High-temperature-resistant endoxylanase EpXYN1, coding genes and application thereof

A technology of endo-xylanase and xylanase, which is applied in the field of genetic engineering, can solve problems such as difficult to withstand high temperature conditions, and achieve the effects of high specific activity, high enzymatic hydrolysis efficiency, and high stability

Inactive Publication Date: 2018-01-16
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, most of the reported xylanases are mesophilic enzymes, whose optimum activity temperature is between 50-55°C, and it is difficult to tolerate high temperature conditions of 65°C or above

Method used

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  • High-temperature-resistant endoxylanase EpXYN1, coding genes and application thereof
  • High-temperature-resistant endoxylanase EpXYN1, coding genes and application thereof
  • High-temperature-resistant endoxylanase EpXYN1, coding genes and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Cloning of the gene encoding the thermostable endo-xylanase EpXYN1 of embodiment 1

[0030] Fungal culture and total RNA extraction: take about 10 9 The spores of the 4-14 strains of Penicillium pumilus were inoculated into 50mL PDA liquid medium, and cultured at 37°C and 180rpm for 4 days. Take 1 mL of the culture into a bottle of solid-state fermentation medium (L.Long, D.Ding, Z.Han, H.Zhao, Q.Lin, S.Ding, Thermotolerant hemicellulolytic and cellulolyticenzymes from Eupenicillium parvum 4-14 display high efficiency upon Release offerulic acid from wheat bran, J.Appl.Microbiol.121(2016) 422-434.), shake well and place it under the conditions of 37°C and 70% humidity for static culture for 3 days. After the white mycelium was rinsed with sterile water, the water was blotted with filter paper, frozen in liquid nitrogen and stored at -70°C. Total bacterial RNA was extracted with TransZolTM Plant Kit (TransGen, Beijing).

[0031] Gene cloning: Take an appropriate amoun...

Embodiment 2

[0033] Example 2 Expression and purification of thermostable endo-xylanase EpXYN1 in Pichia pastoris

[0034] The specific primers enXyn1_f2 and enXyn1_r2 of endoxylanase EpXYN1 were designed and synthesized respectively, as follows:

[0035]enXyn1_f2: 5′-agagaggctgaagctgaattctcaggcctggatacagcggca-3′;

[0036] enXyn1_r2: 5'-gagatgagtttttgttctagatcagtgatggtgatggtgatgcagacattgagagtacca-3'.

[0037] The endoxylanase EpXYN1 gene fragment without signal peptide was amplified from the plasmid containing the endoxylanase EpXYN1 gene by using the pair of primers. The amplified target gene fragment was gel-cut and purified, and passed through HieffClone TM The One Step Cloning Kit (Yeasen, Shanghai) performed homologous recombination with the EcoRI-XbaI digested plasmid pPICZαB to obtain the gene expression plasmid pPIC-EpXyn1. After the recombinant plasmid pPIC-EpXyn1 was linearized with restriction endonuclease SacI and then electroporated into Pichia pastoris KM71H, positive clo...

Embodiment 3

[0038] Example 3 Establishment of an activity assay method for thermostable endoxylanase EpXYN1

[0039] Using Beechwood xylan as substrate, Somogyi-Nelson method was used to measure reducing sugar content and calculate enzyme activity. The 1.5mL reaction system is: 1.0mL 50mM pH 5.0 sodium citrate buffer solution, 400μL 0.5% (w / v) substrate, mix gently and preheat for 10min, then add 100μL crude enzyme solution (pre-diluted to different gradients) multiples), the reaction time is 10min at 75°C. Then take it out one by one, add 500μL Somogyi Reagent solution, put it in a 99°C water bath for 15min, cool it to room temperature, add 0.5mL Nelson Reagent, react for 20min, centrifuge for 10min, draw 200μL onto the microplate, and place it in a microplate reader Measure the absorbance (OD value) of the sample at a wavelength of 520 nm. Simultaneously, prepare different concentrations of xylose solutions, measure the OD of each xylose solution with the above-mentioned method (Somog...

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Abstract

The invention discloses high-temperature-resistant endoxylanase EpXYN1, coding genes and application thereof. The amino-acid sequence of the high-temperature-resistant endoxylanase EpXYN1 is shown asSEQ ID NO.1. The high-temperature-resistant endoxylanase EpXYN1 is new endoxylanase obtained by cloning in fine eupenicillium, the optimum temperature is 75 DEG C, the optimum pH is 5.5, the high-temperature-resistant endoxylanase EpXYN1 can resist high temperature of 65 DEG C, and has good pH stability, the main hydrolysis product for xylan is xylobiose, and therefore, the high-temperature-resistant endoxylanase EpXYN1 has good application prospect in the industrial application. As novel xylanase, the high-temperature-resistant endoxylanase EpXYN1 can be widely applied in the industries suchas biomass energy, foods, feeds, paper making and medical health care and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a high-temperature-resistant endo-xylanase EpXYN1, its coding gene and its application. Background technique [0002] Hemicellulose widely exists in the cell walls of various plants and is one of the most important biomass resources on earth. It is widely used in the preparation of biofuels, high value-added chemicals and bio-based materials. Hetero-xylan is one of the main components of hemicellulose, which consists of a main chain of xylose linked by β-1,4 glycosidic bonds and side chains with different substituents. An important step for the industrial utilization of hemicellulose is to degrade heteroxylans into fermentable monosaccharides, xylooligosaccharides, or other chemicals through enzymatic pathways. The complete degradation of heterogeneous xylan requires the synergy of main chain enzymes (such as endo-xylanase and xylosidase) and side chain enzymes. Endo-β-1,4-xy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/14
Inventor 龙良鲲丁少军徐梅娟
Owner NANJING FORESTRY UNIV
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