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Pectate lyase, coding gene and applications thereof

A technology of pectin lyase and coding gene, which is applied in the field of pectin lyase and its coding gene and application, and can solve the problems of poor thermal stability and low activity of pectin lyase

Active Publication Date: 2016-03-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to solve the technical problems of low activity and poor thermal stability of the existing pectin lyase, and provides a pectin lyase with high activity and good thermal stability, its coding gene and its application

Method used

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  • Pectate lyase, coding gene and applications thereof
  • Pectate lyase, coding gene and applications thereof
  • Pectate lyase, coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 The acquisition of pectin lyase wild type and mutant and its coding gene

[0018] 1. Extract the genomic DNA of Bacillus pumilus (Bacillus pumilus) ATCC7061 (purchased from the Bacteria Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is CGMCCNo.1.3533) and use it as a template, and use 5'-atggctagcatgttgaagaaaaaag-3'(forward ) and 5'-gtgctcgagtacctttccaacaccagc-3'(reverse) were used as primers for PCR amplification. The PCR amplification conditions were as follows: 4 minutes of pre-denaturation at 95°C, followed by 30 cycles of 95°C for 45s, 55°C for 30s, and 72°C for 1 min; finally, extension at 72°C for 10 minutes.

[0019] The above PCR reaction product was recovered and detected by agarose gel electrophoresis, and a band of 1023p was obtained as a result.

[0020] 2. Obtaining of recombinant bacteria containing pectin lyase wild-type coding gene

[0021] The PCR product obtained in the ab...

Embodiment 2

[0032] Example 2 Pectin lyase activity assay

[0033] 1. Obtaining wild-type and mutant recombinant proteins of pectin lyase

[0034] 1. Induced expression

[0035] Inoculate the single colonies of BL21(DE3) / pET28a-pel or pET28a-pel-M3 obtained above into LB liquid medium containing kanamycin (final concentration: 50 μg / ml), culture at 37°C for 12 hours, and collect the fermented The fermentation broth was transferred to 100ml of fresh LB liquid medium according to the inoculum size of 1% (volume percentage content), and cultivated to OD at 37°C 600 When it reached 0.6, sterile IPTG was added to the culture medium so that the final concentration of IPTG in the culture medium was 1 mM and the induction fermentation was carried out at 30°C. After 12 hours, the fermentation was finished, and the fermentation broth was centrifuged at 4000g for 10 minutes to collect the bacteria.

[0036] Resuspend the bacteria in 50ml of 50mM binding buffer (Tris-hydrochloric acid, pH8, 300mMNa...

Embodiment 3

[0051] Example 3 Application of pectin lyase BpPel and its mutant Pel-M3 in ramie degumming

[0052] Experimental method of ramie degumming

[0053] 1 g of ramie was mixed with 10 mg of purified colloid lyase (wild type or mutant), and the reaction system was 20 ml of 100 mM glycine / sodium hydroxide buffer solution (0.3 mM calcium chloride, pH 10.0). Incubate at 50°C for 4h. Subsequently, the treated ramie was washed with water and then dried at 105°C to constant weight. Compare the difference in dry weight of ramie before and after enzyme treatment, that is, the weight loss rate.

[0054] After treated with the purified wild-type BpPel and mutant Pel-M3 by the above method, the weight loss rate of ramie was 17.6% (BpPel) and 23.2% (Pel-M3), respectively.

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Abstract

The present invention relates to a pectate lyase, a coding gene and applications thereof. According to the present invention, the technical problems of not high activity and poor thermal stability of the existing pectate lyase are solved; the amino acid sequence of the pectate lyase and the nucleotide sequence of the coding gene of the pectate lyase are disclosed, and the preparation method of the coding gene and the applications of the pectate lyase in ramie degumming are disclosed; and the preparation method can be widely used in the pectate lyase preparation field.

Description

technical field [0001] The present invention relates to an enzyme and its encoding gene and application, specifically a pectin lyase and its encoding gene and application. Background technique [0002] Pectin is a kind of natural macromolecular compound, which is an important component of plant cell interstitium. It is mainly formed by condensation of galacturonic acid and its methyl ester, and plays a bonding role in plant cell tissue. The main chain of pectin is composed of galacturonic acid connected by α-1,4-glucosidic bonds; the composition of branch chains varies with different sources, including L-rhamnose, arabinose, galactose and xylose, etc. . [0003] Pectinase (Pectinases) refers to the general term for a class of enzymes that decompose pectin. Due to the complex chemical structure of pectin, there are many kinds of pectinases that can catalyze its decomposition. Pectinase can be divided into acid pectinase, neutral pectinase and alkaline pectinase according t...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/10D01C1/00
Inventor 马延和唐双焱梁朝宁桂习武周成薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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