Preparation method of maltooligosyltrehalose hydrolase gene sequence and recombinant protein thereof

A technology based on trehalose hydrolase and maltooligosaccharides, which is applied in the biological field and can solve problems such as limited application and unsatisfactory results

Inactive Publication Date: 2013-06-12
CHENGDU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, researchers have carried out gene cloning and recombinant expression of the two enzymes, but the effect of expressing these two enzymes in E. coli is not satisfactory, which limits the application of enzymatic pathways in the synthesis of trehalose

Method used

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  • Preparation method of maltooligosyltrehalose hydrolase gene sequence and recombinant protein thereof
  • Preparation method of maltooligosyltrehalose hydrolase gene sequence and recombinant protein thereof
  • Preparation method of maltooligosyltrehalose hydrolase gene sequence and recombinant protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Artificial modification and synthesis of malto-oligosaccharyl trehalose hydrolase (MTHase) gene

[0044] According to the above coded amino acid sequence, the forward and reverse strand DNA primers (as shown in SEQ ID NO: 6 to SEQ ID NO: 47) for gene splicing are designed according to the principle of yeast codon preference. XhoI restriction site is added to the 5'end, and EcoR I restriction site sequence is added to the 3'end to facilitate connection with the vector.

[0045] Prepare primers with ultrapure water to make the concentration of primers: 100pmol / μL. Take 1μL of the primers respectively. After mixing, let them react in boiling water at 100℃ for 15 minutes, then immediately bath them on ice; add 5μL of 10X T4DNA ligase Buffer, T4 Polynucleotide Kinase 5μL, and add water to the total The volume is 45μL, 37°C for 30 minutes; 65°C for 20 minutes, ice bath for 2 minutes; 5μL T4 DNALigase is added, 16°C for 2 hours, 65°C for 20 minutes.

[0046] Take 1μL of th...

Embodiment 2

[0047] Example 2 Construction of recombinant expression plasmid and screening of positive clones

[0048] The PCR product finally obtained in Example 1 was purified and recovered, and connected to pMD18-T vector (Takara, Dalian Bao Bioengineering Company). After sequence analysis, the correct positive clone pMD8-T / MTHase ​​was selected, and the sequence obtained was SEQID NO : 4. Double-enzyme digestion with XhoI / EcoRI, isolate and recover the MTHase ​​gene fragment of about 1.7kb, connect it to the pPICZαA yeast expression vector (Invitrogen, USA) digested with XhoI / EcoRI, and screen positive clones pPICZαA / MTHase. After the positive cloning vector was digested with BglII single enzyme, the Pichia pastoris SMD1168H yeast was transformed, and the positive clone strain was selected on the high concentration Zeocin-YPD solid medium ( Pichia pastoris SMD1168H / pPICZαA-MTHase ), and perform further PCR identification and confirmation.

Embodiment 3

[0049] Example 3 Medium and culture expression conditions

[0050] The positive strain described in Example 2 ( Pichia pastoris MD1168H / pPICZαA-MTHase ) Inoculated in YPD medium, cultivated at 30°C for 12-24 hours, and the shaker speed is 240 rpm; when the concentration of the fermentation broth reaches OD 600 =5.0, transfer the inoculum to BMG medium at a volume ratio of 1:100, temperature 30℃, shaker speed 240rpm, culture for 28-35h; centrifuge at 5000g for 5min, collect the bacteria, and wash the bacteria with PBS 1-2 times, transfer to BMM liquid medium, when the concentration of the fermentation broth reaches OD=0.5-1.0, induce culture, the induction temperature is 25℃, the shaker speed is 180rpm, and 5- is added to the medium every 12h. 10mL / L of 0.5% methanol, incubate for 72 hours;

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Abstract

The preparation method of the invention comprises the following steps: the genetic codon of maltooligosyltrehalose hydrolase (MTHase) gene is modified artificially, yeast preferred codon is synthesized and the A / T content of the genetic codon is modified, a MTHase gene sequence of which structure of genetic codon is optimized and A / T content is reduced is obtained; then the complete MTHase gene is cloned to the yeast expression vector pPICZ alpha A, recombinant vector is screened out and introduced in Pichia Pastoris cell to perform recombinant expression; and a yeast strain which can efficiently and stably express MTHase can be obtained. The enzyme activity of MTHase which is secreted by the yeast strain in the supernate of expression culture medium is more than 800mg / L, thus the preparation method of the invention lays a foundation for the industrial production of trehalose which adopts enzyme process and uses starch as raw material.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a synthetically modified malt oligosaccharide trehalose hydrolase gene sequence and a method for preparing a recombinant protein thereof. Background technique [0002] Trehalose [α-D-glucose-(1,1)-α-D-glucoside] is a kind of non-reducing non-reducing compound that is widely found in lower plants, algae, bacteria, fungi, yeasts and insects. Sex disaccharides. In nature, trehalose is not only a storage sugar, but also an important product of stress metabolism. It protects biological cells and biologically active substances from dehydration, drought, high temperature, freezing, high osmotic pressure, and toxic reagents. The function of protecting the activity from being destroyed. Trehalose also has good moisturizing properties. Its sweetness is only half that of sucrose, and it has no reducing aldehyde groups. Therefore, trehalose is widely used in the freezing and preservation of food and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N9/24C12N15/81C12R1/84
Inventor 苟兴华王卫李德华刘达玉张佳敏郭秀兰唐仁勇胡海洋
Owner CHENGDU UNIV
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