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Method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion

A technology of isomaltose oligosaccharide and glucosidase, applied in the field of enzyme engineering, can solve the problems of low conversion rate and long production time, and achieve the effect of remarkable catalytic performance

Active Publication Date: 2016-09-07
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It can be seen that the current IMO production process still has the problems of long production time and low conversion rate

Method used

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  • Method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion
  • Method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion
  • Method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Obtaining of α-glucosidase coding gene fragment

[0045] Total RNA of Aspergillus niger NRRL3135 was extracted with TRNzol total RNA extraction reagent. Using total RNA as a template, referring to the instructions of the RT-PCR kit, using oligo(dT) as a primer to reverse transcribe the first-strand cDNA, and then using the first-strand cDNA as a template, primers F1 (SEQ ID NO: 5) and R1 (SEQ ID NO: 6) carried out PCR amplification to produce Aspergillus niger α-glucosidase gene agD ( figure 1 ). The PCR product was ligated with the plasmid pMD19-T simple vector to obtain the recombinant plasmid pMD-agD, which was transformed into Escherichia coli JM109 competent cells. Positive transformants were screened, and their plasmids were extracted for enzyme digestion verification. The correct recombinant plasmid verified by enzyme digestion was then sequenced (sequence SEQ ID NO: 1, its amino acid sequence is SEQ ID NO: 2).

Embodiment 2

[0046] Embodiment 2: Utilize error-prone PCR method to construct α-glucosidase mutant library

[0047] Nucleotide mutations were introduced into the α-glucosidase gene agD by error-prone PCR in vitro. The reaction conditions for error-prone PCR are as follows:

[0048]

[0049] PCR amplification conditions: 94°C for 3 min; 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1.5 min; 72°C for 10 min. The primers used were primers F1 (SEQ ID NO:5) and R1 (SEQ ID NO:6).

[0050] The error-prone PCR amplification product (named after agDX) was purified by a DNA purification and recovery kit, digested with restriction endonucleases Xba I and Kpn I, and digested with the correspondingly digested plasmid pHY-WZX (Dandan Niu and Zhengxiang Wang. J Ind Microbiol Biotechnol, 2007, 34:357-362) were connected to construct the recombinant plasmid pHY-WZX-agDX. Take 8 μL of recombinant plasmid pHY-WZX-agDX DNA and Bacillus licheniformis CBB3008 competent cells, mix evenly, trans...

Embodiment 3

[0052] Example 3: Screening of thermostable mutant strains

[0053] Pick a single colony on the LB plate, use 96-well plate to culture with LB liquid based on 30-32°C for 55-70 hours, the liquid volume is 200-250 μL, and at the same time, use the initial recombinant bacteria B. licheniformis CBB3008 (pHY-WZX) For control.

[0054] Centrifuge the 96-well plate I to remove bacteria, and transfer 10 μL supernatant from each well to another 96-well plate II. And the 96-well plate was placed at 60°C for heat treatment for 60 minutes. Add 50 μL of 0.2% substrate o-methoxyphenyl-α-D-glucoside to each well, and incubate at 30° C. for 10 minutes. Add 80 μL of 2.5 mol / L NaOH solution to terminate the reaction (in the control group, the stop solution is added first, and then the substrate is added). Prepare diazonium salt solution at the same time: take 0.2g / L NaNO 2 100mL, then add 5mL of 3mol / L HCl to make the solution acidic, incubate at 30°C for 5min, add 10mL of 50g / L aniline, ...

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Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for preparing isomaltooligosaccharides from alpha-glucosidase through synchronous saccharification and glucoside conversion. The half-life t1 / 2 of the alpha-glucosidase is increased by 3.7 times at 66 DEG C; the proportion of core components (IG2 (Immune Globulin 2)+P+IG3 (Immune Globulin 3)) of the isomaltooligosaccharides, which are prepared by adopting the alpha-glucosidase, high-temperature-resistant alpha-amylase, intermediate-temperature alpha-amylase, beta-amylase and pullulanase to carry out starch liquefaction and through the synchronous saccharification and glucoside conversion, in dry substances reaches 52 to 59 percent.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a method for obtaining and efficiently preparing a heat-resistant α-glucosidase mutant, a new method for starch liquefaction, and a process for synchronously performing saccharification and transglycosidation to realize the preparation of high-quality isomaltooligosaccharides. Background technique: [0002] Isomaltooligosaccharides (hereinafter referred to as IMO), also known as branched oligosaccharides, isomaltooligosaccharides, etc., are monosaccharides with at least one α-1,6 glycosidic bond between glucose. It is a class of oligosaccharides ranging from 2 to 6. Its main ingredient is isomaltose (IG 2 ), panose (P), isomaltotriose (IG 3 ) and isomaltotetraose (G n )Wait. Isomaltooligosaccharide is a non-digestible oligosaccharide, which cannot be used as a direct source of energy for the human body, but it can selectively proliferate beneficial bact...

Claims

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Application Information

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IPC IPC(8): C12N9/26C12N15/56C12P19/22C12P19/16C12P19/14
CPCC12N9/2408C12P19/14C12P19/16C12P19/22C12Y302/0102
Inventor 牛丹丹
Owner FUZHOU UNIV
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