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A kind of feed pectinase pla and its gene and application

A pectinase and feed technology, applied in the field of genetic engineering, can solve the problems of low expression level, inappropriate pH or temperature range, etc.

Active Publication Date: 2011-12-28
北京挑战生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some pectinase genes have been cloned, sequenced and expressed (Mertens et al. Fungal Genet Biol 2008; 45: 1616-1624), but many enzymes have some defects in their properties, for example, pH or temperature The scope of action is not suitable, the expression level is low, etc., which cannot meet the needs of practical applications

Method used

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  • A kind of feed pectinase pla and its gene and application
  • A kind of feed pectinase pla and its gene and application
  • A kind of feed pectinase pla and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 Extraction and purification of soil microbial metagenomic DNA

[0099] Soil samples were collected from Guangxi Juice Factory, and the metagenomic DNA of soil microorganisms was extracted by combining physical and chemical cracking methods. The physical method was mainly liquid nitrogen grinding, and the chemical cracking method was mainly based on Brady (Brady SF, Nat Protoc 2007, 2: 1297 -1305), the following is the specific operation process:

[0100] (1) Add 10g of soil to the mortar, pour liquid nitrogen into it to freeze, grind the soil vigorously with a pestle, add liquid nitrogen to grind again after thawing, and repeat three times to make the soil fully ground;

[0101] (2) Add the ground soil into a 50ml centrifuge tube, add 20ml of preheated DNA lysis buffer solution, mix it upside down and put it in a 70°C water bath for 3 hours, during which time the centrifuge tube is turned upside down every 15 minutes Second, the purpose is to fully decompose ...

Embodiment 2

[0111] Example 2 Obtaining of the Conserved Region Sequence of the Pectinase Gene

[0112] According to the conserved (A Q Y P N G G W / F and W A / G Q Q H / Y D E) sequence of the pectin lyase pf09492 gene, degenerate primers pf09492-PF and pf09492-PR were designed and synthesized:

[0113] pf09492-PF: 5'-GCTCAGTACCCNAAYGGNGGNT-3';

[0114] pf09492-PR: 5′-CTCGTCGTRYTGYTGNSCCCA-3′

[0115] Wherein: Y=C / T, R=A / G, S=C / G, N=A / T / G / C.

[0116] Using the above-mentioned degenerate primers to amplify a partial fragment of the target gene by PCR using soil organism metagenomic DNA as a template. The PCR reaction parameters are: 95°C for 5 min; 94°C for 30 sec, 51°C for 30 sec, 72°C for 1 min, after 30 cycles; 72°C for 10 min, agarose electrophoresis detection. A fragment of about 250bp was obtained, and after recovery, it was combined with pEASY-T 3 Carrier ligation was sent to Sanbo Biotechnology Co., Ltd. for sequencing.

Embodiment 3

[0117] Example 3 Obtaining of the full-length sequence of the pectinase gene

[0118] One pectinase gene fragment was randomly selected from a large number of pectinase fragments obtained above to clone the whole gene. According to the known sequence fragments of the target gene, four specific primer pairs SP1, SP2, SP3, and SP4 were designed. Each specific primer pair included a specific upstream primer and a specific downstream primer, and their sequences are shown in Table 1.

[0119] The first round of TAIL-PCR amplification was carried out on the selected pectinase gene fragments with the specific primer pair SP1. Aiming at the low content of the target sequence, a single step specific primer SP1 was added to amplify the target template sequence. to increase the copy number of the target fragment. The second-round TAIL-PCR amplification was performed on the first-round TAIL-PCR product with specific primer pair SP2 and degenerate primers. First, AD primers are added sep...

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Abstract

The invention relates to the field of gene engineering and particularly relates to feed pectin lyase (PLA) and a gene and use thereof. The invention provides the new feed PLA which has an amino acid sequence represented by SEQ ID No.1 or SEQ ID No.2. The invention also provides a gene for coding the feed PLA, which has a nucleotide sequence represented by SEQ ID No.3 or SEQ ID No.4, a recombinantvector and a recombinant strain, which have the gene, and the use of the gene. The optimal pH of the feed PLA provided by the invention is 7.0, and the feed PLA has activity of over 50 percent when the pH value is between 3.0 and 9.0; the stable pH range is between 3.0 and 10.0; and the optimal temperature is 30 DEG C, and the feed PLA has enzyme activity of over 70 percent when the temperature is between 10 to 50 DEG C, the activity at 0 DEG C is still over 30 percent, and the thermal stability under condition of a temperature of 40 DEG C is high. The feed PLA can be used for degrading pectin in plants in industries of feed, food and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a feed pectinase PLA and its gene and application. Background technique [0002] Pectin is mainly a complex polysaccharide composed of D-galacturonic acid residues connected by linear α-1, 4 glycosidic bonds, and is an important component of plant cell walls and mesoplasts (van Buren JP.Academic Press, 1991.p .1-22). Pectinase (pectinases) is a general term for a variety of enzymes that decompose pectin. It exists widely in higher plants and microorganisms. Usually, animal cells cannot synthesize such enzymes. According to the nature of the reaction of decomposing glycosidic bonds or the nature of the degradation substrate, it can be divided into: pectin lyases (pectin lyases, PL), pectin hydrolases (pectin hydrolases), pectin esterases (pectin esterases, PE) and original fruit Gelase (Alkorta I, et al. Process Biochem 1998, 33:21-28). Pectinase is div...

Claims

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Application Information

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IPC IPC(8): C12N9/26C12N15/56C12N15/63C12N15/81C12N1/15C12N1/19C12N15/80A23K1/165C12R1/84A23K20/189
Inventor 黄辉谢宁汤海鸥王海燕王晓睿
Owner 北京挑战生物技术有限公司
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