One-step production method for producing tagatose from starch with extremely low side reaction products

Abstract

The present invention belongs to the field of tagatose production, and provides a one-step production method for producing tagatose with extremely low side reaction product starch. The present invention screens out two enzymes from glucose six phosphate isomerase and tagatose six phosphate isomerase in the high-temperature hot spring metagenome, namely: glucose six phosphate isomerase (Pgi-R) and tagatose six phosphate isomerase (T6PE-P). These two enzymes have extremely low catalytic ability for the side reaction of synthesizing sorbose in the process of catalyzing starch to form tagatose in a one-step method.

Description

Technical Field

[0001] The invention belongs to the field of tagatose production, and particularly relates to a one-step method for producing tagatose with extremely low side reaction product starch. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In the process of producing tagatose by starch one-step catalysis, as tagatose gradually accumulates in the solution system, the side reactions of glucose 6-phosphate isomerase and tagatose 6-phosphate isomerase gradually appear. Glucose 6-phosphate isomerase is originally used to catalyze glucose 6-phosphate to form fructose 6-phosphate; tagatose 6-phosphate isomerase is originally used to catalyze the isomerization of fructose 6-phosphate to form tagatose 6-phosphate; however, when the tagatose content in the system is high, the side reactions produced by these two enzymes can catalyze tagatose to form tagatose (peak 10 in the chromatogram) and sorbose (peak 7 in the chromatogram), such as Figure 1 However, too high a content of sorbose will greatly affect the cost of tagatose separation and crystallization. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a one-step production method for producing tagatose with extremely low side reaction products from starch. The present invention screened out two enzymes from glucose 6-phosphate isomerase and tagatose 6-phosphate isomerase in the high-temperature hot spring metagenome, namely: glucose 6-phosphate isomerase (Pgi-R) and tagatose 6-phosphate isomerase (T6PE-P). These two enzymes have extremely low catalytic ability for the side reaction of synthesizing sorbose in the one-step catalysis of starch to tagatose.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, there is provided a glucose-6-phosphate isomerase (Pgi-R) for producing tagatose in a one-step method with extremely low side reaction product starch, wherein the glucose-6-phosphate isomerase has

[0007] A protein having the amino acid sequence shown in SEQ ID NO:1.

[0008] SEQ ID NO: 1 is as follows:

[0009] mekitfdsskassflseyeinyfegfvkqahemlhsktgpgndflgwvdlplnydreefarikqsaekikadsdvlivi

[0010] giggsylgaraaiemlshsfynmlpksrrktpeiyfvgnnisstyvtdlleliegkevsvnvisksgtttepavafrifrey

[0011] menkygrqearkriyattdkargalkkladeegyesfvipddvggrfsvltavgllpiavcgadidkimqgaadackl

[0012] ysnvdfrnndcyryaaarnalynknktteimvnyepslhfftewwkqlygesegkdqkgifpagvdfttdlhsmgq

[0013] yiqdglrnlfetvihvgkakksitikedkdnidglnflagkemdfvnnkafegtllahtdggvpnfvinvpelneyyfgnlvyffekacgisgyllavnpfdqpgveaykknmfallgkpgyeeqkkqlearlgk.

[0014] In some embodiments, the tagatose glucose-6-phosphate isomerase (Pgi-R) is derived from the bacterium Ruminiclostridium cellobioparum.

[0015] The second aspect of the present invention provides a polynucleotide encoding

[0016] The nucleotide sequence of the protein shown in SEQ ID NO:1.

[0017] The third aspect of the present invention provides the use of the above-mentioned glucose-6-phosphate isomerase (Pgi-R) in reducing the side reactions in the one-step production of tagatose from starch.

[0018] In some embodiments, the side reaction includes catalyzing starch to form sorbose.

[0019] The fourth aspect of the present invention provides a tagatose six phosphate isomerase (T6PE-P) for producing tagatose from starch in a one-step process, wherein the tagatose six phosphate isomerase is a protein having an amino acid sequence shown in SEQ ID NO: 2.

[0020] In some embodiments, the tagatose 6-phosphate isomerase (T6PE-P) is derived from Paraburkholderia phenoliruptrix.

[0021] A fifth aspect of the present invention provides a polynucleotide encoding

[0022] The nucleotide sequence of the protein shown in SEQ ID NO:2.

[0023] SEQ ID NO:2 is as follows:

[0024] Mtnvvsrlsggegtakklkgiysicsahpwvlgaamkqaldddtplliestsnqvdqfggytgmkpadfvrfvhlia

[0025] drtglprtrlilggdhlgpnawrslpaeeamqraealidayvsagftkihldtsmscagdparlsddvvaerasrlcaiae

[0026] aaaerkgnrekpvyiigtevpvpggaaeeletveitspdaaldtvavhrnawrdrglddawqrvialvvqpgvefdht

[0027] kvvdykpelatklsailkelpgmvfeahstdyqtpealaalvqdgfailkvgpgvtfalrealyalaeienelvvpearsn

[0028] lrevvervmlakpgnwekyyhgddrekrllrtysysdrvryywadpeidaaanklisnladidisenvlsrylpeqywqfrrglidatpmsliqskvrevigvyaaacka.

[0029] The sixth aspect of the present invention provides the use of the above-mentioned tagatose six phosphate isomerase (T6PE-P) in reducing the side reactions in the one-step production of tagatose from starch.

[0030] In some embodiments, the side reaction includes catalyzing starch to form sorbose.

[0031] The seventh aspect of the present invention provides a method for producing tagatose in one step by using starch with extremely low side reaction products, comprising:

[0032] α-glucan phosphorylase, phosphoglucomutase, the above-mentioned Pgi-R crude enzyme solution, the above-mentioned T6PE-P crude enzyme solution and 6-phosphate tagatose phosphatase (T6PP) are respectively added to the starch reaction solution, and a catalytic reaction is carried out at a certain temperature to obtain the product.

[0033] In some embodiments, the starch reaction solution includes: dipotassium hydrogen phosphate, magnesium sulfate, and starch liquefaction solution.

[0034] Beneficial effects of the present invention

[0035] (1) The present invention screened and cloned a glucose 6-phosphate isomerase from Ruminiclostridium cellobioparum from the metagenome of a high-temperature hot spring, named Pgi-R. Compared with glucose 6-phosphate isomerase from other sources, the enzyme's ability to catalyze synthetic side reactions is reduced by 70%.

[0036] (2) The present invention screened and cloned a tagatose 6-phosphate isomerase from Paraburkholderia phenoliruptrix from the metagenome of high-temperature hot springs, named T6PE-P. Compared with tagatose 6-phosphate synthase from other sources, the enzyme's ability to catalyze synthetic side reactions is reduced by 85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 This is a chromatogram of a mixed sugar solution formed by catalyzing starch in a one-step process using a traditional method in Example 2, including tagatose (peak 10 of the chromatogram) and sorbose (peak 7 of the chromatogram).

[0039] Figure 2 This is a chromatogram of a mixed sugar solution formed by catalyzing starch using the Pgi-R and T6PE-P one-step method of the present invention in Example 2, including tagatose (peak 10 of the chromatogram) and sorbose (peak 7 of the chromatogram). DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0041] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0042] In the following examples, the experimental materials are as follows:

[0043] Maltodextrin, product of ALDRICH, product number 419672;

[0044] pET41b vector, Novagen, Madison, WI;

[0045] E. coli expression strain BL21 (DE3), Invitrogen, Carlsbad, CA.

[0046] Other raw materials are commercially available products.

[0047] Example 1 Cloning of Pgi-R and T6PE-P genes

[0048] The present invention obtained the amino acid sequence of the possible glucose-6-phosphate isomerase and tagatose-6-phosphate synthase from the sequence obtained from the high-temperature hot spring sediment metagenomics. The present invention then commissioned GenScript Co., Ltd. to design the polynucleotide (DNA) sequence encoding the polypeptide based on the amino acid sequence, and performed codon optimization on the Escherichia coli expression system. The optimized polynucleotide sequence is shown in SEQ.NO.3 and SEQ.NO.4.

[0049] SEQ.NO.3:

[0050]

[0051] SEQ.NO.4:

[0052]

[0053] GenScript Co., Ltd. connected the codon-optimized sequence clone to the pET41b vector to obtain the pET41b-Pgi-R and pET41b-T6PE-P expression vectors. In the expression vector, the T7 promoter and T7 terminator are responsible for gene expression.

[0054] Example 2 Protein expression and activity determination

[0055] (1) Protein expression

[0056] Transform pET41b-Pgi-R and pET41b-T6PE-P into E. coli BL21 (DE3), pick a single clone and add it to 3 ml of LB medium containing 50 μg / ml kanamycin, and culture it at 37°C, 220 rpm overnight. Take 1 ml of overnight bacteria and add it to 200 ml of LB medium containing 50 μg / ml kanamycin, and culture it at 37°C, 220 rpm until the OD600 value reaches about 0.8, add Isopropylβ-D-1-thiogalactopyranoside (IPTG) with a final concentration of 100 μM, and induce protein expression at 37°C and 18°C, respectively. Induce at 37°C for 4 hours and at 18°C ​​for 20 hours. After induction, the bacteria were collected by centrifugation, resuspended in 30 mM phosphate buffer (pH 7.0), and broken by ultrasound to obtain cell lysis solution. The expression level of the enzyme was detected by SDS-PAGE, and the cell lysis solution was centrifuged at high speed (12000 rpm, 10 min) to prepare crude enzyme solution (the crude enzyme solution can be directly added to the catalytic reaction solution according to the percentage ratio).

[0057] (2) Traditional method of catalyzing starch to produce tagatose in one step

[0058] 50U α-glucan phosphorylase (αGP), 50U phosphoglucomutase (PGM), 50U phosphoglucoside isomerase (PGI), 50U tagatose hexaphosphate synthase (T6PE), and 50U 6-phosphate tagatose phosphatase (T6PP) were added to 1L reaction solution (50mM potassium hydrogen phosphate, 5mM magnesium sulfate, 300g / l starch liquefaction solution, pH 7.0) respectively. The reaction was catalyzed at 60℃ for 72h, and then samples were taken to detect tagatose and sorbose in the reaction solution by high performance liquid chromatography ( Figure 1 ).

[0059] (3) One-step production of tagatose from starch using glucose 6-phosphate isomerase (Pgi-R) and tagatose 6-phosphate isomerase (T6PE-P) as catalysts

[0060] In 1L reaction solution (50mM potassium hydrogen phosphate, 5mM magnesium sulfate, 300g / l starch liquefaction solution, pH7.0), 10U α-glucan phosphorylase (αGP), 10U phosphoglucomutase (PGM), 10ml Pgi-R crude enzyme solution, 30ml T6PE-P crude enzyme solution, 50U 6-phosphate tagatose phosphatase (T6PP) were added respectively. After catalytic reaction at 60℃ for 72h, samples were taken and tagatose and sorbose in the reaction solution were detected by high performance liquid chromatography ( Figure 2 ).

[0061] and Figure 1 compared to, Figure 2 The content of sorbose in the starch was reduced by about 85%. It can be seen that the glucose 6-phosphate isomerase (Pgi-R) and tagatose 6-phosphate isomerase (T6PE-P) of the present invention can effectively reduce the occurrence of side reactions in the process of catalyzing starch one-step production of tagatose.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for producing tagatose from starch in one step, characterized in that: include: α-glucan phosphorylase, phosphoglucomutase, crude enzyme solution of glucose 6-phosphate isomerase Pgi-R, crude enzyme solution of tagatose 6-phosphate isomerase T6PE-P and tagatose 6-phosphate phosphatase are respectively added to the starch reaction solution to carry out catalytic reaction, thereby obtaining; The amino acid sequence of the glucose-6-phosphate isomerase Pgi-R is shown in SEQ ID NO: 1; the amino acid sequence of the tagatose-6-phosphate isomerase T6PE-P is shown in SEQ ID NO:

2.

2. The production method according to claim 1, characterized in that The starch reaction liquid comprises: dipotassium hydrogen phosphate, magnesium sulfate and starch liquefaction liquid.

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