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Biological preparation method of mannose

A technology of mannose and glucose, applied in the field of mannose biological preparation, can solve the problems of lack of reference significance in authenticity, conversion rate value not given, low activity, etc.

Active Publication Date: 2022-03-25
天津怡和生物科技有限责任公司
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  • Application Information

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Problems solved by technology

Referring to this idea, the patent WO2018169957A1 involves this route, and at the same time announced a method for synthesizing mannose from cheap raw materials such as starch and sucrose. An example of the synthesis of mannose by an enzyme system, but the specific conversion rate of the system has not been given, and the authenticity lacks reference significance
Patent CN201910126884.8 discloses a method for preparing mannose 6-phosphate phosphatase and mannose. It also uses cheap raw materials such as starch, sucrose, and glucose to realize conversion and synthesis by constructing an in vitro multi-enzyme catalytic system for phosphorylation and dephosphorylation. Mannose, the conversion rate reaches 67%-93%; it has strong reference significance for the large-scale and low-cost preparation of mannose, but the mannose 6-phosphate phosphatase involved in this patent has a low activity of only 1.8 U / mg (Artificially designed routes for the conversion of starch to value-added mannosyl compounds throughcoupling in vitro and in vivo metabolic engineering strategies. Metabolic Engineering, 2020, 61, 215-224.), resulting in the need to add a lot of enzymes in the reaction system to achieve High conversion rate; at the same time, although the conversion rate of mannose in CN201910126884.8 patent reaches 67%, a certain amount of by-products glucose and fructose are still detected in the system, therefore, mannose with high catalytic activity and high substrate specificity is obtained 6-phosphate phosphatase, essential for the production of mannose from starch

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  • Biological preparation method of mannose
  • Biological preparation method of mannose

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Acquisition of M6PP gene and vector construction

[0026] Through database mining and functional characterization, 10 phosphatases from the genus Thermotoga were screened and obtained, which are mannose 6-phosphate phosphatases. The amino acid sequences of the phosphatases in the NCBI database are WP_101513072.1 (SEQID NO.1), WP_101510852.1 (SEQ ID NO.2), WP_041843828.1 (SEQ ID NO.3), WP_012644897.1 (SEQ ID NO.4), WP_008192576.1 (SEQ ID NO.5), WP_165304640.1 (SEQ ID NO. .6), WP_038054438.1 (SEQ ID NO.7), WP_012895992.1 (SEQ ID NO.8), WP_012310432.1 (SEQ ID NO.9), ABQ46309.1 (SEQ ID NO.10).

[0027] After the codon-optimized amino acid sequences of the above-mentioned mannose 6-phosphate phosphatases are codon-optimized, the corresponding gene nucleotide sequences are respectively as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, Shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20. The gene sequence was sent to...

Embodiment 2

[0028] Example 2 Expression and purification of genes

[0029] The recombinant plasmids respectively containing the above 14 mannose 6-phosphate phosphatase genes were transformed into Escherichia coli for exogenous expression and purification.

[0030] Taking the phosphatase whose database number is WP_101513072.1 as an example,

[0031] (1) Transform E. coli expression recombinant plasmid pET-M6PP072 into E. coli BL21(DE3) to obtain recombinant bacteria.

[0032] (2) Pick a single clone into 5mL LB liquid medium, culture at 37°C, 220r / min until OD 600 0.6-0.8. Transfer the bacterial liquid in 5mL LB medium to 800mL 2YT medium, and cultivate to OD at 37°C and 220rpm 600 When the temperature is 0.6-0.8, cool down to 16°C, add IPTG to a final concentration of 0.5mM, and induce expression for 16h.

[0033] (3) Collect the above cultured bacteria into a 50 mL centrifuge tube, and centrifuge at 5500r / min for 15min;

[0034] (4) Discard the supernatant, and resuspend the cells...

Embodiment 3

[0037] Example 3 Determination of M6PP enzyme activity

[0038] For the M6PP purified above, its dephosphorylation activity against mannose 6-phosphate was measured respectively. The enzyme activity assay system included the following: mannose 6-phosphate (20mM), Mg 2+ (1mM), phosphate buffer (pH 7.5, 50 mM), enzyme (0.5 mg / mL), 55°C, react for 10min. Enzyme activity definition (1U): The amount of mannose catalyzed per minute per mg of M6PP is 1 micromole. The samples were detected by high performance liquid chromatography.

[0039] The enzymatic activity of M6PP obtained by calculation and analysis is shown in Table 1. It was found that the 10 phosphatases screened were WP_165304640.1; WP_041843828.1; ABQ46309.1; WP_012644897.1; ; WP_008192576.1; WP_012895992.1 and WP_101513072.1, the catalytic activity of which is derived from Thermotoga maritima published in patent CN201910126884.8 Thermotoga maritima Compared with M6PP (TmM6PP) (Q9WZB9), all have different degrees of i...

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Abstract

The present inventor screened the mannose 6-phosphate phosphatases with higher activity and specificity through database mining. Therefore, the purpose of the present invention is to screen and obtain a phosphatase with high activity and specificity, which can be efficiently used to catalyze the synthesis of mannose from mannose 6-phosphate through the construction of a multi-enzyme system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a biological preparation method of mannose. Background technique [0002] Mannose is a naturally occurring six-carbon monosaccharide widely found in fruits, vegetables, plant materials, and even the human body. Mannose is the only glyconutrient used clinically. It can be used to treat sugar-deficiency glycoprotein syndrome. It has anti-urinary tract infection and anti-tumor activities. At the same time, mannose has been proven to be a prebiotic without increasing blood sugar levels, and mannose is also a secondary humectant widely used in skin care products. Therefore, mannose has various applications in pharmaceuticals, cosmetics, beverages, food, dairy products, confectionary and animal husbandry. However, due to the high price of mannose, its use in daily products is limited. [0003] Mannose preparation methods mainly focus on acid hydrolysis, chemical isomerizatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/02
CPCC12P19/02
Inventor 杨建刚门燕张同孙媛霞马延和
Owner 天津怡和生物科技有限责任公司
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