A high-temperature alpha-L-arabinfuranosidease gene, a high-temperature acetylxylan esterase gene, and protein expression and applications of the genes

An acetyl xylan ester and furanosidase technology, which is applied in the fields of genetic engineering and biomass utilization, can solve the problems of being unsuitable for industrialized large-scale production, complex components of xylan degrading enzymes, harsh growth conditions, etc. Improved biodegradability, high optimum reaction temperature and good thermal stability

Inactive Publication Date: 2016-07-06
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the xylan-degrading enzyme system produced by this bacterium has complex components, is difficult to separate and purify, and its growth conditions are harsh and its growth density is low, so it is not suitable for large-scale industrial production.

Method used

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  • A high-temperature alpha-L-arabinfuranosidease gene, a high-temperature acetylxylan esterase gene, and protein expression and applications of the genes
  • A high-temperature alpha-L-arabinfuranosidease gene, a high-temperature acetylxylan esterase gene, and protein expression and applications of the genes
  • A high-temperature alpha-L-arabinfuranosidease gene, a high-temperature acetylxylan esterase gene, and protein expression and applications of the genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of high-temperature α-L-arabinofuranosidase gene abf51A and acetylxylan esterase gene axeA engineering bacteria

[0036] 1.1 Genomic DNA extraction

[0037] The bacterial genome of C. lactoaceticus6A was extracted with a bacterial genomic DNA extraction kit to obtain genomic DNA, which was frozen at -20°C for future use.

[0038] 1.2 Primer design

[0039] According to the published genome information of C.lactoaceticus6A, the α-L-arabinofuranosidase and acetylxylan esterase genes were predicted, and the following 2 pairs of primers were designed:

[0040] The primers used to amplify the α-L-arabinofuranosidase gene are as follows:

[0041] abf51A-F5'-GCCGCGCGGCAGCATGAAAAAAGCAAAAGTCATCTAC-3'

[0042] abf51A-R5'-GCGGCCGCAAGCGTTTAATTTTCTTCTTCTTTAACCTG-3'

[0043] The primers used to amplify the acetyl xylan esterase gene are as follows:

[0044] axeA-F5'-GCCGCGCGGCAGCATGATACCACTTTGGGAAAATC-3'

[0045] axeA-R5'-GCGGCCGCAAGCGTTTAAAACATTATATCCTA...

Embodiment 2

[0056] Example 2: Expression of recombinant α-L-arabinofuranosidase Abf51A and acetylxylan esterase AxeA in Escherichia coli

[0057] The recombinant bacteria E. coli Rosetta (DE3) were inoculated in 5 ml of LB liquid medium containing 50 μg / ml kanamycin according to the inoculum amount of 1%, and cultured overnight at 37° C. with shaking at 200 rpm. Transfer to 200ml LB liquid medium containing 50μg / ml kanamycin according to the same inoculum amount, shake culture at 200rpm at 37°C until OD 600 When it reaches about 0.4-0.6, add IPTG to a final concentration of 0.1 mM, and continue shaking culture at 200 rpm at 37°C for 4-6 hours. After the cultivation, the cells were collected by centrifugation at 4000 g for 15 min, and resuspended in 30 ml BindingBuffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl) to collect the cells. After sonication, the supernatant obtained by centrifugation at 10,000g at 4°C for 15 minutes is the crude enzyme solution.

Embodiment 3

[0058] Example 3: Expression of recombinant α-L-arabinofuranosidase Abf51A and acetylxylan esterase AxeA in Trichoderma reesei

[0059] Using the C. lactoaceticus6A genome as a template, design primers to amplify to obtain α-L-arabinofuranosidase gene abf51A and acetylxylan esterase gene axeA, respectively, and connect them into vector pSKCST to obtain expression vectors pSKCST-abf51A and pSKCST-axeA respectively . The expression vectors pSKCST-abf51A and pSKCST-axeA were linearized and then transformed into T.reesei protoplasts. After the transformants grew out, the genomic DNA of the transformants was extracted, and the integration of exogenous genes in the transformants was identified by PCR. Use a cutter to cut the colonies of transformants on the selective plate into 0.3 cm diameter bacterial blocks, take 5 blocks from each transformant, transfer them to the EG screening plate, and culture them at 28°C for 2-3 days. Regularly observe and record the growth diameter. Sele...

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Abstract

The invention provides a high-temperature alpha-L-arabinfuranosidease gene abf51A and a high-temperature acetylxylan esterase gene axeA which are derived from caldicellulosiruptor lactoaceticus 6A, and protein expression and applications of the genes. High-temperature alpha-L-arabinfuranosidease abf51A and high-temperature acetylxylan esterase axeA are advantaged by high optimum reaction temperatures, good thermal stability, and the like, and can effectively degrade natural xylan side chains at 65-85 DEG C with pH being 4.5-6.5 to produce arabinose, acetic acid and a small amount of xylose. The two enzymes can effectively eliminate steric hindrance of side chains to xylanase, improve biodegradability of the lignocellulose resource, can be widely used as novel enzyme preparations for feed, food, energy, and other fields, and have potential industrial application value.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biomass utilization, and specifically relates to a high-temperature α-L-arabinofuranosidase gene, a high-temperature acetyl xylan esterase gene and protein expression and application thereof. Background technique: [0002] Hemicellulose, one of the main components of plant cell walls, is one of the most abundant renewable resources on earth. With the increasingly prominent energy problems, the utilization value of hemicellulose resources has gradually been paid attention to by people, and the research on its biodegradation mechanism has become more and more significant. The most abundant type of hemicellulose is xylan, which is a chain of xylopyranose linked by β-1,4-glycosidic bonds, and is usually linked to acetyl, arabinose, and glucose to varying degrees. Groups such as alkyd and ferulic acid ester groups (Scheller, H.V., P. Ulvskov. 2010). The complexity of this structure leads to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/55C12N15/10C12N9/24C12N15/63C12N1/21C12P19/14
Inventor 韩业君贾晓静彭小伟乔玮博米朔甫苏红
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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