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Glucomannanase encoding gene and enzyme, preparation and application thereof

A glucomannanase and coding technology, which can be used in application, genetic engineering, plant genetic improvement, etc., can solve the problems of weak degradation activity of konjac polysaccharide and unsuitable for large-scale application.

Active Publication Date: 2019-06-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The research on mannanase at home and abroad mostly focuses on microorganisms, including bacteria, especially Gram-positive bacteria, including Bacillus and Clostridium, and fungi such as Aspergillus and Trichoderma reesei. ), Streptomyces (Streptomyces) among actinomycetes has been studied more, but the glucomannanase derived from Klebsiella oxytoca (Klebsiella oxytoca) is rarely reported (only four are reported)
The glucomannanases reported so far have weak degradative activities on konjac polysaccharides and are not suitable for large-scale application.

Method used

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  • Glucomannanase encoding gene and enzyme, preparation and application thereof
  • Glucomannanase encoding gene and enzyme, preparation and application thereof
  • Glucomannanase encoding gene and enzyme, preparation and application thereof

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Experimental program
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Embodiment 1

[0053] Embodiment 1 glucomannanase full-length gene cloning

[0054] Genomic DNA of Klebsiella oxytoca was extracted according to the operating steps of the Genomic DNA Purification Kit (Thermo, LOT 00105781). After performing multiple sequence alignment analysis on the glucomannanase gene sequence in The National Center for Biotechnology Information (NCBI) database, degenerate primers were designed KmanA-F: 5'-GATATACATATGGCGGAGCAGTCGCACTTTGAAC-3'; KmanA-R: 5'- GTATAACTCGAGCTCTGCAACCACTTCAATCG-3', using the extracted genomic DNA of Klebsiella oxytoca as a template, amplifies the gene sequence encoding the mature protein of glucomannanase (excluding the signal peptide gene). The PCR reaction conditions were: 94°C for 2 min, 1 cycle; 94°C for 30 s, 68°C for 30 s (0.5°C drop per cycle), 72°C for 2 min 30 s, 30 cycles; 72°C for 5 min, 1 cycle. PCR products were analyzed by agarose gel electrophoresis (see figure 1 ), the target gene was recovered by gel cutting, connected to th...

Embodiment 2

[0055] Embodiment 2 glucomannanase gene sequence analysis

[0056] The sequencing results were analyzed using Basic Local Alignment Search Tool (BLAST) in the GenBank database, DNAMAN software was used for multiple sequence alignment, and Vector NTI was used to analyze sequence information.

[0057]The coding region of the obtained glucomannanase gene (named KmanA) is 2151 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. KmanA encodes 716 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 2, the theoretical protein molecular weight is 79.79kDa, and the predicted isoelectric point is 5.60. The amino acid encoded by KmanA contains multiple structural domains: a COG3934 superfamily domain, a FN3 (Fibronectin type 3) superfamily domain and a CBM (Carbohydrate Binding Module) 6-CBM35-CBM36_Like superfamily domain. Among them, the COG3934 superfamily domain encodes the endo-β-1,4-mannosidase domain; the FN3 domain has also been reported in bact...

Embodiment 3

[0058] Recombinant expression and purification of embodiment 3 KmanA gene in Escherichia coli

[0059] In order to facilitate the recombinant expression of the gene, NdeI and XhoI enzyme cutting sites were respectively introduced into the designed upstream and downstream primers. The PCR cleaning product KmanA and the expression vector pET21a were double-digested with NdeI and XhoI, respectively, and the digested products were cleaned and recovered, and then used T 4 DNA ligase connection (ligation system: (5μLT 4 DNA Ligase 0.5μL, 10×T 4 DNA LigaseBuffer 0.5 μL, pET21a 2 μL, PCR product 2 μL), ligation conditions: ligate overnight at room temperature. ). Take 5 μL of the ligation product to transform E.coli TOP10 competent cells, spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and incubate at 37°C for 12-16 h. Pick a single clone, use degenerate primers for colony PCR verification, insert the correctly amplified single clone into liquid Luria-Bertani...

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Abstract

The invention discloses a preparation method and an application of a glucomannanase gene derived from Klebsiella oxytoca and an enzyme thereof, which relates to a technical method using genetic engineering. The gene of the glucomannanase is cloned into an E. coli expression vector to obtain a recombinant strain of Escherichia coli which can heterologously express the enzyme. The glucomannanase prepared by heterologous expression of the strain can efficiently degrade konjac polysaccharide (also known as konjac glucomannan). The glucomannanase provided by the invention can be widely applied to the fields of agriculture, food, feed additives, medicine and preparation of glucomannan oligosaccharides.

Description

technical field [0001] The invention relates to a gene sequence of glucomannanase, its preparation method and application. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the glucomannanase and its application in polysaccharide degradation. The glucomannanase provided by the invention can be widely used in fields such as agriculture, food, feed addition, medicine and oligosaccharide preparation. Background technique [0002] Konjac (Amorphophallus konjac), commonly known as konjac, is a perennial herbaceous plant belonging to Araceae Konjac. The main ingredient in konjac is konjac polysaccharide (also known as konjac glucomannan), which is composed of glucose and mannose. Konjac glucomannan is formed by linking β-D-glucose and β-D-mannose at a molar ratio of 1:1.6 or 1:1.69 through β-1,4 pyranoside bonds, and every 19 sugar residues There is an acetyl group, and its special structure makes it have a variety of biological functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/24C12N15/63C12P19/14C12P19/02
Inventor 尹恒李悝悝王文霞李倩
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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