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Preparation method of isomalto-oligosaccharide and catalyst of isomalto-oligosaccharide

A technology of isomaltooligosaccharide and maltose syrup, which is applied in the manufacture of isomaltooligosaccharide and its catalyst field, can solve the problems of complex equipment and high operability, and achieve the effect of low enzyme loss, high catalytic activity and remarkable effect

Active Publication Date: 2016-01-06
森大(天津)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

[0005] However, after the catalytic reaction is completed, the immobilized enzyme needs to be recycled and reused. At present, column type, membrane reactor and other devices are commonly used in factories to realize the recovery of immobilized enzyme. This operation requires complicated equipment and high operability.

Method used

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  • Preparation method of isomalto-oligosaccharide and catalyst of isomalto-oligosaccharide
  • Preparation method of isomalto-oligosaccharide and catalyst of isomalto-oligosaccharide
  • Preparation method of isomalto-oligosaccharide and catalyst of isomalto-oligosaccharide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Obtaining of α-glucosidase coding gene fragment

[0052] The mRNA of Aspergillus niger (Aspergillus niger) CBS513.88 strain was extracted by liquid nitrogen grinding method, under the action of reverse transcriptase, RT-PCR was carried out with Oligo(dT) as primer, and cDNA was prepared by reverse transcription. With this cDNA as a template, according to the α-glucosidase-related genes published by NCBI—agA, agB, agC, agD, agE, agF gene sequence, the upstream and downstream primers AGA-1 (SEQ ID NO: 3) and AGA-2 ( SEQ ID NO: 4), AGB-1 (SEQ ID NO: 5) and AGB-2 (SEQ ID NO: 6), AGC-1 (SEQ ID NO: 7) and AGC-2 (SEQ ID NO: 8), AGD-1 (SEQ ID NO: 9) and AGD-2 (SEQ ID NO: 10), AGE-1 (SEQ ID NO: 11) and AGE-2 (SEQ ID NO: 12), AGF-1 (SEQ ID NO: 13) and AGF-2 (SEQ ID NO: 14), and by the method of PCR The gene fragments of agA of 1815bp, agB of 2892bp, agC of 2229bp, agD of 2958bp, agE of 1764bp and agF of 1968bp were amplified respectively.

Embodiment 2

[0053] Example 2: Construction of α-glucosidase single gene integrated expression vector

[0054] The gene fragment obtained in Example 1 was cloned into the SnaBI site of the pPIC9k plasmid to obtain recombinant plasmids pPIC9k-agA, pPIC9k-agB, pPIC9k-agC, pPIC9k-agD, pPIC9k-agE, and pPIC9k-agF.

Embodiment 3

[0055] Example 3: Construction of α-glucosidase double gene tandem integration expression vector

[0056] Using the recombinant plasmid in Example 2 as a template to design upstream and downstream universal primers AOX1 (SEQ ID NO: 15) and AOX2 (SEQ ID NO: 16), obtain the promoter sequence and terminator sequence derived from pPIC9k by PCR amplification. α-glucosidase-related gene fragments, AOX-agA, AOX-agB, AOX-agC, AOX-agD, AOX-agE, AOX-agF. And respectively cloned into the XbaI site of the corresponding plasmid in step 2 (the corresponding plasmid was digested by XbaI and pfu and other DNA polymerases were filled in, and the corresponding fragments were connected), to obtain the recombinant plasmid pPIC9k-agAB of tandem expression of α-glucosidase-related genes, pPIC9k-agAC, pPIC9k-agAD, pPIC9k-agAE, pPIC9k-agAF, pPIC9k-agBC, pPIC9k-agBD, pPIC9k-agBE, pPIC9k-agBF, pPIC9k-agCD, pPIC9k-agCE, pPIC9k-agCF, pPIC9k-agDE, pPIC9k- agDF, pPIC9k-agEF.

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Abstract

The invention provides a method of producing isomalto-oligosaccharide by an enzyme method through taking starch and a derivative thereof as raw materials. The isomalto-oligosaccharide is formed by performing combinational expression on an alpha-glucosidase encoding gene from aspergillus niger and performing catalyzed synthesis on immobilized alpha-glucosidase, and quick separation and recycling of a product-immobilized enzyme mixture are realized by a non-aqueous double-phase system. According to the method, the isomalto-oligosaccharide is catalytically produced by adding immobilized alpha-glucosidase microspheres through the double-phase reaction system; after a reaction, the immobilized enzyme can be automatically distributed into an organic phase, thus realizing separation of the immobilized enzyme from a product. The method has the characteristics of mild reaction condition, no need of adding other crosslinking agents, good economy, reduction of the production cost, high enzyme activity recycling rate of the immobilized enzyme, good operation stability and the like. The isomalto-oligosaccharide product obtained by the method can be widely applied to the important fields of medicines, foods, feeds and the like.

Description

Technical field: [0001] The invention relates to a production method of isomaltose oligosaccharide, in particular to a method for breeding a catalyst-producing strain, a method for preparing an enzyme preparation for catalysis by a microbial fermentation method, a method for immobilizing a catalyst, a method for effectively separating a catalyst, a method for reusing it, and a low-cost Process for the continuous production of polyisomaltose. Background technique: [0002] Isomaltooligosaccharides (hereinafter referred to as IMO), also known as branched oligosaccharides, isomaltooligosaccharides, etc., are monosaccharides with at least one a-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, b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/26C12N11/14C12N11/08C12N11/04C12P19/14C12P19/12C12P19/04C12P19/00C12R1/84
CPCY02P20/50
Inventor 王正祥路福平牛丹丹
Owner 森大(天津)生物科技有限公司
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