Technological process for synthesizing mycose by enzyme process

A technology of enzymatic synthesis and process method, applied in DNA preparation, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc., can solve problems such as high cost, achieve significant economic benefits and reduce costs.

Inactive Publication Date: 2006-03-01
李宝健
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Currently, the cost of trehalose production methods is relatively high, and...

Method used

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  • Technological process for synthesizing mycose by enzyme process
  • Technological process for synthesizing mycose by enzyme process
  • Technological process for synthesizing mycose by enzyme process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Cloning and Sequence Analysis of Grifola frondosa Trehalose Synthase Gene

[0061] A pair of primers were designed according to the gene sequence of Grifola frondosa trehalose synthase gene recorded on the gene bank website: primer 1: 5′CC GAA TTC ATG GCT CCT CCC CAC CAG-3′, primer 2: 5′GC TCT AGA TCCCTG CAC ATG CAG TTC-3 ', utilized the RT-PCR method to amplify an about 2.2kb fragment from the total RNA of Grifola frondosa, and this fragment was connected to the pMD-T vector (named as PMD-T-TR), and carried out sequencing. The fragment amplified from Grifola frondosa RNA is 2199bp in full length and encodes 732 amino acids. According to DNASIS software analysis, compared with the reported sequence, there are 98.5% of the bases identical, and the amino acid sequence has 99.2% similarity.

Embodiment 2

[0062] Example 2 Cloning and sequence analysis of Escherichia coli sucrose phosphorylase gene

[0063] Extract the total DNA of Escherichia coli, use primer 1: 5'CC GAA TTC ATG AAA CAG AAAATT ACG-3', primer 2: 5'GC TCT AGA TTT AAT CCA CAT AACCTG-3' for PCR amplification, and get about 1.7kb The fragments were connected to the PMD-T carrier (named as PMD-T-SUC) and sequenced. The gene has a full length of 1680bp and encodes 559 amino acids. According to DNASIS software analysis, the sequencing result is exactly the same as the sequence of Escherichia coli sucrose phosphorylase in gene BANK.

Embodiment 3

[0064] Example 3 Construction of Grifola frondosa Trehalose Synthase Gene Yeast Expression Vector

[0065] Such as figure 1 As shown, the trehalose synthase gene on the PMD-T-TR vector was double-digested with EcoRI and BamHI, and ligated with the pPICZα plasmid after the double-digestion, and the ligated plasmid was named pPICZα-TR. Such as figure 2 As shown, both the results of single enzyme digestion and double enzyme digestion indicate that the trehalose synthase gene has been connected to the carrier. The PCR reaction was carried out using the carrier as a template, and the target fragment was also amplified, and the sequencing results showed that it was the same as the previous sequencing results.

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Abstract

This invention discloses method synthesizing trehalose by enzymes. It includes steps as follows: a. clone huishuhua trehalose synthesizing enzyme gene and E. coli sucrose phosphorylase gene separately; b. construct expression vector of huishuhua trehalose synthesizing enzyme gene and E. coli sucrose phosphorylase gene separately; c. transform and express the expression vector of huishuhua trehalose synthesizing enzyme gene and E. coli sucrose phosphorylase gene separately in yeast, construct and purify the recombinant protein; d. add the said purified recombinant protein to synthesize trehalose in the reaction solution which has sucrose and glucose as main component.

Description

technical field [0001] The invention relates to a method for preparing trehalose, in particular to a process for synthesizing trehalose by applying genetic engineering, enzyme engineering and fermentation engineering. Background technique [0002] Trehalose (trehalose) is a stable non-reducing disaccharide, which is composed of two glucopyranose molecules linked by 1,1 glycosidic bonds (Sugimoto Toshiyuki, 1994). Trehalose can exist in several solid forms, the most common being dihydrate, with a melting point of 97°C. When heated to 130°C to make it lose crystal water and become anhydrous crystals, the melting point can reach 214-216°C. Its aqueous solution is stable, colorless and odorless, with a slightly sweet taste and no caramelization. The physical and chemical properties of trehalose are very stable, and it cannot reduce Fehling's reagent, nor can it be hydrolyzed by α-glucosidase, but it can be hydrolyzed into two glucose molecules under strong acid conditions. [...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/81C12P19/12C12P19/14C12N15/10C12N15/63C12N1/19
Inventor 李宝健徐志祥
Owner 李宝健
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