阅读说明：本技术 一种酶法制备5’-鸟苷酸的方法 (Method for preparing 5' -guanylic acid by enzyme method ) 是由 张玲 夏海洋 于 2019-08-28 设计创作，主要内容包括：本发明公开了一种酶法制备5’-鸟苷酸的方法,属于食品加工技术领域。所述制备方法包括：以氨基酸序列如SEQ ID NO.1所示的酸性磷酸转移酶突变体纯酶为催化剂,添加到含有鸟苷和焦磷酸钠的pH为3.6～9.0的反应溶液中,在25～60℃条件下进行催化反应,反应结束后,获得含5’-鸟苷酸的混合液,再经分离纯化获得5’-鸟苷酸。本发明选用的酸性磷酸转移酶突变体具有磷酸转移酶活性高,水解酶活性低,底物特异性高的优点；方法工艺环保,反应条件温和,成本低,得到的目标产物收率高,制备方法简单,适用于5’-鸟苷酸的工业化生产,应用前景广阔。(The invention discloses a method for preparing 5' -guanylic acid by an enzymatic method, belonging to the technical field of food processing. The preparation method comprises the following steps: adding pure acid phosphotransferase mutant enzyme with an amino acid sequence shown as SEQ ID NO.1 as a catalyst into a reaction solution containing guanosine and sodium pyrophosphate and having a pH value of 3.6-9.0, carrying out catalytic reaction at 25-60 ℃, obtaining a mixed solution containing 5 '-guanylic acid after the reaction is finished, and separating and purifying to obtain the 5' -guanylic acid. The acid phosphotransferase mutant selected by the invention has the advantages of high phosphotransferase activity, low hydrolase activity and high substrate specificity; the method has the advantages of environment-friendly process, mild reaction conditions, low cost, high yield of the obtained target product, simple preparation method, suitability for industrial production of 5' -guanylic acid and wide application prospect.)

1. The application of the acid phosphotransferase mutant with the amino acid sequence shown in SEQ ID NO.1 in catalyzing guanosine to prepare 5' -guanylic acid.

2. A method for preparing 5' -guanylic acid by an enzymatic method, which is characterized by comprising the following steps: adding pure acid phosphotransferase mutant enzyme with an amino acid sequence shown as SEQ ID NO.1 as a catalyst into a reaction solution containing guanosine and sodium pyrophosphate and having a pH value of 3.6-9.0, carrying out catalytic reaction at 25-60 ℃, obtaining a mixed solution containing 5 '-guanylic acid after the reaction is finished, and separating and purifying to obtain the 5' -guanylic acid.

3. The method according to claim 2, wherein the concentration of guanosine is 1 to 20mg/mL, the concentration of sodium pyrophosphate is 150 to 300mg/mL, and the concentration of pure acid phosphotransferase mutant enzyme is 20 to 120. mu.g/mL in the reaction system.

4. The enzymatic method for preparing 5' -guanylic acid according to claim 2, wherein a sodium acetate buffer solution having a pH of 3.6 to 5.8 is used as a reaction medium in the reaction solution.

5. The enzymatic process of claim 4 wherein the reaction medium is sodium acetate buffer at pH 4.0.

6. The enzymatic method of producing 5' -guanylic acid according to claim 2, wherein the temperature of the catalytic reaction is 25 to 40 ℃.

7. The enzymatic method of preparing 5' -guanylic acid according to claim 6, wherein the temperature of the catalytic reaction is 35 ℃.

8. The enzymatic method for preparing 5' -guanylic acid according to claim 2, wherein the time for catalytic reaction is 6-8 hours.

9. The enzymatic method for preparing 5' -guanylic acid according to claim 2, wherein the method for preparing the pure enzyme of the acid phosphotransferase mutant comprises culturing recombinant Escherichia coli containing the gene encoding the acid phosphotransferase mutant to OD ₆₀₀ of 0.6-0.8, adding IPTG with a final concentration of 1mM, inducing at 25 ℃ for 10-12 h, collecting the bacterial cells, performing ultrasonic disruption to obtain cell lysate, centrifuging to obtain supernatant, and further separating and purifying to obtain the pure enzyme of the acid phosphotransferase mutant.

Technical Field

The invention relates to the technical field of food processing, in particular to a method for preparing 5' -guanylic acid by an enzymatic method.

Background

5 ' -guanylic acid (Guanosine-5 ' -monophosphate, i.e., 5 ' -GMP) is a nucleotide seasoning widely used in foods, and it is used in combination with sodium glutamate in the process of food processing to constitute a basic seasoning for foods, and has a remarkable flavor-enhancing effect, and therefore, it is also called as a flavor-providing nucleotide. The prior production methods of the flavor-developing nucleotide mainly comprise four methods: chemical synthesis, microbial fermentation, enzymolysis, and biocatalysis.

The chemical synthesis method mainly uses nucleoside as raw material to make phosphorylation reaction. The commonly used phosphorylating agents are mainly phosphoric acid or reactive derivatives of pyrophosphoric acid, such as acid chloride pyrophosphate, bis-p-nitrophenyl pyrophosphate, monochloro and dichloro derivatives of phosphoric acid, etc., while phosphorus oxychloride is widely used in industry. In order to obtain 5 '-nucleotides by introducing a phosphate group into the 5' -hydroxyl group of a nucleoside, the 2 '-and 3' -hydroxyl groups of the ribose of the nucleoside are protected with protecting groups before phosphorylation reaction, and the protecting groups are removed after phosphorylation is completed. The overall reaction yield of the method is reduced due to the protection and deprotection steps. The reagents used in the chemical synthesis method are expensive and toxic, so that the reagents are generally used for producing some 5' -nucleotide and derivatives thereof with special purposes, and the reagents are difficult to be used for industrial production.

Microbial fermentation processes are divided into one-step and two-step processes. One-step method, namely direct fermentation method: directly producing 5 '-inosinic acid and 5' -xanthylic acid by microbial fermentation. The two-step method is a method combining a fermentation method and a chemical method: firstly, the nucleoside is produced by microbial fermentation, and then is phosphorylated by a chemical method, and finally, the 5 '-inosinic acid and the 5' -guanylic acid are obtained. The microbial fermentation method has few byproducts and low cost, but the application of the method is greatly limited by the characteristics of microorganisms.

the enzymolysis method has become a classical method for preparing various natural nucleotides, has the longest history and the most mature technology, and most of the nucleotides are produced by the enzymolysis method at present. The mixture of 4 nucleotides can be obtained by preparing the nucleotides by an enzymolysis method once, the utilization rate of the enzyme is high, but the difficulty of obtaining 4 high-purity products by extraction in the later period is high, so that the production period is long, the separation and purification process is complicated, and the product purity is not high.

A biocatalytic method: the biocatalytic nucleoside phosphorylation method mainly utilizes biocatalytic catalysis. Currently, enzymes available for catalyzing the synthesis of 5' -guanylic acid are mainly kinases, acid phosphotransferase (AP/PTAse), and the like.

Kinases are a class of phosphotransferases that transfer phosphate groups from ATP to other phosphate acceptors, and can be used to synthesize a variety of 5' -nucleotides, according to the following equation: ATP + nucleoside ADP + 5' -nucleotide. The disadvantage of this method is that ATP is always consumed in the catalytic process, so that it is necessary to culture ATP-consuming microorganisms capable of regenerating reaction or to add a large amount of ATP with high price to complete the reaction, which is expensive, and thus the method is limited.

The method for synthesizing 5 '-guanylic acid by catalyzing guanosine and phosphate group donor with acid phosphotransferase has the characteristics of simplicity, high efficiency, short period, low cost, environmental protection requirement conformity and the like, is suitable for industrial production, and has great development potential compared with the disclosed process for preparing 5' -guanylic acid. At present, the technology for producing guanosine by a fermentation method in China is mature and industrialization is realized, and the key for further developing and producing 5' -guanylic acid on the basis of the technology is how to realize the specific phosphorylation of enzyme catalytic guanosine. Therefore, a production process which is simple and safe to operate, low in cost, high in product purity and environment-friendly is urgently needed.

Disclosure of Invention

The invention aims to provide an acid phosphotransferase capable of specifically catalyzing guanosine phosphorylation, which is used for preparing 5' -guanylic acid by an enzyme method, and provides a production process which is simple and safe to operate, mild in reaction conditions, low in production cost, high in yield and purity and suitable for industrial production.

In order to achieve the purpose, the invention adopts the following technical scheme:

The application of the acid phosphotransferase mutant with the amino acid sequence shown in SEQ ID NO.1 in catalyzing guanosine to prepare 5' -guanylic acid.

The acid phosphotransferase mutant is obtained by modifying acid phosphotransferase (AP/PTase) from Morganella morganii, and the specific modification method comprises the following steps: the first 20 amino acid sequences were deleted and site-directed mutated at 2 amino acid positions (G72D, I151T) using the acid phosphotransferase primary amino acid sequence published by Genebank (Genebank accession number P28581).

The research of the invention shows that the modified acid phosphotransferase mutant shows higher substrate specificity for catalyzing guanosine, has the characteristics of high phosphotransferase activity and low hydrolase activity, and can effectively convert guanosine to synthesize 5' -guanylic acid. The reaction formula is as follows:

In the reaction formula, Acid Phosphotransferase is Acid Phosphotransferase; acid Phosphotase is an Acid phosphohydrolase. The acid phosphotransferase mutant has a bidirectional effect.

The invention also provides a coding gene of the acid phosphotransferase mutant, codon optimization is carried out according to the codon frequency of escherichia coli, and the optimized nucleotide sequence is shown as SEQ ID NO. 2.

The invention also provides a method for preparing 5' -guanylic acid by an enzymatic method, which comprises the following steps: adding pure acid phosphotransferase mutant enzyme with an amino acid sequence shown as SEQ ID NO.1 as a catalyst into a reaction solution containing guanosine and sodium pyrophosphate and having a pH value of 3.6-9.0, carrying out catalytic reaction at 25-60 ℃, obtaining a mixed solution containing 5 '-guanylic acid after the reaction is finished, and separating and purifying to obtain the 5' -guanylic acid.

Preferably, in the reaction system, the concentration of guanosine is 1 to 20mg/mL, the concentration of sodium pyrophosphate is 150 to 300mg/mL, and the concentration of pure acid phosphotransferase mutant enzyme is 20 to 120. mu.g/mL. More preferably, the molar conversion of guanosine in the reaction system under the conditions that the concentration of guanosine was 1mg/mL, the concentration of sodium pyrophosphate was 150mg/mL, and the concentration of pure acid phosphotransferase mutant enzyme was 20. mu.g/mL, was 97%.

Preferably, sodium acetate buffer solution with the pH value of 3.6-5.8 is adopted as a reaction medium in the reaction solution.

more preferably, the reaction medium is a sodium acetate buffer at pH 4.0. The research shows that the acidic phosphotransferase mutant catalyzes 5' -guanylic acid to synthesize at the fastest pH condition of 4.0, and the pH value is the optimal reaction pH value of the enzyme.

Preferably, the temperature of the catalytic reaction is 25-40 ℃. More preferably, the temperature of the catalytic reaction is 35 ℃. The research shows that the temperature condition that the acid phosphotransferase mutant catalyzes 5' -guanylic acid to synthesize at the fastest speed is 35 ℃.

Preferably, the time of the catalytic reaction is 6-8 h. Research shows that in the initial stage of the catalytic reaction, the concentration of the 5 '-guanylic acid of the reaction product is approximately linearly increased along with the extension of the reaction time, and when the reaction time reaches 6-8 hours, the concentration of the 5' -guanylic acid reaches the highest.

Preferably, the preparation method of the pure enzyme of the acid phosphotransferase mutant comprises the steps of culturing recombinant escherichia coli containing coding genes of the acid phosphotransferase mutant until OD ₆₀₀ is 0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM, inducing at 25 ℃ for 10-12 h, collecting thalli, performing ultrasonic disruption to obtain cell lysate, centrifuging to obtain supernatant, and further separating and purifying to obtain the pure enzyme of the acid phosphotransferase mutant.

The method of the invention is not only suitable for preparing 5 '-guanylic acid, but also can be universally used for preparing 5' -monophosphate nucleotide ester compounds.

The invention has the beneficial effects that:

(1) The invention adopts a new enzyme method process to prepare 5' -guanylic acid, successfully solves the technical problems existing in the prior art, and the selected acid phosphotransferase mutant has the advantages of high phosphotransferase activity, low hydrolase activity and high substrate specificity; the preparation method is simple, greatly simplifies the industrial production steps of 5' -guanylic acid, and successfully solves the technical problems of raw material toxicity, complex process and the like in the existing chemical synthesis process.

(2) The raw material source is wide, and the requirement on equipment is low.

(3) The method has the advantages of environment-friendly process, mild reaction conditions, low cost, high yield of the obtained target product, suitability for industrial production of 5' -guanylic acid and wide application prospect.

Drawings

FIG. 1 shows the diagram of the purification electrophoresis of acid phosphotransferase, wherein M is protein Marker, lane 1 is whole cell, lane 2 is supernatant after cell disruption, lane 3 is pellet after cell disruption, and lane 4 is purified protein.

FIG. 2 is a graph showing the effect of pH on the catalytic performance of acid phosphotransferases.

FIG. 3 is a graph showing the effect of temperature on the catalytic performance of acid phosphotransferases.

FIG. 4 shows the phosphotransferase activity studies at different substrate concentrations.

FIG. 5 is a graph showing a study of phosphotransferase activity amplification at guanosine concentrations of 10mg/mL and 20 mg/mL.

Detailed Description

The technical solution and the effects of the present invention are further described by the following specific examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Simple modifications of the invention applying the inventive concept are within the scope of the invention as claimed.

In the following examples, all starting materials are commercially available unless otherwise indicated.

guanosine was purchased from bio-engineering (shanghai) gmbh;

Expression vectors pET28a and e.coli BL21(DE3) were purchased from invitrogen.