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Biological preparation method of mannitol

A technology of mannitol and derivatives, applied in the field of biological preparation of mannitol, can solve the problems of difficult metabolic transformation, metabolic by-products, low selectivity of chemical catalysis, single mannitol and the like

Active Publication Date: 2021-04-20
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has a stable source of raw materials and an unlimited production period. However, the selectivity of the chemical catalytic method is low, and a large amount of by-product D-sorbitol is produced in the reaction process.
For example, in the actual product obtained by using sucrose aqueous formic acid hydrolysis to glucose and fructose and then carrying out catalytic hydrogenation reaction, sorbitol accounts for 80%, while mannitol only accounts for 20% (Wu Guoquan et al. 2004. Chemical Technology Economics (04): 4-6.) Extracting mannitol from natural substances such as seaweed and kelp is another method for industrial production of mannitol. This method can obtain a single mannitol, but it is limited by raw material resources, extraction yield, climatic conditions, and energy consumption. and other restrictions
The production of mannitol by microbial fermentation has the advantages of high reaction selectivity, mild reaction conditions, and low energy consumption, but it has the disadvantages of difficult metabolic transformation of microbial engineering bacteria and easy production of metabolic by-products.

Method used

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  • Biological preparation method of mannitol
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  • Biological preparation method of mannitol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Embodiment 1: Enzyme activity assay in the enzymatic synthesis pathway of mannitol

[0137] Catalytic pathway for the conversion of starch to mannitol by an in vitro multi-enzyme catalytic system see figure 2 . In this example, (1) starch phosphorylase (α-glucan phosphorylase, EC 2.4.1.1, αGP) is derived from Escherichia coli (Escherichia coli, UniProt number P00490); (2) glucose phosphomutase (phosphoglucomutase, EC 5.4.2.2, PGM) is derived from Clostridium thermocellum (Clostridium thermocellum, UniProt number A3DEW8); (3) phosphoglucose isomerase (phosphoglucose isomerase, EC 5.3.1.9, PGI) is derived from Clostridium thermocellum (Clostridium thermocellum , UniProt number A3DBX9); (4) Mannitol 1-phosphate 5-dehydrogenase (mannitol1-phosphate 5-dehydrogenase, EC 1.1.1.17, M1PDH) derived from Escherichia coli (Escherichiacoli, UniProt number P09424); (5) Mannitol 1 -Phosphatase (mannitol 1-phosphatase, EC 3.1.3.22, M1Pase) derived from Eimeria tenella (UniProt number...

Embodiment 2

[0149] Example 2: Synthesis of mannitol from soluble starch catalyzed by multiple enzymes in vitro

[0150] In this example, enzymes are used to catalyze the synthesis of mannitol from soluble starch in vitro. First, six kinds of enzymes were recombinantly expressed (Example 1): αGP derived from Escherichia coli, PGM derived from Clostridium thermocellum, PGI derived from Clostridium thermocellum, M1PDH derived from Escherichia coli, derived from A. tenenella M1Pase from Coccidia, FDH from Thiobacillus.

[0151] Mannitol was quantitatively analyzed by high performance liquid chromatography (HPLC). The chromatographic column used was Bio-Rad Aminex HPX-87H, the mobile phase was 5mM sulfuric acid, the flow rate was 0.6mL / min, the column temperature was 60°C, and the detector used was a differential refractive index detector. Standard sample testing such as Figure 4 As shown in A, the retention time of mannitol is about 10.0 minutes. The concentration of mannitol is directly...

Embodiment 3

[0155] Example 3 In vitro multi-enzyme catalyzed synthesis of mannitol from soluble starch treated with isoamylase

[0156] Starch is a polysaccharide linked by α-1,4 and α-1,6 glycosidic bonds, which cannot be completely phosphorylated by starch phosphorylase. Isoamylase (IA, EC 3.2.1.68) can hydrolyze α-1,6 glycosidic bonds in starch, thereby helping starch phosphorylase to phosphorylate substrates and increasing the yield of mannitol.

[0157] In this example, the isoamylase is derived from Sulfolobus tokodaii (Sulfolobus tokodaii, UniProt number Q973H3). The expression vector pET20b-StIA reported in the literature (Cheng et al.2015. Scientific Reports 5:13184.) was introduced into E. coli BL21(DE3), and recombinant E. coli BL21(DE3) / pET20b-StIA, BL21 containing pET20b-StIA was obtained. (DE3) / pET20b-StIA can express the recombinant isoamylase StIA-his shown in sequence 7 in the sequence list. The protein expression of the recombinant isoamylase StIA-his was carried out a...

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Abstract

The invention discloses a biological preparation method of mannitol. The method for preparing the mannitol through the in-vitro multi-enzyme catalytic reaction comprises the steps that starch and / or a starch derivative serves as a substrate, and the composition for preparing the mannitol is adopted to perform the multi-enzyme catalytic reaction to obtain the mannitol. The composition for preparing mannitol is a composition X, a composition Y or a composition Z. The composition X contains an enzyme X, and the enzyme X is a composition of which the active ingredients are starch phosphorylase, glucose phosphate mutase, phosphoglucose isomerase, mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase and formate dehydrogenase. According to the biological preparation method of mannitol, mannitol is prepared through in-vitro multi-enzyme catalysis, the mannitol can be obtained with the yield of 99% by using various cheap and easily available raw materials, and the biological preparation method is low in production cost, high in substrate conversion rate and product yield, environment-friendly and suitable for popularization.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a biological preparation method of mannitol. Background technique [0002] D-mannitol (hereinafter referred to as mannitol), also known as D-mannitol, hexyl alcohol, cordycepic acid, D-wood honey alcohol, is a natural compound widely present in a variety of bacteria, fungi, algae and in plants. [0003] Mannitol is widely used in food, pharmaceutical, chemical and other fields. In the food industry, mannitol has low water absorption and has a refreshing sweetness, which can be used for anti-sticking of chewing gum, rice cakes and other foods; in the pharmaceutical industry, mannitol can increase plasma osmotic pressure after entering the body, dehydrate tissues, and It is also a good diuretic to reduce intracranial pressure and intraocular pressure; in the chemical industry, polymannitol-oxypropylene ether made of mannitol as a starter is widely used in the plastics industry, and s...

Claims

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Application Information

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IPC IPC(8): C12P7/18C12N9/90C12N9/92C12N9/04C12N9/16C12N15/55C12N9/44C12N9/10C12N9/12C12N9/02
Inventor 游淳魏欣蕾
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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