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Beta-mannanase, gene, preparation method, vector and host cell

A mannanase, recombinant host cell technology, applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of long fermentation cycle, no affinity tag, and difficult to achieve mannan polymerization The problem of high-efficiency expression of carbohydrase gene to achieve the effect of convenient purification

Inactive Publication Date: 2009-03-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yet the mannanase gene of Aspergillus niger AS2710 bacterial strain has not been cloned yet, and its expression is mainly fermented by Aspergillus niger AS2710 bacterial strain, and this bacterium fermentation period is long (more than 5 days), because there is not effective promoter control in addition, also can not It is easy to achieve high-efficiency expression of mannanase gene, and the enzyme does not have an effective affinity tag, which is not conducive to rapid and convenient purification

Method used

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  • Beta-mannanase, gene, preparation method, vector and host cell
  • Beta-mannanase, gene, preparation method, vector and host cell
  • Beta-mannanase, gene, preparation method, vector and host cell

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1. Construction of recombinant plasmid pUC1.17 and transformed strain DH5α (pUC1.17) containing the β-mannanase gene shown in SEQ ID: NO.1

[0036] 2L of water samples were collected from hot springs in Tengchong, Yunnan (pH2, 90°C), filtered through a 0.22 μm microporous membrane (Milipore Company) to collect the bacteria in the water samples, and washed with STE buffer (0.1M NaCl; 10mM Tris-HCl, pH8 .0; 1mM EDTA, pH8.0) to wash the bacteria on the filter membrane, centrifuge at 4000rpm for 10min, and obtain the fresh wet weight of the bacteria about 0.5g. UltraClean for bacteria sediment TM Soil DNA Kit was used to extract total DNA (100 μl, 4 μg / μl), and the ratio of absorbance was determined: A260 / A280=1.947, A260 / A230=2.15. Take 50 μl of the above total DNA solution (containing about 200 μg DNA), and digest it with the restriction enzyme Sal I. The enzyme digestion system contains 5 μl Sal I enzyme (20 U / μl), 5 μl Buffer1 (10 mM KCl, 10 mM Tris-HCl, 0.1 m...

Embodiment 2

[0037] The construction of embodiment 2.β-mannanase expression vector and transformed bacterial strain:

[0038] According to the nucleotide sequence (manA sequence) shown in SEQ ID: NO.1, synthesize the forward and reverse primers, and the forward primer is 5'-GTGC GCTAGC ATGGGACG-3', the underlined part is the restriction site of NheI, and the reverse primer is 5'-GCG AAGCTT TCATCGATTTG-3', the underlined part is the restriction site of HindIII. Using these two primers, use the plasmid pUC1.17 as a template, and use the following PCR reaction system (Taq DNA polymerase and its buffer, and dNTP are products from Tiangen Company):

[0039] 10X buffer 5μl

[0040] dNTP 4μl

[0041] Taq DNA polymerase 0.5μl

[0042] Forward primer (25pM) 1μl

[0043] Reverse primer (25pM) 1μl

[0044] pUC1.17 template (1μg / μl) 1μl Reaction conditions: 94°C pre-denaturation for 4 minutes, then 94°C denaturation for 30 seconds-50°C annealing for 30 seconds-72°C extension for 1min 30 cycles...

Embodiment 3

[0045] Example 3. Preparation of Mannanase Gene Mutants

[0046] Using the expression vector pETMan-HA963 of Example 2 as a template, using Stratagene's site-directed mutagenesis kit (QuikChange site-directed mutagenesis kit), referring to the instructions, by introducing mutant bases into the primers and performing PCR amplification, the SEQ ID : The nucleotide sequence shown in NO.1 was mutated from A to C at position 476, and the vector pETMan-E159A containing the mutant was obtained. The nucleotide sequence of the mutant was sequenced by Beijing Nuosai Genome Research Center Co., Ltd., and the result is shown as SEQ ID: NO.3, which realized the preset point mutation, that is, compared with SEQ ID: NO.1, the The nucleotide sequence of the mutant gene is mutated from A to C at position 476, the triplet codon GAA is changed to GCA, and the amino acid Glu at position 159 of the corresponding amino acid sequence (as shown in SEQ ID: NO.4) is changed to Ala. According to the same...

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Abstract

The invention provides a Beta-mannose which has amino acid sequences shown in SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6 and SEQ ID: NO.8. Or substitution, depletion or addition of one or multiple amino acids is carried out to the amino acid sequences shown in the SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6 or SEQ ID: NO.8 to obtain amino acid sequences of Beta-mannose with the same activity. The invention also provides a gene for coding the Beta-mannose, a recombinant vector containing the gene and a host cell containing the recombinant vector. The invention further provides a preparation method of the Beta-mannose, including the culture of the host cell provided by the invention. The Beta-mannose provided by the invention has heat-resisting property and acid resistance and a prokaryotic expression system established by the invention can be utilized for production. Furthermore, six histidine tags can be utilized for purification.

Description

technical field [0001] The invention relates to a β-mannanase, its coding gene and preparation method, as well as a recombinant vector and a recombinant host cell containing the gene. Background technique: [0002] Mannanase is a kind of endonuclease that can hydrolyze the second largest component in hemicellulose - isomannan. It has been widely used in many fields. For example: mannanase can be used as a feed enzyme additive. Adding mannanase in feed can supplement endogenous digestive enzyme deficiency, eliminate and degrade antinutritional factors in feed raw materials, promote the growth of livestock and poultry, and reduce breeding pollution (Lee, et al. al., Poultry Science, 82: 1925-1931, 2003); can hydrolyze mannan-type plant gums (such as carob gum, guar gum, konjac, etc.) to generate oligomeric manna with physiological effects of proliferating bifidobacteria Sugar has a great market prospect. [0003] The sources of β-mannanase are very extensive, including Baci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N1/21C12R1/19
Inventor 马延和张跃灵薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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