A kind of acid β-mannanase and its gene and application

Abstract

The invention relates to the field of genetic engineering, and in particular relates to acid beta-mannase Man5DB as well as a gene and an application of the acid beta-mannase, wherein an amino acid sequence of the acid beta-mannase is represented as either SEQ ID NO. 1 or SEQ ID NO. 2. The enzyme has the following natures: optimum pH is 5.0, and the enzyme can keep enzyme activity at above 60% within a pH range of 3.5-6.0; optimum reaction temperature is 65 DEG C, and the enzyme can keep enzyme activity at above 50% at 45-70 DEG C; after being processed for 60min at 37 DEG C, the enzyme can keep enzyme activity at above 80% within a pH range of 4.0-9.0, thus showing that the enzyme is excellent in pH stability; and after being processed for 60min at 50 DEG C, the enzyme activity is free from loss and can be still kept at above 20% after being processed for 5min at 65 DEG C. In addition, specific activity is 795Umg<-1>. The acid beta-mannase is excellent in heat resistance, and is applicable to such industries as feed, food and medicine. According to the technical scheme disclosed by the invention, mannase that is excellent in nature and applicable to industrial application can be produced through genetic engineering means.

Description

technical field

[0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acid-producing beta-mannanase and its gene and application. Background technique

[0002] Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Mannan is an important component of plant hemicellulose. It is a linear polymer connected by β-1,4-D-mannose. The side chains of the polysaccharide mainly contain glucosyl, acetyl and hemicellulose. Lactosyl and other substituent groups. β-mannanase (β-mannanase EC3.2.1.78) is an endohydrolase that hydrolyzes mannan. It degrades the β-1,4 glycosidic bond of the mannose backbone in an endo-cutting manner and releases a short β -l,4 mannan oligosaccharides.

[0003] In recent years, with the discovery of the physiological functions of mannan oligosaccharides, the rise of green feed and the enhancement of people's awareness of environmental protection, the research and utilization of ene...

Examples

Embodiment 1

[0136] Cloning of embodiment 1β-mannanase encoding gene man5D

[0137] Extraction of Staphylotrichum coccosporum genomic DNA

[0138] Staphylotrichum coccosporum NBRC31817 was purchased from NBRC (NITE biological resource center) in Japan.

[0139] The bacteria cultured in the enzyme-producing medium for 3 days were centrifuged at 12,000 rpm for 10 minutes, and the collected mycelium was added to a high-temperature sterilized mortar, and quickly ground to powder with liquid nitrogen, and then the ground bacteria were transferred to a new 50mL centrifuge tube filled with 15ml CTAB lysate, gently invert up and down to mix, place in a 70°C water bath for 3 hours, every 20min, invert up and down and gently mix once to fully lyse the bacteria. Centrifuge at 12,000 rpm at 4°C for 10 min, pipette the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Take the...

Embodiment 2

[0145] Example 2 Obtaining of β-mannanase cDNA

[0146] Extraction of Staphylotrichum coccosporum total RNA using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, and then design primers F and R to amplify the open reading frame (see Table 1), amplify the single-stranded cDNA, obtain the cDNA sequence of mannanase, and amplify the product After recovery, they were sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0147] After comparing the genome sequence and cDNA sequence of mannanase, it was found that the gene contained 4 introns, the cDNA was 1362 bp long, encoded 453 amino acids and a stop codon, and the N-terminal 16 amino acids were its signal peptide sequence. The comparison proves that the gene encoding mannanase isolated and cloned from Staphylotrichum coccosporum is a new gene.

Embodiment 3

[0148] The construction of embodiment 3 β-mannanase engineering strains

[0149] (1) Construction of expression vector and expression in yeast

[0150] Using the correctly sequenced mannanase Man5DB cDNA as a template, the expression primers F and R with EcoR I and Not I restriction sites were designed and synthesized (see Table 1) for the coding region of the mature protein of Man5DB Amplify. And utilize EcoR I and Not I to digest the PCR product, connect into the expression vector pPIC9 (Invitrogen, San Diego), the sequence of β-mannanase Man5DB mature protein is inserted into the downstream of the signal peptide sequence of the above expression vector, and signal peptide The correct reading frame was formed, and the yeast expression vector pPIC9-man5DB was constructed to transform E. coli competent cell JM109. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant pl...

technical field

[0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acid-producing beta-mannanase and its gene and application. Background technique

[0002] Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Mannan is an important component of plant hemicellulose. It is a linear polymer connected by β-1,4-D-mannose. The side chains of the polysaccharide mainly contain glucosyl, acetyl and hemicellulose. Lactosyl and other substituent groups. β-mannanase (β-mannanase EC3.2.1.78) is an endohydrolase that hydrolyzes mannan. It degrades the β-1,4 glycosidic bond of the mannose backbone in an endo-cutting manner and releases a short β -l,4 mannan oligosaccharides.

[0003] In recent years, with the discovery of the physiological functions of mannan oligosaccharides, the rise of green feed and the enhancement of people's awareness of environmental protection, the research and utilization of ene...

Examples

Embodiment 1

[0136] Cloning of embodiment 1β-mannanase encoding gene man5D

[0137] Extraction of Staphylotrichum coccosporum genomic DNA

[0138] Staphylotrichum coccosporum NBRC31817 was purchased from NBRC (NITE biological resource center) in Japan.

[0139] The bacteria cultured in the enzyme-producing medium for 3 days were centrifuged at 12,000 rpm for 10 minutes, and the collected mycelium was added to a high-temperature sterilized mortar, and quickly ground to powder with liquid nitrogen, and then the ground bacteria were transferred to a new 50mL centrifuge tube filled with 15ml CTAB lysate, gently invert up and down to mix, place in a 70°C water bath for 3 hours, every 20min, invert up and down and gently mix once to fully lyse the bacteria. Centrifuge at 12,000 rpm at 4°C for 10 min, pipette the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Take the...

Embodiment 2

[0145] Example 2 Obtaining of β-mannanase cDNA

[0146] Extraction of Staphylotrichum coccosporum total RNA using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, and then design primers F and R to amplify the open reading frame (see Table 1), amplify the single-stranded cDNA, obtain the cDNA sequence of mannanase, and amplify the product After recovery, they were sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0147] After comparing the genome sequence and cDNA sequence of mannanase, it was found that the gene contained 4 introns, the cDNA was 1362 bp long, encoded 453 amino acids and a stop codon, and the N-terminal 16 amino acids were its signal peptide sequence. The comparison proves that the gene encoding mannanase isolated and cloned from Staphylotrichum coccosporum is a new gene.

Embodiment 3

[0148] The construction of embodiment 3 β-mannanase engineering strains

[0149] (1) Construction of expression vector and expression in yeast

[0150] Using the correctly sequenced mannanase Man5DB cDNA as a template, the expression primers F and R with EcoR I and Not I restriction sites were designed and synthesized (see Table 1) for the coding region of the mature protein of Man5DB Amplify. And utilize EcoR I and Not I to digest the PCR product, connect into the expression vector pPIC9 (Invitrogen, San Diego), the sequence of β-mannanase Man5DB mature protein is inserted into the downstream of the signal peptide sequence of the above expression vector, and signal peptide The correct reading frame was formed, and the yeast expression vector pPIC9-man5DB was constructed to transform E. coli competent cell JM109. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant pl...

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