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Acid xylanase mutants with improved enzyme activity and heat resistance, coding gene and application thereof

An acid xylanase and xylanase mutation technology, applied in the field of genetic engineering, can solve the problems of easy inactivation, denaturation inactivation of neutral xylanase, affecting the use effect, etc., and achieve good application potential, enzyme Vitality and thermal stability enhancement effect

Active Publication Date: 2019-07-12
青岛红樱桃生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] During the production of feed, the enzyme preparation and the feed must be granulated at high temperature after being mixed evenly, during which the enzyme is volatile and inactivated; at the same time, the enzyme preparation added to the feed usually needs to play a role in the gastrointestinal tract, and the acidic environment of the gastric juice is very easy It denatures and inactivates neutral xylanase, which affects the use effect; therefore, the development of heat-resistant acid xylanase is conducive to its application in the feed industry
At present, most xylanases on the market have an optimum reaction temperature between 40-70°C, an optimum pH between 5.0-7.0, and are easily inactivated after experiencing high temperature and gastric acid. A method to solve such problems It is the use of coating agents and carriers to improve the stability of enzymes, but this will undoubtedly increase the production cost of enzyme preparations, and the use of coating treatment enzyme preparations will seriously affect their bioavailability; another economical and effective method is to The gene for the enzyme improves its stability

Method used

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  • Acid xylanase mutants with improved enzyme activity and heat resistance, coding gene and application thereof
  • Acid xylanase mutants with improved enzyme activity and heat resistance, coding gene and application thereof
  • Acid xylanase mutants with improved enzyme activity and heat resistance, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: error-prone PCR constructs the mutant library of acid xylanase XYNTFO

[0043] Referring to the amino acid sequence of acid xylanase XYNTFO (SEQ ID NO: 1), optimize its DNA sequence (SEQ ID NO: 2) according to the codon preference of Pichia pastoris, synthesize the optimized gene, and design EcoR at the 5' end I restriction enzyme cutting site, Not I restriction enzyme cutting site is designed at the 3' end.

[0044] SEQ ID No: 1

[0045]

[0046] SEQ ID No: 2

[0047]

[0048] Use the GeneMorph II Random Mutation PCR Kit and use the xylan XYNTFO gene as a template to perform random mutations. The primer sequences used are as follows:

[0049] XYNTF0F:A GAATTC TTCCCTTCAGAATTG;

[0050] XYNTFOR:A GCGGCCGC TCATGAGACAGTG.

[0051] The amplified random mutation PCR product was double-digested with EcoR I and Not I, purified and recovered, then connected to the pET-21a(+) vector, transformed into Escherichia coli BL21-DE3, and positive clones were...

Embodiment 2

[0059] Example 2: The second round of error-prone PCR constructs a mutant library of xylanase XYNTF01

[0060] Using the xylanase gene XYNTF01 screened in Example 1 as a template, the second round of random mutation was carried out. The mutation library construction process, materials, reagents, and operating conditions were the same as in Example 1; mutants were cultivated and screened with XYNTF01 As a control, the remaining activity of the xylanase mutant was detected after heat treatment (80°C water bath for 3 minutes), and the mutant gene with a higher remaining enzyme activity than XYNTF01 was sequenced.

[0061] The following mutants with improved enzyme activity and heat resistance were finally screened:

[0062] The mutation method of XYNTF02 is P84T / S14R, and the amino acid sequence is shown in SEQ ID No: 5;

[0063] The mutation method of XYNTF03 is P84T / L121E, and the amino acid sequence is shown in SEQ ID No: 7;

[0064] The mutation method of XYNTF04 is P84T / S1...

Embodiment 3

[0084] Example 3: Expression verification of xylanase mutants with improved enzyme activity and heat resistance in Pichia pastoris

[0085] The xylanase mutant genes screened above were connected to the pPIC9K plasmid with EcoR I and Not I double restriction sites and transformed into Escherichia coli DH5α, and the expression vector pPIC- XYNm, sequence verified. The expression vectors of each mutant were linearized by digestion with Sal I and then electroporated into Pichia pastoris GS115. The transformants of the expression strains of each mutant were screened on the MD plate and transferred to the YPD plate for activation (each mutant was picked 48 transformants). The activated transformants were placed in 48 deep-well plates (each well containing 1 mL of BMGY medium), cultured with shaking at 30°C for 18 hours, then induced by adding 1% methanol, and continued shaking culture, after which 1% methanol of the culture volume was added every 24 hours. After inducing expressi...

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Abstract

The invention provides acidic xylanase mutants with improved enzyme activity and heat resistance, a coding gene and application thereof. Based on the acid xylanase XYNTF0 derived from Talaromyces, xylanase mutants XYNTF01, XYNTF02, XYNTF03, XYNTF04 and XYNTF05 are obtained through a large amount of mutation and screening; compared with the unmutated xylanase, the enzyme activity and heat resistance of the xylanase mutants obtained are remarkably improved, and are beneficial to the development and application in the field of feeds.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a kind of acid xylanase mutant with improved enzyme activity and heat resistance, its coding gene and application. Background technique [0002] Xylan is a polypentose sugar present in the plant cell wall and is the main component of hemicellulose, accounting for about 15% to 35% of the dry weight of plant cells. Xylan is formed by polymerizing xylose monomers through β-1,4-glycosidic linkages. Endo-1,4-β-D-xylanase, referred to as xylanase (xylanase), generates woody Sugars, or xylooligosaccharides, are key enzymes in the degradation of xylan. [0003] In the breeding industry, xylan in feed cannot be effectively degraded in the animal digestive system, and it will also affect the absorption and utilization of other nutrients, greatly reducing the conversion efficiency of feed. Therefore, adding xylanase to the feed to convert the anti-nutritional factor xylan i...

Claims

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Application Information

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IPC IPC(8): A23K20/189C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCA23K20/189C12N9/2482C12N15/815C12Y302/01008
Inventor 肖志壮薛海曌
Owner 青岛红樱桃生物技术有限公司
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