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Method for synthesizing 6-hexose phosphate by employing enzymic method

A technology of hexose phosphate and enzymatic synthesis, applied in the directions of transferase, fermentation, introduction of foreign genetic material using a carrier, etc., can solve problems such as unfavorable large-scale production, high ATP price, and instability, and achieve industrialized production. , Easy industrial production, fast growth effect

Inactive Publication Date: 2015-06-03
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has been reported in the literature that hexokinase is used to catalyze the synthesis of M-6-P from mannose, but the reaction process requires ATP to provide phosphate groups. Because ATP is expensive and not very stable, it is not easy to store
Based on the above factors, this process is not conducive to large-scale production of M-6-P

Method used

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  • Method for synthesizing 6-hexose phosphate by employing enzymic method
  • Method for synthesizing 6-hexose phosphate by employing enzymic method
  • Method for synthesizing 6-hexose phosphate by employing enzymic method

Examples

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Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 - Preparation of Inorganic Polyphosphate Hexokinase

[0044] Wherein, the inorganic polyphosphate hexokinase in the present invention can be prepared by the following method:

[0045] (1) Cloning the gene expressing inorganic polyphosphate hexokinase in mycobacteria;

[0046] The deoxyribonucleic acid (DNA) of the gene expressing inorganic polyphosphate-type hexokinase in mycobacteria was synthesized by chemical synthesis. Using the DNA as a template, primers were designed according to the sequence of the DNA sequence database (GenBank), and corresponding enzyme cutting sites (NdeI and HindIII) and protective bases were added. The primers used for PCR amplification of the inorganic polyphosphate-type hexokinase gene are as follows:

[0047] Upstream primer: 5'-GCATATGATGACCGCCACCGATTC-3'

[0048] Downstream primer: 5'-GAAGCTTCCGGTTGGTAGCTGTGAC-3'

[0049] The PCR reaction system contained 0.2 μL of inorganic polyphosphate-type hexokinase DNA, 0.5 μL of ups...

Embodiment 2-5

[0057] Prepare a reaction solution, wherein hexose, phosphate donor polyphosphate and / or adenosine triphosphate are used as substrates, recombinant inorganic polyphosphate-type hexokinase and cofactors are added, in a certain pH buffer system, at a certain Reaction under temperature, pH, reaction time. After the reaction, the 6-hexose phosphate was quantitatively detected by high performance anion chromatography, using 25mmol / L sodium hydroxide solution as the mobile phase, isocratic elution, and the 6-hexose phosphate was detected under alkaline conditions. Its content is calibrated by external standard method, and the conversion rate of the corresponding reactant is obtained after calculation. The amount of enzyme added in the synthesis reaction was calibrated by Coomassie brilliant blue protein assay. Reaction condition and result are as shown in table 1 in each embodiment:

[0058] Table 1: Embodiment 2-5 reaction condition and result

[0059]

[0060] Remarks: The a...

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Abstract

The invention relates to a method for synthesizing 6-hexose phosphate by employing an enzymic method. The method comprises the following steps: with hexose as a substrate, polyphosphates and / or triphosadenine salt as phosphate donors, putting a reactant into a pH buffer system; adding inorganic polyphosphoric acid hexokinase and cofactors to the buffer system; and synthesizing the 6-hexose phosphate under the catalysis action of the inorganic polyphosphoric acid hexokinase. Compared with a chemical method, the method for synthesizing the 6-hexose phosphate by employing the enzymic method is green and safe; compared with other methods for synthesizing the 6-hexose phosphate by other enzymic methods, the polyphosphates and / or triphosadenine salt can be used as phosphate donors by the inorganic polyphosphoric acid hexokinase disclosed by the invention. The triphosadenine salt with high cost is replaced with polyphosphates, so that the problem of high production cost is solved.

Description

[0001] This application relates to a Sequence Listing. [0002] This application contains a Sequence Listing in computer readable form, which is hereby incorporated by reference. technical field [0003] The invention belongs to the field of biochemical industry, in particular to a method for enzymatically producing hexose-6-phosphate. Background technique [0004] During protein metabolism, proteins associated with endosomes, cell membranes, and extracellular secretions must be modified in order to be transported to their final location in the cell. Mannose-6-phosphate (mannose-6-phosphate, M-6-P) is a phosphorylated sugar compound that widely exists in living organisms and is closely related to protein modification. M-6-P binds to its corresponding receptor 6-phosphate receptor (mannose-6-phosphate receptor, M-6-P receptor), which plays an important role in the process of lysosomal enzyme effect. M-6-P is the sorter and deliveryman of lysosomal enzymes. [0005] Lysosom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12N9/12C12N15/70
Inventor 陈必强喻小春刘珞谭天伟
Owner BEIJING UNIV OF CHEM TECH
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