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 unsatisfactory results and limited application

Inactive Publication Date: 2010-11-10
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
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Effect test

Embodiment 1

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

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

[0045] Prepare primers with ultra-pure water so that the primer concentration is 100 pmol / μL, take 1 μL of primers respectively, mix and react in boiling water at 100 ° C for 15 minutes, and immediately put in ice bath; add 5 μL 10X T4 DNA ligase Buffer, T4 Polynucleotide Kinase 5 μL, add water to the total The volume is 45 μL, 37°C for 30 minutes; 65°C for 20 minutes, ice bath for 2 minutes; add 5 μL T4 DNALigase, react for 2 hours at 16°C, 65°C for 20 minutes.

[00...

Embodiment 2

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

[0048] The PCR product finally obtained in Example 1 was purified and recovered, connected to pMD18-T vector (Takara, Dalian Bao Biological Engineering Company), after sequence analysis and selection of the correct positive clone pMD8-T / MTHase, the obtained sequence is SEQ ID NO : 4. Carry out double 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) through XhoI / EcoRI double digestion, and screen out positive clones pPICZαA / MTHase. After the positive cloning vector was digested with BglII, it was transformed into Pichia pastoris SMD1168H yeast, and the positive cloning strain was screened on high-concentration Zeocin-YPD solid medium ( Pichia pastoris SMD1168H / pPICZαA-MTHase ) and further confirmed by PCR.

Embodiment 3

[0049] Example 3 Culture medium and culture expression conditions

[0050] The positive bacterial strain described in embodiment 2 ( Pichia pastoris MD1168H / pPICZαA-MTHase ) was inoculated in YPD medium, cultured at 30°C for 12-24h, and the shaker speed was 240rpm; when the concentration of the fermentation broth reached OD 600 When = 5.0, transfer the inoculum to BMG culture medium according to the volume ratio of 1:100 for cultivation, the temperature is 30°C, the rotating speed of the shaker is 240rpm, cultivate for 28-35h; centrifuge at 5000g for 5min, collect the thalline, and wash the thalline with PBS 1-2 times, transfer to BMM liquid medium, and when the concentration of the fermentation broth reaches OD=0.5-1.0, induce culture, the induction temperature is 25°C, the shaker speed is 180rpm, and 5- 0.5% methanol at 10mL / L, cultured 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 method for preparing artificially synthesized maltoligosaccharide-based trehalose hydrolase gene sequence and its recombinant protein. Background technique [0002] Trehalose [α-D-glucose-(1,1)-α-D-glucoside] is a non-reducing substance with multiple functions widely present in lower plants, algae, bacteria, fungi, yeast and insects, etc. Sexual disaccharides. In nature, trehalose is not only a storage sugar, but also an important product of stress metabolism, which can protect biological cells and biologically active substances in adverse environmental conditions such as 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 of that of sucrose, and it has no reducing aldehyde groups, so trehalose is widely us...

Claims

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Application Information

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Patent Type & Authority Applications(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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