CN117402866A - Application of L-arabinose isomerase mutant in preparation of D-tagatose - Google Patents
Application of L-arabinose isomerase mutant in preparation of D-tagatose Download PDFInfo
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- CN117402866A CN117402866A CN202311620368.3A CN202311620368A CN117402866A CN 117402866 A CN117402866 A CN 117402866A CN 202311620368 A CN202311620368 A CN 202311620368A CN 117402866 A CN117402866 A CN 117402866A
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- tagatose
- arabinose isomerase
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- LKDRXBCSQODPBY-OEXCPVAWSA-N D-tagatose Chemical compound OCC1(O)OC[C@@H](O)[C@H](O)[C@@H]1O LKDRXBCSQODPBY-OEXCPVAWSA-N 0.000 title claims abstract description 49
- 108010018080 L-arabinose isomerase Proteins 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 87
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 32
- 239000008101 lactose Substances 0.000 claims abstract description 32
- 230000035772 mutation Effects 0.000 claims abstract description 10
- 239000000758 substrate Substances 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 20
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 150000001413 amino acids Chemical group 0.000 claims description 8
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 7
- 229960000310 isoleucine Drugs 0.000 claims description 7
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 7
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 7
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 6
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 5
- 235000004279 alanine Nutrition 0.000 claims description 5
- 235000013922 glutamic acid Nutrition 0.000 claims description 5
- 239000004220 glutamic acid Substances 0.000 claims description 5
- 229930182817 methionine Natural products 0.000 claims description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 3
- 235000003704 aspartic acid Nutrition 0.000 claims description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 3
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 3
- 230000000813 microbial effect Effects 0.000 claims description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 59
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- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 32
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 21
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- 238000001514 detection method Methods 0.000 description 7
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000006911 enzymatic reaction Methods 0.000 description 6
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- 238000004128 high performance liquid chromatography Methods 0.000 description 6
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- 102100026189 Beta-galactosidase Human genes 0.000 description 5
- 108010005774 beta-Galactosidase Proteins 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 238000002741 site-directed mutagenesis Methods 0.000 description 5
- 108090000769 Isomerases Proteins 0.000 description 4
- 102000004195 Isomerases Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
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- 244000063299 Bacillus subtilis Species 0.000 description 3
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- 241000588724 Escherichia coli Species 0.000 description 3
- 108010059881 Lactase Proteins 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
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- 239000012634 fragment Substances 0.000 description 3
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 description 3
- 229940116108 lactase Drugs 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000003765 sweetening agent Substances 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
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- 238000012163 sequencing technique Methods 0.000 description 2
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- 235000021092 sugar substitutes Nutrition 0.000 description 2
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- 238000003786 synthesis reaction Methods 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 208000007976 Ketosis Diseases 0.000 description 1
- ZAQJHHRNXZUBTE-UCORVYFPSA-N L-ribulose Chemical compound OC[C@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-UCORVYFPSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001323 aldoses Chemical class 0.000 description 1
- -1 alkylene glycol Chemical compound 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
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- 101150035354 araA gene Proteins 0.000 description 1
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- 150000002083 enediols Chemical class 0.000 description 1
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
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- 238000007871 hydride transfer reaction Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
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- C12P19/02—Monosaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
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Abstract
The invention discloses an application of an L-arabinose isomerase mutant in preparing D-tagatose, and belongs to the technical field of enzyme engineering. The invention obtains the L-arabinose isomerase series single-point mutant with obviously improved lactose conversion rate through site-directed mutation and superposition combination construction, and then carries out superposition combination mutation on the single-point mutant and the single-point mutant to obtain the catalytic effectThe combination mutation Y19E/F279I with increased rate. The preparation conditions of the D-tagatose are optimized, and the pH is 6.0 and 500 g.L at 55 DEG C ‑1 Lactose concentration and enzyme addition amount of 1.0 mg.mL ‑1 And contains Mn 2+ With Co 2+ Under the condition of (2), the conversion rate can reach 23.01 percent, which is greatly improved compared with the wild 14.56 percent, and has better industrialized application potential.
Description
Technical Field
The invention relates to an application of an L-arabinose isomerase mutant in preparing D-tagatose, belonging to the technical field of enzyme engineering.
Background
Along with the promotion of Chinese health actions and the improvement of national health consciousness, people increasingly recognize that chronic diseases such as obesity, diabetes and the like caused by high-sugar and high-heat diet have serious harm to human health. Therefore, development of sugar substitute sweeteners has become a research hotspot. In recent years, D-tagatose has been widely used in the fields of foods, medicines, cosmetics, and the like as a natural functional sugar substitute because of its numerous excellent properties.
D-tagatose was approved by the european union for sale, was first introduced into the commercial area in 8 months in the year, and was subsequently approved for use by the food sanitation departments such as united states, australia, japan, korea, etc., and was used in a large amount as a substitute for white sugar in health drinks and products such as yogurt, juice, etc. The public announcement of 6 new food materials such as tagatose (No. 10 in 2014) issued by the national health committee in 2014 has clarified that D-tagatose is a new food material that can be used in foods other than infant foods.
D-tagatose can be obtained in various modes, the most main production mode in the market at present is to prepare D-tagatose by taking lactose as a raw material, the method is produced by a two-step process, namely, the lactose is hydrolyzed by beta-galactosidase (EC 3.2.1.23) to obtain D-galactose, and then the D-galactose is catalyzed by L-arabinose isomerase (L-AI) to be subjected to isomerization conversion to obtain the D-tagatose. L-AI (EC 5.3.1.4), an intracellular enzyme encoded by the araA gene in microorganisms, belongs to the family of aldehyde-ketone isomerase (EC5.3.1), a key enzyme in the pentose phosphate pathway of microorganisms, is considered an important biocatalyst in the production of rare sugars. Various scholars have proposed two reaction mechanisms of ketol isomerase by exploring and analyzing the three-dimensional structure and active center of the enzyme: an alkylene glycol mechanism and a hydride transfer mechanism. Furthermore, the catalytic mechanisms of these enzymes all require metal ion mediated proton exchange, effecting the conversion of aldose to ketose from the intermediate of the enediol. The L-arabinose isomerase can not only isomerize L-arabinose to produce L-ribulose, but also convert catalyzed D-galactose into D-tagatose.
Since the isomerization reaction of D-galactose to D-tagatose is a reversible reaction, the reaction process is more biased to move towards the product direction in order to obtain higher D-tagatose yield, and the affinity of the L-arabinose isomerase for D-galactose is improved. Currently, strategies for increasing the affinity of L-arabinose isomerase substrates are mainly as follows: firstly, searching more proper enzyme seeds; secondly, the active site of enzymes is engineered by protein engineering, and this is one of the most common means at present.
Although the L-arabinose isomerase from different sources has different properties, the L-arabinose isomerase can be widely applied to the industries of foods, health care products and medicines, and has important industrial value. However, the existing demands cannot be met by the only wild-type enzyme in nature, and the application of the L-arabinose isomerase is limited by the short plates with certain properties, so that the L-arabinose isomerase is modified by molecular means, the properties of the L-arabinose isomerase are improved by directed evolution, and the gene meeting the demands of industrial application is obtained, so that the L-arabinose isomerase can be better applied to industrial production.
Disclosure of Invention
The invention provides an L-arabinose isomerase mutant, which is characterized in that the 19 th site is mutated from tyrosine to glutamic acid or aspartic acid or the 185 th site is mutated from methionine to alanine on the basis of a starting sequence; or mutating position 279 from phenylalanine to isoleucine.
In one embodiment, the starting sequence is set forth in SEQ ID NO. 1.
In one embodiment, the mutant is a mutant Y19D obtained by mutating tyrosine 19 of the amino acid sequence shown in SEQ ID NO.1 to aspartic acid to obtain a mutant Y19D shown in SEQ ID NO. 2.
In one embodiment, the mutant is a mutant Y19E obtained by mutating tyrosine 19 of the amino acid sequence shown in SEQ ID NO.1 to glutamic acid.
In one embodiment, the mutant is obtained by mutating phenylalanine at position 279 of the amino acid sequence shown in SEQ ID NO.1 to isoleucine to obtain mutant F279I shown in SEQ ID NO. 4.
In one embodiment, the mutant is a mutant M185A as shown in SEQ ID NO.5, obtained by mutating methionine at position 185 of the amino acid sequence shown in SEQ ID NO.1 to alanine.
In one embodiment, the mutant is obtained by mutating tyrosine 19 of the amino acid sequence shown in SEQ ID NO.1 to glutamic acid and mutating phenylalanine 279 to isoleucine to obtain mutant Y19E/F279I shown in SEQ ID NO. 6.
In one embodiment, the mutant is obtained by mutating methionine at position 185 of the amino acid sequence shown in SEQ ID NO.1 to alanine and mutating phenylalanine at position 279 to isoleucine to obtain mutant M185A/F279I shown in SEQ ID NO. 7.
The invention also provides a gene for encoding the L-arabinose isomerase mutant.
In one embodiment, the nucleotide sequence of the gene is shown in any one of SEQ ID NOS.9 to 14.
The invention also provides a vector carrying the gene.
The invention also provides a microbial cell carrying the gene or the vector.
In one embodiment of the invention, the microbial cells include, but are not limited to, bacterial cells or fungal cells.
The invention also provides a method for preparing D-tagatose, which is to add the L-arabinose isomerase mutant into a lactose-containing reaction system for reaction.
In one embodimentWherein the reaction system takes lactose as a substrate and also contains Mn 2+ And/or Co 2+ 。
In one embodiment of the invention, the enzyme addition amount of the L-arabinose isomerase mutant is more than or equal to 0.2mg/mL; the method specifically comprises the following steps: 0.2-1.4 mg/mL.
In one embodiment, the concentration of lactose as substrate is 200-700 g/L.
In one embodiment, the pH of the reaction system is from 5.0 to 7.5.
In one embodiment, the temperature in the reaction system is 50 to 70 ℃.
In one embodiment, the reaction time in the reaction system is 36 to 60 hours.
The invention also provides an application of the L-arabinose isomerase mutant or the method in preparing D-tagatose.
The beneficial effects are that:
(1) The invention constructs the mutant with improved D-galactose catalytic efficiency, and the conversion rate of the prepared D-tagatose is obviously improved compared with that of a wild type mutant. The conversion rate of D-tagatose prepared from D-galactose by wild type is 28.35%, and the conversion rates of D-tagatose prepared from mutant Y19E, M185A and F279I are 40.31%, 37.94% and 44.72%, respectively; the conversion of wild-type to lactose for D-tagatose was 14.56% and the conversions of mutants Y19E/F279I and M185A/F279I were 22.43% and 20.89%, respectively.
(2) The invention optimizes the method for producing D-tagatose by catalyzing the mutant, and the pH value is 6.0 and 500 g.L at 55 DEG C -1 Lactose concentration and enzyme addition amount of 1.0 mg.mL -1 And contains Mn 2+ With Co 2+ Under the condition of (2), the conversion rate of D-tagatose can reach 23.01 percent.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of purified proteins of wild-type and mutant L-arabinose isomerase; wherein M is a protein molecular weight standard; 1, wild-type WT;2, mutant M185A/F279I;3, mutant Y19E/F279I.
FIG. 2 shows the effect of reaction temperature on D-tagatose production by mutant Y19E/F279I.
FIG. 3 shows the effect of reaction pH on D-tagatose production by mutant Y19E/F279I.
FIG. 4 shows the effect of lactose concentration on D-tagatose production by mutant Y19E/F279I.
FIG. 5 shows the effect of enzyme addition on D-tagatose production by mutant Y19E/F279I.
Detailed Description
The invention is further illustrated below in conjunction with specific examples.
D-galactose, lactose and the like referred to in the following examples were purchased from national pharmaceutical chemicals Co., ltd.
The following examples relate to the following media:
LB liquid medium: yeast powder 5 g.L -1 Tryptone 10 g.L -1 ，NaCl 10g·L -1 。
LB solid medium: on the basis of LB liquid medium, agar is added: 20 g.L -1 。
TB medium: yeast powder 24 g.L -1 Glycerol 5 g.L -1 Tryptone 12 g.L -1 ，K 2 HPO 4 ·3H 2 O 16.43g·L -1 ，KH 2 PO 4 2.31g·L -1 。
The detection method involved in the following examples is as follows:
determination of L-arabinose isomerase Activity:
the reaction system of 1mL contains 150 g.L -1 D-galactose, 250. Mu.L crude enzyme solution and 50 mmol.L -1 Sodium phosphate buffer (pH 6.5) at 1.0 mmol.L respectively -1 And 2.0 mmol.L -1 Will Mn at the final concentration of (C) 2+ And Co 2+ Added into the reaction mixture, reacted at 60 ℃ for 30min after mixing, and immediately boiled in boiling water for 10-15min to terminate the enzyme reaction.
Definition of enzyme activity: one enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1. Mu. Mol of D-tagatose per minute.
The method for detecting the d-tagatose content comprises the following steps:
the components in the reaction system are detected by HPLC, and the detection method is specifically as follows:
determining the amount of reaction product in the sample by high performance liquid chromatography (high performance liquid chromatography, HPLC); the detection conditions are as follows: agilent-Ca chromatographic column with ultrapure water as mobile phase and flow rate of 0.5 mL-min -1 The column temperature is 80 ℃, the detection temperature is 40 ℃ by a differential detector, and the sample injection amount is 10 mu L.
Calculation of product conversion: conversion (%) = concentration of product D-tagatose x 100/concentration of all sugars in the product.
Example 1: expression of wild-type L-arabinose isomerase Gene
The wild type enzyme utilizes a chemical synthesis method to synthesize a gene LfAI of encoding L-arabinose isomerase with a nucleotide sequence shown as SEQ ID NO.8 onto a vector pET-24a (+) to directly obtain a recombinant plasmid pET-24a (+) -LfAI, then amplifying a target gene LfAI fragment thereof, connecting the target gene LfAI fragment with a pHY300PLK vector fragment to obtain a bacillus subtilis expression recombinant plasmid, and transforming the obtained recombinant plasmid into Escherichia coli JM109 to obtain the recombinant plasmid; the transformation product was then spread on LB solid medium (containing 100 mg.mL) -1 Ampicillin), and inversely culturing in a constant temperature incubator at 37 ℃ for 8-12 hours to obtain a transformant; picking up the transformant, inoculating the transformant into an LB liquid medium, shaking and culturing for 8-12 hours at 37 ℃ and 200rpm, extracting plasmids, and carrying out sequencing verification to verify correctness to obtain the recombinant plasmid pHY300PLK-LfAI.
Example 2: construction and expression of L-arabinose isomerase single mutant
(1) Preparation of mutants
According to the optimized gene sequence (SEQ ID NO. 8) of the L-arabinose isomerase given by a synthesis company, respectively designing and synthesizing Y19E, Y19D, M185A, F279I mutant primers, carrying out site-directed mutagenesis on the L-arabinose isomerase LfAI, and respectively sequencing to confirm whether the coding genes of the L-arabinose isomerase mutant are correct; and introducing the vector carrying the mutant gene into bacillus subtilis for expression to obtain the single mutation L-arabinose isomerase.
PCR amplification of the site-directed mutant encoding genes: the rapid PCR technique is utilized, and an expression vector pHY300PLK-LfAI carrying a gene encoding L-arabinose isomerase is used as a template.
The site-directed mutagenesis primer for introducing F279I mutation is:
forward primer: 5'-ATGCATTTACAGATAATATTCAAGATCTTGAAGGCCT-3';
reverse primer: 5'-AGGCCTTCAAGATCTTGAATATTATCTGTAAATGCAT-3';
the site-directed mutagenesis primer for introducing the M185A mutation is as follows:
forward primer: 5'-CAAGATTTGGCGATACAGCAAGAGATGTTGCAGTTAC-3';
reverse primer: 5'-GTAACTGCAACATCTCTTGCTGTATCGCCAAATCTTG-3';
the site-directed mutagenesis primer for introducing the Y19E mutation is:
forward primer: 5'-TTGGCTCACAACCGCTGGAAGGCCCGGAAGCACTGGC-3';
reverse primer: 5'-GCCAGTGCTTCCGGGCCTTCCAGCGGTTGTGAGCCAA-3';
the site-directed mutagenesis primer for introducing the Y19D mutation is:
forward primer: 5'-TTGGCTCACAACCGCTGGATGGCCCGGAAGCACTGGC-3';
reverse primer: 5'-GCCAGTGCTTCCGGGCCCTACAGCGGTTGTGAGCCAA-3';
the PCR reaction system is as follows: 2x pfx mix 25. Mu.L, forward primer (10. Mu. Mol. L -1 ) 1. Mu.L, reverse primer (10. Mu. Mol.L) -1 ) 1. Mu.L of template DNA 1. Mu.L, distilled water was added to 50. Mu.L.
The PCR amplification procedure was set as follows: firstly, pre-denaturation at 98 ℃ for 4min; then enter 30 cycles: denaturation at 98℃for 30s, annealing at 55℃for 30s, and extension at 72℃for 8min; finally, the mixture is extended for 10min at 72 ℃ and is kept at 4 ℃. The PCR products were detected by 1% agarose gel electrophoresis.
Dpn I was added to the PCR product to verify correct, the template was degraded in a water bath at 37℃for 2 hours, and then E.coli JM109 competent cells were transformed, and the transformed product was coated to a solution containing 100 mg.mL -1 Ampicillin LB solid medium, culturing at 37 ℃ for 10-12h, picking positive cloning, and culturing in LB liquid medium for 8-10h. Mutant with correct sequencingFrom glycerol joint to LB medium, overnight culture, extraction of plasmid, transformation of expression host bacillus subtilis WS9 competent cells, obtaining recombinant strain capable of expressing mutant Y19E, Y19D, M185A, F279I.
(2) Expression of mutants
Inoculating mutant Y19E, Y19D, M185A, F279I in LB liquid medium (containing 100 mg.mL) -1 Ampicillin) for 8h, inoculating the seeds into 50mL TB liquid fermentation medium (containing 20 mug.mL) according to 5% inoculum size -1 Tetracycline hydrochloride). E.coli BL21 is cultivated for 2 hours at 37 ℃, and is transferred into a shaking table at 33 ℃ to continue cultivation and fermentation for 48 hours, a certain volume of fermentation liquor is centrifuged for 10 minutes at 4 ℃ and 12000rpm, the supernatant is discarded, bacterial cells are collected, 50mL of 50mM potassium dihydrogen phosphate-disodium hydrogen phosphate buffer with pH of 5.0 is added into the bacterial cells, after the bacterial cells are fully resuspended, a high-pressure homogenizer is used for breaking walls, after centrifugation for 20 minutes at 10000rpm, the wall-broken supernatant is collected to obtain crude enzyme solutions, and the enzyme activities of the crude enzyme solutions are shown in Table 1.
(3) Superposition and validation of mutants
According to the same strategy, the mutants Y19E, M185A, F279I are respectively overlapped and combined to form Y19E/F279I, M A/F279I and Y19E/M185A, and crude enzyme liquid enzyme activities are detected, wherein the crude enzyme liquid enzyme activities are shown in Table 1.
TABLE 1 enzyme Activity of expression mutants
Example 3: purification of L-arabinose isomerase
(1) To 500mL of the wall-broken supernatant of the recombinant bacterium obtained in example 2, 175g of solid ammonium sulfate was added and salted out for 12 hours;
(2) Centrifuging the salted crude enzyme solution at 4 ℃ and 10000rpm for 20min, dissolving the precipitate in a proper amount of buffer solution A containing 50mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole and pH 7.4, dialyzing in the buffer solution A for 10h, and filtering through a 0.22 mu M membrane to prepare a sample;
(3) After the Ni affinity column is equilibrated with buffer A, the sample is sucked into the Ni column to makeAfter complete adsorption, 100mL of buffer A containing 25mM imidazole, 100mL of buffer A containing 50mM imidazole, 100mL of buffer A containing 75mM imidazole, 100mL of buffer A containing 500mM imidazole were used in this order at a flow rate of 1 mL.min -1 Eluting the target protein L-arabinose isomerase by using buffer solution A containing 500mM imidazole, and collecting part of eluent;
(4) The active component (buffer A containing 500mM imidazole) was dialyzed against 50mM phosphate buffer, pH5.0, for 10 hours to obtain a purified L-arabinose isomerase-purified enzyme solution.
(5) After purification, the recombinant L-arabinose isomerase reaches electrophoretic purity, and the apparent molecular weight is 542600 daltons. The purification electrophoresis chart is shown in FIG. 1.
Example 4: application of L-arabinose isomerase mutant in preparation of D-tagatose
The method comprises the following specific steps:
(1) D-galactose is used as a substrate, and the final concentration of the substrate is 200 g.L respectively added into a reaction system -1 D-galactose and L-arabinose isomerase wild enzyme/mutant, wherein the enzyme adding amount of the L-arabinose isomerase wild enzyme/mutant is 1.40mg/mL, D-tagatose is prepared by reaction in a water bath shaking table under the reaction conditions of 200rpm, pH6.5 and 60 ℃, and the conversion rate of the wild enzyme WT and the mutants Y19E, M185A and F279I for producing D-tagatose is detected.
(2) Lactose is used as a substrate, and the final concentration of the substrate is 400 g.L -1 Lactose and L-arabinose isomerase wild enzyme/mutant, and the enzyme adding amount of the L-arabinose isomerase wild enzyme/mutant is 0.7mg.mL -1 D-tagatose was prepared by reaction in a water bath shaker at 200rpm, pH6.0 and 55℃and the conversion of wild-type enzyme WT and mutant Y19E/F279I, M A/F279I to D-tagatose was examined.
Results as shown in table 2, the D-tagatose conversion for the production of D-galactose with mutants Y19E, M185A and F279I increased from 28.35% to 40.31%, 37.94% and 44.72%, respectively, for the wild type; the D-tagatose conversion rate of the mutants Y19E/F279I and M185A/F279I prepared by lactose is increased from 14.56% of the wild type to 22.43% and 20.89% respectively.
TABLE 2 conversion of mutant enzyme preparation D-tagatose (%)
Example 5: enzymatic reaction kinetics of wild-type and mutant enzymes
The kinetics of the enzymatic reaction of the enzyme was determined using D-galactose as substrate, and the enzyme was purified using different concentrations of D-galactose and wild-type and different mutants and incubated for 30min at its optimal temperature and detected by HPLC. K (K) m And V max Nonlinear fitting curve analysis with Origin software yields k cat The values were calculated by combining the results of the fit analysis with the molecular weight of the protein.
K of Y19E/F279I cat /K m There was a significant increase compared to the Wild Type (WT), 5.92X 10 by wild type enzyme -3 Increased to 1.25X10 -1 The catalytic efficiency is improved by 21.11 times; the specific enzyme activity (11.5+/-0.36U/mg) is improved by 4.23 times compared with the wild type (2.2+/-0.42U/mg).
TABLE 3 kinetics of enzymatic reactions of mutant enzymes
Example 6: influence of temperature on preparation of D-tagatose by catalyzing lactose by optimal superposition mutant Y19E/F279I
The influence of different reaction temperatures on the preparation of D-tagatose by the catalysis of the superposition mutant Y19E/F279I is explored. The initial enzyme adding amount of the superposition mutant Y19E/F279I is 0.5 mg.mL -1 Lactose concentration 400g/L, pH6.0, mn 2+ And Co 2+ The reaction system was added at a final concentration of 1.0mmol/L, and commercial lactase was selected from solid food grade lactase FDG-2252 (available from Xia Cheng (Beijing) Biotechnology development Co., ltd.) in an amount of 0.2mg/g lactose. The reaction is carried out on a constant-temperature water bath shaking table, and the initial rotating speed is 200 r.min -1 Setting temperature gradients of 50, 55, 60, 65 and 70 ℃, sampling at intervals untilUntil the reaction is equilibrated. Boiling the obtained sample in boiling water for 15min, and inactivating. The supernatant was centrifuged to be diluted to a proper concentration, and the concentration of each component in the enzymatic conversion reaction solution was measured by High Performance Liquid Chromatography (HPLC), and the conversion was calculated by the following formula. Enzyme conversion experiments were performed in 30mL glass vials with three sets of replicates for each temperature.
The results are shown in FIG. 2. At a reaction temperature of 55 ℃, the conversion rate can reach 21.27 percent. At higher temperatures, carrying out the isomerisation reaction increases the rate of the reaction, shifts the equilibrium of the reaction towards the formation of D-tagatose, and even reduces the viscosity of the substrate. However, too high a temperature, the catalytic efficiency of the enzyme is also affected, resulting in a decrease in the conversion, so that the conversion at 70℃is only 12.27%. During the reaction, the conversion rate is closely related to the reaction temperature and the thermal stability of the enzyme.
Example 7: effect of pH on the preparation of D-tagatose by lactose catalyzed by the superposition mutant Y19E/F279I
The reaction pH was optimized according to the reaction temperature of 55℃in example 6. The initial enzyme adding amount of the superposition mutant is 0.5 mg.mL -1 Lactose concentration of 400g/L, temperature of 55deg.C, mn 2+ And Co 2+ The reaction system was added at a final concentration of 1.0mmol/L, and the commercial lactase was weighed according to the instructions and dissolved in the buffer solution to add, the amount added being the same as in example 6. The reaction is carried out on a constant-temperature water bath shaking table, and the initial rotating speed is 200 r.min -1 Gradients of pH5.0, 5.5, 6.0, 6.5 and 7.0 were set and samples were taken at intervals until the reaction equilibrated. Sample treatment and detection after the reaction were the same as in example 6. Three sets of replicates were set for each pH.
The effect of lactose production of d-tagatose at different pH conditions is shown in FIG. 3. In the reaction for preparing D-tagatose from lactose, the acidic condition is favorable for the hydrolysis reaction (D-galactose is generated), but the peracid environment influences the enzyme reaction rate, so that the experimental result shows that the conversion rate is obviously reduced under the condition of pH 5.0. On the contrary, the highest conversion rate of D-tagatose obtained by the preparation of the D-tagatose is 22.48% at the pH of 6.0, and the conversion rate is lower under the condition of being higher or lower than the pH of 6.0, which proves that the weak acid environment is really favorable for the conversion of lactose to D-tagatose. In addition, the Maillard reaction at high temperature is increased under the alkaline condition, and the conversion rate is also lower.
Example 8: effect of substrate concentration on preparation of D-tagatose by lactose catalyzed by superposition mutant Y19E/F279I
The effect of substrate concentration on the enzyme reaction was investigated by setting a lactose concentration gradient of 200-700g/L according to the optimized reaction conditions of examples 6 and 7 at 55℃and pH 6.0. Three sets of replicates were set for each lactose concentration. Sample treatment and detection after the reaction were the same as in example 6.
The results are shown in FIG. 4. The conversion rate is 22.92% when the lactose concentration is 500 g/L; the conversion rate tends to decrease to various degrees with increasing substrate concentration, and analysis is due to too poor system fluidity at too high substrate concentration, and the inhibition of hydrolysis reaction, resulting in reduced D-galactose production and insufficient isomerization reaction. Under the condition that the enzyme concentration is enough, when the substrate concentration is reduced, the reaction speed is increased along with the increase of the lactose concentration, so that when the lactose concentration is lower than 500g/L, the conversion rate of the reaction in the earlier stage of each substrate concentration is obviously increased, and the conversion rate gradually approaches equilibrium along with the extension of the reaction time.
Example 9: effect of substrate concentration on preparation of D-tagatose by lactose catalyzed by superposition mutant Y19E/F279I
The effect of enzyme addition on the enzyme reaction was investigated by setting the concentration gradient of isomerase at a final concentration of 0.2-1.4mg/mL according to the optimized reaction temperature, pH and substrate concentration of examples 6-8, and the conditions of 55℃and pH6.0, lactose final concentration of 500 g/L. Three groups of enzyme adding amounts are arranged in parallel. Sample treatment and detection after the reaction were the same as in example 6.
As a result, as shown in FIG. 5, the conversion rate was increased in proportion to the amount of enzyme added 48 hours before the reaction was equilibrated, and the higher the enzyme concentration, the faster the rate of substrate conversion, while other conditions were maintained and the substrate concentration was sufficient to maintain the whole reaction progress; in the actual reaction process, when the enzyme concentration is high, the enzyme concentration and the substrate conversion rate are not in the above-mentioned growth relationshipThe reaction curve is gradually gentle. The amount of the isomerase added was 1.0mg.mL as the amount of the enzyme added was increased -1 When the highest conversion was 23.01%; and the enzyme adding amount is 1.4 mg.mL -1 When the conversion rate is basically equal to 1.0 mg.mL -1 It is likely that further increases do not improve conversion and affect reaction balance due to the sufficient amount of enzyme added.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
- An l-arabinose isomerase mutant characterized by having at least one mutation based on the starting sequence:(1) Mutating the 19 th position from tyrosine to glutamic acid or aspartic acid;(2) Mutating position 185 from methionine to alanine;(3) Mutation of phenylalanine to isoleucine at position 279.
- 2. The mutant according to claim 1, wherein the starting sequence is as shown in SEQ ID No. 1.
- 3. The mutant according to claim 1, wherein the mutant is (a) or (b):(a) Mutating tyrosine 19 of the amino acid sequence shown in SEQ ID NO.1 to glutamic acid, and mutating phenylalanine 279 to isoleucine;(b) Methionine at position 185 of the amino acid sequence shown in SEQ ID NO.1 was mutated to alanine and phenylalanine at position 279 was mutated to isoleucine.
- 4. A gene encoding the L-arabinose isomerase mutant according to any one of claims 1 to 3.
- 5. A vector carrying the gene of claim 4.
- 6. A microbial cell expressing a mutant according to any one of claims 1 to 3, or carrying a gene according to claim 4, or comprising a vector according to claim 6.
- 7. A method for producing D-tagatose, characterized in that the L-arabinose isomerase mutant according to any one of claims 1 to 3 is reacted in a reaction system containing lactose.
- 8. The method of claim 7, wherein the reaction system further comprises Mn 2+ And/or Co 2+ 。
- 9. The method according to claim 7 or 8, wherein the content of the L-arabinose isomerase mutant in the reaction system is not less than 0.2g/L and the concentration of lactose as a substrate is not less than 200g/L.
- 10. Use of an L-arabinose isomerase mutant according to any one of claims 1 to 3 or a method according to any one of claims 7 to 9 for the preparation of D-tagatose or a D-tagatose containing product.
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