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Application of novel fructosidase coding gene

A technology of fructosidase and fructanase, applied in the direction of glycosylase, enzyme, hydrolase, etc., can solve the problem of insufficient types of fructosidase

Inactive Publication Date: 2016-02-24
BRIGHT DAIRY & FOOD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 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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  • Application of novel fructosidase coding gene
  • Application of novel fructosidase coding gene
  • Application of novel fructosidase coding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Determination of the position of β-fructofuranosidase in Lactobacillus plantarum and extraction of crude enzyme

[0044] After culturing Lactobacillus plantarum (Lactobacillus plantarum) ST-III at 37° C. for 24 h, the supernatant and cells were collected by centrifugation (4° C., 12,000×g, 10 min). 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 cells were washed twice by centrifugation with 50 mMPBS (pH=6.0), lysozyme was added to 2 mg / mL, and then acted at 37° C. for 1 h. 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

[0049] Cloning expression and property determination of embodiment 2 fructosidase

[0050] 1. Construction of recombinant expression plasmids

[0051] 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 template, PCR reaction was carried out. The PCR reaction system is 20.0μL, and the final concentration of each reaction is: 1×PCRbuffer, 1mmol / L Mg 2+ , the dNTP of 0.2mmol / L, the upstream and downstream primers (SEQIDNo.3, SEQIDNo.4) of 0.4μmol / L, the TaqDNA polymerase of 0.1U / μL and the DNA template of 2.0ng / μL, use 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 ...

Embodiment 3

[0064] The determination of embodiment 3β-fructofuranosidase properties

[0065] 1. Determination of β-fructofuranosidase enzyme activity

[0066] The β-fructofuranosidase assay uses kestose as the substrate, and the system includes: 50 μL 20 mM kestose, 50 μL 50 mMPBS (pH 6.0), 15 μL enzyme solution (11 μg / mL), and the volume is made up to 200 μL with water. 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.

[0067] 2. Determination of optimum pH of β-fructofuranosidase

[0068] Prepare different pH buffers (pH3.0–8.0, 0.2MNa 2 HPO 4 / 0.1M citrate buffer; pH8.5–9.0, 50mM Tris–HCl buffer) t...

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Abstract

The invention discloses an application of a novel fructosidase coding gene. Fructosidase has the amino acid sequence shown as SEQ.ID NO:1 in a sequence table. The coding gene has the nucleotide sequence shown as SEQ.ID NO:2 in the sequence table. The fructosidase originates from Lactobacillus plantarum, compared with fructosidase originating from other sources, the enzyme has the enzymatic activity of decomposing FOS (fructooligosaccharides) and freeing fructose radicals from the FOS as well as the inulinase activity and levanase activity; lactic acid bacteria without the capability of utilizing the FOS can have the capability of utilizing the FOS when the enzyme is heterogeneously expressed in the other lactic acid bacteria.

Description

[0001] This application is a divisional application of a patent application with the application number 201310521419.7, the title of the invention is "A Novel Fructosidase and Its Encoding Gene and Application", and the filing date is October 28, 2013. technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to the application of a novel fructosidase coding gene. Background technique [0003] 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, fructooligosaccharides 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 fatt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26
CPCC12N9/2431C12Y302/01026
Inventor 陈臣刘景张红发王渊龙周方方任婧顾瑾麟董懿樱
Owner BRIGHT DAIRY & FOOD CO LTD
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