WO2024045796A1 - 一种溶剂耐受性提高的环糊精葡萄糖基转移酶及其制备 - Google Patents

一种溶剂耐受性提高的环糊精葡萄糖基转移酶及其制备 Download PDF

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WO2024045796A1
WO2024045796A1 PCT/CN2023/102014 CN2023102014W WO2024045796A1 WO 2024045796 A1 WO2024045796 A1 WO 2024045796A1 CN 2023102014 W CN2023102014 W CN 2023102014W WO 2024045796 A1 WO2024045796 A1 WO 2024045796A1
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mutant
tolerance
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cyclodextrin
amino acid
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韩瑞枝
倪晔
姜钰琳
樊朝悦
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Jiangnan University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)

Definitions

  • the invention relates to a cyclodextrin glycosyltransferase with improved solvent tolerance and its preparation, and belongs to the technical fields of enzyme engineering and genetic engineering.
  • Cyclodextrin glycosyltransferase (CGTase for short, EC 2.4.1.19) belongs to the ⁇ -amylase family. It has a variety of transglycosylation activities (cyclization, disproportionation, and coupling activities) and is of great commercial value. At present, this enzyme is mainly used in industry to produce cyclodextrin. In addition, this enzyme also contains partial hydrolysis activity and plays an important role in the short-chain glycosylation reaction process. In recent years, good progress has been made in using its transglycosylation activity to improve the properties of natural substrates, such as stevioside, rutin, genistein, etc.
  • Glycosylation treatment effectively solves the problems of poor solubility, difficulty in storage, and instability of these substances in practical applications, and most of the glycosylation products maintain the excellent properties and value of the substances themselves, so they are used in many fields such as food and chemical industries. It has good applications in all fields.
  • the purpose of the present invention is to provide a cyclodextrin glycosyltransferase mutant with improved solvent tolerance, which is beneficial to expanding the glycosylation application of cyclodextrin glycosyltransferase to natural hydrophobic substrates.
  • the mutant is obtained by mutating the amino acids at positions 146, 147, and 539 of the cyclodextrin glycosyltransferase whose amino acid sequence is shown in SEQ ID NO. 1.
  • the cyclodextrin glycosyltransferase is derived from Paenibacillus softenans (Paenibacillus macerans).
  • nucleotide sequence of the cyclodextrin glycosyltransferase is shown in SEQ ID NO. 2.
  • the mutant is to mutate the arginine at position 146 of the cyclodextrin glycosyltransferase whose amino acid sequence is shown in SEQ ID NO. 1 to phenylalanine, and is named It is R146F, its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.
  • the mutant is a glycine at position 539 of the cyclodextrin glycosyltransferase whose amino acid sequence is shown in SEQ ID NO. 1, mutated to isoleucine, and is named G539I , its amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.6.
  • the mutant is to mutate the glycine at position 539 of the cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO. 1 to isoleucine, and at the same time, Arginine at position 146 was mutated to phenylalanine, named G539I/R146F. Its amino acid sequence is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.8.
  • the mutant is to mutate the glycine at position 539 of the cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO. 1 to isoleucine; at the same time, the glycine at position 539 is mutated into isoleucine;
  • the arginine at position 146 was mutated to phenylalanine; the aspartic acid at position 147 was mutated to asparagine; it was named G539I/R146F/D147N.
  • Its amino acid sequence is shown in SEQ ID NO.9, and the core The nucleotide sequence is shown in SEQ ID NO.10.
  • the present invention also provides a gene encoding the above mutant.
  • the invention also provides a recombinant vector carrying the above gene.
  • the vector is: pET series vector, pUT series vector or pBAD series vector.
  • the invention also provides a recombinant cell that expresses the above mutant, carries the above gene, or carries the above recombinant vector.
  • the recombinant cells use fungi or bacteria as host cells.
  • the host cell is: Escherichia coli or Bacillus subtilis.
  • the invention also provides a method for preparing the cyclodextrin glycosyltransferase mutant, and the specific steps include:
  • design primers for site-directed mutation use the plasmid containing the cyclodextrin glycosyltransferase gene as a template to perform full plasmid PCR, obtain the recombinant plasmid carrying the mutated gene after digesting the template, and transfer it into the host bacteria, Screen for kanamycin resistance, select bacteria for culture, and send samples for sequencing;
  • the collected fermentation broth is centrifuged to collect the bacterial cells, ultrasonically disrupted, and centrifuged again.
  • the collected supernatant is the crude enzyme solution of the mutant.
  • the recombinant plasmid uses pET-28a(+) as an expression vector.
  • the host bacterium is E. coli BL21 (DE3).
  • the organic solvent includes: dimethyl sulfoxide (DMSO), ethanol, methanol and acetone.
  • DMSO dimethyl sulfoxide
  • the present invention also provides a method for improving the tolerance of cyclodextrin glycosyltransferase to organic solvents.
  • the method is to combine the amino acid sequence of the cyclodextrin glycosyltransferase shown in SEQ ID NO.1 Glycine at position 539 was mutated to isoleucine;
  • the organic solvent includes one or more of dimethyl sulfoxide (DMSO), ethanol, methanol and acetone.
  • DMSO dimethyl sulfoxide
  • the invention also provides a method for producing long-chain glycosylated genistein, which is characterized in that the method is: adding a cyclodextrin glycosyltransferase mutant to a reaction system containing soluble starch and genistein. Carry out the reaction to obtain a reaction liquid; separate the reaction liquid to obtain long-chain glycosylated genistein.
  • the present invention also provides the application of the above-mentioned mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell in the glycosylation of natural hydrophobic substrates, or in the glycosylation of some organic solvent reaction systems.
  • the present invention constructs four cyclodextrin glycosyltransferase mutants with improved organic solvent tolerance, R146F, G539I, G539I/R146F, G539I/R146F/D147N, which can directly The original activity measured was maintained at 90% to 120% of the WT activity.
  • the present invention has obtained two cyclodextrin glycosyltransferase mutants with improved organic solvent tolerance, which promotes the glycosylation efficiency of hydrophobic substrates, helps expand the industrial application scope of CGTase, and has extremely high Great application prospects.
  • Figure 1 Protein gel images of wild-type and mutant pure enzymes; among them, M: Marker; 1: R146F; 2:, G539I; 3: G539I/R146F; 4: G539I/R146F/D147N; 5: wild-type WT.
  • LB liquid medium yeast powder 5.0g ⁇ L -1 , tryptone 10.0g ⁇ L -1 , NaCl 10.0g ⁇ L -1 .
  • LB solid medium Add 2% agar to LB liquid medium.
  • TB liquid medium yeast powder 24.0g ⁇ L -1 , tryptone 12.0g ⁇ L -1 , KH 2 PO 3 2.3g ⁇ L -1 , K 2 HPO 3 16.4g ⁇ L -1 , glycerol 5g ⁇ L - 1 .
  • Methyl orange method to measure cyclization activity Take 50 ⁇ L of enzyme solution diluted to a suitable concentration, add 200 ⁇ L of maltodextrin (concentration of 10 g ⁇ L -1 prepared in advance with 50 mM phosphate buffer (pH 6.0)), and incubate at 40°C. React in a water bath shaker for 10 minutes, immediately add 250 ⁇ L hydrochloric acid (1.0M) to terminate the reaction, then add 150 ⁇ L methyl orange (prepared with 50mM phosphate buffer to a concentration of 0.5mM), and let stand for 20 minutes at room temperature (20°C). Measure the absorbance. The group without enzyme solution was used as a blank control.
  • Enzyme activity definition The amount of enzyme required to generate 1 ⁇ mol ⁇ -cyclodextrin per minute under these conditions is defined as one enzyme activity unit.
  • E.coli BL21(DE3)/pET-20b(+)-cgt preserved in the laboratory.
  • the strain E.coli BL21(DE3)/pET-20b(+)-cgt is pET-20b( +) and the cyclodextrin glycosyltransferase cgt whose nucleotide sequence is shown in SEQ ID NO.2, ligated to prepare the recombinant plasmid after enzyme digestion, and the recombinant plasmid was introduced into E.coli BL21 (DE3) to prepare Sufficient bacteria, its specific construction process can be found in the reference literature: Han, R., Ge, B., Jiang, M. et al. High production of genistein diglucoside derivative using cyclodextrin glycosyltransferase from Paenibacillus macerans.J Ind Microbiol Biotechnol 44,1343– 1354(2017);
  • E.coli BL21(DE3)/pET-20b(+)-cgt is streaked and activated, a single colony is picked and inoculated into LB liquid medium containing ampicillin (100 mg/L), and cultured at 37°C for 10 hours. Finally, use a kit to extract plasmid pET-20b(+)-cgt.
  • One-step cloning and replacement vector design primers, the primer sequence is, F:CAGCAAATGGGTCGCGGATCCTCACCGGACACCTCAGTGGA
  • the amino acid sequence of the cyclodextrin glycosyltransferase in the present invention is as shown in SEQ ID NO.1 (the nucleotide sequence of the gene is as shown in SEQ ID NO.2), and primers are designed according to the selected mutation site to The extracted recombinant plasmid pET28a(+)-cgt was used as a template for whole plasmid PCR.
  • the primer sequences were as follows:
  • Reverse primer 5'-CGGATTGTC AAA ATCTGCCGGAGA-3', the mutated bases are underlined.
  • Reverse primer 5'-AATACCACT AAT GGTGACCGCGGT-3', the mutated bases are underlined.
  • Forward primer 1 5'-TCTCCGGCAGAT TTT GACAATCCG-3', the mutated base is underlined;
  • Reverse primer 1 5'-CGGATTGTC AAA ATCTGCCGGAGA-3', the mutated bases are underlined.
  • Forward primer 2 5'-ACCGCGGTCACC ATT AGTGGTATT-3', the mutated base is underlined;
  • Reverse primer 2 5'-AATACCACT AAT GGTGACCGCGGT-3', the mutated bases are underlined.
  • Forward primer 1 5'-TCTCCGGCAGAT TTTAAT AATCCG-3', the mutated base is underlined;
  • Reverse primer 1 5'-CGGATT ATTAAA ATCTGCCGGAGA-3', the mutated bases are underlined.
  • Forward primer 2 5'-ACCGCGGTCACC ATT AGTGGTATT-3', the mutated base is underlined;
  • Reverse primer 2 5'-AATACCACT AAT GGTGACCGCGGT-3', the mutated bases are underlined.
  • the PCR reaction systems are all: 5 ⁇ PrimeSTAR Buffer (Mg 2+ Plus) 5 ⁇ L, 2.5mM dNTPs 4 ⁇ L, 10 ⁇ M forward primer 1 ⁇ L, 10 ⁇ M reverse primer 1 ⁇ L, template DNA 1 ⁇ L, 2.5U/ ⁇ L PrimeSTAR Taq HS 0.5 ⁇ L, plus Make up to 50 ⁇ L with double distilled water;
  • the PCR product amplification conditions were: pre-denaturation at 98°C for 5 min; 98°C for 10 s, 50°C for 15 s, 68°C for 4 min, 25 cycles followed by incubation at 68°C for 10 min, and finally storage at 16°C.
  • E.coli BL21(DE3)/pET28a(+)-R146F E.coli BL21(DE3 )/pET28a(+)-G539I
  • E.coli BL21(DE3)/pET28a(+)-G539I/R146F E.coli BL21(DE3)/pET28a(+)-G539I/R146F/D147N;
  • the recombinant E. coli containing the mutant gene prepared in step 1 and the wild-type strain E.coli BL21(DE3)/pET-28a(+)-cgt were respectively inserted into LB liquid culture medium (containing kanamycin, 50 mg). /L), culture at 37°C for 10 hours, and prepare the seed liquid;
  • the resulting seed liquid was inserted into TB liquid medium (containing kanamycin, 50 mg/L) at an inoculation amount of 1% (v/v), and cultured on a shaking table at 37°C until OD 600 reached 0.6 to 0.8, and the final concentration was added.
  • the expression is induced by 0.1mM IPTG at a temperature of 16°C. After fermentation and culture for 18 hours, the fermentation broth is prepared;
  • the crude enzyme solution of the above mutant and the crude enzyme solution of the wild enzyme were subjected to agarose gel electrophoresis respectively to verify that the protein expression was successful.
  • the above-mentioned crude enzyme solutions were passed through membranes and purified by Ni column affinity chromatography.
  • First use buffer A (containing 20mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole, pH 7.4) to balance the Ni column. After equilibrium, load the protein sample. After loading the sample multiple times to ensure complete adsorption, use different concentrations in sequence. (20 ⁇ 500mM) imidazole solution is gradient eluted and the flow-through is collected. After the end, a 10% protein gel is prepared to detect the target protein.
  • the imidazole solution containing the target protein is combined and concentrated using an ultrafiltration tube and a phosphate buffer of pH 6.0. Replacement, and finally the obtained protein samples were aliquoted, quick-frozen in liquid nitrogen, and stored at -80°C for later use.
  • Pure enzyme solutions containing wild-type CGTase, pure enzyme solutions containing R146F, pure enzyme solutions containing G539I, pure enzyme solutions containing G539I/R146F, and pure enzyme solutions containing G539I/R146F/D147N were prepared respectively.
  • the organic solvent tolerance of cyclodextrin glucosyltransferase is analyzed by measuring its residual enzyme activity after incubation in organic solvents of different concentrations for a certain period of time.
  • DMSO dimethyl sulfoxide
  • step (3) After incubating the system obtained in step (1) for 1 hour at 4°C, add 50 ⁇ L of the maltodextrin solution prepared in step (2). After reacting at 40°C for 10 min, immediately add 250 ⁇ L hydrochloric acid (1.0M) to terminate. react, then add 150 ⁇ L of the methyl orange solution prepared in step (2), let it stand at room temperature for 20 minutes, and measure the absorbance at 505 nm.
  • the relative activity of the other three mutants did not have much impact compared with the WT.
  • the mutant T599D/N600D/Y601H although the DMSO resistance of this mutant was greatly improved, its original The decrease in activity is serious, but the activity of the triple mutant G539I/R146F/D147N is 20% higher than that of WT. Therefore, the triple mutant G539I/R146F/D147N may have more advantages in practical applications.
  • the concentrations of ethanol were selected as 0% and 12% (v/v) respectively.
  • the incubation time was controlled at 1 h, and the activity of the pure enzyme obtained was measured using the methyl orange method (the methyl orange method was appropriately improved for convenience of detection).
  • step (3) After incubating the system obtained in step (1) for 1 hour at 4°C, add 50 ⁇ L of the maltodextrin solution prepared in step (2). After reacting at 40°C for 10 min, immediately add 250 ⁇ L hydrochloric acid (1.0M) to terminate. react, then add 150 ⁇ L of the methyl orange solution prepared in step (2), let it stand at room temperature for 20 minutes, and measure the absorbance at 505 nm.
  • mutant T599D/N600D/Y601H in 12% ethanol is similar to the above two combined mutations, and the effect is not obvious, which may also be related to the greater decrease in the original activity of this mutant.
  • the relative activity of the four mutants in the present invention is not significantly lower than that of WT, and the relative activity is maintained at 87-115%.
  • the activity of the triple mutant G539I/R146F/D147N is improved compared with WT.
  • the concentrations of methanol were selected as 0% and 12% (v/v) respectively.
  • the incubation time was controlled at 1 h, and the activity of the pure enzyme obtained was measured using the methyl orange method (the methyl orange method was appropriately improved for convenience of detection).
  • step (3) After incubating the system obtained in step (1) for 1 hour at 4°C, add 50 ⁇ L of the maltodextrin solution prepared in step (2). After reacting at 40°C for 10 min, immediately add 250 ⁇ L hydrochloric acid (1.0M) to terminate. react, then add 150 ⁇ L of the methyl orange solution prepared in step (2), let it stand at room temperature for 20 minutes, and measure the absorbance at 505 nm.
  • the mutation R146F plays an important role. Comparing the mutant T599D/N600D/Y601H, its tolerance in 12% methanol was 6.9% higher than that of WT, but the effect was far less obvious than the optimal triple mutation G539I/R146F/D147N.
  • the concentrations of acetone were selected as 0% and 15% (v/v) respectively.
  • the incubation time was controlled at 1 h, and the activity of the pure enzyme obtained was measured using the methyl orange method (the methyl orange method was appropriately improved for convenience of detection).
  • step (3) After incubating the system obtained in step (1) for 1 hour at 4°C, add 50 ⁇ L of the maltodextrin solution prepared in step (2). After reacting at 40°C for 10 min, immediately add 250 ⁇ L hydrochloric acid (1.0M) to terminate. react, then add 150 ⁇ L of the methyl orange solution prepared in step (2), let it stand at room temperature for 20 minutes, and measure the absorbance at 505 nm.
  • the pure enzyme solutions of mutants R146F, G539I, G539I/R146F, G539I/R146F/D147N and wild-type WT prepared in Example 1, as well as the mutant T599D/N600D/Y601H The pure enzyme solution is appropriately diluted, and soluble starch is used as the glycosyl donor and genistein is used as the glycosyl acceptor for glycosylation reaction.
  • the four mutants in the present invention have a certain improvement in the glycosylation efficiency of genistein compared with WT.
  • the best one is the triple mutant G539I/R146F/D147N, which has a higher conversion rate than WT. 16%, the remaining single-shot R146F, G539I and double mutant G539I/R146F increased by 12%, 9% and 13% respectively compared with WT.
  • the tolerance of mutant T599D/N600D/Y601H in DMSO was also greatly improved, it may be due to the decrease in its enzyme activity. Many, the performance in conversion rate is not obvious, it is similar to WT, and there is no obvious improvement.

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Abstract

本发明公开了一种溶剂耐受性提高的环糊精葡萄糖基转移酶及其制备，属于酶工程和基因工程技术领域。本发明构建了四个有机溶剂耐受性提高的环糊精葡萄糖基转移酶突变体。其中，对DMSO和甲醇耐受性最好的突变体为G539I/R146F/D147N，较WT分别提高了1.6和1.7倍；对乙醇耐受性最好的突变体为R146F，较WT提高了1.4倍；对丙酮耐受性最好的突变体为G539I/R146F，较WT提高了1.5倍。本发明有助于拓展糖基转移酶在有机反应体系中的应用，提高CGTase对天然疏水底物的酶促效率，具有极大的应用前景。

Description

一种溶剂耐受性提高的环糊精葡萄糖基转移酶及其制备 技术领域
本发明涉及一种溶剂耐受性提高的环糊精葡萄糖基转移酶及其制备，属于酶工程和基因工程技术领域。
背景技术
环糊精糖基转移酶(Cyclodextrin glycosyltransferase，简称CGTase，EC 2.4.1.19)，属于α-淀粉酶家族，因其具有多种转糖基活力(环化、歧化、耦合活力)，极具商业价值，目前该酶在工业上主要用于生产环糊精，此外，该酶还含有部分水解活力，在短链糖基化反应过程中起到重要作用。近年来利用其转糖基活力改善天然底物的性质研究取得了较好的进展，如甜菊苷、芦丁、染料木素等。糖基化处理有效解决了这些物质在实际应用中溶解性差、不易储存、不稳定等多种问题，且糖基化产物多数保持了物质本身的优良性质和价值，因而在食品、化工等多个领域均有较好的应用。
然而，在应用过程中，新的问题也随之出现亟待解决。在实际应用中，直接依靠挖掘的天然环糊精糖基转移酶往往很难满足生产要求。究其原因，天然酶多数都存在酶活低，不稳定，底物混杂性差等问题，且由于环糊精糖基转移酶复杂的催化机理，常产生多种产物，产物特异性差，因此，目前对于该酶的分子改造多集中于催化活性，热稳定性，专一性等方面，并取得了不错的进展。然而，对于酶学反应，其催化效率除酶本身外，与酶所处环境也有很大的关系。在工业生产中，有机溶剂的使用是极为重要的一环，很多疏水底物的生物转化都依赖于有机溶剂，但有机溶剂往往对多数酶存在不利影响，使酶活性降低甚至失活，严重限制了生产效率。
与多数酶类似，天然的环糊精糖基转移酶在有机溶剂环境中的酶活及酶稳都会受到极大影响，因此，为了突破这一瓶颈，通过分子改造策略提高环糊精糖基转移酶的有机溶剂耐受性及其在有机溶剂中的酶活具有极大的意义。
发明内容
本发明的目的在于提供了一种对溶剂耐受性提高的环糊精葡萄糖基转移酶突变体，有利于扩大环糊精糖基转移酶对天然疏水性底物的糖基化应用。
所述突变体是将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第146位、第147位、第539位的氨基酸突变得到的。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶来源于软化类芽孢杆菌 (Paenibacillus macerans)。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶的核苷酸序列如SEQ ID NO.2所示。
在本发明的一种实施方式中，所述突变体是将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第146位的精氨酸突变为苯丙氨酸，命名为R146F，其氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。
在本发明的一种实施方式中，所述突变体是将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸，命名为G539I，其氨基酸序列如SEQ ID NO.5所示，核苷酸序列如SEQ ID NO.6所示。
在本发明的一种实施方式中，所述突变体是将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸，同时将第146位的精氨酸突变为苯丙氨酸，命名为G539I/R146F，其氨基酸序列如SEQ ID NO.7所示，核苷酸序列如SEQ ID NO.8所示。
在本发明的一种实施方式中，所述突变体是将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸；同时将第146位的精氨酸突变为苯丙氨酸；同时将第147位的天冬氨酸突变为天冬酰胺；命名为G539I/R146F/D147N，其氨基酸序列如SEQ ID NO.9所示，核苷酸序列如SEQ ID NO.10所示。
本发明还提供了一种编码上述突变体的基因。
本发明还提供了一种携带上述基因的重组载体。
在本发明的一种实施方式中，所述载体为：pET系列载体、pUT系列载体或pBAD系列载体。
本发明还提供了一种表达上述突变体，或携带上述基因，或携带上述重组载体的重组细胞。
在本发明的一种实施方式中，所述重组细胞是以真菌或细菌为宿主细胞。
在本发明的一种实施方式中，所述宿主细胞为：大肠杆菌或枯草芽孢杆菌。
本发明还提供了一种制备所述环糊精葡萄糖基转移酶突变体的方法，具体步骤包括：
(1)首先设计定点突变的引物，以含有所述环糊精葡萄糖基转移酶基因的质粒为模板进行全质粒PCR，经消化模板后获得携带突变基因的重组质粒，将其转入宿主菌，用卡那霉素抗性筛选后挑菌培养，并送样测序；
(2)将测序正确的菌株进行培养，37℃培养12h后按1％的接种量转接到TB培养基， 37℃培养至OD长至0.8左右后诱导表达，诱导温度为16℃，诱导18h；
(3)结束培养后将收集的发酵液进行离心收集菌体，进行超声破碎，再次离心，收集的上清液即为所述突变体的粗酶液。
在本发明的一种实施方式中，所述重组质粒以pET-28a(+)为表达载体。
在本发明的一种实施方式中，所述宿主菌为E.coli BL21(DE3)。
在本发明的一种实施方式中，所述有机溶剂包括：二甲基亚砜(DMSO)、乙醇、甲醇及丙酮。
本发明还提供了一种提高环糊精葡萄糖基转移酶对有机溶剂的耐受性的方法，所述方法为，将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸；
或将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸，同时将第146位的精氨酸突变为苯丙氨酸；
或将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸；同时将第146位的精氨酸突变为苯丙氨酸；同时将第147位的天冬氨酸突变为天冬酰胺。
在本发明的一种实施方式中，所述有机溶剂包括二甲基亚砜(DMSO)、乙醇、甲醇及丙酮中的一种或多种。
本发明还提供了一种生产长链糖基化染料木素的方法，其特征在于，所述方法为，向含有可溶性淀粉和染料木素的反应体系中加入环糊精葡萄糖基转移酶突变体进行反应，得到反应液；将反应液进行分离，获得长链糖基化染料木素。
本发明还提供了上述突变体，或上述基因，或上述重组载体，或上述重组细胞，在天然疏水性底物的糖基化应用，或在部分有机溶剂反应体系中的糖基化应用。
有益效果
(1)本发明构建了四个有机溶剂耐受性提高的环糊精葡萄糖基转移酶突变体，R146F、G539I、G539I/R146F、G539I/R146F/D147N，其在不含有机溶剂的体系中直接测活的原始活力保持在WT活力的90％～120％。
(2)在对二甲基亚砜的耐受性测定中，WT在25％的DMSO体系中孵育1h后的残余活力剩21.3％，而突变体R146F、G539I、G539I/R146F、G539I/R146F/D147N的耐受性均有提高，其中，最优突变体为G539I/R146F/D147N，较WT的DMSO耐受性提高了1.6倍。
(3)在对乙醇的耐受性测定中，WT在12％的乙醇中孵育1h后的残余活力为17.9％，对上述四种突变体在相同条件下进行测定发现，其中最优的突变体为R146F，较WT的乙醇 耐受性提高了1.4倍。
(4)在对甲醇的耐受性测定中，WT在12％的甲醇中孵育1h后的残余活力为27.4％，对上述四种突变体在相同条件下进行测定发现，其中最优的突变体为G539I/R146F/D147N，较WT的甲醇耐受性提高了1.7倍。
(5)在对丙酮的耐受性测定中，WT在15％的丙酮中孵育1h后的残余活力为22.3％，对上述四种突变体在相同条件下进行测定发现，其中最优的突变体为G539I/R146F，较WT的丙酮耐受性提高了1.5倍。
(6)本发明获得了两个有机溶剂耐受性提高的环糊精糖基转移酶突变体，促进了对疏水性底物的糖基化效率，有助于拓展CGTase的工业应用范围，具有极大的应用前景。
附图说明
图1：野生型及突变体纯酶的蛋白胶图；其中，M：Marker；1：R146F；2：、G539I；3：G539I/R146F；4：G539I/R146F/D147N；5：野生型WT。
具体实施方式
本发明的具体实施例仅用作进一步的说明，不能作为本发明的限制内容和范围。
下述实施例中所涉及的引物序列如表1所示：
表1：引物
下述实施例中涉及的培养基如下：
LB液体培养基：酵母粉5.0g·L-1、胰蛋白胨10.0g·L-1、NaCl 10.0g·L-1。
LB固体培养基：在LB液体培养基的基础上添加2％琼脂。
TB液体培养基：酵母粉24.0g·L-1、胰蛋白胨12.0g·L-1、KH2PO3 2.3g·L-1、K2HPO3 16.4g·L-1、甘油5g·L-1。
下述实施例中所涉及的检测方法如下：
环糊精糖基转移酶的活力检测：
甲基橙法测环化活力：取50μL稀释至合适浓度的酶液，加入200μL麦芽糊精(预先用50mM磷酸缓冲液(pH 6.0)配制的浓度为10g·L-1)，在40℃的水浴摇床中反应10min，立即加入250μL盐酸(1.0M)终止反应，再加入150μL甲基橙(用50mM磷酸缓冲液配制成浓度为0.5mM)，室温(20℃)静置20min，在505nm下测定吸光度。以不加酶液组作为空白对照。
酶活定义：在该条件下每分钟生成1μmolα-环糊精所需的酶量定义为一个酶活力单位。
下述实施例中所涉及的T599D/N600D/Y601H突变体纯酶的构建方法记载于公开号为CN113817704A的中国发明专利申请文本中。
实施例1：环糊精葡萄糖基转移酶突变体的制备及表达
1、定点突变
(1)pET28a(+)-cgt的构建
具体步骤如下：
取实验室保藏的甘油菌E.coli BL21(DE3)/pET-20b(+)-cgt，所述菌株E.coli BL21(DE3)/pET-20b(+)-cgt，是将pET-20b(+)与核苷酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶cgt，酶切后连接制备得到重组质粒，将重组质粒导入到E.coli BL21(DE3)中，制备得到充足菌，其具体的构建过程可见参考文献：Han,R.,Ge,B.,Jiang,M.et al.High production of genistein diglucoside derivative using cyclodextrin glycosyltransferase from Paenibacillus macerans.J Ind Microbiol Biotechnol 44,1343–1354(2017)；
将E.coli BL21(DE3)/pET-20b(+)-cgt将进行划线活化后，挑取单菌落接种于含氨苄青霉素(100mg/L)的LB液体培养基中，在37℃培养10h后，用试剂盒提取质粒pET-20b(+)-cgt。
一步克隆更换载体：设计引物，引物序列为，F:CAGCAAATGGGTCGCGGATCCTCACCGGACACCTCAGTGGA
R:GTGGTGGTGGTGGTGCTCGAGATTTTGCCAATCCACCGTCA；以质粒 pET-20b(+)-cgt为模板利用PCR技术获得大量克隆目的片段，经胶回收后与经双酶切(BamH I和Xho I)后的质粒pET-28a(+)进行连接，转入大肠杆菌BL21后进行涂布培养，待菌落明显长出后挑取接入到LB液体培养基培养10h，提取质粒并送样测序，测序正确的质粒标记为pET-28a(+)-cgt。
(2)突变体的构建
本发明中的环糊精葡萄糖基转移酶的氨基酸序列如SEQ ID NO.1所示(基因的核苷酸序列如SEQ ID NO.2所示)，根据选定的突变位点设计引物，以提取的重组质粒pET28a(+)-cgt作为模板进行全质粒PCR，引物序列如下：
突变体R146F所用引物：
正向引物：5'-TCTCCGGCAGATTTTGACAATCCG-3'，下划线为突变碱基；
反向引物：5'-CGGATTGTCAAAATCTGCCGGAGA-3'，下划线为突变碱基。
突变体G539I所用引物：
正向引物：5'-ACCGCGGTCACCATTAGTGGTATT-3'，下划线为突变碱基；
反向引物：5'-AATACCACTAATGGTGACCGCGGT-3'，下划线为突变碱基。
突变体G539I/R146F所用引物：
正向引物1：5'-TCTCCGGCAGATTTTGACAATCCG-3'，下划线为突变碱基；
反向引物1：5'-CGGATTGTCAAAATCTGCCGGAGA-3'，下划线为突变碱基。
正向引物2：5'-ACCGCGGTCACCATTAGTGGTATT-3'，下划线为突变碱基；
反向引物2：5'-AATACCACTAATGGTGACCGCGGT-3'，下划线为突变碱基。
突变体G539I/R146F/D147N所用引物：
正向引物1：5'-TCTCCGGCAGATTTTAATAATCCG-3'，下划线为突变碱基；
反向引物1：5'-CGGATTATTAAAATCTGCCGGAGA-3'，下划线为突变碱基。
正向引物2：5'-ACCGCGGTCACCATTAGTGGTATT-3'，下划线为突变碱基；
反向引物2：5'-AATACCACTAATGGTGACCGCGGT-3'，下划线为突变碱基。
PCR反应体系均为：5×PrimeSTAR Buffer(Mg2+Plus)5μL，2.5mM dNTPs 4μL，10μM正向引物1μL，10μM反向引物1μL，模板DNA 1μL，2.5U/μL PrimeSTAR Taq HS 0.5μL，加双蒸水补足至50μL；
PCR产物扩增条件均为：98℃预变性5min；98℃10s，50℃15s，68℃4min，25个循环后68℃保温10min，最后16℃保存。
用1％琼脂糖凝胶电泳检测PCR产物，检测正确后加消化酶(Dpn I)消化模板，37℃ 下消化1h，将消化后的产物转入感受态大肠杆菌BL21，在含浓度为50mg/L的卡那霉素的LB固体培养基上过夜培养，挑取阳性克隆接入LB液体培养基中培养10h后，提取质粒送样测序，测序正确即获得可表达突变体的重组大肠杆菌，即制备得到重组大肠杆菌：E.coli BL21(DE3)/pET28a(+)-R146F，E.coli BL21(DE3)/pET28a(+)-G539I，E.coli BL21(DE3)/pET28a(+)-G539I/R146F，E.coli BL21(DE3)/pET28a(+)-G539I/R146F/D147N；
(3)含有野生型CGTase的重组菌的构建
将重组质粒pET28a(+)-cgt按照上述方法导入E.coli BL21(DE3)中，即得到：E.coli BL21(DE3)/pET-28a(+)-cgt。
2、突变体的表达
分别将步骤1制备得到的含有突变体基因的重组大肠杆菌、及野生型菌株E.coli BL21(DE3)/pET-28a(+)-cgt接入LB液体培养基(含卡那霉素，50mg/L)，于37℃培养10h，制备得到种子液；
将所得种子液按1％(v/v)的接种量接入TB液体培养基(含卡那霉素，50mg/L)中，37℃摇床培养至OD600达到0.6～0.8，加终浓度为0.1mM的IPTG诱导表达，温度为16℃，发酵培养18h后，制备得到发酵液；
将制备得到的发酵液离心收菌，4℃、8000r/min离心10min，倒掉上清液，剩余的沉淀用磷酸盐缓冲液(pH 6.0，50mM)重悬后进行超声破碎，300W破碎10min，将破碎液于4℃，8000r/min离心30min，所得上清液，即为含有突变体的粗酶液，即得到含有野生型CGTase的粗酶液、含有R146F的粗酶液，含有G539I的粗酶液，含有G539I/R146F的粗酶液，含有G539I/R146F/D147N的粗酶液。
分别将上述突变体粗酶液和野生酶粗酶液进行琼脂糖凝胶电泳，验证蛋白表达成功。
3、突变体和野生型酶的纯化
分别将上述粗酶液过膜处理，进行Ni柱亲和层析纯化。先用缓冲液A(含20mM磷酸钠、0.5M氯化钠、20mM咪唑、pH 7.4)平衡Ni柱，待平衡后进行蛋白样品上样，多次上样使之完全吸附后，依次用不同浓度(20～500mM)的咪唑溶液进行梯度洗脱并收集流穿，结束后配置10％的蛋白胶检测目的蛋白，合并含有目的蛋白的咪唑溶液，并用超滤管及pH 6.0的磷酸缓冲液进行浓缩置换，最后将获得的蛋白样品进行分装，并用液氮速冻，于-80℃保藏备用。
分别制备得到含有野生型CGTase的纯酶液、含有R146F的纯酶液、含有G539I的纯酶液、含有G539I/R146F的纯酶液、含有G539I/R146F/D147N的纯酶液。
分别将上述突变体和野生型的酶液进行琼脂糖凝胶电泳，结果如图1所示，在74kDa(Marker的第三条带大小为70kDa)处有相应的条带。
实施例2：环糊精葡萄糖基转移酶的有机溶剂耐受性检测
在本发明的实施方案中，环糊精葡萄糖基转移酶的有机溶剂耐受性通过测定其在不同浓度的有机溶剂中孵育一定时间后的残余酶活进行分析。
残余活力的计算＝孵育后的酶活(含不同浓度的有机溶剂体系中测得的酶活)/未孵育的酶活(仅含PBS的体系中测得的酶活)
具体步骤如下：
1、环糊精葡萄糖基转移酶的有机溶剂二甲基亚砜(DMSO)耐受性检测
将二甲基亚砜(DMSO)的浓度分别选用0％，25％(v/v)，孵育时间控制在1h，利用甲基橙法(为方便检测，对甲基橙法进行了适当改进)对获得的纯酶进行测活。
具体方法如下：
(1)分别将实施例1制备得到的纯酶液、T599D/N600D/Y601H突变体纯酶液稀释至酶活为0.01-0.02mg/mL后，取100μL，加入到装有100μL含DMSO的磷酸缓冲液(50mM，pH 6.0)中，其中，含DMSO的磷酸缓冲液中DMSO的初始体积分数分别为0％，50％；
(2)配制麦芽糊精溶液：用50mM磷酸缓冲液(pH 6.0)，将麦芽糊精配制成浓度为40g·L-1的麦芽糊精溶液；
配制甲基橙溶液：用50mM磷酸缓冲液将甲基橙配制成浓度为0.5mM甲基橙溶液。
(3)分别将步骤(1)得到的体系于4℃孵育1h后，加入50μL步骤(2)制备得到的麦芽糊精溶液，在40℃下反应10min后，立即加入250μL盐酸(1.0M)终止反应，再加入150μL步骤(2)制备得到的甲基橙溶液后，室温静置20min，在505nm下测定吸光度。
以0％(v/v)的DMSO组中测得的酶活为原始活力，在25％(v/v)的DMSO中孵育后测得的野生型WT和突变体的残余活力及相对活力列于表2。
表2野生型WT与突变体的残余活力及相对活力比较
从表2可以看出：在25％的DMSO体系中于4℃孵育1h后测活，WT的残余活力为21.3％。相比于WT，四个突变体的DMSO耐受性均有一定的提升，单突变体R146F及G539I相比于WT在25％的DMSO中孵育1h后耐受性提高了6～7％，组合后的双突变体G539I/R146F的耐受性有所增加，较WT提高了10.5％，最优的三突变体G539I/R146F/D147N在相同孵育条件下较WT提高了13.8％，是WT的1.6倍。突变体T599D/N600D/Y601H在25％的DMSO体系中也具有较好的耐受性，效果与最优的三突G539I/R146F/D147N相近。
此外，除突变体G539I外，其余三个突变体的相对活力较WT没有太大的影响，而对比突变体T599D/N600D/Y601H，该突变体的DMSO抗性虽然也有较大提升，但其原始活力下降比较严重，而三突变体G539I/R146F/D147N的活力较WT提高了20％，因此，三突变体G539I/R146F/D147N在实际应用中可能更具优势。
2、环糊精葡萄糖基转移酶的有机溶剂乙醇耐受性检测
将乙醇的浓度分别选用0％，12％(v/v)。孵育时间控制在1h，利用甲基橙法(为方便检测，对甲基橙法进行了适当改进)对获得的纯酶进行测活。
具体方法如下：
(1)分别将实施例1制备得到的纯酶液、T599D/N600D/Y601H突变体纯酶液稀释至酶活为0.01-0.02mg/mL后，取100μL，加入到装有100μL含乙醇的磷酸缓冲液(50mM，pH6.0)中，其中，含乙醇的磷酸缓冲液中乙醇的体积分数分别为0％，24％；
(2)配制麦芽糊精溶液：用50mM磷酸缓冲液(pH 6.0)，将麦芽糊精配制成浓度为40g·L-1的麦芽糊精溶液；
配制甲基橙溶液：用50mM磷酸缓冲液将甲基橙配制成浓度为0.5mM甲基橙溶液。
(3)分别将步骤(1)得到的体系于4℃孵育1h后，加入50μL步骤(2)制备得到的麦芽糊精溶液，在40℃下反应10min后，立即加入250μL盐酸(1.0M)终止反应，再加入150μL步骤(2)制备得到的甲基橙溶液后，室温静置20min，在505nm下测定吸光度。
以0％(v/v)的乙醇组中测得的酶活为原始活力，在12％(v/v)的乙醇中孵育后测得的野生型WT和突变体的残余活力及相对活力列于表3。
表3野生型WT与突变体的残余活力及相对活力比较

从表3可以看出：在12％的乙醇体系中于4℃孵育1h后测活，WT的残余活力为17.9％。相比于WT，两个单突变体的乙醇耐受性有较为明显的提升，最优的单突变体R146F较WT提高了7.4％，是WT的1.4倍，单突变体G539I较WT的乙醇耐受性提高了5.9％，而两个组合突变在乙醇中的耐受性较WT没有明显改善。突变体T599D/N600D/Y601H在12％的乙醇中的耐受性与上述两个组合突变类似，效果也不明显，这可能也与该突变体的原始活力下降较多有关。此外，对比DMSO的数据，发现该酶对乙醇的耐受性较差。
此外，在酶活方面，本发明中的四个突变体的相对活力较WT没有明显降低，相对活力保持在87～115％，三突变体G539I/R146F/D147N的活力较WT有所提高。
3、环糊精葡萄糖基转移酶的有机溶剂甲醇耐受性检测
将甲醇的浓度分别选用0％，12％(v/v)。孵育时间控制在1h，利用甲基橙法(为方便检测，对甲基橙法进行了适当改进)对获得的纯酶进行测活。
具体方法如下：
(1)分别将实施例1制备得到的纯酶液、T599D/N600D/Y601H突变体纯酶液稀释至酶活为0.01-0.02mg/mL后，取100μL，加入到装有100μL含甲醇的磷酸缓冲液(50mM，pH6.0)中，其中，含甲醇的磷酸缓冲液中甲醇的体积分数分别为0％，24％；
(2)配制麦芽糊精溶液：用50mM磷酸缓冲液(pH 6.0)，将麦芽糊精配制成浓度为40g·L-1的麦芽糊精溶液；
配制甲基橙溶液：用50mM磷酸缓冲液将甲基橙配制成浓度为0.5mM甲基橙溶液。
(3)分别将步骤(1)得到的体系于4℃孵育1h后，加入50μL步骤(2)制备得到的麦芽糊精溶液，在40℃下反应10min后，立即加入250μL盐酸(1.0M)终止反应，再加入150μL步骤(2)制备得到的甲基橙溶液后，室温静置20min，在505nm下测定吸光度。
以0％(v/v)的甲醇组中测得的酶活为原始活力，在12％(v/v)的甲醇中孵育后测得的野生型WT和突变体的残余活力及相对活力列于表4。
表4野生型WT与突变体的残余活力及相对活力比较

从表4可以看出：在12％的甲醇体系中于4℃孵育1h后测活，WT的残余活力为27.4％。相比于WT，单突变体R146F，双突变体G539I/R146F及三突变体G539I/R146F/D147N的甲醇耐受性改善较为明显，其中三突的效果最好，较WT提高了近1.7倍，提高了18.9％，其次是单突变体R146F及双突G539I/R146F，较WT分别提高了12.7％和11.3％。然而，单突变体G539I对甲醇的耐受性不如WT，此外，对比单突R146F及双突G539I/R146F的耐受性数据，叠加上G539I后，甲醇的耐受性有所下降，因此，对于甲醇的耐受性检验中，突变R146F起重要作用。对比突变体T599D/N600D/Y601H，其在12％的甲醇中耐受性较WT提升了6.9％，但效果远不如最优的三突G539I/R146F/D147N明显。
在酶活方面，四个突变体的相对活力较WT没有明显降低，相对活力保持在89～113％，三突变体G539I/R146F/D147N的活力较WT有所提高。
4、环糊精葡萄糖基转移酶的有机溶剂丙酮耐受性检测
将丙酮的浓度分别选用0％，15％(v/v)。孵育时间控制在1h，利用甲基橙法(为方便检测，对甲基橙法进行了适当改进)对获得的纯酶进行测活。
具体方法如下：
(1)分别将实施例1制备得到的纯酶液、T599D/N600D/Y601H突变体纯酶液稀释至酶活为0.01-0.02mg/mL后，取100μL，加入到装有100μL含丙酮的磷酸缓冲液(50mM，pH6.0)中，其中，含丙酮的磷酸缓冲液中乙醇的体积分数分别为0％，30％；
(2)配制麦芽糊精溶液：用50mM磷酸缓冲液(pH 6.0)，将麦芽糊精配制成浓度为40g·L-1的麦芽糊精溶液；
配制甲基橙溶液：用50mM磷酸缓冲液将甲基橙配制成浓度为0.5mM甲基橙溶液。
(3)分别将步骤(1)得到的体系于4℃孵育1h后，加入50μL步骤(2)制备得到的麦芽糊精溶液，在40℃下反应10min后，立即加入250μL盐酸(1.0M)终止反应，再加入150μL步骤(2)制备得到的甲基橙溶液后，室温静置20min，在505nm下测定吸光度。
以0％(v/v)的丙酮组中测得的酶活为原始活力，在15％(v/v)的丙酮中孵育后测得的野生型WT和突变体的残余活力及相对活力列于表5。
表5野生型WT与突变体的残余活力及相对活力比较

从表5可以看出：在15％的丙酮体系中于4℃孵育1h后测活，WT的残余活力为22.3％。相比于WT，四个突变体的丙酮耐受性均有提升，最优的是双突变体G539I/R146F，较WT提高了12％，是WT的1.5倍。此外，单突变体R146F及三突G539I/R146F/D147N的丙酮耐受性效果接近，较WT提高了7～8.4％，而单突变体G539I及T599D/N600D/Y601H的丙酮耐受性较WT提升不明显，仅提高了4％左右。
在酶活方面，与前面的结果一致，四个突变体的相对活力较WT没有明显降低，相对活力保持在87～117％，三突变体G539I/R146F/D147N的活力较WT有所提高。
实施例3：对染料木素的糖基化应用
以染料木素为例，分别将实施例1制备得到的突变体R146F、G539I、G539I/R146F、G539I/R146F/D147N纯酶液及野生型WT的纯酶液，以及突变体T599D/N600D/Y601H纯酶液进行适当稀释，分别以可溶性淀粉为糖基供体，以染料木素为糖基受体进行糖基化反应。
具体步骤如下：
(1)用磷酸缓冲液(pH 6.0，50mM)配制浓度为40g/L的可溶性淀粉溶液；
(2)用DMSO溶液溶解染料木素，得到染料木素溶液，浓度为7.5g/L。
(3)按照可溶性淀粉溶液：染料木素溶液：纯酶液(v:v:v)＝6：2：2的比例进行混合(加酶量控制在0.15～0.2U/mL)，在40℃摇床中反应16～18h后加热终止反应。
对样品进行离心过膜处理后进行高效液相色谱(HPLC)分析，具体结果如表6所示。
表6野生型WT与各突变体对染料木素的糖基化效率比较
由上表可知：本发明中的四个突变体较WT对染料木素的糖基化效率均有一定的提升，其中，最好的是三突变体G539I/R146F/D147N，转化率较WT提高了16％，其余单突R146F、 G539I，双突G539I/R146F较WT分别提高了12％，9％及13％，突变体T599D/N600D/Y601H在DMSO中的耐受性虽然也有较大提升，但可能是由于其酶活下降较多，在转化率上表现不明显，与WT差不多，没有明显提升。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种环糊精葡萄糖基转移酶突变体，其特征在于，所述突变体是将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸得到的；
    或将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸，同时将第146位的精氨酸突变为苯丙氨酸得到的；
    或将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸；同时将第146位的精氨酸突变为苯丙氨酸；同时将第147位的天冬氨酸突变为天冬酰胺得到的。
  2. 编码权利要求1所述突变体的基因。
  3. 携带权利要求2所述基因的重组载体。
  4. 如权利要求3所述的重组载体，其特征在于，以pET系列载体、pUT系列载体或pBAD系列载体为表达载体。
  5. 表达权利要求1所述突变体，或携带权利要求2所述基因，或携带权利要求3或4所述重组载体的重组细胞。
  6. 如权利要求5所述的重组细胞，其特征在于，所述重组细胞以细菌或真菌为宿主细胞。
  7. 一种提高环糊精葡萄糖基转移酶对有机溶剂的耐受性的方法，其特征在于，所述方法为，将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸；
    或将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸，同时将第146位的精氨酸突变为苯丙氨酸；
    或将氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第539位的甘氨酸突变为异亮氨酸；同时将第146位的精氨酸突变为苯丙氨酸；同时将第147位的天冬氨酸突变为天冬酰胺。
  8. 如权利要求7所述的方法，其特征在于，所述有机溶剂包括二甲基亚砜(DMSO)、乙醇、甲醇及丙酮中的一种或多种。
  9. 一种生产长链糖基化染料木素的方法，其特征在于，所述方法为，向含有可溶性淀粉和染料木素的反应体系中加入权利要求1所述的环糊精葡萄糖基转移酶突变体进行反应，得到反应液；将反应液进行分离，获得长链糖基化染料木素。
  10. 权利要求1所述的突变体，或权利要求2所述的基因，或权利要求3或4所述的重组载体，或权利要求5或6所述的重组细胞，在天然疏水性底物的糖基化应用，或在有机溶剂反应体系中的糖基化应用。
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KNEGTEL RONALD M A, WIND RICHE `LE D, ¨TTE HENRIE, ROZEBOOM J, KALK KOR H, BUITELAAR REINETTA M, DIJKHUIZEN LUBBERT, DIJKSTRA BAUK: "Crystal Structure at 2.3 Å Resolution and Revised Nucleotide Sequence of the Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 256, no. 3, 1 March 1996 (1996-03-01), United Kingdom , pages 611 - 622, XP093143707, ISSN: 0022-2836, DOI: 10.1006/jmbi.1996.0113 *

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