medicine & Materia Medica Research.
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[2] Shen C, Li R, Hu T & Yan K. 2011. Determination of xylanase activity with DNS method. Dyeing & Finishing, 2: 35-39.
  
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[3] Zhang S, Pei X & Wu Z. 2009. Cloning and expression of feruloyl esterase A from Aspergillus niger, and establishment of fast activity detection methods. Chinese Journal of Applied & Environmental Biology, 2: 276-279.
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2617000 parameters</partinfo>
 
<partinfo>BBa_K2617000 parameters</partinfo>
 
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Latest revision as of 11:09, 17 October 2018


Xyn10D-Fae1A：Bifunctional xylanase/ferulic acid esterase

Xyn10D-fae1A is a bifunctional enzyme, which has the activity of ferulic esterase and xylanase. Feruloyl esterase can hydrolyze ferulic acid ester groups, which are responsible for attaching in complex cellular cell wall structures. Xylanase can hydrolyze plant cell wall component xylan. This part is responsible for pretreatment of straw.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 534
    Illegal AgeI site found at 381
    Illegal AgeI site found at 628
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1236

Characterization

Molecular weight

This gene codes for a protein of 726 amino acids with a molecular mass of 82.3 kDa.

Detection of ferulic acid

To detect the expression of Xyn10D-Fae1A gene(piGEM2018-Module001), we converted carrying Xyn10D-Fae1A gene to co-culture with straw culture medium. Gas chromatography was used to detect ferulic acid [1]. The ferulic acid production was monitored by periodically taking samples from the fermentation liquid of E. coli BL21(DE3) carrying Xyn10D-Fae1A gene. GC results showed that the peak of ferulic acid was appeared after fermentation for 24h with the strain carrying Xyn10D-Fae1A gene, while no ferulic acid was detected in the sample of E. coli BL21(DE3) without vector (Fig. 1).

Fig. 1 The production of ferulic acid determined using GC. The supernatants of fermented liquid of E. coli BL21(DE3) with or without Xyn10D-Fae1A gene were used for the determination of ferulic acid, respectively. Samples were taken from reaction mixtureat 0h, 24h.

The ferulic acid peak was further analyzed by using GC-MS (Fig. 2).

Fig. 2 Mass spectrums of ferulic acid derivative. Sample was the supernatants of E. coli BL21(DE3) carrying Xyn10D-Fae1A gene after fermentation for 24h.

Enzyme activity of xylanase

Xylanase can decompose xylan to xylose. Comparing with wild type,our E. coli carrying Xyn10D-Fae1A gene take effect [2] (Fig. 3).


Fig. 3 Xylanase Activity on Xylan. Data were measured inphosphate buffer solution at pH 6.0 and 40℃.

Enzyme activity of ferulic acid esterase

Ferulic acid esterase can decompose 4-nitrophenyl trans-ferulate to produce p-nitrophenol and ferulic acid. We used crude extract fraction of strain to detect enzyme activity [3].

Fig. 4 Enzyme Activity of Ferulic acid esterase. Data were measured in phosphate buffer solution at pH 6.4 and 40℃.

The results (Fig. 4) showed that the ferulic acid enzyme activity of our strain was much higher than that of the wild type.

References

[1] Qiu-Yi, L. I., Gan, G. P., Wang, G. Z., & Liu, Y. W. (2007). Determination of ferulic acid in ligusticum chuanxiong hort. oil by gc. Lishizhen Medicine & Materia Medica Research.

[2] Shen C, Li R, Hu T & Yan K. 2011. Determination of xylanase activity with DNS method. Dyeing & Finishing, 2: 35-39.

[3] Zhang S, Pei X & Wu Z. 2009. Cloning and expression of feruloyl esterase A from Aspergillus niger, and establishment of fast activity detection methods. Chinese Journal of Applied & Environmental Biology, 2: 276-279.