Method for producing tagatose by starch with extremely low side reaction product through one-step methodTechnical Field
The invention belongs to the field of tagatose production, and in particular relates to a one-step method for producing tagatose by using starch with extremely low side reaction products.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
In the process of catalyzing and producing tagatose by a starch one-step method, along with the gradual accumulation of tagatose in a solution system, the side reaction of glucose hexaphosphate isomerase and tagatose hexaphosphate isomerase is gradually shown. The glucose hexaphosphate isomerase is originally used for catalyzing glucose hexaphosphate to form fructose hexaphosphate, tagatose hexaphosphate isomerase is originally used for catalyzing fructose hexaphosphate to form tagatose hexaphosphate, however, side reactions generated by the two enzymes can catalyze tagatose to form tagatose (peak No. 10 of a chromatogram) and sorbose (peak No. 7 of the chromatogram) when the tagatose content is high in a system, as shown in fig. 1. However, too high a level of sorbose can greatly impact the cost of separation and crystallization of tagatose.
Disclosure of Invention
In order to solve the problems, the invention provides a production method for producing tagatose by a starch one-step method with extremely low side reaction products. The invention screens out two enzymes, namely glucose hexaphosphate isomerase (Pgi-R) and tagatose hexaphosphate isomerase (T6 PE-P), from glucose hexaphosphate isomerase and tagatose hexaphosphate isomerase in a hot spring metagenome, and the two enzymes have extremely low side reaction catalysis capability for synthesizing sorbose in the process of catalyzing starch to form tagatose by a one-step method.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect of the present invention, there is provided a glucose hexaphosphate isomerase (Pgi-R) for the one-step production of tagatose from starch, which is an extremely low side reaction product, said glucose hexaphosphate isomerase having
The protein with the amino acid sequence shown in SEQ ID NO. 1.
SEQ ID NO. 1 is as follows:
mekitfdsskassflseyeinyfegfvkqahemlhsktgpgndflgwvdlplnydreefarikqsaekikadsdvlivi
giggsylgaraaiemlshsfynmlpksrrktpeiyfvgnnisstyvtdlleliegkevsvnvisksgtttepavafrifrey
menkygrqearkriyattdkargalkkladeegyesfvipddvggrfsvltavgllpiavcgadidkimqgaadackl
ysnvdfrnndcyryaaarnalynknktteimvnyepslhfftewwkqlygesegkdqkgifpagvdfttdlhsmgq
yiqdglrnlfetvihvgkakksitikedkdnidglnflagkemdfvnnkafegtllahtdggvpnfvinvpelneyyfgnlvyffekacgisgyllavnpfdqpgveaykknmfallgkpgyeeqkkqlearlgk.
In some embodiments, the tagatose glucose hexaphosphate isomerase (Pgi-R) is derived from a bacterium Ruminiclostridium cellobioparum.
In a second aspect of the invention, there is provided a polynucleotide encoding a polypeptide such as
The nucleotide sequence of the protein shown in SEQ ID NO. 1.
In a third aspect of the invention, there is provided the use of glucose hexaphosphate isomerase (Pgi-R) as described above for reducing the side reactions of a starch one-step process for the production of tagatose.
In some embodiments, the side reaction includes catalyzing the formation of sorbose from the starch.
In a fourth aspect of the present invention, there is provided a tagatose hexaphosphate isomerase (T6 PE-P) for use in a one-step process for producing tagatose from starch, the tagatose hexaphosphate isomerase being a protein having an amino acid sequence shown in SEQ ID NO. 2.
In some embodiments, (T6 PE-P) of tagatose hexaphosphate isomerase is derived from a bacterium Paraburkholderia phenoliruptrix.
In a fifth aspect of the invention, there is provided a polynucleotide encoding a polypeptide such as
The nucleotide sequence of the protein shown in SEQ ID NO. 2.
SEQ ID NO. 2 is as follows:
Mtnvvsrlsggegtakklkgiysicsahpwvlgaamkqaldddtplliestsnqvdqfggytgmkpadfvrfvhlia
drtglprtrlilggdhlgpnawrslpaeeamqraealidayvsagftkihldtsmscagdparlsddvvaerasrlcaiae
aaaerkgnrekpvyiigtevpvpggaaeeletveitspdaaldtvavhrnawrdrglddawqrvialvvqpgvefdht
kvvdykpelatklsailkelpgmvfeahstdyqtpealaalvqdgfailkvgpgvtfalrealyalaeienelvvpearsn
lrevvervmlakpgnwekyyhgddrekrllrtysysdrvryywadpeidaaanklisnladidisenvlsrylpeqywqfrrglidatpmsliqskvrevigvyaaacka.
In a sixth aspect, the invention provides the use of (T6 PE-P) of tagatose hexaphosphate isomerase as described above for reducing side reactions in the one-step production of tagatose from starch.
In some embodiments, the side reaction includes catalyzing the formation of sorbose from the starch.
In a seventh aspect of the present invention, there is provided a method for producing tagatose from starch, which is an extremely low side reaction product, by a one-step process, comprising:
And respectively adding alpha glucan phosphorylase, phosphoglucomutase, the Pgi-R crude enzyme liquid, the T6PE-P crude enzyme liquid and the tagatose-6 phosphate phosphatase (T6 PP) into the reaction liquid of the starch, and carrying out catalytic reaction at a certain temperature to obtain the modified starch.
In some embodiments, the starch reaction solution comprises dipotassium hydrogen phosphate, magnesium sulfate and starch liquefaction solution.
The beneficial effects of the invention are that
(1) The invention screens and clones a glucose hexaphosphate isomerase from Ruminiclostridium cellobioparum from a high Wen Requan metagenome, which is named Pgi-R. Compared with glucose hexaphosphate isomerase from other sources, the capability of the enzyme for catalyzing synthesis side reaction is reduced by 70%.
(2) The invention screens and clones a tagatose hexaphosphate isomerase from Paraburkholderia phenoliruptrix from a high Wen Requan metagenome, which is named as T6PE-P. Compared with tagatose hexaphosphate synthetase from other sources, the enzyme has 85% lower capability of catalyzing synthesis side reaction.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a chromatogram of a mixed sugar solution for catalyzing starch formation in a one-step process using the conventional method in example 2, tagatose (peak No. 10 of the chromatogram) and sorbose (peak No. 7 of the chromatogram).
FIG. 2 is a chromatogram of a mixed sugar solution for catalyzing starch formation using the Pgi-R, T PE-P one-step process of the invention in example 2, tagatose (peak No. 10 of the chromatogram) and sorbose (peak No. 7 of the chromatogram).
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
In the following examples, the experimental materials are as follows:
maltodextrin, ALDRICH company product, product number 419672;
pET41b vector, novagen, madison, wis;
Coli expressing strain BL21 (DE 3), invitrogen, carlsbad, calif.
Other raw materials are all commercial products.
EXAMPLE 1 cloning of Pgi-R and T6PE-P genes
The amino acid sequences of the glucose hexaphosphate isomerase and tagatose hexaphosphate synthase which can be obtained are obtained from sequences obtained under the metagenome measurement of the hot spring sediment. The invention then entrusts the golden sri company to design the polynucleotide (DNA) sequence for encoding the polypeptide according to the amino acid sequence, and optimizes the codon of the escherichia coli expression system, and the optimized polynucleotide sequence is shown as SEQ.NO.3 and SEQ.NO. 4.
SEQ.NO.3:
ATGGAGAAGATCACCTTCGATAGCAGTAAGGCCAGTAGTTTTCTGAGCGAATACGAAATCAACTACTTCGAGGGTTTCGTGAAGCAGGCCCATGAAATGCTGCATAGTAAAACCGGTCCGGGCAATGATTTTCTGGGTTGGGTGGATCTGCCGCTGAATTATGATCGCGAAGAATTTGCACGCATCAAGCAGAGCGCCGAAAAAATTAAGGCCGATAGCGATGTTCTGATCGTGATTGGTATCGGCGGCAGTTATCTGGGTGCCCGTGCAGCCATTGAAATGCTGAGTCATAGCTTTTACAACATGCTGCCGAAGAGTCGTCGTAAAACCCCGGAAATCTATTTCGTGGGTAACAATATCAGCAGCACCTATGTTACCGACCTGCTGGAACTGATTGAAGGTAAAGAAGTGAGCGTGAACGTGATTAGTAAGAGCGGCACCACCACCGAACCGGCAGTGGCATTTCGTATTTTTCGCGAATACATGGAGAACAAGTACGGTCGTCAGGAAGCCCGCAAACGCATCTATGCAACCACCGATAAAGCCCGTGGTGCCCTGAAAAAACTGGCAGATGAAGAAGGTTACGAGAGTTTTGTGATCCCGGATGATGTTGGTGGTCGCTTTAGTGTTCTGACCGCAGTGGGTCTGCTGCCGATTGCCGTTTGTGGTGCAGATATTGATAAGATCATGCAGGGCGCCGCCGATGCATGCAAACTGTATAGTAATGTGGACTTCCGCAACAACGACTGTTATCGTTATGCCGCCGCACGCAATGCCCTGTATAATAAGAATAAGACCACCGAGATCATGGTGAACTACGAACCGAGCCTGCATTTCTTTACCGAATGGTGGAAACAGCTGTACGGTGAAAGCGAAGGTAAAGATCAGAAGGGTATCTTCCCGGCAGGCGTGGATTTTACCACCGATCTGCATAGCATGGGCCAGTATATTCAGGATGGCCTGCGTAATCTGTTCGAAACCGTTATTCACGTGGGCAAAGCAAAAAAGAGCATCACCATTAAGGAGGACAAGGATAACATCGACGGTCTGAATTTCCTGGCCGGCAAAGAAATGGATTTCGTTAATAACAAGGCATTCGAGGGCACCCTGCTGGCACATACCGATGGCGGTGTGCCGAATTTTGTTATTAACGTTCCGGAGCTGAACGAGTATTACTTTGGCAATCTGGTGTACTTCTTCGAGAAGGCCTGCGGTATTAGCGGTTATCTGCTGGCAGTGAATCCGTTTGATCAGCCGGGCGTGGAAGCATATAAAAAGAATATGTTCGCACTGCTGGGTAAACCGGGCTATGAAGAACAGAAAAAGCAGCTGGAAGCGCGCCTGGGCAAA
SEQ.NO.4:
ATGACCAACGTGGTGAGTCGCCTGAGTGGCGGCGAAGGTACCGCAAAAAAGCTGAAAGGCATCTATAGCATCTGCAGTGCCCATCCGTGGGTTCTGGGTGCAGCCATGAAACAGGCCCTGGATGATGATACCCCGCTGCTGATTGAAAGCACCAGTAATCAGGTGGATCAGTTTGGCGGCTATACCGGTATGAAACCGGCCGATTTTGTGCGTTTTGTTCATCTGATCGCCGATCGTACCGGTCTGCCGCGTACCCGCCTGATTCTGGGTGGCGATCATCTGGGCCCGAATGCATGGCGCAGCCTGCCGGCAGAAGAAGCAATGCAGCGCGCAGAAGCCCTGATTGATGCATACGTTAGTGCCGGTTTTACCAAAATCCACCTGGATACCAGTATGAGTTGCGCAGGTGACCCGGCACGCCTGAGCGATGATGTTGTGGCAGAACGCGCAAGCCGCCTGTGCGCAATTGCAGAAGCCGCAGCCGAACGTAAAGGCAATCGTGAAAAACCGGTTTACATCATCGGCACCGAAGTGCCGGTGCCGGGTGGCGCAGCTGAAGAACTGGAAACCGTGGAAATTACCAGTCCGGATGCCGCCCTGGATACCGTGGCCGTTCATCGTAATGCCTGGCGCGATCGTGGTCTGGATGATGCATGGCAGCGCGTTATTGCACTGGTTGTTCAGCCGGGCGTTGAATTTGATCATACCAAAGTGGTGGACTACAAGCCGGAACTGGCAACCAAACTGAGCGCAATTCTGAAAGAACTGCCGGGTATGGTTTTCGAAGCCCATAGTACCGATTACCAGACCCCGGAAGCACTGGCCGCCCTGGTTCAGGATGGTTTTGCCATTCTGAAAGTGGGTCCGGGTGTTACCTTTGCCCTGCGCGAAGCACTGTATGCCCTGGCCGAAATTGAAAATGAACTGGTGGTGCCGGAAGCCCGTAGCAATCTGCGCGAAGTTGTGGAACGCGTGATGCTGGCCAAACCGGGTAATTGGGAAAAATATTACCACGGCGACGACCGCGAAAAACGCCTGCTGCGTACCTATAGCTATAGTGATCGTGTTCGCTACTACTGGGCCGATCCGGAAATTGATGCAGCCGCCAATAAGCTGATTAGCAATCTGGCAGATATCGACATCAGCGAAAACGTGCTGAGTCGTTATCTGCCGGAACAGTATTGGCAGTTTCGTCGTGGCCTGATTGATGCCACCCCGATGAGTCTGATTCAGAGCAAAGTGCGTGAAGTGATTGGCGTTTATGCCGCCGCATGCAAAGCA
And (3) connecting the sequence clone pET41b vector with optimized codons by the Kirschner limited company to obtain pET41b-Pgi-R and pET41b-T6PE-P expression vectors. In this expression vector, the expression of the gene is responsible for by elements such as the T7 promoter (T7 promotor) and the T7terminator (T7 terminator).
Example 2 protein expression and Activity assay
(1) Protein expression
PET41b-Pgi-R and pET41b-T6PE-P were transformed into E.coli BL21 (DE 3), and the single clone was picked up to 3ml of LB medium containing 50. Mu.g/ml kanamycin, cultured at 37℃and 220rpm overnight. 1ml of overnight bacteria was taken into 200ml of LB medium containing 50. Mu.g/ml kanamycin, at 37℃and 220rpm until the OD600 reached about 0.8, and Isopropyl beta-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 100. Mu.M, and protein expression was induced at 37℃and 18℃respectively. Induction was for 4 hours at 37 ℃,20 hours at 18 ℃. After the induction, the cells were collected by centrifugation, resuspended in 30mM phosphate buffer (pH 7.0), and subjected to ultrasonic disruption to obtain a cell disruption solution, the expression level of the enzyme was detected by SDS-PAGE, and the cell disruption solution was subjected to high-speed centrifugation (12000 rpm,10 min) to prepare a crude enzyme solution (the crude enzyme solution may be directly added to the catalytic reaction solution in a percentage ratio).
(2) One-step production of tagatose by catalytic starch using traditional method
To 1L of the reaction solution (50 mM dipotassium hydrogen phosphate, 5mM magnesium sulfate, 300g/L starch liquefied solution, pH 7.0), 50U of alpha glucan phosphorylase (. Alpha.GP), 50U of Phosphoglucomutase (PGM), 50U of glucose phosphate isomerase (PGI), 50U of tagatohexaphosphate synthase (T6 PE), 50U of tagatose phosphate phosphatase (T6 PP) were added, and after the reaction was catalyzed at 60℃for 72 hours, samples were taken, and tagatose and sorbose in the reaction solution were detected by high performance liquid chromatography (FIG. 1).
(3) One-step production of tagatose using glucose hexaphosphate isomerase (Pgi-R) and tagatose hexaphosphate isomerase (T6 PE-P) catalytic starch
To 1L of the reaction solution (50 mM dipotassium hydrogen phosphate, 5mM magnesium sulfate, 300g/L starch liquefied solution, pH 7.0), 10U of alpha glucan phosphorylase (. Alpha.GP), 10U of glucose mutase Phosphate (PGM), 10ml of Pgi-R crude enzyme solution, 30ml of T6PE-P crude enzyme solution, 50U of tagatose phosphate phosphatase (T6 PP) were added, and after catalytic reaction at 60℃for 72 hours, samples were taken, and tagatose and sorbose in the reaction solution were detected by high performance liquid chromatography (FIG. 2).
The content of sorbose in fig. 2 is reduced by about 85% compared to fig. 1. From the results, the glucose hexaphosphate isomerase (Pgi-R) and tagatose hexaphosphate isomerase (T6 PE-P) of the invention can effectively reduce the occurrence of side reactions in the process of producing tagatose by a one-step method of catalyzing starch.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.