High temperature xylanase gene and high temperature alpha-glucuronidase gene, and protein expression methods and applications thereof

A technology of xylanase and aldolidase, which is applied in the fields of genetic engineering and biomass utilization, can solve the problems of poor stability, low yield, and low activity of xylanase preparations, and achieve good thermal stability and optimum The effect of high reaction temperature

Inactive Publication Date: 2015-06-24
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

[0008] The purpose of the present invention is to make up for the low efficiency of the existing natural xylan enzymatic hydrolysis process, and the problems of low activity, low yield and poor stability of xylanase preparations, and provide a high-temperature xylanase gene Xyn10A and an α- Glucuronidase Agu67A and its recombinant vector and recombinant engineering bacteria, as well as the expression method and application of these two enzymes

Method used

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  • High temperature xylanase gene and high temperature alpha-glucuronidase gene, and protein expression methods and applications thereof
  • High temperature xylanase gene and high temperature alpha-glucuronidase gene, and protein expression methods and applications thereof
  • High temperature xylanase gene and high temperature alpha-glucuronidase gene, and protein expression methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of high temperature xylanase gene Xyn10A and α-glucuronidase gene Agu67A engineering bacteria

[0032] 1.1 Genomic DNA extraction

[0033] The bacterial genome of Caldicellulosiruptorlactoaceticus 6A was extracted with a bacterial genomic DNA extraction kit to obtain genomic DNA, which was frozen at -20°C for future use.

[0034] 1.2 Primer design

[0035] According to the published C.lactoaceticus 6A genome information, the xylanase and α-glucuronidase genes were predicted, and the following 2 pairs of primers were designed:

[0036] The primers used to amplify the xylanase gene are as follows, and the two ends of the primers are respectively introduced with Nhe I and Hind III restriction sites:

[0037] Calla_1331-F 5'-CTA GCTAGC ATGGCTAATTATGAGCATC-3'

[0038] Calla_1331-R 5'-CCC AAGCTT TTAAAGAGTAATTTCAATAAACTTG-3'

[0039] The primers used to amplify the α-glucuronidase gene are as follows:

[0040] Calla_1259-F 5'-GCCGCGCGGCAGCATGAT...

Embodiment 2

[0054] Example 2: Induced expression of recombinant xylanase Xyn10A and α-glucuronidase Agu67A

[0055] Recombinant bacteria were inoculated in 5ml LB liquid medium containing 50μg / ml kanamycin at 1% inoculum size, and cultured overnight at 37°C with shaking at 200rpm. Transfer to 100ml 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, take out the culture medium and bathe in ice for 10 minutes. Add IPTG to a final concentration of 0.1 mM, shake at 200 rpm at 16°C overnight. The next day, centrifuge at 4000g for 15min, add 3ml Binding Buffer (50mM Tris-HCl pH7.5, 300mMNaCl) to resuspend and collect the bacteria. After sonication, the supernatant obtained by centrifugation at 10,000g at 4°C for 15 minutes was the crude enzyme solution.

Embodiment 3

[0056] Example 3: Purification of recombinant xylanase Xyn10A and α-glucuronidase Agu67A

[0057] Using recombinant enzyme C, the N-terminus contains His-Tag, the recombinant protein was separated by heat inactivation at 65°C and Ni-NAT affinity chromatography. The crude enzyme solution was heat-treated at 65°C for 30 minutes to remove heat-labile proteins, and centrifuged at 10,000 g at 4°C for 15 minutes to obtain the supernatant. After 600μl Ni-NAT affinity chromatography column was equilibrated with 6ml Binding Buffer, the supernatant was passed through the column. After passing through the column, wash with 6ml Binding Buffer, then elute with 400μl Elution Buffer (50mM Tris-HCl pH7.5, 300mMNaCl, 150mM imidazole), and collect the eluted sample solution. A 30ml Superdex200 Sephadex column (GE Healthcare) was pre-equilibrated with citric acid buffer (50mM citrate pH6.0, 150mM NaCl), and the eluted sample solution was passed through the column, and the protein was collected ...

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Abstract

The invention provides a high temperature xylanase gene and a high temperature alpha-glucuronidase gene, and protein expression methods and applications thereof. The high temperature xylanase gene and the high temperature alpha-glucuronidase gene are from Caldicellulosiruptor lactoaceticus 6A, and are strongly expressed in a recombinant manner. High temperature xylanase and high temperature alpha-glucuronidase respectively have the advantages of high reaction temperature, good thermal stability and wide pH adaptability. The xylanase can effectively degrade natural xylan and lignocellulose at 80-85DEG C under a pH value of 6.0-8.0 to generate xylooligosaccharide, and the degradation rate reaches 80-98%. The xylanase and the alpha-glucuronidase can synergistically degrade natural xylan and lignocellulose raw materials to generate xylobiose, xylose and glucuronic acid. The xylanase and the alpha-glucuronidase can be used in the fields of foods, feeds and biological energy.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biomass utilization, and specifically relates to a high-temperature xylanase gene and a high-temperature alpha-glucuronidase gene and protein expression and application thereof. Background technique: [0002] Plant cell walls are the most important renewable resources in nature, and these resources are getting more and more attention in the context of the world's energy shortage. Plant cell walls mainly consist of cellulose, hemicellulose and lignin. Xylan is the polysaccharide in plant cell wall which is second only to cellulose in content. It is the main component of hemicellulose and has become the most potential renewable resource due to its large quantity and easy extraction of components. Xylan is a hybrid high polymer composed of xylopyranose linked by β-1,4-glycosidic bonds, with a degree of polymerization of 150-200 (Scheller, H.V., P. Ulvskov. 2010). Xylan from different sources ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N9/24C12N15/10C12N15/70C12N1/21C12P19/14C12P19/12C12P19/02C12R1/19C12R1/01
Inventor 韩业君贾晓静彭小伟米朔甫乔玮博
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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