阅读说明：本技术 一种d-阿洛酮糖3-差向异构酶突变体及其应用 (D-psicose3-epimerase mutant and application thereof ) 是由 柳志强 贾东旭 孙晨奕 彭晨 金利群 郑裕国 陈德水 廖承军 程新平 李勉 毛宝 于 2019-11-29 设计创作，主要内容包括：本发明涉及一种D-阿洛酮糖3-差向异构酶突变体,及其在微生物催化D-果糖异构化制备D-阿洛酮糖中的应用。所述D-阿洛酮糖3-差向异构酶突变体,由所示氨基酸经定点突变而得,所述点突变位点为下列中的一个或多个：(1)第242位、(2)第105位、(3)第210位、(4)第147位、(5)第184位。本发明有益效果主要体现在：本发明提供了一种全新的D-阿洛酮糖3-差向异构酶及其突变体,该突变体具有高最适反应温度85℃,解决了现有酶无法在高温生产D-psicose的技术难题。使用本突变体生产D-psicose,产物得率最高可达40.1％,优于原始酶和其他突变酶的转化效果,具有较好工业应用前景。(The invention relates to a D-psicose3-epimerase mutant and application thereof in preparing D-psicose by catalyzing D-fructose isomerization by microorganisms. The D-psicose3-epimerase mutant is obtained by site-directed mutagenesis of the amino acids shown, wherein the site of: (1) 242 th bit, (2) 105 th bit, (3) 210 th bit, (4) 147 th bit, and (5) 184 th bit. The invention has the following beneficial effects: the invention provides a brand-new D-psicose3-epimerase and a mutant thereof, wherein the mutant has a high optimal reaction temperature of 85 ℃, and solves the technical problem that the existing enzyme cannot produce D-psicose at high temperature. The mutant is used for producing D-psicose, the yield of the product can reach 40.1 percent at most, the conversion effect of the D-psicose is better than that of the original enzyme and other mutant enzymes, and the D-psicose has better industrial application prospect.)

1. A D-psicose3-epimerase mutant obtained by site-directed mutagenesis of an amino acid sequence shown in SEQ ID NO.5 at one or more of the following sites: (1) 242 th bit, (2) 105 th bit, (3) 210 th bit, (4) 147 th bit, and (5) 184 th bit.

2. The mutant according to claim 1, characterized in that the point mutation is one or more of the following: (1) valine at position 242 is mutated to lysine, leucine, tyrosine, threonine or asparagine; (2) glycine 105 is mutated to asparagine, aspartic acid or glutamic acid; (3) isoleucine at position 210 is mutated to threonine, phenylalanine, glutamine or serine; (4) leucine at position 147 is mutated to lysine, asparagine, arginine or cysteine; (5) threonine 184 is mutated to tyrosine or phenylalanine.

3. The mutant according to claim 1, wherein the amino acid sequence of the mutant is as set forth in SEQ ID No. 7.

4. Use of the mutant of any one of claims 1 to 3 in the preparation of D-psicose by microbial catalysis of D-fructose isomerization.

5. The use according to claim 4, wherein the catalysis is carried out at a temperature of 60 to 85 ℃.

6. The use according to claim 5, characterized in that the use is: wet thallus obtained by fermentation culture of engineering bacteria containing D-psicose3-epimerase mutant gene is used as enzyme source, D-fructose is used as substrate, cobalt salt is used as assistant, and Na is used₂HPO₄/NaH₂PO₄And (3) taking the buffer solution as a reaction medium, and reacting at 65-85 ℃ under the condition of 100-300 r/min to prepare the D-psicose.

7. The use of claim 6, wherein: in the reaction system, the initial concentration of the substrate is 300-700 g/L, the dosage of the wet thalli is 10-50 g/L, and the initial concentration of the cobalt salt is 0.5-5 mM.

(I) technical field

The invention relates to a D-psicose3-epimerase mutant and application thereof in preparing D-psicose by catalyzing D-fructose isomerization by microorganisms.

(II) background of the invention

D-psicose is a C-3 epimer of D-fructose, belongs to a rare sugar family, and is an ideal sucrose substitute due to high sweetness and low energy.

Bioconversion is a process of converting a substrate into a product using one or more specific extracellular or intracellular enzymes produced by a microorganism as biocatalysts. The biotransformation has the characteristics of mild reaction conditions and high utilization rate of raw materials, and meanwhile, the transformation process has excellent chemoselectivity, regioselectivity and stereoselectivity, and can ensure the efficient synthesis of the target compound. At present, the preparation of various carbohydrate compounds by using isomerase or cells containing the isomerase as biocatalyst through isomerization reaction has become an important economic growth point of sugar industry.

D-psicose3-epimerase (D-psicose3-epimerase, DPE for short) belongs to the family of ketose 3-epimerases and is used for catalyzing the isomerization of D-fructose to generate D-psicose. The current D-psicose production technology is mainly focused on the organizations such as the Japan rare sugar research center, the Seoul national university, and the like, and the DPE used is mainly derived from wild bacteria such as Agrobacterium tumefaciens ATCC33970, Clostridium cellulolyticum H10, Rhodobacterium sphaeroides SK011 (Kim T et al, PLoS ONE,2016,11(7): e 0160044). The DPE does not belong to heat-resistant enzyme, catalytic reaction can be carried out only at the isomerization temperature of 50-60 ℃, and the conversion rate of D-fructose is 25-35%.

It has been reported that the DPE-mediated D-fructose isomerization process is a thermodynamic equilibrium reaction, and the isomerization reaction is promoted to the D-psicose direction along with the increase of the isomerization temperature. If the catalyst can be catalyzed at a high temperature, such as 70 ℃ or higher, isomerization temperature, the equilibrium is promoted to be carried out forward, the conversion rate of D-fructose is improved, a high-concentration product is obtained, and the production cost, the extraction cost and the like are greatly reduced. At present, the DPE enzyme source of the D-psicose used for synthesis is less, and the enzyme capable of meeting the high-temperature catalytic preparation of high-concentration D-psicose is rare. Under the background that the high-temperature-resistant enzyme preparation is not successfully put on the market, the research and development of the novel high-temperature-resistant DPE have important significance for meeting the increasing sugar intake requirements of the people.

Disclosure of the invention

The invention aims to provide a D-psicose3-epimerase mutant with high catalytic activity at the temperature of more than 70 ℃ and application thereof in preparing D-psicose by catalyzing D-fructose isomerization by microorganisms.

The technical scheme adopted by the invention is as follows:

a D-psicose3-epimerase mutant obtained by site-directed mutagenesis of an amino acid sequence shown in SEQ ID NO.5 at one or more of the following sites: (1) 242 th bit, (2) 105 th bit, (3) 210 th bit, (4) 147 th bit, and (5) 184 th bit.

Specifically, the point mutation is one or more of the following: (1) valine V at position 242 is mutated into lysine K, leucine L, tyrosine Y or asparagine N; (2) glycine G at position 105 is mutated into asparagine N, aspartic acid D or glutamic acid E; (3) isoleucine I at position 210 is mutated to threonine T, phenylalanine F, glutamine Q or serine S; (4) leucine L at position 147 is mutated into lysine K, asparagine N or cysteine C, and threonine T at position 184 is mutated into tyrosine Y or phenylalanine F.

Preferably, the amino acid sequence of the mutant is shown as SEQ ID NO. 7.

The invention excavates and screens novel DPE and carries out site-directed mutagenesis, constructs high-expression genetic engineering bacteria for producing D-psicose at high temperature through genetic engineering technology, improves the synthesis level of the existing sugar isomer compound, has important theoretical significance and application and development potential, and has great significance for filling the market blank lacking high-temperature resistant enzyme.

The invention also relates to application of the mutant in preparing D-psicose by microbial catalysis of D-fructose isomerization.

Preferably, the catalysis is carried out at 70-85 ℃.

Specifically, the application is as follows: wet thallus obtained by fermentation culture of engineering bacteria containing D-psicose-3-epimerase mutant gene is used as enzyme source, D-fructose is used as substrate, cobalt salt is used as assistant, Na is used as auxiliary agent₂HPO₄/NaH₂PO₄And (3) taking the buffer solution as a reaction medium, and reacting at 75-85 ℃ under the condition of 100-300 r/min to prepare the D-psicose.

Preferably, in the reaction system, the initial concentration of the substrate is 300-700 g/L, the dosage of the wet bacteria is 10-50 g/L, and the initial concentration of the cobalt salt is 0.5-5 mM.

The invention has the following beneficial effects: the invention provides a brand-new DPE and a mutant thereof, wherein the mutant has a high optimal reaction temperature of 85 ℃, and solves the technical problem that the existing enzyme cannot produce D-psicose at high temperature. The D-psicose produced by the NtDPE mutant has the conversion rate of 50.12 percent under the conversion condition of 80 ℃, is higher than the conversion effect of original enzyme and other mutant enzymes, and has better application prospect.

(IV) description of the drawings

FIG. 1 is a schematic diagram showing the optimal temperature of a recombinase;

FIG. 2 is a schematic diagram showing the effect of metal ions on the activity of recombinant enzymes.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: