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Genetically engineered bacterium for producing linear malt oligosaccharide generating enzyme and application of genetically engineered bacterium

A malto-oligosaccharide and genetically engineered bacteria technology, applied in the field of microbial engineering, can solve problems such as unfavorable enzyme separation and purification applications, and achieve the effects of industrial application value, high starch conversion rate and product purity, and reduced production and processing costs.

Active Publication Date: 2016-09-21
JIANGNAN UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, researchers at home and abroad have been trying to heterologously express linear maltooligosaccharide-producing enzymes in order to greatly increase their production and break through the secretion limitation of natural enzymes. Glycogenases are cloned and expressed in Escherichia coli, and they are mainly located in the cytoplasm or periplasmic space, which is not conducive to the separation and purification of enzymes and their applications in food, medicine and other fields

Method used

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  • Genetically engineered bacterium for producing linear malt oligosaccharide generating enzyme and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing linear malt oligosaccharide generating enzyme and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing linear malt oligosaccharide generating enzyme and application of genetically engineered bacterium

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Effect test

Embodiment 1

[0023] Example 1 Construction of Bacillus subtilis secretion expression system

[0024] Design the upstream primer (including EcoR I restriction site) according to the gene of linear maltooligosaccharide producing enzyme: 5'-GTATAACGAATTCCAACCGCGAACCG-3'; the downstream primer (including BamH I restriction site): 5'-CCTTAGGATCCAAAGCTTCCCGTTGGG-3 '.

[0025] The target gene SEQ ID NO.1 containing the coding signal peptide sequence was cloned, and the PCR system was: 10×Ex Taq buffer (Mg 2+ Plus) 5 μL, dNTPs (2.5 mM each) 5 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, Ex Taq HS (5U / μL) 0.25 μL, add double distilled water to 50 μL. PCR amplification conditions were: pre-denaturation at 94°C for 4 minutes; further 35 cycles (94°C for 30s, 55°C for 30s, 72°C for 2min); and finally, incubation at 72°C for 10 minutes.

[0026] The PCR product and plasmid pMD 18-T simple were subjected to double enzyme digestion and then gel-cut to recover, ligate...

Embodiment 2

[0029] Embodiment 2 Fermentative production of recombinant linear maltooligosaccharide generating enzyme

[0030] Pick a single colony of Bacillus subtilis containing the expression vector mfa / pST from the preservation plate and inoculate it into 50 mL of LB medium containing 5 μg / mL kanamycin and gibberellin (1% tryptone, 0.5% yeast extract, 1% Sodium chloride, pH 7.0), culture overnight at 37°C and 200rpm in shake flasks; transfer to 50mL TB medium containing 5μg / mL kanamycin and gibberellin (1.2% tryptone , 2.4% yeast extract, 0.4% glycerol, 17mM KH 2 PO 4 , 72mM K 2 HPO 4 , pH 6.0), 25°C, 200rpm shake flask culture for 60h; the obtained fermentation broth was centrifuged at 10000rpm for 10min to remove bacteria, and the obtained fermentation supernatant was the crude enzyme solution. The hydrolytic activity of the crude enzyme solution was determined to reach 36.6U / mL.

Embodiment 3

[0031] Example 3 Determination of Hydrolytic Activity of Recombinant Linear Maltooligosaccharide Generating Enzyme

[0032]Prepare a 1% (w / v) soluble starch solution with citric acid-disodium hydrogen phosphate buffer (20mM, pH 6.5) as the substrate, add 0.2mL of appropriately diluted enzyme solution to 1.8mL of the substrate, and set the temperature at 45°C After reacting for 15 minutes, 2 mL of 3,5-dinitrosalicylic acid (DNS) solution was added to terminate the enzymatic reaction. After boiling in water for 5 minutes, the mixture was cooled in an ice bath. The absorbance was measured at 540 nm and compared with the glucose standard curve. Define the amount of enzyme needed to generate 1 μmol of reducing sugar (calculated as glucose) per minute as 1 enzyme activity unit (U).

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Abstract

The invention discloses a genetically engineered bacterium for producing a linear malt oligosaccharide generating enzyme and application of the genetically engineered bacterium, and belongs to the technical field of microbial engineering. According to the genetically engineered bacterium, secretory expression of the linear malt oligosaccharide generating enzyme in bacillus subtilis is achieved, a method is safe and efficient, the hydrolytic activity reaches 36.6 U / mL, and the enzyme producing capacity is 10 times or above of that of an original bacterium; the obtained recombinant enzyme is an extracellular enzyme, convenience is brought for extraction and purification of large-scale production in the later period, and the product meets the food requirement. Starch is hydrolyzed by the purified recombinant linear malt oligosaccharide generating enzyme to generate linear malt oligosaccharide syrup which does not contain glucose and contains a high proportion of maltopentaose, and the linear malt oligosaccharide syrup can be directly used for producing functional food and also can be used for producing products such as high-purity linear malt oligosaccharides.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing straight-chain maltooligosaccharide-producing enzymes and an application thereof, belonging to the technical field of microbial engineering. Background technique [0002] Linear maltooligosaccharides usually refer to a polymer composed of 3-10 glucose units connected by α-1,4 glycosidic bonds. It is a new sugar source integrating nutrition and function. It has low sweetness, It has the characteristics of good taste and strong moisture retention, and can play a role in reducing sweetness and improving texture in food. [0003] It is particularly important that linear maltooligosaccharides have many unique physiological health functions and can be used as raw materials for functional foods. The main performances are: 1) easy to digest and absorb, it does not need to be digested by salivary amylase and pancreatic amylase, It can be directly hydrolyzed and absorbed by maltase in intesti...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/56C12N15/75C12P19/20A23L33/125C12R1/125
Inventor 李兆丰顾正彪李才明丁宁尹健潘思惠程力洪雁
Owner JIANGNAN UNIV
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