CN113201512A - High-yield kestose inulin sucrase mutant - Google Patents

High-yield kestose inulin sucrase mutant Download PDF

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CN113201512A
CN113201512A CN202110613892.2A CN202110613892A CN113201512A CN 113201512 A CN113201512 A CN 113201512A CN 202110613892 A CN202110613892 A CN 202110613892A CN 113201512 A CN113201512 A CN 113201512A
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沐万孟
张文立
倪大伟
徐炜
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Abstract

The invention discloses an inulin sucrase mutant for high yield of kestose, belonging to the technical field of enzyme genetic engineering. The invention constructs a single-point mutant enzyme R425W by taking the inulin sucrase which is derived from the microorganism Lactobacillus reuteri 121 and has a partial sequence truncation as a parent. R425W changes the chain length of the product significantly, which makes the inulosucrase lose the ability to synthesize polysaccharide and accumulate a large amount of fructooligosaccharide, especially kestose. By optimizing the reaction conditions, the yield of the kestose produced by R425W can reach 206g/L at most. The production conditions were phosphate buffer pH 6.5, 45 ℃, 700g/L sucrose, 15. mu.g/mL enzyme addition, and 36h reaction time. The discovery has important research value for the industrial preparation of kestose and the industrial application of the inulosucrase.

Description

High-yield kestose inulin sucrase mutant
Technical Field
The invention relates to an inulin sucrase mutant for high yield of kestose, belonging to the technical field of enzyme genetic engineering.
Background
The Inulosucrase (EC 2.4.1.10) belongs to glycoside hydrolase GH68 family, and takes sucrose as sole substrate, and performs hydrolysis reaction to generate glucose and fructose, and performs transglycosylation reaction to generate fructo-oligosaccharide and inulin. Both fructo-oligosaccharide and inulin are widely accepted prebiotics and have significant effects on promoting the proliferation of intestinal probiotics. When the inulosucrase takes cane sugar as a substrate, fructosyl is continuously transferred to an extended inulin chain, and the product system contains inulins with different polymerization degrees. By controlling the extension of inulin chain, the inulosucrase loses the ability of synthesizing long-chain inulin, the chain length of the product is controlled in a targeted way, and the fructo-oligosaccharide with specific polymerization degree can be produced.
Kestose is the smallest fructooligosaccharide, and is a trisaccharide formed by connecting one fructose molecule and one sucrose molecule through beta- (2,1) or beta- (2, 6). As a main fructooligosaccharide ingredient, it shows a more significant effect in promoting the growth of probiotics including clostridium flexneri prausnitzii, Bifidobacterium bifidum than fructooligosaccharides of other polymerization degrees. Kestose is present in many plants, but its low content and difficult separation and purification are major obstacles limiting its industrial application. The production of kestose by enzymatic synthesis and microbial fermentation has the potential to facilitate its production and industrial application.
Disclosure of Invention
The technical problem is as follows:
currently, the production of fructooligosaccharides is mainly performed by β -fructofuranosidase (EC 3.2.1.26)) and endoinulinase (EC3.2.1.7). The content of kestose is limited by enzymes from different sources and a stable industrial production process cannot be obtained. The studies on the regulation of the chain length of the inulosucrase only enabled the inulosucrase to produce fructooligosaccharides with different degrees of polymerization, and there were no studies on the production of kestose.
The invention provides a mutant enzyme of inulosucrase for high yield of kestose, which has important practical significance for industrial preparation of kestose.
The technical scheme is as follows:
in order to solve the technical problems, the invention carries out molecular modification on the inulosucrase (Lare121-ISase) from the microorganism Lactobacillus reuteri 121 by a site-directed mutagenesis method.
The first purpose of the invention is to provide an inulin sucrase mutant, and the amino acid sequence of the mutant is shown as SEQ ID NO. 4.
In one embodiment, the mutant is an inulin sucrase with the amino acid sequence shown in SEQ ID No.2, in which arginine at position 425 is replaced by tryptophan.
It is a second object of the invention to provide a gene encoding said mutant inulosucrase.
In one embodiment, the gene has the sequence shown in SEQ ID NO. 3.
The third purpose of the invention is to provide a vector carrying the gene.
In one embodiment, the vector includes, but is not limited to, a pET series vector.
In one embodiment, the vector comprises pET-22b (+).
The fourth purpose of the invention is to provide a genetic engineering bacterium for expressing the mutant of the inulosucrase.
In one embodiment, the genetically engineered bacterium is a host Escherichia coli, and pET-22b (+) is a vector.
The fifth purpose of the invention is to provide a method for producing kestose, which takes the inulin sucrase mutant or the genetic engineering bacteria containing the inulin sucrase mutant as a catalyst and takes sucrose as a substrate to prepare the kestose.
In one embodiment, the preparation is with a sodium phosphate buffer as the buffer system.
In one embodiment, the sucrose is added in an amount of 600-800 g/L.
In one embodiment, the addition amount of the mutant inulosucrase in the method is 10-30 mug/mL
In one embodiment, the production conditions of the method are 40-50 ℃ and the reaction time is 10-50 h.
The invention also provides the application of the mutant or the kestose produced by the genetic engineering bacteria in the fields of medicine production and food.
Has the advantages that:
the single-site mutant enzyme R425W obviously changes the chain length of a product, so that the inulosucrase loses the capability of synthesizing polysaccharide and accumulates a large amount of fructo-oligosaccharide, especially kestose. Through reaction condition optimization, the yield of the kestose produced by R425W reaches 206 g/L.
Drawings
FIG. 1 liquid phase diagram of wild enzyme and mutant R425W.
FIG. 2 conditions optimization of the production of kestose by mutant R425W (A) optimization of enzyme addition (B) optimization of sucrose concentration (C) production of kestose as a function of reaction time.
Detailed Description
Example 1: three-dimensional structure analysis of inulin sucrase and construction of mutant plasmid
(1) And (4) determining mutation points.
The number of the partial sequence truncated inulosucrase (Lare121-IS delta 121-701) derived from the microorganism Lactobacillus reuteri 121 in NCBI database IS AAN05575.1, the nucleotide sequence IS shown as SEQ ID NO.1, and the amino acid sequence IS shown as SEQ ID NO. 2. Three-dimensional modeling IS carried out on Lare121-IS delta 121-701, single-point mutation IS rationally designed by combining substrate pocket conformation, calculation simulation and interaction of a substrate and an enzyme molecule, and arginine at position 425 IS selected and mutated into tryptophan with the largest side chain group.
(2) And (5) constructing a plasmid.
1) Construction of the original plasmid pET-22b (+) -Lare 121-IS. DELTA.121-701
Synthesizing the inulosucrase (Lare121-IS delta 121-701) with the nucleotide sequence shown as SEQ ID NO.1, inserting the inulosucrase into the multiple cloning site of the vector pET-22b (+), and obtaining the original plasmid pET-22b (+) -Lare121-IS delta 121-701 through sequencing verification.
2) Construction of the mutant plasmid pET-22b (+) -R425W
Designing a site-directed mutagenesis primer (Table 2), carrying out single-point mutagenesis by using an original plasmid pET-22b (+) -Lare121-IS delta 121-701-carrying Lare121-IS delta 121-701-containing gene as a template, and replacing the 425 th arginine of the inulin sucrase (Lare121-IS delta 121-701-containing gene) in the step (1) with tryptophan to construct a mutagenesis plasmid pET-22b (+) -R425W. Mutation is verified by PCR and template digestion reaction, and sequencing of the product of template digestion. The nucleotide sequence of the mutant enzyme is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
TABLE 1 PCR reaction System
Figure BDA0003097216150000031
And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 3min for 40s, 32 cycles, and storage at 4 ℃.
TABLE 2 primer sequence Listing
Figure BDA0003097216150000032
TABLE 3 template digestion reaction System
Figure BDA0003097216150000033
Reaction conditions are as follows: the reaction was carried out at 37 ℃ for 90 min.
Example 2: construction of engineering strain and expression and purification of mutant enzyme
(1) And (5) construction of an engineering strain.
The mutant plasmid pET-22b (+) -R425W and the original plasmid pET-22b (+) -Lare121-IS delta 121-701 obtained in example 1 were transformed into E.coli (E.coli) BL21(DE3) competent cells, spread on LB solid medium containing 100. mu.g/mL ampicillin, and cultured at 37 ℃ for 12 hours to obtain plates with recombinant engineered bacteria E.coli BL21/pET-22b (+) -R425W and E.coli BL21/pET-22b (+) -Lare121-IS delta 121-701, respectively.
(2) Expression of the mutant enzyme.
Selecting a single colony on the plate in the step (1) to 4mL of LB liquid culture medium containing 50 mug/mL of ampicillin, culturing at 37 ℃ for 12h to obtain a seed solution, transferring the seed solution into 200mL of LB liquid culture medium containing 50 mug/mL of ampicillin, and culturing at 37 ℃ for 2-3 h until OD is achieved600The value is 0.6-0.8, IPTG with the final concentration of 1mmol/L is added to induce the expression of protein, and the fermentation liquid is obtained after the culture is carried out for 6-8 h at the temperature of 28 ℃. The fermentation broth was centrifuged at 8000rpm for 15min at 4 ℃ to collect the cells.
(3) And (5) purifying the mutant enzyme.
Adding 20mL of a disruption buffer (50mmol/L Tris-HCl, 200mmol/L NaCl, pH 7.0) to the cells obtained in step (2), fully suspending the cells, then carrying out ultrasonication, centrifuging at 8000rpm for 15min at 4 ℃, and collecting the supernatant, i.e., a crude enzyme solution.
The crude enzyme solution was purified using a nickel ion affinity column. First, the column was equilibrated with equilibration buffer (50mmol/LTris-HCl, 500mmol/LNaCl, pH 7.0); then, adding the obtained crude enzyme solution into a column; next, the heteroprotein was washed with a buffer containing a low concentration of imidazole (50mmol/L Tris-HCl, 500mmol/L NaCl, 50mmol/L imidazole, pH 7.0); finally, elution was performed with a buffer solution containing high concentration of imidazole (50mmol/L Tris-HCl, 500mmol/L NaCl, 500mmol/L imidazole, pH 7.0) to obtain the mutant enzyme R425W and the parent enzyme Lare 121-IS. DELTA.121-701.
(4) The enzyme activity of the inulosucrase is determined under the conditions of optimal temperature and pH.
10. mu.g of the mutant enzyme R425W obtained in step (3) was added to sucrose (300 g/L) as a substrate under reaction conditions of pH 6.5 and 55 ℃ for 20 min. Adding NaOH with the final concentration of 100mmol/L into the reaction system, carrying out ice bath for 20min to terminate the reaction, and adding HCl with the final concentration of 100mmol/L to neutralize the system. Finally, the enzyme activity is 170U/mg.
Example 3: condition optimization of mutant enzyme R425W for producing kestose
And (3) product spectrum comparison: using 300g/L sucrose as substrate, 10. mu.g of the mutant enzyme R425W obtained in example 2 and the parent enzyme Lare121-IS Δ 121-Asca 701 were added to 1mL of the reaction system (pH 6.5), respectively, and reacted at 45 ℃ for 12 hours. And after the reaction is finished, adding NaOH with the final concentration of 100mmol/L into the reaction system, carrying out ice bath for 20min to terminate the reaction, and adding HCl with the final concentration of 100mmol/L to neutralize the system. The liquid chromatogram of the reaction product is shown in FIG. 1. The parent enzyme Lare121-IS delta 121-701 produces sugars with different degrees of polymerization, while the mutant enzyme R425W can only accumulate pentasaccharides at most and accumulates kestose in large quantities (FIG. 1).
Optimizing the conditions for producing kestose:
(1) optimizing the enzyme adding amount. Using 300g/L of sucrose as a substrate, 2, 4, 6, 8, 10, 15, 20, 25, and 30. mu.g of the mutant enzyme R425W were added to 1mL of the reaction system (pH 6.5), and the reaction was carried out at 45 ℃ for 12 hours. And after the reaction is finished, adding NaOH with the final concentration of 100mmol/L into the reaction system, carrying out ice bath for 20min to terminate the reaction, and adding HCl with the final concentration of 100mmol/L to neutralize the system. As a result, as shown in FIG. 2, the yield of kestose was 40g/L or more when the amount of enzyme added was 10 to 30. mu.g/mL.
(2) The sucrose concentration is optimized. Using 100, 200, 300, 400, 500, 600, 700, and 800g/L sucrose as a substrate, 15. mu.g of the enzyme was added to 1mL of the reaction system (pH 6.5) and reacted at 45 ℃ for 12 hours. And after the reaction is finished, adding NaOH with the final concentration of 100mmol/L into the reaction system, carrying out ice bath for 20min to terminate the reaction, and adding HCl with the final concentration of 100mmol/L to neutralize the system. As shown in FIG. 2, the yield of kestose was 160g/L or more when the amount of sucrose added was 600 to 800. mu.g/mL.
(3) Effect of reaction time on kestose yield. Using 700g/L of sucrose as a substrate, 15. mu.g of the mutant enzyme R425W was added to 1mL of the reaction system (pH 6.5) and reacted at 45 ℃ for 3, 5, 7.5, 10, 12, 15, 20, 24, 30, 36, 42, and 48 hours. And after the reaction is finished, adding NaOH with the final concentration of 100mmol/L into the reaction system, carrying out ice bath for 20min to terminate the reaction, and adding HCl with the final concentration of 100mmol/L to neutralize the system. As shown in FIG. 2, the yield of kestose can reach 180g/L or more when the reaction time is 10 to 50 hours.
By optimizing the reaction conditions, the yield of the kestose produced by R425W can reach 206g/L at most. The production conditions were phosphate buffer pH 6.5, 45 ℃, 700g/L sucrose, 15. mu.g/mL enzyme addition, and 36h reaction time.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> inulin sucrase mutant with high yield of kestose
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gatggctatt actaccaaac ttatgatcaa tggaaagcta ctaacaaagg tgccgataat 900
attgcaatgc gtgatgctca tgtaattgaa gatgataatg gtgatcggta ccttgttttt 960
gaagcaagta ctggtttaga aaattatcaa ggcgaggacc aaatttataa ctggttaaat 1020
tatggcggag atgacgcatt taatatcaag agcttattta gaattctttc caatgatgat 1080
attaagagtc gggcaacttg ggctaatgca gctatcggta tcctcaaact aaataaggac 1140
gaaaagaatc ctaaggtggc agagttatac tcaccattaa tttctgcacc aatggtaagc 1200
gatgaaattg agcgaccaaa tgtagttaaa ttgggtaata aatattactt atttgccgct 1260
acccgtttaa attggggaag taatgatgat gcttggatga atgctaatta tgccgttggt 1320
gataatgttg caatggtcgg atatgttgct gatagtctaa ctggatctta taagccatta 1380
aatgattctg gagtagtctt gactgcttct gttcctgcaa actggcgaac agcaacttat 1440
tcatattatg ctgtccccgt tgccggaaaa gatgaccaag tattagttac ttcatatatg 1500
actaatagaa atggagtagc gggtaaagga atggattcaa cttgggcacc gagtttctta 1560
ctacaaatta acccggataa cacaactact gttttagcta aaatgactaa tcaaggggat 1620
tggatttggg atgattcaag cgaaaatctt gatatgattg gtgatttaga ctccgctgct 1680
ttacctggcg aacgtgataa acctgttgat tgggacttaa ttggttatgg attaaaaccg 1740
catgatcacc accaccacca ccac 1764
<210> 4
<211> 588
<212> PRT
<213> Artificial sequence
<400> 4
Met Asp Ser Lys Ala Ala Gln Glu Asn Thr Asn Thr Ala Lys Asn Asp
1 5 10 15
Asp Thr Gln Lys Ala Ala Pro Ala Asn Glu Ser Ser Glu Ala Lys Asn
20 25 30
Glu Pro Ala Val Asn Val Asn Asp Ser Ser Ala Ala Lys Asn Asp Asp
35 40 45
Gln Gln Ser Ser Lys Lys Asn Thr Thr Ala Lys Leu Asn Lys Asp Ala
50 55 60
Glu Asn Val Val Lys Lys Ala Gly Ile Asp Pro Asn Ser Leu Thr Asp
65 70 75 80
Asp Gln Ile Lys Ala Leu Asn Lys Met Asn Phe Ser Lys Ala Ala Lys
85 90 95
Ser Gly Thr Gln Met Thr Tyr Asn Asp Phe Gln Lys Ile Ala Asp Thr
100 105 110
Leu Ile Lys Gln Asp Gly Arg Tyr Thr Val Pro Phe Phe Lys Ala Ser
115 120 125
Glu Ile Lys Asn Met Pro Ala Ala Thr Thr Lys Asp Ala Gln Thr Asn
130 135 140
Thr Ile Glu Pro Leu Asp Val Trp Asp Ser Trp Pro Val Gln Asp Val
145 150 155 160
Arg Thr Gly Gln Val Ala Asn Trp Asn Gly Tyr Gln Leu Val Ile Ala
165 170 175
Met Met Gly Ile Pro Asn Gln Asn Asp Asn His Ile Tyr Leu Leu Tyr
180 185 190
Asn Lys Tyr Gly Asp Asn Glu Leu Ser His Trp Lys Asn Val Gly Pro
195 200 205
Ile Phe Gly Tyr Asn Ser Thr Ala Val Ser Gln Glu Trp Ser Gly Ser
210 215 220
Ala Val Leu Asn Ser Asp Asn Ser Ile Gln Leu Phe Tyr Thr Arg Val
225 230 235 240
Asp Thr Ser Asp Asn Asn Thr Asn His Gln Lys Ile Ala Ser Ala Thr
245 250 255
Leu Tyr Leu Thr Asp Asn Asn Gly Asn Val Ser Leu Ala Gln Val Ala
260 265 270
Asn Asp His Ile Val Phe Glu Gly Asp Gly Tyr Tyr Tyr Gln Thr Tyr
275 280 285
Asp Gln Trp Lys Ala Thr Asn Lys Gly Ala Asp Asn Ile Ala Met Arg
290 295 300
Asp Ala His Val Ile Glu Asp Asp Asn Gly Asp Arg Tyr Leu Val Phe
305 310 315 320
Glu Ala Ser Thr Gly Leu Glu Asn Tyr Gln Gly Glu Asp Gln Ile Tyr
325 330 335
Asn Trp Leu Asn Tyr Gly Gly Asp Asp Ala Phe Asn Ile Lys Ser Leu
340 345 350
Phe Arg Ile Leu Ser Asn Asp Asp Ile Lys Ser Arg Ala Thr Trp Ala
355 360 365
Asn Ala Ala Ile Gly Ile Leu Lys Leu Asn Lys Asp Glu Lys Asn Pro
370 375 380
Lys Val Ala Glu Leu Tyr Ser Pro Leu Ile Ser Ala Pro Met Val Ser
385 390 395 400
Asp Glu Ile Glu Arg Pro Asn Val Val Lys Leu Gly Asn Lys Tyr Tyr
405 410 415
Leu Phe Ala Ala Thr Arg Leu Asn Trp Gly Ser Asn Asp Asp Ala Trp
420 425 430
Met Asn Ala Asn Tyr Ala Val Gly Asp Asn Val Ala Met Val Gly Tyr
435 440 445
Val Ala Asp Ser Leu Thr Gly Ser Tyr Lys Pro Leu Asn Asp Ser Gly
450 455 460
Val Val Leu Thr Ala Ser Val Pro Ala Asn Trp Arg Thr Ala Thr Tyr
465 470 475 480
Ser Tyr Tyr Ala Val Pro Val Ala Gly Lys Asp Asp Gln Val Leu Val
485 490 495
Thr Ser Tyr Met Thr Asn Arg Asn Gly Val Ala Gly Lys Gly Met Asp
500 505 510
Ser Thr Trp Ala Pro Ser Phe Leu Leu Gln Ile Asn Pro Asp Asn Thr
515 520 525
Thr Thr Val Leu Ala Lys Met Thr Asn Gln Gly Asp Trp Ile Trp Asp
530 535 540
Asp Ser Ser Glu Asn Leu Asp Met Ile Gly Asp Leu Asp Ser Ala Ala
545 550 555 560
Leu Pro Gly Glu Arg Asp Lys Pro Val Asp Trp Asp Leu Ile Gly Tyr
565 570 575
Gly Leu Lys Pro His Asp His His His His His His
580 585

Claims (10)

1. An inulosucrase mutant R425W, wherein the amino acid sequence of the mutant is shown as SEQ ID NO. 4.
2. A gene encoding the mutant inulosucrase of claim 1.
3. A vector carrying the gene of claim 2.
4. A genetically engineered bacterium expressing the gene of claim 2 or the vector of claim 3.
5. The genetically engineered bacterium of claim 4, wherein E.coli is used as a host and pET-22b (+) is used as a vector.
6. A method for preparing kestose, which is characterized in that the method takes the inulosucrase mutant as claimed in claim 1 or the genetically engineered bacterium as claimed in claim 4 or 5 as a catalyst, and takes cane sugar as a substrate to prepare kestose.
7. The method of claim 6, wherein the method is carried out by preparing a sodium phosphate buffer as a buffer system.
8. The method according to claim 6, wherein the sucrose is added in an amount of 600-800 g/L and 10-30 μ g/mL.
9. The method according to claim 6, wherein the production conditions of the method are 40-50 ℃ and 10-50 h of reaction.
10. Use of the mutant inulosucrase according to claim 1 or kestose produced by the genetically engineered bacterium according to claim 4 or 5 in the fields of pharmaceutical production and food.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6635460B1 (en) * 2000-05-25 2003-10-21 Nederlandse Organisatie Voor Toegepast - Natuurwetenschappelijk Onderzoek Tno Fructosyltransferases
CN105506034A (en) * 2016-01-26 2016-04-20 江南大学 Method for efficient synthesis of difructose anhydride III
CN110396512A (en) * 2019-07-26 2019-11-01 江南大学 A kind of Inulosucrase mutant and its application
US20200354481A1 (en) * 2017-11-03 2020-11-12 Kaleido Biosciences, Inc. Methods of producing glycan polymers

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6635460B1 (en) * 2000-05-25 2003-10-21 Nederlandse Organisatie Voor Toegepast - Natuurwetenschappelijk Onderzoek Tno Fructosyltransferases
CN105506034A (en) * 2016-01-26 2016-04-20 江南大学 Method for efficient synthesis of difructose anhydride III
US20200354481A1 (en) * 2017-11-03 2020-11-12 Kaleido Biosciences, Inc. Methods of producing glycan polymers
CN110396512A (en) * 2019-07-26 2019-11-01 江南大学 A kind of Inulosucrase mutant and its application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MUNIR A. ANWAR 等: ""The role of conserved inulosucrase residues in the reaction and product specificity of Lactobacillus reuteri inulosucrase"", 《THE FEBS JOURNAL》 *
THANAPON CHAROENWONGPAIBOON 等: ""Modulation of fructooligosaccharide chain length and insight into the product binding motif of Lactobacillus reuteri 121 inulosucrase"", 《CARBOHYDRATE POLYMERS》 *
倪大伟 等: ""微生物菊糖蔗糖酶及在食品中的应用研究进展"", 《食品科学》 *

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