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Novel fructosidase as well as encoding gene and applications thereof

An amino acid, a technology in the sequence table, applied in the application, glycosylase, genetic engineering and other directions, can solve the problem of insufficient type of fructosidase

Active Publication Date: 2014-02-05
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a new type of fructosidase, its coding gene, a recombinant expression vector containing the gene and Its transformant and the application of the enzyme, the fructosidase is derived from plantarum lactobacillus, compared with other sources of fructosidase, the enzyme not only has the enzyme activity of decomposing fructooligosaccharides and free fructosyl therefrom, but also Has inulinase activity and fructanase activity; heterologous expression of this enzyme in other lactic acid bacteria can enable lactic acid bacteria that do not have the ability to utilize fructooligosaccharides to obtain fructooligosaccharides

Method used

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  • Novel fructosidase as well as encoding gene and applications thereof
  • Novel fructosidase as well as encoding gene and applications thereof
  • Novel fructosidase as well as encoding gene and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Determination of the position of β-fructofuranosidase in Lactobacillus plantarum and extraction of crude enzyme

[0043] After culturing Lactobacillus plantarum (Lactobacillus plantarum) ST-III at 37°C for 24h, the supernatant and cells were collected by centrifugation (4°C, 12,000×g, 10min). After the supernatant was sterilized by 0.45 μm membrane filtration, it was concentrated to 1 / 20 of the original volume by ultrafiltration through a 25 kDa ultrafiltration membrane, which was the supernatant of the fermentation broth. After the bacterial cells were washed twice by centrifugation with 50mM PBS (pH=6.0), lysozyme was added to 2mg / mL, and then acted at 37°C for 1h. Sonicate intermittently in an ice bath for 8 minutes (400W, ultrasonic 5s, intermittent 5s), and then centrifuge (4°C, 12,000×g, 10min) to collect the supernatant and precipitate respectively. The pellet was resuspended in an equal volume of the above-mentioned PBS, which was the broken cell pell...

Embodiment 2

[0047] Cloning expression and property determination of embodiment 2 fructosidase

[0048] 1. Construction of recombinant expression plasmids

[0049] Using SEQ ID No. 3: 5'-GGAATTCCATATGGAGCTCATCGTCATGATATGGAAT-3' and SEQ ID No. 4: 5'-CCGCTCGAGCCGCTAGCTTCGCAACATC-3' as primers, and Lactobacillus plantarum ST-III genome as a template, a PCR reaction was performed. The PCR reaction system is 20.0μL, and the final concentration of each reaction is: 1×PCR buffer, 1mmol / L Mg 2+ , 0.2mmol / L dNTP, 0.4μmol / L upstream and downstream primers (SEQ ID No.3, SEQ ID No.4), 0.1U / μL Taq DNA polymerase and 2.0ng / μL DNA template, with ddH 2 O to make up to 20.0 μL. The PCR reaction parameters were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, 35 cycles; extension at 72°C for 7 min. Amplified products were detected by 1% agarose gel electrophoresis. Ligate the purified target gene fragments to -T Easy y vector (...

Embodiment 3

[0062] The determination of embodiment 3β-fructofuranosidase properties

[0063] 1. Determination of β-fructofuranosidase enzyme activity

[0064] The β-fructofuranosidase assay uses kestose as the substrate, and the system includes: 50 μL of 20 mM kestose, 50 μL of 50 mM PBS (pH6.0), 15 μL of enzyme solution (11 μg / mL), and make up the volume with water to 200 μL. Mix well at 37°C for about 10 minutes, and then in a water bath at 100°C for 5 minutes. The fructose produced was detected by a fructose assay kit (BioVision, USA). Definition of β-fructofuranosidase enzyme activity: Under the conditions of 37°C and pH 6.0, 1 μmol of fructose produced by hydrolyzing kestose per minute is defined as a unit. And the specific enzyme activity of the enzyme was calculated by measuring the protein content.

[0065] 2. Determination of optimum pH of β-fructofuranosidase

[0066] Prepare different pH buffers (pH3.0–8.0, 0.2M Na 2 HPO 4 / 0.1M citrate buffer solution; pH8.5–9.0, 50mM Tr...

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Abstract

The invention discloses novel fructosidase, an encoding gene of the novel fructosidase, a recombinant expression vector containing the encoding gene, a recombinant expression transformant containing the encoding gene, and applications of novel fructosidase. The novel fructosidase has an amino acid sequence as shown in a sequence table SEQ.ID NO:1. The encoding gene has a nucleotide sequence as shown in a sequence table SEQ.ID NO:2. The invention also provides the recombinant expression vector and the recombinant expression transformant which contain the nucleotide sequence of the encoding gene. The novel fructosidase is derived from lactobacillus plantarum; compared with fructosidase of other sources, the novel fructosidase not only has an enzyme activity of decomposing fructo-oligosaccharide and liberating fructosyl from the fructo-oligosaccharide, but also has an inulase activity and a levan hydrolase activity; lactic acid bacteria without capability of utilizing the fructo-oligosaccharide can obtain the capability of utilizing the fructo-oligosaccharide, by means of heterologously expressing the novel fructosidase in other lactic acid bacteria.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel fructosidase, its coding gene and application. Background technique [0002] Fructooligosaccharides (FOS), also known as fructooligosaccharides, refers to the formation of 1 to 9 fructosyl groups connected to the D-fructosyl groups of sucrose with β-2,1 bonds or β-2,6 bonds. mixture. As a widely recognized prebiotic substance, fructooligosaccharide cannot be directly digested and absorbed by the human body. When it reaches the human gastrointestinal tract, it can selectively stimulate beneficial bacteria in the intestine (such as bifidobacteria and some lactic acid bacteria) Proliferation, and the short-chain fatty acids produced by metabolism can reduce the pH value in the intestine, inhibit the growth and reproduction of exogenous pathogenic bacteria and inherent fungi in the intestine, and play a role in regulating the intestinal flora. [0003] The reason wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/70C12N15/74C12N1/21C12R1/19C12R1/225C12R1/25
CPCC12N9/2431C12Y302/01026
Inventor 陈臣周方方任婧刘景张红发顾瑾麟王渊龙董懿樱
Owner BRIGHT DAIRY & FOOD
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