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A kind of β-mannosidase and its application

A technology of mannosidase and mannose, applied in the field of bioengineering, can solve problems such as limiting the application of β-mannosidase and difficult expression, and achieve the effect of potential industrial application value

Active Publication Date: 2021-10-26
东莞泛亚太生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There have been a few reports on the cloning and expression of β-mannosidase genes from microbial sources abroad (ChauhanPS, GuptaN. Insight into microbial mannosidases: a review [J]. Critical Reviews in Biotechnology, 2016, 8: 1-12.), but in the literature Most of the reported β-mannosidases have relatively large molecular weights, ranging from 90 to 130 KDa, and are difficult to express, which limits the application of β-mannosidases

Method used

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  • A kind of β-mannosidase and its application
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  • A kind of β-mannosidase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning of β-mannosidase LrMan5 gene

[0037] Extract the genomic DNA of Lichtheimia ramose, use the genomic DNA as a template, design primer LrMan5-F / R, the sequence is as shown in Table 1 (SEQ ID: 3 / SEQ ID: 4), obtain β-mannosidase by conventional PCR method (LrMan5) gene, the gene sequence is shown in SEQ ID NO:1.

[0038] Table 1: LrMan5 amplification primer sequences

[0039]

[0040] The PCR reaction system for obtaining the LrMan5 gene is shown in Table 2, and the reaction conditions are shown in Table 3.

[0041] Table 2: PCR reaction system

[0042]

[0043] Table 3: PCR reaction conditions

[0044]

Embodiment 2

[0045] Example 2 Construction and verification of Pichia pastoris expression vector comprising the gene encoding LrMan5

[0046] (1) LrMan5 gene and plasmid pPICZαA were double digested

[0047] Table 4 shows the double enzyme digestion reaction system of LrMan5 gene, and Table 5 shows the double enzyme digestion reaction system of plasmid pPICZαA.

[0048] Table 4: LrMan5 Gene Double Digestion Reaction System

[0049]

[0050] Table 5: Plasmid pPICZαA double enzyme digestion reaction system

[0051]

[0052] (2) Connection of pPICZαA vector and LrMan5 gene

[0053] The connection system is shown in Table 6, and the expression vector pPICZαA-LrMan5 was constructed (the map of the expression vector is shown in figure 1 shown), transform Escherichia coli DH5a, and carry out vector screening and amplification.

[0054] Table 6: Connection system between pPICZαA vector and LrMan5

[0055]

[0056] (3) Conversion

[0057] a. Preparation of Pichia pastoris Competent Cel...

Embodiment 3

[0079] Example 3 Expression and purification of recombinant β-mannosidase LrMan5

[0080] A single colony of Pichia pastoris that grew well was picked, inoculated into a finger flask containing 5 mL of BMG, and cultured overnight at 30°C with shaking. Transfer the cultured bacterial solution overnight to a 500mL Erlenmeyer flask containing 50mL YPDG, seal it with eight layers of gauze to ensure a good air-permeable environment, and culture overnight at 28°C and 250rpm. Centrifuge the cultured bacterial solution overnight at 1500g for 3min, resuspend 50mL of fresh BMMY culture in a 500mL Erlenmeyer flask, seal with eight layers of gauze, and cultivate at 28°C with shaking at 250rpm. Methanol was added every 6h to a final concentration of 1.2-1.5% methanol. Take an appropriate amount of culture fluid every 12 hours to measure the OD600 value and enzyme activity, so as to determine the best harvest time for recombinant protein expression. The fermentation product was centrifuge...

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Abstract

The invention discloses a β-mannosidase and its application. The sequence of the polypeptide of β-mannosidase of the present invention is: (1a) the amino acid sequence shown in SEQ ID NO: 1; or (2a) having at least 75% sequence identity with SEQ ID NO: 1 and having the same sequence as SEQ ID NO: 1 ID NO: 1 Amino acid sequence of β-mannosidase with the same function. The invention also discloses an expression vector and host cell of β-mannosidase. The β-mannosidase disclosed by the present invention has good stability of mannosidase enzyme activity at pH 3.5-8 and temperature 30-65°C. When the β-mannosidase of the present invention is used to produce mannose, the yield of mannose can reach 7.1 mg / mL, which is about 6 times that of not adding the β-mannosidase of the present invention, and has potential industrial application value.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a β-mannosidase and application thereof. Background technique [0002] β-mannosidase (EC3.2.1.25) belongs to an exonuclease of the hemicellulase system, which can catalyze the hydrolysis of 1,4-β-D glucosidase, cut off mannose at the non-reducing end, and is used in food , pharmaceutical, petroleum and biotransformation industries have a wide range of applications (Mccleary B V, Nurthen E, Taravel F R, et al.Characterisation of oligosaccharides produced on hydrolysis of galactomannan with β-D-m annanase[J]. JUL): 91-109) especially as an alternative to chemically produced mannose. Mannose has good solubility and is not easy to crystallize, so it has potential application value in food and pharmaceutical industries. Another application of β-mannosidase is to use its transglycosidic ability to synthesize manno-oligosaccharides to replace chemical methods to produce funct...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12P19/02C12R1/84
CPCC12N9/2491C12P19/02C12Y302/01025
Inventor 王耀辉谢敏谢建华何志梅徐莉敏王铮
Owner 东莞泛亚太生物科技有限公司
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