CN114015671A - Histone alcohol phosphate aminotransferase mutant 66, engineering bacteria and application - Google Patents
Histone alcohol phosphate aminotransferase mutant 66, engineering bacteria and application Download PDFInfo
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Abstract
The invention discloses a histidinol phosphate aminotransferase mutant 66, engineering bacteria and application, and belongs to the technical field of biology. The histidinol phosphotransferase gene is derived from Streptomyces luteus strain HY61, and has a nucleotide sequence shown in SEQ ID NO.1 and an amino acid sequence shown in SEQ ID NO. 2. The mutant is represented by SEQ ID NO:2, the screened mutant enzyme activity is improved to 103.2 percent compared with the original mutant enzyme activity which is obtained by single mutation at the 66 th site of the amino acid sequence shown in the 2. The histidinol phosphate aminotransferase mutant engineering strain constructed by the invention is matched with nitrogen-filling protection and a stabilizer, so that the stability of the 3-indole pyruvic acid is effectively improved, the raw materials are cheap and easy to obtain, the production process is simple and easy to implement, the production cost is lower, and the high-yield green production of the 3-indole pyruvic acid is realized.
Description
Technical Field
The invention relates to a histidinol phosphate aminotransferase mutant, engineering bacteria and application, belonging to the technical field of biology.
Background
3-Indole Pyruvic Acid (IPA) is an important intermediate compound, is a key precursor for manufacturing substances such as 3-indole acetic acid (auxin), 3-indole ethanol, DL-tryptophan, D-tryptophan and the like, is a therapeutic drug for nervous systems, has wide application in the fields of agriculture, food and medicine, and has wide application prospect. The traditional chemical method for synthesizing IPA needs to consume a large amount of alkali and pyridine aldehyde, is expensive, has harsh reaction conditions, complicated product purification and low yield, and has the risk of environmental pollution caused by byproducts. Oxidative deamination by using L-amino acid oxidase is also an important method for synthesizing IPA, but the degradation of IPA is caused by a byproduct of hydrogen peroxide, and the yield and the purity are difficult to ensure. Patent CN200880104143.X utilizes Escherichia coli engineering to express amino acid oxidase from Providencia rettgeri AJ2770 strain, and converts L-tryptophan to produce IPA, wherein in a reaction system of 200mmol/L substrate tryptophan, the highest concentration of IPA is only 129mM, and the yield of the product is low.
Transaminase (Transaminase), also called Aminotransferase (Aminotransferase), catalyzes transaminases between amino groups and keto groups, can produce IPA by transamination using L-tryptophan which is a cheap raw material, does not need to be introduced with oxygen in the reaction process, does not produce byproducts (such as hydrogen peroxide) which can destroy the stability of keto acid, and has a wide application prospect.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a histidinol phosphate aminotransferase mutant, a recombinant strain and a whole cell thereof as a catalyst for synthesizing 3-indole pyruvic acid.
The invention is realized by the following technical scheme:
the invention provides a histidinol phosphate aminotransferase mutant, wherein a histidinol phosphate aminotransferase gene is derived from Streptomyces luteovirticus HY61(Streptomyces luteovirticus) CGMCC15060, the nucleotide sequence of the mutant is shown as SEQ ID NO.1, and the amino acid sequence of the mutant is shown as SEQ ID NO. 2. The mutant is represented by SEQ ID NO:2, the amino acid sequence is obtained by single mutation or multiple point mutation at the 66 th position, the 138 th position, the 197 th position, the 226 th position and the 311 th position of the amino acid sequence shown in the sequence table.
Further, the mutant is one of histidinol phosphate aminotransferase mutant in which valine at position 66 is mutated to isoleucine (hisC-V66I), asparagine at position 138 is mutated to glutamine (hisC-N138Q), serine at position 197 is mutated to histidine (hisC-S197H) or proline at position 226 is mutated to phenylalanine and arginine at position 311 is mutated to tyrosine (hisC-P226F-R311Y), and more preferably, the mutant is hisC-P226F-R311Y.
The invention also relates to a coding gene of the histidinol phosphate aminotransferase mutant.
The invention provides an engineering bacterium containing the coding gene.
The invention also provides application of the histidinol phosphate aminotransferase mutant in catalyzing L-tryptophan to synthesize 3-indole pyruvic acid.
Further, the application method comprises the steps of taking bacterial cells obtained by fermenting and centrifuging engineering bacteria containing histidinol phosphate aminotransferase mutant genes as a whole-cell catalyst, taking L-tryptophan as a substrate, taking pyruvic acid with the molar concentration of 1.2 times of that of the L-tryptophan as an amino receptor, adding 25mg/L coenzyme pyridoxal phosphate and 1g/L of a stable protective agent into a conversion system, adjusting the pH to 7.5 by ammonia water, introducing nitrogen to drive oxygen, and reacting at the temperature of 30-40 ℃ to produce the 3-indolypyruvic acid.
Furthermore, in the transformation system, the dosage of the catalyst is 10-40 g/L in terms of the weight of wet bacteria, the concentration of the substrate is 50-200 mM, and the concentration of pyruvic acid is 60-240 mM.
Further, the engineering bacteria containing histidinol phosphate aminotransferase mutant genes are prepared by the following method: inoculating the recombinant escherichia coli containing the histidinol phosphate aminotransferase mutant gene into an LB slant culture medium to be cultured for 12-18 h at 37 ℃; inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shaking culture at 37 ℃ and 180r/min for 4-10 h; 2.0L of culture medium is filled in a 3L fermentation tank, the seed liquid is inoculated into the fermentation culture medium according to the inoculation amount of 2-12% of the volume ratio, the initial rotating speed is 220r/min, the initial aeration flow is 1.0L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value at 20-30% of air saturation, ammonia water with the mass volume ratio of 25% is used for adjusting the pH value to be stabilized at 6.7, lactose is added after the culture is carried out for 4-12 h at 37 ℃ until the final concentration is 1-5 g/L, and the temperature is reduced to 20-30 ℃ for induced expression; and feeding 500g/L of glycerol solution in the fermentation process to maintain the concentration of the glycerol at 10000r/min and 4 ℃ for centrifugation for 10min after the fermentation is finished, collecting the thalli, and washing the thalli twice by using sterile normal saline to obtain the histidinol phosphate aminotransferase mutant whole-cell catalyst.
Further, the components and final concentrations of the LB slant culture medium are as follows: 5g/L of yeast extract powder, 10g/L of peptone, 5g/L of NaCl, 100mg/L of ampicillin, 20g/L of agar, pH 7.0-7.2, and performing high-pressure steam sterilization at 121 ℃ for 20 min;
further, the components and final concentrations of the LB liquid seed culture medium are as follows: 5g/L of yeast extract powder, 10g/L of peptone, 5g/L of NaCl, 100mg/L of ampicillin, 7.0-7.2 of pHs, and sterilizing for 20min by high-pressure steam at 121 ℃;
further, the components and final concentrations of the fermentation medium are as follows: 20g/L of glycerol, 20g/L of peptone, 5g/L of yeast extract powder and MgSO4 2g/L，KH2PO4 12.5g/L，(NH4)2SO42g/L, 1g/L of citric acid, 3g/L of corn steep liquor dry powder and CaCl21g/L, 20 mu g/L of D-biotin, pH 6.7-7.0, and high-pressure steam sterilization at 121 ℃ for 20 min.
Further, the stabilizing and protecting agent is one or the combination of sodium sulfite and phytic acid.
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts error-prone PCR technology to successfully mutate histidinol phosphate aminotransferase derived from streptomyces luteorus and construct engineering strains, the enzyme activity of the screened mutant is improved by 120 percent compared with the original enzyme activity, the mutant is used for the enzymatic synthesis of 3-indole pyruvic acid, the stability of the 3-indole pyruvic acid is effectively improved by the matching use of nitrogen charging protection and stabilizing agent, the product concentration can reach 179.8mM to the maximum, and the conversion rate can reach 89.9 percent to the maximum. Meanwhile, the raw materials used by the production method are cheap and easy to obtain, the production process is simple and easy to implement, and the production cost is low. The established whole-cell conversion system solves the problems of harsh reaction conditions, complicated product purification, low yield and environmental pollution risk of byproducts in the synthesis of IPA by a chemical method and the damage influence of the byproduct hydrogen peroxide of L-amino acid oxidase on the products, and realizes the high-yield green production of IPA.
Detailed Description
The technical solution of the present invention is further explained by the following examples, but the scope of the present invention is not limited in any way by the examples.
Example 1: expression vector and engineering bacterium construction
An upstream primer hisC-NcoI-F containing an NcoI enzyme cutting site was designed according to Streptomyces luteus strain HY61 with the biological preservation number of CGMCC15060 (the strain was biologically deposited before filing date and disclosed in patent 2018100327172) gene hisC sequence (SEQ ID No. 1):
and a downstream primer hisC-XhoI-R containing an XhoI cleavage site:
The recombinant vector pET32a-hisC was used as a template for amplification by an error-prone PCR method. The error-prone PCR reaction system is as follows: PCR Grade Water 39. mu.L, 10 XTIONIUM Taq Buffer 5. mu.L, MnSO4(8mM) 1. mu.L, dGTP (2mM) 1. mu.L, 50 XDersify dNTP Mix 1. mu.L, Primer Mix 1. mu.L, template 1. mu.L, TITANIUM Taq Polym 1. mu.L, for a 50. mu.L system (where the Primer Mix in the reaction system is a mixed solution of 0.5. mu.L of each of the upstream and downstream primers as described in example 1). The error-prone PCR reaction conditions were: 30s at 94 ℃ for 1 cycle; 30s at 94 ℃, 1min at 68 ℃ and 30s, and 25 cycles; 1min at 68 ℃ for 1 cycle. Error-prone PCR amplification products were detected on a 1% agarose gel and purified using the AxyPrep DNA gel recovery kit. The vector pET32a and the amplification product are respectively cut by Nco I and Xho I, then the amplification product is connected with the vector by T4 DNA ligase, the connected recombinant vector is hot shocked and transferred into E.coli BL21(DE3) competent cells, and is cultured for 1h at 37 ℃ and 200r/min for activation, the activated recombinant cells are coated on an LB plate containing 0.1mg/mL ampicillin resistance, and are inversely cultured at 37 ℃ overnight, and the histidinol phosphate aminotransferase mutant expression library is obtained.
From the colonies of the obtained histidinol phosphate aminotransferase mutant expression library, high-activity mutant strains are screened from the single colonies by using a high-throughput screening method. The specific screening method comprises the following steps: randomly selecting 600 single colonies from a flat plate, inoculating the single colonies into a 96-deep-hole plate, culturing for 9 hours at 37 ℃ and 180r/min by using an LB liquid culture medium, centrifugally collecting thalli, removing a supernatant culture medium, adding a fermentation culture medium, culturing for 6 hours at 37 ℃ and 220r/min, adding lactose to a final concentration of 2g/L, and cooling to 20 ℃ for induced expression; the cells were collected by centrifugation and the enzyme activity was measured. And (3) a standard enzyme activity detection system: 25g/L wet thallus, 50mM substrate L-tryptophan, 25mg/L coenzyme pyridoxal phosphate, 60mM pyruvic acid, and the reaction medium is phosphate buffer solution with pH 7.5, and the total volume is 1 mL. Definition of unit enzyme activity: under standard reaction conditions, the amount of enzyme required to produce 1. mu. mol of 3-indolylpyruvic acid per minute is one enzyme activity unit U. 4 high-activity histidinol phosphate aminotransferase mutant strains are obtained by screening, and are respectively shown as SEQ ID NO:2 (hisC-V66I), glutamine 138 (hisC-N138Q), serine 197 (hisC-S197H), proline 266 (phenylalanine) and arginine 311 (tyrosine) 311 (hisC-P226F-R311Y), and E.coli BL21(DE3)/pET32a-hisC-V66I, E.coli BL21(DE3)/pET32a-hisC-N138Q, E.coli BL21(DE3)/pET32a-hisC-S197H and E.coli BL21(DE3)/pET32a-hisC 46311-P46226-R Y. The most active of them was the engineered strain E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y (Table 1).
TABLE 1 histidinol phosphate aminotransferase mutant enzyme Activity
Example 2: production of histidinol phosphate aminotransferase mutant (hisC-N138Q, hisC-P226F-R311Y) by fermentation of recombinant bacteria and production of IPA by enzymatic conversion of L-tryptophan
(1) Respectively inoculating E.coli BL21(DE3)/pET32a-hisC-N138Q and E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y into LB slant culture medium for culturing at 37 ℃ for 18 h; inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shaking culture at 37 deg.C and 180r/min for 9 h; 2.0L of culture medium is filled in a 3L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by the inoculation amount of 4 percent of the volume ratio, the initial rotating speed is 220r/min, the initial aeration flow is 1.0L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value at 20-30 percent of air saturation, the pH value is adjusted by ammonia water with the mass volume ratio of 25 percent and is stabilized at 6.7, the lactose is added after the culture is carried out for 6h at 37 ℃ until the final concentration is 2g/L, and the temperature is reduced to 20 ℃ for induced expression; and in the fermentation process, 500g/L of glycerol solution is fed in to maintain the glycerol concentration at 10000R/min and 4 ℃ for 10min after the 5-10 g/L fermentation is finished, the thalli are collected by centrifugation, and are washed twice by sterile normal saline to respectively obtain the whole-cell catalyst of E.coli BL21(DE3)/pET32a-hisC-N138Q and E.coli BL21(DE3)/pET32 a-hisC-P226F-R311Y.
(2) The histidinol phosphate aminotransferase mutant is used for producing the 3-indole pyruvic acid by whole-cell catalysis, and the transformation conditions are as follows: preparing 2L of 50mM L-tryptophan solution, adding pyruvic acid with the molar concentration 1.2 times that of tryptophan and pyridoxal phosphate with the molar concentration 25mg/L, phytic acid with the concentration 1g/L, ammonia water to adjust the pH value to 7.5, adding 10g of wet thalli, introducing nitrogen at the flow rate of 0.5L/min to drive oxygen for 15min, maintaining the temperature at 35 ℃, converting for 3h on a 3L fermentation tank, and taking E.coli BL21(DE3)/pET32a-hisC-N138Q as a catalyst to ensure that the IPA concentration is 45.7mM and the molar conversion rate is 91.4%; the IPA concentration using E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y as a catalyst was 47.6mM, and the molar conversion was 95.2%.
Example 3: production of histidinol phosphate aminotransferase mutant (hisC-P226F-R311Y) by fermentation of recombinant bacteria and production of IPA by enzymatic conversion of L-tryptophan
(1) Inoculating genetically engineered bacterium E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y into LB slant culture medium for culturing for 15h at 37 ℃; inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shaking culture at 37 deg.C and 180r/min for 10 h; 2.0L of culture medium is filled in a 3L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by the inoculation amount of 5 percent of the volume ratio, the initial rotating speed is 220r/min, the initial aeration flow is 1.0L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value at 20-30 percent of air saturation, the pH value is adjusted by ammonia water with the mass volume ratio of 25 percent and is stabilized at 6.7, the lactose is added after the culture is carried out for 4 hours at 37 ℃ until the final concentration is 1g/L, and the temperature is reduced to 22 ℃ for induced expression; and feeding 500g/L of glycerol solution in the fermentation process to maintain the concentration of the glycerol at 5-10 g/L, centrifuging at 10000r/min and 4 ℃ for 10min after the fermentation is finished, collecting thalli, and washing the thalli twice by using sterile normal saline to obtain the whole-cell catalyst.
(2) The histidinol phosphate aminotransferase mutant (hisC-P226F-R311Y) is used for producing the 3-indole pyruvic acid by whole cell catalysis, and the transformation conditions are as follows: 2L of 100mM L-tryptophan solution is prepared, pyruvic acid with the molar concentration 1.2 times that of tryptophan and pyridoxal phosphate with the molar concentration 25mg/L, 1g/L of sodium sulfite and ammonia water are added to adjust the pH value to 7.5, 20g of wet thalli are added, nitrogen is introduced at the flow rate of 0.5L/min to drive oxygen for 15min, the temperature is maintained at 35 ℃, the conversion is carried out for 5h on a 3L fermentation tank, the IPA concentration is 87.4mM, and the molar conversion rate is 87.4%.
Example 4: production of histidinol phosphate aminotransferase mutant (hisC-P226F-R311Y) by fermentation of recombinant bacteria and production of IPA by enzymatic conversion of L-tryptophan
(1) Inoculating genetically engineered bacterium E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y into LB slant culture medium for culturing for 15h at 37 ℃; inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shaking culture at 37 deg.C and 180r/min for 10 h; 2.0L of culture medium is filled in a 3L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by the inoculation amount of 5 percent of the volume ratio, the initial rotating speed is 220r/min, the initial aeration flow is 1.0L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value at 20-30 percent of air saturation, the pH value is adjusted by ammonia water with the mass volume ratio of 25 percent and is stabilized at 6.7, the lactose is added after the culture is carried out for 4 hours at 37 ℃ until the final concentration is 1g/L, and the temperature is reduced to 22 ℃ for induced expression; and feeding 500g/L of glycerol solution in the fermentation process to maintain the concentration of the glycerol at 5-10 g/L, centrifuging at 10000r/min and 4 ℃ for 10min after the fermentation is finished, collecting thalli, and washing the thalli twice by using sterile normal saline to obtain the whole-cell catalyst.
(2) The histidinol phosphate aminotransferase mutant (hisC-P226F-R311Y) is used for producing the 3-indole pyruvic acid by whole cell catalysis, and the transformation conditions are as follows: 2L of 100mM L-tryptophan solution is prepared, pyruvic acid with the molar concentration of 1.2 times of that of tryptophan, pyridoxal phosphate with the molar concentration of 25mg/L, sodium sulfite with the molar concentration of 1g/L, phytic acid with the molar concentration of 1g/L and ammonia water are added to adjust the pH to be 7.5, 20g of wet thalli are added, nitrogen is introduced at the flow rate of 0.5L/min to expel oxygen for 15min, the temperature is maintained at 35 ℃, the conversion is carried out for 5h on a 3L fermentation tank, the concentration of IPA is 92.2mM, and the molar conversion rate is 92.2%. Example 5: production of histidinol phosphate aminotransferase mutant (hisC-P226F-R311Y) by fermentation of recombinant bacteria and production of IPA by enzymatic conversion of L-tryptophan
(1) Inoculating genetically engineered bacterium E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y into LB slant culture medium for culturing at 37 ℃ for 14 h; inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shaking culture at 37 deg.C and 180r/min for 8 h; 2.0L of culture medium is filled in a 3L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by the inoculation amount of 5 percent of the volume ratio, the initial rotating speed is 220r/min, the initial aeration flow is 1.0L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value at 20-30 percent of air saturation, the pH value is adjusted by ammonia water with the mass volume ratio of 25 percent and is stabilized at 6.7, the lactose is added after the culture is carried out for 4 hours at 37 ℃ until the final concentration is 4g/L, and the temperature is reduced to 20 ℃ for induced expression; and feeding 500g/L of glycerol solution in the fermentation process to maintain the concentration of the glycerol at 5-10 g/L, centrifuging at 10000r/min and 4 ℃ for 10min after the fermentation is finished, collecting thalli, and washing the thalli twice by using sterile normal saline to obtain the whole-cell catalyst.
(2) The histidinol phosphate aminotransferase mutant (hisC-P226F-R311Y) is used for producing the 3-indole pyruvic acid by whole cell catalysis, and the transformation conditions are as follows: 2L of 200mM L-tryptophan solution is prepared, pyruvic acid with the molar concentration 1.2 times that of tryptophan, pyridoxal phosphate with the concentration 25mg/L, sodium sulfite with the concentration 1g/L, phytic acid with the concentration 1g/L and ammonia water are added to adjust the pH to be 7.5, 30g of wet thalli are added, nitrogen is introduced at the flow rate of 0.5L/min to expel oxygen for 15min, the temperature is maintained at 37 ℃, the conversion is carried out for 12h on a 3L fermentation tank, the IPA concentration is 179.8mM, and the molar conversion rate is 89.9%.
SEQ ID No.1:
Atgacgcgattacacgagctccggctgcacctcaacgagaacccctacccgccactgcccgaggtccgcgaggccctcgcggcgcagctggacgcggtcaaccgctatccggagttcaccccggtcacgctcgtcgggatgatcgccgactggctgggggtcgcccgtgaggcggtggcggtcggcaacggttcggtcgggatcgcgctccaggtcctcgacctgtgcacgggaccgggcgacgaggtggtgtacgggtggcggtcgttcgacgcgtatcccatcatcacgcggatggccggggccgaggccgtgcaggtgccgctgacggccgccggtgagcaggacctcgacggaatgctcgccgcggtcacaccgcggaccagggtcgtggtgctgtgcaacccgcacaacccgacgggcacggtcttcgaccgcatcgcgctgaagtccttcctcgcggccctgccggagcgcgtcacggtcgtcctggacgaggcgtaccacgagttcgcccgcgaccccgggatccccgacgggctggaccacctcgcggaccaccccaacctcctggtcctgcgcaccttctccaaggcctacgggctggccgcgctgcgcgtcggctactgcgtcgcggcgccggagctcgcggggcggctgcgggaggcgtcgctgccgtacggcatcagcccgctcgcccaggtcggggtggcggcgtcgctgggggcgcgggagcagctcacggcgcgcgtcgacgcgatcgtcggcgagcgcgaccggctgcgcgaggggctcgcccgtctcggctggcacagcttcccgagccacggcaacttcctgtggctcggggccggggacgcgagcgagcgcctcgcggcggcgctgggcggggccggggcgctggtgcgctgctacccgggcgaaggcgtgcggctgaccgtgggcctgcccgaggccaacgacctcgtcctgcgcgccgccggaccggcggccgcctga；
SEQ ID No.2：
MTRLHELRLHLNENPYPPLPEVREALAAQLDAVNRYPEFTPVTLVGMIADWLGVAREAVAVGNGSVGIALQVLDLCTGPGDEVVYGWRSFDAYPIITRMAGAEAVQVPLTAAGEQDLDGMLAAVTPRTRVVVLCNPHNPTGTVFDRIALKSFLAALPERVTVVLDEAYHEFARDPGIPDGLDHLADHPNLLVLRTFSKAYGLAALRVGYCVAAPELAGRLREASLPYGISPLAQVGVAASLGAREQLTARVDAIVGERDRLREGLARLGWHSFPSHGNFLWLGAGDASERLAAALGGAGALVRCYPGEGVRLTVGLPEANDLVLRAAGPAAA。
Sequence listing
<110> Shandong Yangcheng Biotech Co., Ltd
<120> histidinol phosphate aminotransferase mutant 66, engineering bacteria and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 999
<212> DNA
<213> Streptomyces luteovirticus strain HY61(Streptomyces luteoviterillitatus HY61)
<400> 1
atgacgcgat tacacgagct ccggctgcac ctcaacgaga acccctaccc gccactgccc 60
gaggtccgcg aggccctcgc ggcgcagctg gacgcggtca accgctatcc ggagttcacc 120
ccggtcacgc tcgtcgggat gatcgccgac tggctggggg tcgcccgtga ggcggtggcg 180
gtcggcaacg gttcggtcgg gatcgcgctc caggtcctcg acctgtgcac gggaccgggc 240
gacgaggtgg tgtacgggtg gcggtcgttc gacgcgtatc ccatcatcac gcggatggcc 300
ggggccgagg ccgtgcaggt gccgctgacg gccgccggtg agcaggacct cgacggaatg 360
ctcgccgcgg tcacaccgcg gaccagggtc gtggtgctgt gcaacccgca caacccgacg 420
ggcacggtct tcgaccgcat cgcgctgaag tccttcctcg cggccctgcc ggagcgcgtc 480
acggtcgtcc tggacgaggc gtaccacgag ttcgcccgcg accccgggat ccccgacggg 540
ctggaccacc tcgcggacca ccccaacctc ctggtcctgc gcaccttctc caaggcctac 600
gggctggccg cgctgcgcgt cggctactgc gtcgcggcgc cggagctcgc ggggcggctg 660
cgggaggcgt cgctgccgta cggcatcagc ccgctcgccc aggtcggggt ggcggcgtcg 720
ctgggggcgc gggagcagct cacggcgcgc gtcgacgcga tcgtcggcga gcgcgaccgg 780
ctgcgcgagg ggctcgcccg tctcggctgg cacagcttcc cgagccacgg caacttcctg 840
tggctcgggg ccggggacgc gagcgagcgc ctcgcggcgg cgctgggcgg ggccggggcg 900
ctggtgcgct gctacccggg cgaaggcgtg cggctgaccg tgggcctgcc cgaggccaac 960
gacctcgtcc tgcgcgccgc cggaccggcg gccgcctga 999
<210> 2
<211> 332
<212> PRT
<213> Streptomyces luteovirticus strain HY61(Streptomyces luteoviterillitatus HY61)
<400> 2
Met Thr Arg Leu His Glu Leu Arg Leu His Leu Asn Glu Asn Pro Tyr
1 5 10 15
Pro Pro Leu Pro Glu Val Arg Glu Ala Leu Ala Ala Gln Leu Asp Ala
20 25 30
Val Asn Arg Tyr Pro Glu Phe Thr Pro Val Thr Leu Val Gly Met Ile
35 40 45
Ala Asp Trp Leu Gly Val Ala Arg Glu Ala Val Ala Val Gly Asn Gly
50 55 60
Ser Val Gly Ile Ala Leu Gln Val Leu Asp Leu Cys Thr Gly Pro Gly
65 70 75 80
Asp Glu Val Val Tyr Gly Trp Arg Ser Phe Asp Ala Tyr Pro Ile Ile
85 90 95
Thr Arg Met Ala Gly Ala Glu Ala Val Gln Val Pro Leu Thr Ala Ala
100 105 110
Gly Glu Gln Asp Leu Asp Gly Met Leu Ala Ala Val Thr Pro Arg Thr
115 120 125
Arg Val Val Val Leu Cys Asn Pro His Asn Pro Thr Gly Thr Val Phe
130 135 140
Asp Arg Ile Ala Leu Lys Ser Phe Leu Ala Ala Leu Pro Glu Arg Val
145 150 155 160
Thr Val Val Leu Asp Glu Ala Tyr His Glu Phe Ala Arg Asp Pro Gly
165 170 175
Ile Pro Asp Gly Leu Asp His Leu Ala Asp His Pro Asn Leu Leu Val
180 185 190
Leu Arg Thr Phe Ser Lys Ala Tyr Gly Leu Ala Ala Leu Arg Val Gly
195 200 205
Tyr Cys Val Ala Ala Pro Glu Leu Ala Gly Arg Leu Arg Glu Ala Ser
210 215 220
Leu Pro Tyr Gly Ile Ser Pro Leu Ala Gln Val Gly Val Ala Ala Ser
225 230 235 240
Leu Gly Ala Arg Glu Gln Leu Thr Ala Arg Val Asp Ala Ile Val Gly
245 250 255
Glu Arg Asp Arg Leu Arg Glu Gly Leu Ala Arg Leu Gly Trp His Ser
260 265 270
Phe Pro Ser His Gly Asn Phe Leu Trp Leu Gly Ala Gly Asp Ala Ser
275 280 285
Glu Arg Leu Ala Ala Ala Leu Gly Gly Ala Gly Ala Leu Val Arg Cys
290 295 300
Tyr Pro Gly Glu Gly Val Arg Leu Thr Val Gly Leu Pro Glu Ala Asn
305 310 315 320
Asp Leu Val Leu Arg Ala Ala Gly Pro Ala Ala Ala
325 330
Claims (10)
1. A histidinol phosphate aminotransferase mutant 66 is characterized in that the histidinol phosphate aminotransferase gene is derived from Streptomyces luteus strain HY61, the nucleotide sequence of which is shown as SEQ ID NO.1, and the amino acid sequence of which is shown as SEQ ID NO. 2; the mutant 66 is represented in SEQ ID NO:2, the 66 th valine of the amino acid sequence shown in the specification is mutated into isoleucine.
2. A gene encoding histidinol phosphate aminotransferase mutant 66 of claim 1.
3. An engineered bacterium comprising the coding gene of claim 2.
4. Use of the histidinol phosphate aminotransferase mutant 66 of claim 1 to catalyze the synthesis of 3-indolylpyruvic acid from L-tryptophan.
5. The application of the 3-indolylpyruvic acid as claimed in claim 4, wherein the application method comprises the steps of taking bacterial cells obtained by fermenting and centrifuging engineering bacteria containing histidinol phosphate aminotransferase mutant 66 genes as a whole-cell catalyst, taking L-tryptophan as a substrate, taking pyruvic acid with the molar concentration of 1.2 times of that of the L-tryptophan as an amino acceptor, adding 25mg/L of coenzyme pyridoxal phosphate and 1g/L of a stable protective agent into a conversion system, adjusting the pH to be 7.5 by ammonia water, introducing nitrogen to expel oxygen, and reacting at the temperature of 30-40 ℃ to produce the 3-indolylpyruvic acid.
6. The use according to claim 5, wherein the catalyst is used in an amount of 10 to 40g/L based on the weight of wet cells, the substrate concentration is 50 to 200mM, and the pyruvic acid concentration is 60 to 240mM in the conversion system.
7. The preparation method of the engineering bacteria of claim 3, which is characterized by comprising the following steps: inoculating recombinant escherichia coli containing histidinol phosphate aminotransferase mutant 66 genes into an LB slant culture medium to be cultured for 12-18 h at 37 ℃; inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shaking culture at 37 ℃ and 180r/min for 4-10 h; 2.0L of culture medium is filled in a 3L fermentation tank, the seed liquid is inoculated into the fermentation culture medium according to the inoculation amount of 2-12% of the volume ratio, the initial rotating speed is 220r/min, the initial aeration flow is 1.0L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value at 20-30% of air saturation, ammonia water with the mass volume ratio of 25% is used for adjusting the pH value to be stabilized at 6.7, lactose is added after the culture is carried out for 4-12 h at 37 ℃ until the final concentration is 1-5 g/L, and the temperature is reduced to 20-30 ℃ for induced expression; and (3) feeding 500g/L of glycerol solution in the fermentation process to maintain the concentration of the glycerol at 10000r/min and 4 ℃ for centrifugation for 10min after the fermentation is finished, collecting the thalli, and washing the thalli twice by using sterile normal saline to obtain the histidinol phosphate aminotransferase mutant 66 whole-cell catalyst.
8. The method according to claim 7, wherein the LB slant medium has the following composition and final concentration: 5g/L of yeast extract powder, 10g/L of peptone, 5g/L of NaCl, 100mg/L of ampicillin, 20g/L of agar, pH 7.0-7.2, and performing high-pressure steam sterilization at 121 ℃ for 20 min.
9. The method according to claim 7, wherein the LB liquid seed medium has the following composition and final concentration: 5g/L of yeast extract powder, 10g/L of peptone, 5g/L of NaCl, 100mg/L of ampicillin, 7.0-7.2 of pHs, and high-pressure steam sterilization at 121 ℃ for 20 min.
10. The method of claim 5, wherein the fermentation medium comprises the following components in final concentrations: 20g/L of glycerol, 20g/L of peptone, 5g/L of yeast extract powder and MgSO4 2g/L，KH2PO4 12.5g/L，(NH4)2SO42g/L, 1g/L of citric acid, 3g/L of corn steep liquor dry powder and CaCl21g/L, 20 mu g/L of D-biotin, pH 6.7-7.0, and sterilizing for 20min by high-pressure steam at 121 ℃; the stabilizing and protecting agent is one or the combination of sodium sulfite and phytic acid.
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