CN111763696A - Application of Protein PfuPGM as Glucose Phosphomutase in the Production of Inositol - Google Patents

Abstract

The invention discloses application of protein PfuPGM as glucose phosphoglucomutase in producing inositol. The amino acid sequence of the protein PfuPGM is shown as SEQ ID NO: 1 is shown. Experiments have shown that the protein pfuPGM has the activity of converting glucose 1 phosphate to glucose 6 phosphate, i.e., glucose phosphoglucomutase activity. The in vitro multi-enzyme catalytic system constructed by the proteins PfuPGM, alpha GP, IPS and IMP, and the whole cell system constructed by the cells expressing the proteins PfuPGM, alpha GP, IPS and IMP can produce inositol from starch, and the conversion rate of the inositol is more than 55%. The invention has important application value.

Description

Application of Protein PfuPGM as Glucose Phosphomutase in the Production of Inositol

technical field

The invention belongs to the field of biotechnology, in particular to the application of protein PfuPGM as a glucose phosphate mutase in the production of inositol.

Background technique

Inositol, also known as cyclohexanol, belongs to the B group of water-soluble vitamins. Inositol is an essential substance for the growth of humans, animals and microorganisms, and is widely used in medicine, food, feed and other industries.

In vitro multi-enzyme catalyzed preparation of inositol has received extensive attention from researchers. The patent (CN 106148425 B) discloses a preparation method of inositol, which uses starch, cellulose or its derivatives as substrates, and in a multi-enzyme reaction system, the substrate is converted into muscle by in vitro multi-enzyme efficient catalysis. alcohol. Haruyuki Atomi et al. disclosed an in vitro multi-enzyme-catalyzed starch conversion method to produce inositol, which can obtain a molar conversion rate of 96% by supplementing NAD ⁺ . The patent (CN 105925643 A) discloses a method for preparing inositol by enzymatic catalysis, which takes polysaccharide or oligosaccharide with glucose as the substrate as the substrate, and produces inositol through multi-step enzymatic conversion. The researchers are devoted to the optimization and modification of each enzyme involved in the reaction pathway, in order to improve the robustness of the multi-enzyme catalyzed production of inositol in vitro.

SUMMARY OF THE INVENTION

The object of the present invention is the production of inositol.

The present invention first protects the application of protein PfuPGM, which can be any of a-d:

a Application as glucose phosphomutase;

The application of b in the preparation of glucose phosphomutase;

c in the production of inositol;

d Use in the manufacture of products for the production of inositol.

The protein PfuPGM can be e or f or g or k as follows:

The amino acid sequence of e is the protein shown in SEQ ID NO: 1;

f amino acid sequence is the protein shown in SEQ ID NO: 3;

g fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 1 or SEQ ID NO: 3;

k is a protein with glucose phosphomutase activity obtained by substituting and/or deleting and/or adding one or several amino acid residues to the protein indicated by e or f or g.

Among them, SEQ ID NO: 1 consists of 455 amino acid residues. SEQ ID NO: 3 consists of 463 amino acid residues.

In order to facilitate purification of the protein in e or f, a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 1 or SEQ ID NO: 3.

For the protein in the above k, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The protein in the above k can be artificially synthesized, or can be obtained by first synthesizing its encoding gene and then carrying out biological expression.

The gene encoding the protein in the above k can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and/or carrying out missense mutation of one or several base pairs, And/or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

The present invention also protects the application of a nucleic acid molecule encoding any of the above-mentioned proteins PfuPGM or "an expression cassette, recombinant vector or recombinant microorganism containing a nucleic acid molecule encoding any of the above-mentioned proteins PfuPGM", which can be any of a-d :

a Application as glucose phosphomutase;

The application of b in the preparation of glucose phosphomutase;

c in the production of inositol;

d Use in the manufacture of products for the production of inositol.

In the above-mentioned application, the nucleic acid molecule encoding any of the above-mentioned proteins PfuPGM can be a DNA molecule shown in x or y or m or n:

The x coding region is the DNA molecule shown in SEQ ID NO: 2;

The y nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

m has 75% or more identity with the nucleotide sequence defined by x or y, and encodes a DNA molecule of any of the above-mentioned proteins PfuPGM;

n A DNA molecule that hybridizes to a nucleotide sequence defined by x or y under stringent conditions and encodes any of the proteins described above, PfuPGM.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Wherein, SEQ ID NO: 2 consists of 1368 nucleotides, and the nucleotide sequence shown in SEQ ID NO: 2 encodes the amino acid sequence shown in SEQ ID NO: 1.

Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein PfuPGM of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 80% or higher identity to the nucleotide sequence of the protein PfuPGM isolated by the present invention, as long as they encode the protein PfuPGM, are derived from the nucleotide sequence of the present invention and are equivalent sequences of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 80% or higher, or 85% or higher with the nucleotide sequence of the present invention encoding the protein PfuPGM consisting of the amino acid sequence shown in SEQ ID NO: 1, or 90% or more, or 95% or more identical nucleotide sequences. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above-mentioned application, the recombinant vector containing the nucleic acid molecule encoding any of the above-mentioned proteins PfuPGM can specifically be the recombinant plasmid pET-20b-PfuPGM-CO; The small DNA fragment between the restriction endonucleases NdeI and XhoI is replaced with the DNA molecule shown in SEQ ID NO: 2 to obtain a recombinant plasmid.

In the above application, the recombinant microorganism containing the nucleic acid molecule encoding any of the above-mentioned proteins PfuPGM may be a recombinant bacteria obtained by introducing the recombinant plasmid pET-20b-PfuPGM-CO into the starting microorganism. The starting microorganism may be Escherichia coli. The Escherichia coli can specifically be Escherichia coli BL21 (DE3).

In any of the above-mentioned applications, the production of inositol is based on starch as a substrate.

The present invention also protects methods of producing inositol.

The method for producing myo-inositol protected by the present invention can specifically be method one, comprising the steps:

h Preparation of reaction system 1 (in vitro multi-enzyme catalytic system); reaction system 1 contains buffer, divalent magnesium ions, glucan phosphorylase (αGP), any of the above-mentioned proteins PfuPGM, inositol 3-phosphate synthesis enzymes (IPS), inositol monophosphatase (IMP) and starch;

i Take reaction system 1, and react at 65-75°C (eg 65-70°C, 70-75°C, 65°C, 70°C or 75°C) for more than 12h (eg 12h, 24h, 36h, 48h) to obtain reaction product 1;

jIsolation of myo-inositol from reaction product 1.

In the step h, the buffer may be a phosphate buffer. The phosphate buffer can be specifically 40mM phosphate buffer (pH 7.0). The starch can be soluble starch.

In the step h, the reaction system 1 may contain 30-50 mM (eg 30-40 mM, 40-50 mM, 30 mM, 40 mM or 50 mM) phosphate buffer (pH 7.0), 4-6 mM (eg 4-5 mM, 5-6mM, 4mM, 5mM or 6mM) of divalent magnesium ions, 0.8-1.2U/ml (eg 0.8-1.0U/ml, 1.0-1.2U/ml, 0.8U/ml, 1.0U/ml or 1.2U /ml) of αGP, 0.8-1.2U/ml (such as 0.8-1.0U/ml, 1.0-1.2U/ml, 0.8U/ml, 1.0U/ml or 1.2U/ml) of protein PfuPGM, 0.8-1.2 U/ml (eg 0.8-1.0U/ml, 1.0-1.2U/ml, 0.8U/ml, 1.0U/ml or 1.2U/ml) of IPS, 0.8-1.2U/ml (eg 0.8-1.0U/ml) ml, 1.0-1.2U/ml, 0.8U/ml, 1.0U/ml or 1.2U/ml) of IMP and 90-110g/L (eg 90-100g/L, 100-110g/L, 90g/L, 100g/L or 110g/L) of soluble starch.

In the step h, reaction system 1 specifically contains 40mM phosphate buffer (pH7.0), 5mM divalent magnesium ions, 1U/ml αGP, 1U/ml protein PfuPGM, 1U/ml IPS, 1U /ml of IMP and 100g/L of soluble starch.

In the above method, the reaction system 1 may further contain isoamylase (IA). The concentration of isoamylase in reaction system 1 is 0.8-1.2U/ml (eg 0.8-1.0U/ml, 1.0-1.2U/ml, 0.8U/ml, 1.0U/ml or 1.2U/ml).

Specifically, reaction system 1 contains 40 mM phosphate buffer (pH 7.0), 5 mM divalent magnesium ions, 1 U/ml αGP, 1 U/ml protein PfuPGM, 1 U/ml IPS, 1 U/ml IMP, 1 U/ml of IA and 100 g/L of soluble starch.

In the above method, αGP is derived from Thermotogamaritima , and the gene number on KEGG is TM1168. IPS is derived from Archaeoglobus fulgidus , and the gene number on KEGG is AF1794. IMP is derived from Thermotogamaritima , and the gene number on KEGG is TM1415. IA is derived from Sulfolobustokodaii , and the gene number on KEGG is ST0928. All enzymes were heterologously expressed and purified in E. coli.

In the step i, the reaction at 65-75°C (eg 65-70°C, 70-75°C, 65°C, 70°C or 75°C) for more than 12h (eg 12h, 24h, 36h, 46h, 48h) can be completed in one step, It can also be recycled multiple times.

The method for producing myo-inositol protected by the present invention can be specifically method two, comprising the steps:

o Prepare reaction system 2 (which is a whole-cell system); reaction system 2 contains buffer solution, divalent magnesium ions, cells expressing glucan phosphorylase (αGP), cells expressing any of the above-mentioned proteins PfuPGM, expressing Cells expressing inositol 3-phosphate synthase (IPS), cells expressing inositol monophosphatase (IMP), and starch;

p take reaction system 2, react at 65-75°C (eg 65-70°C, 70-75°C, 65°C, 70°C or 75°C) for more than 12h (eg 12h, 24h, 36h, 48h) to obtain reaction product 2;

q Isolation of myo-inositol from reaction product 2.

In the step o, the buffer can be a phosphate buffer. The phosphate buffer may specifically be a 10mM phosphate buffer (pH 7.0). The starch can be soluble starch.

In the step o, the reaction system 2 may contain 8-12mM (such as 8-10mM, 10-12mM, 8mM, 10mM or 12mM) of phosphate buffer (pH7.0), 4-6mM (such as 4-5mM, 5-6mM, 4mM, 5mM or 6mM) of divalent magnesium ions, 0.12-0.16g DCW/L (eg 0.12-0.14g DCW/L, 0.14-0.16g DCW/L, 0.12g DCW/L, 0.14g DCW/ L, 0.16g DCW/L) of αGP-expressing cells, 0.06-0.10g DCW/L (eg 0.06-0.08g DCW/L, 0.08-0.10g DCW/L, 0.06gDCW/L, 0.08g DCW/L or 0.10g DCW/L) cells expressing the protein PfuPGM, 0.4-0.6g DCW/L (such as 0.4-0.5g DCW/L, 0.5-0.6g DCW/L, 0.4g DCW/L, 0.5g DCW/L or 0.6g DCW/L) g DCW/L) cells expressing IPS, 0.06-0.10g DCW/L (eg 0.06-0.08g DCW/L, 0.08-0.10g DCW/L, 0.06g DCW/L, 0.08g DCW/L or 0.10g DCW /L) IMP-expressing cells and 90-110 g/L (eg, 90-100 g/L, 100-110 g/L, 90 g/L, 100 g/L, or 110 g/L) of soluble starch.

In the above method, the reaction system 2 may further contain cells expressing isoamylase (IA).

In the step o, the reaction system 2 may contain 8-12mM (such as 8-10mM, 10-12mM, 8mM, 10mM or 12mM) of phosphate buffer (pH7.0), 4-6mM (such as 4-5mM, 5-6mM, 4mM, 5mM or 6mM) of divalent magnesium ions, 0.12-0.16g DCW/L (eg 0.12-0.14g DCW/L, 0.14-0.16g DCW/L, 0.12g DCW/L, 0.14g DCW/ L, 0.16g DCW/L) of αGP-expressing cells, 0.06-0.10g DCW/L (eg 0.06-0.08g DCW/L, 0.08-0.10g DCW/L, 0.06gDCW/L, 0.08g DCW/L or 0.10g DCW/L) cells expressing the protein PfuPGM, 0.4-0.6g DCW/L (such as 0.4-0.5g DCW/L, 0.5-0.6g DCW/L, 0.4g DCW/L, 0.5g DCW/L or 0.6g DCW/L) g DCW/L) cells expressing IPS, 0.06-0.10g DCW/L (eg 0.06-0.08g DCW/L, 0.08-0.10g DCW/L, 0.06g DCW/L, 0.08g DCW/L or 0.10g DCW /L) IMP expressing cells, 0.06-0.10g DCW/L (eg 0.06-0.08g DCW/L, 0.08-0.10g DCW/L, 0.06g DCW/L, 0.08g DCW/L or 0.10g DCW/L) IA-expressing cells and 90-110 g/L (eg, 90-100 g/L, 100-110 g/L, 90 g/L, 100 g/L, or 110 g/L) of soluble starch.

Specifically, reaction system 2 contains 10 mM phosphate buffer (pH 7.0), 5 mM divalent magnesium ions, 0.14 g DCW/L cells expressing αGP, 0.08 g DCW/L cells expressing protein PfuPGM, 0.5 g DCW/L L IPS expressing cells, 0.08 g DCW/L IMP expressing cells, 0.08 g DCW/L IA expressing cells and 100 g/L soluble starch.

Any of the glucose phosphomutases described above can convert glucose 1 phosphate into glucose 6 phosphate.

Experiments have shown that the protein PfuPGM has the activity of converting glucose 1 phosphate into glucose 6 phosphate, that is, glucose phosphate mutase activity. The specific activity of glucose phosphate mutase reaches 16.2U/mg, and has high stability and circulation. usability. The in vitro multi-enzyme catalytic system constructed by protein PfuPGM and glucan phosphorylase (αGP), inositol 3-phosphate synthase (IPS) and inositol monophosphatase (IMP), cells expressing protein PfuPGM and glucan Phosphorylase (αGP) cells, inositol 3-phosphate synthase (IPS)-expressing cells and inositol monophosphatase (IMP)-expressing cells jointly construct a whole-cell system, which can produce inositol, inositol from starch The conversion rate is over 55%. The invention has important application value.

Description of drawings

Figure 1 is a schematic diagram of the structure of the recombinant plasmid pET-20b-PfuPGM-CO.

Figure 2 is the SDS-PAGE detection result of step 2 in Example 1.

Fig. 3 is the measurement result of the glucose phosphomutase activity of the protein PfuPGM of Example 2.

Figure 4 shows the thermal stability results of the protein PfuPGM in Example 3.

Figure 5 is the HPLC detection result of protein PfuPGM, αGP, IPS and IMP catalyzed starch production of inositol in Example 4.

FIG. 6 shows the recycling effect of protein PfuPGM to prepare inositol in Example 5. FIG.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.

The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

In the following examples, a high performance liquid chromatography (HPLC) analyzer equipped with a US Bio-Rad HPX-87H column can be used to distinguish inositol, glucose, glucose 1 phosphate, and glucose 6 phosphate in the reaction solution; And the inositol can be quantified, and the concentration of inositol is proportional to the intensity of the characteristic peak of inositol in the HPLC chart.

Example 1. Expression and purification of protein PfuPGM

1. Construction of recombinant plasmid pET-20b-PfuPGM-CO

The inventor of the present invention entrusted Wuxi Qinglan Biotechnology Co., Ltd. to construct the recombinant plasmid pET-20b-PfuPGM-CO. Specific steps are as follows:

1. The amino acid sequence of the protein PfuPGM is shown in SEQ ID NO:1. According to the amino acid sequence of protein PfuPGM, design and optimize (E. coli expression system) the DNA molecule encoding protein PfuPGM. The optimized DNA molecule is shown in SEQ ID NO:2.

2. Artificially synthesize the DNA molecule shown in SEQ ID NO: 2.

3. Replace the small DNA fragment between the restriction endonucleases NdeI and XhoI of the pET-20b vector (a product of Youbao Biotechnology Co., Ltd., product catalog number VT1196) with the DNA molecule shown in SEQ ID NO: 2, other The steps were unchanged, and the recombinant plasmid pET-20b-PfuPGM-CO was obtained.

The recombinant plasmid pET-20b-PfuPGM-CO expresses the protein PfuPGM shown in SEQ ID NO:3.

The schematic diagram of the structure of the recombinant plasmid pET-20b-PfuPGM-CO is shown in Figure 1.

2. Expression and purification of protein PfuPGM

1. Transform the recombinant plasmid pET-20b-PfuPGM-CO into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli.

2. After step 1 is completed, the recombinant Escherichia coli monoclone is inoculated into 3 ml of LB liquid medium containing 100 μg/ml ampicillin, and cultured at 37° C. and 220 rpm overnight to obtain culture liquid 1.

3. After completing step 2, inoculate 1 ml of cultured bacterial solution 1 into 200 ml of LB liquid medium containing 100 μg/ml ampicillin, and cultivate at 37° C. and 220 rpm to obtain cultured bacterial solution 2 with an OD _600nm value of about 0.8.

4. After step 3 is completed, IPTG is added to the culture solution 2 to obtain a culture system; in the culture system, the concentration of IPTG is 100 μM.

5. After completing step 4, take the culture system and induce 4 hours at 37°C or 20 hours at 18°C.

6. After completing step 5, centrifuge to collect the bacteria.

7. After completing step 6, take the bacterial cells, resuspend them with 30 mM phosphate buffer (pH 7.0), and then ultrasonically break the walls to obtain a cell disrupting liquid.

8. After completing step 7, take the cell disruption liquid, centrifuge at 12000 rpm for 10 min, and collect the supernatant.

9. Perform SDS-PAGE on the cell disrupted liquid obtained in step 7 and the supernatant collected in step 8.

SDS-PAGE detection results are shown in Figure 2 (M is the protein marker, 1 is the cell disrupted solution induced at 37°C for 4 h, 2 is the cell disrupted solution induced at 18 °C for 20 h, 4 is the supernatant induced at 37 °C for 4 h, 5 is the 18 The supernatant was induced for 20 h at ℃). The results showed that the protein PfuPGM was almost always expressed in soluble form regardless of whether it was induced at 18°C for 20 hours or at 37°C for 4 hours.

10. Take the supernatant collected in step 8, and treat it at 80°C for 20 minutes (the purpose is to purify) to obtain a purified product; then detect it by SDS-PAGE.

SDS-PAGE detection results are shown in Figure 2 (M is the protein marker, 3 is the purified product induced at 37 °C for 4 h, and 6 is the purified product induced at 18 °C for 20 h). The results showed that the protein PfuPGM was only partially purified, but the purity was not high.

11. Take the purified product obtained in step 10 and purify it with a Ni-NTA column to obtain the purified protein PfuPGM, which is used for subsequent experiments.

According to the above method, the DNA molecule shown in SEQ ID NO: 2 was replaced with the TmPGM gene, and other steps remained unchanged to obtain the protein TmPGM. The TmPGM gene encodes the protein TmPGM. The protein TmPGM is derived from Thermotogamaritima , and the gene number on KEGG is TM0769.

Example 2. Determination of the enzyme activity of the glucose phosphate mutase (converting glucose 1 phosphate into glucose 6 phosphate) of the protein PfuPGM

The method for determining the activity of glucose phosphomutase is as follows:

1. Prepare the reaction system. The reaction system contained 100 mM HEPES buffer, 10 mM glucose 1 phosphate, 5 mM magnesium sulfate, and 0.0005 g/l or 0.001 g/l protein PfuPGM.

2. Take the reaction system prepared in step 1 and react at 70 °C for 10 min.

3. After completing step 2, the reaction was terminated in an ice bath; then the increase of glucose 6 phosphate was measured by coupling of glucose 6 phosphate dehydrogenase, and the glucose phosphate mutase activity of the protein PfuPGM was obtained according to the increase of glucose 6 phosphate.

Glucose phosphomutase activity was defined as: the amount of enzyme required to convert glucose 1 phosphate to 1 μmol of glucose 6 phosphate per minute at 70°C was 1 U.

The glucose 6 phosphate produced during the reaction is shown in Figure 3A. The results show that the enzymatic reaction is a linear reaction. That is, the glucose 6 phosphate produced and the reaction time are proportional to the enzyme concentration.

The enzyme concentration was plotted against the rate of glucose 6 phosphate production, see Figure 3B. The results showed that the specific activity of glucose phosphomutase of protein PfuPGM was 16.2 U/mg at 70℃.

According to the above method, the protein PfuPGM was replaced with the protein TmPGM, and other steps were unchanged. The results showed that the specific activity of glucose phosphomutase of protein TmPGM was 16.6U/mg at 70℃.

Example 3. Stability of protein PfuPGM

1. Add protein PfuPGM to 30 mM phosphate buffer (pH 7.0) containing 5 mM magnesium sulfate ions to obtain a sample solution. In this sample solution, the concentration of protein PfuPGM was 0.1 mg/ml.

2. Take the sample solution obtained in step 1 and treat it at 70°C for 4h, 8h, 16h, 20h or 32h to obtain a treatment solution.

3. According to the method for measuring glucose phosphomutase activity in Example 2, replace "0.0005g/l or 0.001g/l protein PfuPGM" with 5 μl or 10 μl of the treatment solution to obtain the glucose phosphomutase activity of the treatment solution; "0.0005 g/l or 0.001 g/l protein PfuPGM" was replaced with 5 μl or 10 μl of the sample solution, and the glucose phosphomutase activity of the sample solution was obtained.

The test results are shown in Figure 4. The results showed that the t _1/2 time of the protein PfuPGM at 70°C (that is, the time when the enzyme activity lost half of the enzyme activity) was 31.8h, and it had good stability.

According to the above method, the protein PfuPGM was replaced with the protein TmPGM, and other steps were unchanged.

The test results are shown in Figure 4. The results showed that the t _1/2 time of protein TmPGM at 70°C (ie the time when the enzyme activity lost half) was 8.9h.

Example 4. Application of protein PfuPGM in the preparation of inositol

To construct an in vitro multi-enzyme catalytic system containing glucan phosphorylase (αGP), glucose phosphate mutase (ie protein PfuPGM), inositol 3-phosphate synthase (IPS) and inositol monophosphatase (IMP), starch Produces inositol for substrates. In the in vitro multi-enzyme catalytic system, αGP is used to produce glucose 1 phosphate from starch and inorganic phosphorus ions, protein PfuPGM is used to convert glucose 1 phosphate to glucose 6 phosphate, IPS is used to convert glucose 6 phosphate to inositol 1 phosphate, IMP Used to convert inositol 1-phosphate to inositol, releasing inorganic phosphorus. Phosphorus ions are in equilibrium throughout the reaction. αGP is derived from Thermotogamaritima , and the gene number on KEGG is TM1168. IPS is derived from Archaeoglobus fulgidus , and the gene number on KEGG is AF1794. IMP is derived from Thermotogamaritima , and the gene number on KEGG is TM1415. All enzymes were heterologously expressed and purified in E. coli.

1. Prepare a 1ml reaction system; the reaction system contains 40mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, 1U/ml αGP, 1U/ml protein PfuPGM, 1U/ml IPS, 1U/ml ml of IMP and 100 g/L of soluble starch.

2. Take the reaction system prepared in step 1 and react at 70°C for 0h, 24h or 48h.

3. Use a high performance liquid chromatography (HPLC) analyzer equipped with a US Bio-Rad HPX-87H column to detect the components in the reaction system after completing step 2. The mobile phase for HPLC was 5 mM dilute sulfuric acid.

The test results are shown in Figure 5. The results showed that the concentration of inositol increased with the increase of reaction time; when the reaction was carried out at 70℃ for 48h, the concentration of inositol was 55g/L, and the conversion rate was 55%. It can be seen that the protein PfuPGM has the activity of converting glucose 1 phosphate into glucose 6 phosphate, and can construct an in vitro multi-enzyme catalytic system with other enzymes for the reaction of producing inositol from starch.

Embodiment 5. The recycling effect of protein PfuPGM to prepare myo-inositol

Relative inositol content = inositol content in filtrate N / inositol content in filtrate 1 × 100%, where N is 2, 3, 4, or 5.

1. The recycling effect of protein PfuPGM to prepare inositol

1. Prepare a 1ml reaction system; the reaction system contains 40mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, 1U/ml αGP, 1U/ml protein PfuPGM, 1U/ml IPS, 1U/ml ml of IMP and 100 g/L of soluble starch.

2. Take the reaction system prepared in step 1, react at 70°C for 12 hours; ultrafiltration to obtain filtrate 1 and concentrate 1.

The content of inositol in filtrate 1 was determined by HPLC. The inositol content was recorded as 100%.

3. Take the concentrate 1, wash it twice with buffer by ultrafiltration, then add phosphate buffer (pH 7.0) with a final concentration of 40 mM, 5 mM divalent magnesium ions and 100 g/L soluble starch, and react at 70 °C 12h; ultrafiltration to obtain filtrate 2 and concentrate 2.

The content of inositol in filtrate 2 was detected by HPLC, and the relative content of inositol was calculated.

4. Take the concentrate 2, wash it twice with buffer by ultrafiltration, then add phosphate buffer (pH 7.0) with a final concentration of 40 mM, 5 mM divalent magnesium ions and 100 g/L soluble starch, and react at 70 °C 12h; ultrafiltration to obtain filtrate 3 and concentrate 3.

The content of inositol in filtrate 3 was detected by HPLC, and the relative content of inositol was calculated.

5. Take the concentrated solution 3, wash it twice with buffer solution by ultrafiltration, then add phosphate buffer (pH 7.0) with a final concentration of 40mM, 5mM divalent magnesium ion and 100g/L soluble starch, and react at 70℃ 12h; ultrafiltration to obtain filtrate 4 and concentrate 4.

The content of inositol in filtrate 4 was detected by HPLC, and the relative content of inositol was calculated.

6. Take concentrate 4, wash it twice with buffer by ultrafiltration, then add phosphate buffer (pH 7.0) with a final concentration of 40 mM, 5 mM divalent magnesium ions and 100 g/L soluble starch, and react at 70 °C 12h; ultrafiltration to obtain filtrate 5 and concentrate 5.

The content of inositol in the filtrate 5 was detected by HPLC, and the relative content of inositol was calculated.

The test results are shown in Figure 6. The results showed that the relative content of inositol in filtrate 5 could reach 49.5%.

2. The recycling effect of protein TmPGM to prepare inositol

Replace the protein PfuPGM with the protein TmPGM according to the methods 1 to 4 in step 1, and the other steps remain unchanged.

The test results are shown in Figure 6. The results showed that when the protein TmPGM was used to prepare inositol, the relative content of inositol in filtrate 3 was only 9.5%.

It can be seen that the stability and reusability of the multi-enzyme catalytic system containing protein PfuPGM have been greatly improved.

Example 6. Application of isoamylase (IA) in the preparation of inositol

In order to improve the utilization rate of starch, the present invention adds isoamylase (IA) to the reaction system for preparing inositol. IA is derived from Sulfolobustokodaii , and the gene number on KEGG is ST0928.

1. Prepare a 1ml reaction system; the reaction system contains 40mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, 1U/ml αGP, 1U/ml protein PfuPGM, 1U/ml IPS, 1U/ml ml of IMP, 1 U/ml of IA and 100 g/L of soluble starch.

2. Take the reaction system prepared in step 1 and react at 70°C for 48h.

3. Use a high performance liquid chromatography (HPLC) analyzer equipped with a US Bio-Rad HPX-87H column to detect the components in the reaction system after completing step 2. The mobile phase for HPLC was 5 mM dilute sulfuric acid.

The results showed that the concentration of inositol increased with the increase of reaction time; after the reaction, the concentration of inositol was 70g/L, and the conversion rate was 70%.

According to the above method, the protein PfuPGM was replaced with the protein TmPGM, and other steps were unchanged. The results showed that the concentration of inositol increased with the increase of reaction time; after the reaction, the concentration of inositol was 69 g/L, and the conversion rate was 69%.

It can be seen that adding IA helps to increase the production of inositol.

Example 7. Application of whole cell system containing protein PfuPGM in the preparation of inositol

Cells expressing α-glucan phosphorylase (αGP), cells expressing glucose phosphomutase (ie protein PfuPGM), cells expressing inositol 3-phosphate synthase (IPS), and cells expressing inositol monophosphatase ( IMP) cells and cells expressing isoamylase (IA); permeabilization treatment was performed after inducing the expression of the enzyme. After the permeabilized cells were mixed together, inorganic phosphate and starch were added to carry out the whole-cell catalytic reaction.

The way of permeabilization treatment is heat treatment. Specifically: collect the whole cells containing the enzyme, add 10mM phosphate buffer (pH7.0) to wash once, discard the supernatant; then add 10mM phosphate buffer (pH7.0) to resuspend; the resuspended cells 75 After 15min treatment at ℃, the permeabilization treatment was completed.

Specific steps are as follows:

1. Prepare a 1ml reaction system; the reaction system contains 10mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, 0.14g DCW/L cells expressing αGP, and 0.08g DCW/L cells expressing protein PfuPGM , 0.5 g DCW/L IPS expressing cells, 0.08 g DCW/L IMP expressing cells, 0.08 g DCW/L IA expressing cells and 100 g/L soluble starch.

2. Take the reaction system prepared in step 1 and react at 70°C for 46h.

3. Use a high performance liquid chromatography (HPLC) analyzer equipped with a US Bio-Rad HPX-87H column to detect the components in the reaction system after completing step 2. The mobile phase for HPLC was 5 mM dilute sulfuric acid.

The results showed that the concentration of inositol increased with the increase of reaction time; after the reaction, the concentration of inositol was 65.3g/L, and the conversion rate was 65.3%. It can be seen that the cells expressing the protein PfuPGM also have the activity of catalyzing the conversion of glucose 1 phosphate to glucose 6 phosphate, and can form a whole-cell catalytic system with other cells expressing the enzyme to produce inositol from starch.

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> Application of Protein PfuPGM as Glucose Phosphomutase in the Production of Inositol

<160> 3

<170> PatentIn version 3.5

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Asp Thr Arg Val Ser Gly Glu Met Leu Lys Ser Ala Leu Ile Ser Gly

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Leu Leu Ser Val Gly Cys Asp Val Ile Asp Val Gly Ile Ala Pro Thr

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Pro Ala Ile Gln Trp Ala Thr Asn His Leu Lys Ala Asp Gly Gly Ala

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Val Ile Thr Ala Ser His Asn Pro Pro Glu Tyr Asn Gly Ile Lys Leu

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Leu Glu Pro Asn Gly Met Gly Leu Lys Lys Glu Arg Glu Ala Ile Val

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Glu Glu Ile Phe Phe Lys Glu Asp Phe Asp Arg Val Glu Trp His Glu

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Ile Gly Glu Val Arg Glu Val Asp Ile Ile Lys Pro Tyr Ile Glu Ala

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Ile Lys Ser Lys Val Asp Val Glu Ala Ile Lys Lys Arg Arg Pro Phe

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Leu Leu Arg Glu Leu Gly Cys Lys Val Val Ser Val Asn Ala His Pro

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Arg Phe Ile Gln Gly Asp Lys Thr Phe Ala Leu Val Ala Asp Ala Val

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Leu Arg Glu Asn Gly Gly Gly Leu Leu Val Thr Thr Val Ala Thr Ser

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Asn Leu Leu Asp Asp Ile Ala Lys Lys His Gly Ala Lys Val Met Arg

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Thr Lys Val Gly Asp Leu Ile Val Ala Arg Ala Leu Tyr Glu Asn Asn

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Phe Ala Lys Ser Gly Lys Lys Phe Ser Glu Leu Ile Asp Glu Leu Pro

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Ala Ile Val Asn Lys Val Ala Glu Met Ala Arg Glu Arg Gly Tyr Thr

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Val Asp Thr Thr Asp Gly Ala Lys Ile Ile Phe Glu Asp Gly Trp Val

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Ala Lys Ser Glu Glu Lys Ala Gln Glu Tyr Leu Asp Leu Gly Ile Glu

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Leu Leu Glu Lys Ala Leu Ser

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agccataatc cgccggaata caacggcatt aagctgctgg agccgaatgg catgggtctg 360

aaaaaggaac gcgaggccat cgtggaagag atcttcttca aggaggattt cgatcgcgtt 420

gagtggcatg aaatcggcga ggtgcgtgag gtggacatca tcaaaccgta tatcgaagcg 480

atcaagagca aggtggacgt tgaggccatc aagaaacgcc gcccgtttgt ggtggttgat 540

accagcaatg gtgcgggtag tctgacgctg ccatatctgc tgcgcgaact gggttgcaaa 600

gtggtgagcg ttaacgcgca tccagatggc catttcccag cgcgcaatcc ggaaccgaac 660

gaggaaaatc tgaagggctt catggagatc gtgaaagcgc tcggcgcgga ctttggcgtt 720

gcccaagatg gtgatgccga ccgtgccgtg ttcatcgacg agaatggccg cttcatccaa 780

ggcgacaaaa cgtttgcgct ggtggccgat gcggtgctcc gtgaaaatgg cggtggtctg 840

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aaagtgatgc gcaccaaagt tggcgatctg atcgtggcgc gtgcgctgta tgagaataat 960

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Asp Ile Thr Pro Glu Phe Ala Leu Lys Ile Gly Met Ala Phe Gly Thr

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Leu Leu Arg Arg Glu Gly Lys Lys Lys Pro Val Val Val Val Gly Arg

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Asp Thr Arg Val Ser Gly Glu Met Leu Lys Ser Ala Leu Ile Ser Gly

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Leu Leu Ser Val Gly Cys Asp Val Ile Asp Val Gly Ile Ala Pro Thr

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Pro Ala Ile Gln Trp Ala Thr Asn His Leu Lys Ala Asp Gly Gly Ala

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Val Ile Thr Ala Ser His Asn Pro Pro Glu Tyr Asn Gly Ile Lys Leu

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Leu Glu Pro Asn Gly Met Gly Leu Lys Lys Glu Arg Glu Ala Ile Val

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Glu Glu Ile Phe Phe Lys Glu Asp Phe Asp Arg Val Glu Trp His Glu

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Ile Gly Glu Val Arg Glu Val Asp Ile Ile Lys Pro Tyr Ile Glu Ala

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Val Val Val Asp Thr Ser Asn Gly Ala Gly Ser Leu Thr Leu Pro Tyr

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Leu Leu Arg Glu Leu Gly Cys Lys Val Val Ser Val Asn Ala His Pro

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Asp Gly His Phe Pro Ala Arg Asn Pro Glu Pro Asn Glu Glu Asn Leu

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Lys Gly Phe Met Glu Ile Val Lys Ala Leu Gly Ala Asp Phe Gly Val

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Ala Gln Asp Gly Asp Ala Asp Arg Ala Val Phe Ile Asp Glu Asn Gly

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Arg Phe Ile Gln Gly Asp Lys Thr Phe Ala Leu Val Ala Asp Ala Val

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Leu Arg Glu Asn Gly Gly Gly Leu Leu Val Thr Thr Val Ala Thr Ser

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Asn Leu Leu Asp Asp Ile Ala Lys Lys His Gly Ala Lys Val Met Arg

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Thr Lys Val Gly Asp Leu Ile Val Ala Arg Ala Leu Tyr Glu Asn Asn

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Val Leu Gly Arg Asp Gly Ala Met Thr Val Ala Lys Val Val Glu Ile

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Phe Ala Lys Ser Gly Lys Lys Phe Ser Glu Leu Ile Asp Glu Leu Pro

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Lys Tyr Tyr Gln Ile Lys Thr Lys Arg His Val Glu Gly Asp Arg Tyr

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Ala Ile Val Asn Lys Val Ala Glu Met Ala Arg Glu Arg Gly Tyr Thr

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Val Asp Thr Thr Asp Gly Ala Lys Ile Ile Phe Glu Asp Gly Trp Val

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Leu Val Arg Ala Ser Gly Thr Glu Pro Ile Ile Arg Ile Phe Ser Glu

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Ala Lys Ser Glu Glu Lys Ala Gln Glu Tyr Leu Asp Leu Gly Ile Glu

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Claims (10)

1. Application of protein PfuPGM, any one of a-d: a Application as glucose phosphomutase; The application of b in the preparation of glucose phosphomutase; c in the production of inositol; d use in the preparation of products for the production of inositol; The protein PfuPGM is e or f or g as follows: The amino acid sequence of e is the protein shown in SEQ ID NO: 1; f amino acid sequence is the protein shown in SEQ ID NO: 3; g A fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 1 or SEQ ID NO: 3. 2. The nucleic acid molecule encoding the protein PfuPGM in claim 1, the expression cassette containing the nucleic acid molecule encoding the protein PfuPGM in claim 1, the recombinant vector containing the nucleic acid molecule encoding the protein PfuPGM in claim 1 or containing The application of the recombinant microorganism of the nucleic acid molecule of the protein PfuPGM coding in claim 1, is any one in a-d: a Application as glucose phosphomutase; The application of b in the preparation of glucose phosphomutase; c in the production of inositol; d Use in the manufacture of products for the production of inositol. 3. application as claimed in claim 2, is characterized in that: the nucleic acid molecule of described protein PfuPGM of described coding claim 1 is the DNA molecule shown in x or y: The x coding region is the DNA molecule shown in SEQ ID NO: 2; The y nucleotide sequence is the DNA molecule shown in SEQ ID NO:2. 4. application as claimed in claim 2, is characterized in that: described recombinant carrier is recombinant plasmid pET-20b-PfuPGM-CO; The small DNA fragment between Dicer NdeI and XhoI is replaced with the DNA molecule shown in SEQ ID NO: 2 to obtain a recombinant plasmid. 5. The application according to any one of claims 1 to 4, wherein the production of inositol is based on starch as a substrate. 6. A method for producing inositol, comprising the steps: h Preparation of reaction system 1; the reaction system 1 contains 30-50mM pH7.0 phosphate buffer, 4-6mM divalent magnesium ion, 0.8-1.2U/ml αGP, 0.8-1.2U/ml claim 1 The protein described in PfuPGM, 0.8-1.2U/ml IPS, 0.8-1.2U/ml IMP and 90-110g/L soluble starch; i take reaction system 1 and react at 65-75°C for more than 12 hours to obtain reaction product 1; jIsolation of myo-inositol from reaction product 1. 7 . The method of claim 6 , wherein the reaction system 1 further contains isoamylase; the concentration of isoamylase in the reaction system 1 is 0.8-1.2 U/ml. 8 . 8. A method for producing inositol, comprising the steps of: o Prepare reaction system 2; reaction system 2 contains 8-12mM pH7.0 phosphate buffer, 4-6mM divalent magnesium ions, 0.12-0.16g DCW/L cells expressing αGP, 0.06-0.10g DCW/L cells expressing the protein PfuPGM of claim 1, 0.4-0.6 g DCW/L cells expressing IPS, 0.06-0.10 g DCW/L cells expressing IMP, and 90-110 g/L soluble starch; p take reaction system 2, and react at 65-75 °C for more than 12 hours to obtain reaction product 2; q Isolation of myo-inositol from reaction product 2. 9 . The method of claim 8 , wherein the reaction system 2 further contains cells expressing isoamylase. 10 . 10. The method of claim 9, wherein the reaction system 2 contains 8-12mM phosphate buffer at pH 7.0, 4-6mM divalent magnesium ions, 0.12-0.16g DCW/L expressing αGP 0.06-0.10g DCW/L cells expressing protein PfuPGM, 0.4-0.6g DCW/L cells expressing IPS, 0.06-0.10g DCW/L cells expressing IMP, 0.06-0.10g DCW/L expressing isoamyloid Enzymatic cells and 90-110g/L of soluble starch.

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