L-ribose isomerase and application thereof in preparation of L-ribose by biological method
阅读说明：本技术 一种l-核糖异构酶及其在生物法制备l-核糖中的应用 (L-ribose isomerase and application thereof in preparation of L-ribose by biological method ) 是由 徐虹 刘超 徐铮 王笑 李莎 冯小海 于 2019-10-18 设计创作，主要内容包括：本发明公开了一种L-核糖异构酶,其氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.2所示。该L-核糖异构酶具有较好的热稳定性和pH稳定性,对L-核酮糖的催化效率能达到85％以上。本发明还公开了包含上述L-核糖异构酶的重组菌以及该重组菌的构建方法,该重组菌对L-核酮糖的催化效率能达到81％以上。本发明描述的L-核糖异构酶对于L-核糖的工业化生产具有很好的应用前景与经济价值。(The invention discloses L-ribose isomerase, wherein the amino acid sequence of the L-ribose isomerase is shown as SEQ ID NO.1, and the nucleotide sequence of the L-ribose isomerase is shown as SEQ ID NO. 2. The L-ribose isomerase has good thermal stability and pH stability, and the catalytic efficiency of the L-ribose isomerase on L-ribulose can reach more than 85%. The invention also discloses a recombinant strain containing the L-ribose isomerase and a construction method of the recombinant strain, and the catalytic efficiency of the recombinant strain on L-ribulose can reach more than 81%. The L-ribose isomerase described by the invention has good application prospect and economic value for industrial production of L-ribose.)
1. An L-ribose isomerase, which has the following amino acid sequence:
2. a gene encoding the L-riboisomerase of claim 1, which has the following nucleotide sequence:
3. a recombinant plasmid containing the L-riboisomerase gene according to claim 2.
4. A recombinant bacterium comprising the L-riboisomerase gene according to claim 2.
5. The method for constructing the recombinant strain according to claim 4, wherein the method comprises the steps of:
(1) cloning a nucleotide sequence shown in SEQ ID NO.2 onto a plasmid to obtain a recombinant plasmid;
(2) and (3) transforming the recombinant plasmid into host bacteria to obtain recombinant bacteria.
6. The method for constructing the recombinant strain according to claim 5, wherein the plasmid is pET-28a (+), and the host strain is Escherichia coli BL21(DE 3).
7. Use of the L-ribose isomerase of claim 1 for the preparation of L-ribose.
8. The use of claim 7, wherein 1-100 g/L of L-ribulose is used as a substrate, and L-ribose isomerase is added to perform an enzyme conversion reaction, wherein the amount of the enzyme is 10-500U, the reaction temperature is 30-70 ℃, and the conversion time is 1-20 h.
9. Use of the recombinant bacterium of claim 4 for the production of L-ribose.
10. The application of the recombinant strain as claimed in claim 9, wherein 1-100 g/L of L-ribulose is used as a substrate, the recombinant strain is added for conversion reaction, the addition amount of the recombinant strain is 10-100 g/L, the reaction temperature is 30-70 ℃, and the conversion time is 1-48 h.
Technical Field
The invention belongs to the technical field, and particularly relates to L-ribose isomerase from actinomyces fermentum (Actinotaceae NX-1) and application thereof.
Background
L-ribose belongs to rare monosaccharide, is an important medical intermediate, has important medical value, is various L-ribose derivatives synthesized by L-ribose, and is widely applied to the field of antivirus and antitumor.
At present, the preparation of the L-ribose mainly adopts a chemical method, needs to undergo at least 7 steps of complex reaction, and has the problems of more byproducts, low yield and the like. The L-ribose isomerase (L-RI) can prepare L-ribose by taking L-ribulose as a raw material, has the characteristics of mild reaction, high yield and the like, and the L-ribulose can be obtained by catalyzing L-arabinose by a biological method. The literature currently reports only three L-ribose isomerases, respectively from Acinetobacter sp.DL-28, Geodermatophilus obscurus DSM43160 and Cellulomonas parahominis MB 426. However, the catalytic efficiency of L-RI on L-ribulose has not been found to be high, so that the mining of L-RI having a high catalytic activity on L-ribulose is of great importance for the production of L-ribose.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an L-ribose isomerase with high catalytic capability on L-ribulose.
The technical problem to be solved by the invention is to provide a genetically engineered bacterium containing the L-ribose isomerase and a construction method thereof.
The invention finally aims to solve the technical problem of providing the L-ribose isomerase and the application of the genetic engineering bacteria in preparing L-ribose.
In order to solve the technical problems, the invention adopts the following technical scheme:
an L-ribose isomerase, the amino acid sequence of which is shown in SEQ ID NO. 1. The L-ribose isomerase is derived from Actinotalea fermentans NX-1. The amino acid sequence of the L-ribose isomerase is as follows:
Met Thr Arg Thr Tyr Val Thr Arg Arg Glu Tyr Asp Glu Trp Leu Gln
Glu Ala Ala Ser Leu Ala Arg Ala Leu Arg Tyr Pro Ile Thr Pro Asp
Met Val Asn Asp Ser Ala Gly Ile Val Trp Gly Asp Asp Gln Tyr Ala
Ala Phe Glu Asn Gly Leu Trp Ser Arg Glu Pro Tyr Glu Leu Met Val
Ile Phe Glu Ala Leu Asn Glu Pro Ala Val Asp Gly Leu Pro Val Ser
Ala Ala His Gly Ser Glu Tyr Ser Gly Leu Cys Asp Asp Leu Met Ile
Val His Pro Gly Lys Phe Cys Pro Pro His Tyr His Asn Arg Lys Thr
Glu Ser Tyr Glu Val Val Leu Gly Glu Met Asp Val Phe Tyr Gly Pro
Glu Pro Val Arg Val Glu Asp Glu Glu Thr Val Thr Trp Ser Pro Met
Pro Ala Gly Ser Pro Trp Pro Asp Gly Val Ala Leu Pro Ala Gly Arg
Glu Asp Thr Tyr Ala Ala Leu Thr Ser Tyr Val Arg Leu Ser Ala Gly
Asp Pro Lys Phe Val Met His Arg Lys His Leu His Thr Phe Arg Cys
Pro Ala Glu Ala Thr Thr Pro Leu Val Val Arg Glu Val Ser Thr Tyr
Ser His Glu Pro Thr Glu His Ala Ala Asp Asp Pro Ala Pro Leu Pro
Thr Trp Ala Gly Leu His Asp Asn Ala Trp Leu Ser Pro Ala Ala Gln
Thr Gly Arg Leu Val Thr His Ile Arg。
a gene encoding the L-riboisomerase of claim 1, which has the following nucleotide sequence:
atgacccgta cgtatgtgac ccgtcgcgaa tacgatgaat ggctgcagga agcagcaagt
ctggcacgtg cactgcgcta tccgattacg ccggatatgg tcaacgactc cgcgggcatc
gtgtggggtg atgaccagta tgcagctttc gaaaatggtc tgtggtcacg cgaaccgtac
gaactgatgg ttatttttga agcactgaat gaaccggctg tggatggtct gccggtttcg
gcagcacatg gtagcgaata ttctggtctg tgcgacgatc tgatgatcgt gcacccgggt
aaattctgtc cgccgcatta tcacaatcgt aaaaccgaaa gttacgaagt ggttctgggt
gaaatggatg tgttttacgg cccggaaccg gttcgcgtcg aagacgaaga aaccgttacg
tggagcccga tgccggcagg ttctccgtgg ccggatggtg tcgcgctgcc ggccggccgt
gaagacacct atgcagctct gacgtcatac gttcgcctgt cggcgggcga tccgaaattt
gtcatgcatc gtaaacatct gcacaccttc cgttgcccgg cagaagctac cacgccgctg
gtcgtgcgtg aagttagtac ctattcccat gaaccgacgg aacacgcagc agatgatccg
gcaccgctgc cgacctgggc aggtctgcat gacaacgcgt ggctgtctcc ggcagctcag
accggccgtc tggtgacgca cattcgctaa。
a recombinant plasmid containing the above-mentioned L-riboisomerase gene.
A recombinant bacterium containing the above-mentioned L-ribose isomerase gene.
The construction method of the recombinant bacterium comprises the following steps:
(1) cloning a nucleotide sequence shown in SEQ ID NO.2 onto a plasmid to obtain a recombinant plasmid;
(2) and (3) transforming the recombinant plasmid into host bacteria to obtain recombinant bacteria.
Wherein, the plasmid is pET-28a (+), and the host bacterium is Escherichia coli BL21(DE 3).
The specific plasmid construction method and the recombinant bacterium construction method are as follows:
(1) constructing an expression plasmid: amplifying the nucleotide sequence shown as SEQ ID NO.2 by using the following primer sequences:
primer 1: 5' -CGGGATCC(BamH I)ATGACCCGTACGTATGTGACCCGTC-3’；
Primer 2: 5' -CCCAAGCTT(Hind III)TTAGCGAATGTGCGTCACCAGACGG-3’；
The PCR amplification system is as follows: genomic DNA 2. mu.L, primer1 and primer2 each 1. mu.L, dNTP 2. mu.L, 10 XTag buffer 2.5. mu.L, Extag polymerase 0.5. mu.L, ddH2O14μL；
The PCR reaction program is: pre-denaturation at 94 ℃ for 5min and denaturation at 94 ℃ for 30 s; then annealing at 54 deg.C for 2min, extending at 72 deg.C for 5min, circulating for 30 times, and storing at 4 deg.C;
recovering PCR amplification products, carrying out double enzyme digestion by restriction enzymes BamH I and Hind III, and connecting with plasmid pET-28a subjected to the same double enzyme digestion under the action of T4 ligase to obtain recombinant plasmid pET-28 a-afri;
(2) constructing a recombinant bacterium: transforming the recombinant plasmid pET-28a-afri into competent escherichia coli BL21(DE3), coating the competent escherichia coli BL21 on an LB solid culture medium containing 25 mu g/mL kanamycin, and culturing at 37 ℃ for 18-24 h to obtain a primary positive clone; screening by a resistance culture medium to obtain positive clones: respectively picking the primary positive clones in 5mL LB liquid culture medium containing 25 mug/mL kanamycin, culturing overnight at 37 ℃ and 200rpm, extracting plasmids, digesting the plasmids by restriction enzymes BamH I and Hind III, and judging the plasmids have the sequence table SEQ ID NO: 1 is recombinant plasmid pET-28a-afri, and the colony with the plasmid is positive clone, namely recombinant bacterium.
The sequencing of the recombinant plasmid pET-28a-afri shows that the inserted fragment is a protein containing 747bp and encoding 249 amino acids.
The application of the L-ribose isomerase in preparing L-ribose is provided.
Wherein 1-100 g/L of L-ribulose is used as a substrate, the concentration of the L-ribulose is preferably 100g/L, L-ribose isomerase is added for carrying out enzyme conversion reaction, the dosage of the enzyme is 10-500U, and the addition amount of the L-ribose isomerase is preferably 20U; the reaction temperature is 30-70 ℃, and the preferable reaction temperature is 37 ℃; the conversion time is 1-12 h, and the conversion time is preferably 3 h.
Wherein the enzyme activity of the L-ribose isomerase is defined by the following method: the amount of L-ribose isomerase required to catalyze the production of 1. mu. mol of L-ribose per unit time using L-ribulose as a substrate was defined as 1U.
The preparation method of the L-ribose isomerase comprises the following steps:
inoculating the recombinant strain with the nucleotide sequence shown in SEQ ID NO.2 into LB liquid culture medium added with 25 mug/mL kanamycin, and carrying out shake culture at 37 ℃ overnight; then transferring the strain to LB culture medium containing 25 mug/mL kanamycin with the inoculation amount of 5% (v/v), and fermenting and culturing for 2-3 h at 37 ℃ until OD is reached600When the concentration is 0.6, 0.2-1 mmol/L isopropyl-beta-D-thiogalactoside or 0.5-20 g/L lactose is added, induction expression is continued for 6-20 h, and then the thalli are centrifugally collected.
The obtained cells were suspended in a pH7.0 potassium phosphate buffer, cells were disrupted by ultrasonic waves, and centrifuged to collect the supernatant (crude enzyme solution), and the crude enzyme solution was filtered through a 0.22 μm filter and purified with Ni-NTA affinity resin to obtain L-ribose isomerase purified enzyme. The present invention can utilize purified enzyme or crude enzyme solution to add to the reaction system.
The recombinant bacterium is applied to the preparation of L-ribose.
Wherein 1-100 g/L of L-ribulose is used as a substrate, and the addition amount of the L-ribulose is 100 g/L; adding a recombinant bacterium for conversion reaction, wherein the addition amount of the recombinant bacterium is 10-100 g/L (calculated by wet weight of the bacterium), and the addition amount of the recombinant bacterium is 50g/L of wet bacterium; the reaction temperature is 30-70 ℃, and the preferable reaction temperature is 37 ℃; the conversion time is 1-48 h, and the conversion time is preferably 3 h.
The preparation method of the recombinant bacterium comprises the following steps:
inoculating the recombinant strain with the nucleotide sequence shown in SEQ ID NO.2 into LB liquid culture medium added with 25 mug/mL kanamycin, and carrying out shake culture at 37 ℃ overnight; then transferring the strain to LB culture medium containing 25 mug/mL kanamycin with the inoculation amount of 5% (v/v), and fermenting and culturing for 2-3 h at 37 ℃ until OD is reached600When the concentration is 0.6, 0.2-1 mmol/L isopropyl-beta-D-thiogalactoside or 0.5-20 g/L lactose is added, induction expression is continued for 6-20 h, and then the thalli are centrifugally collected.
Has the advantages that: the invention provides L-ribose isomerase, which has good stability, the reaction temperature is 30-70 ℃, the reaction pH is 5.5-10, and the L-ribose isomerase shows extremely high catalytic efficiency on L-ribulose. The L-ribose isomerase with high catalytic capacity to the L-ribulose has the conversion rate to the L-ribulose of over 75 percent under the most suitable catalytic condition, and has wide application prospect and economic value to the industrial production of the L-ribose.
Drawings
FIG. 1 is a schematic diagram of the construction of recombinant plasmid pET-28-afri.
FIG. 2 is a reaction curve for preparing L-ribose by L-ribose isomerase.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.