Maltooligosyl Trehalose Trehalohydrolase (MTHase) Mutant and Application Thereof

Abstract

The present invention discloses a maltooligosyl trehalose trehalohydrolase (MTHase) mutant and application thereof, belonging to the technical fields of gene engineering and enzyme engineering. The present invention provides a series of MTHase mutants to prepare trehalose, having a better effect. Further, the MTHase mutant is expressed in Escherichia coli BL21 (DE3) and the enzyme is optimized by fermentation, which can significantly increase the yield of enzymes.

Claims

1. A mutant of a maltooligosyl trehalose trehalohydrolase, comprising an amino acid sequence with mutations selected from the group consisting of mutation of methionine (Met) at position 140, mutation of leucine (Leu) at position 202, mutation of leucine (Leu) at position 218, mutation of tyrosine (Tyr) at position 323, mutation of phenylalanine (Phe) at position 338 and mutation of isoleucine (Ile) at position 404; wherein the mutations are relative to a parent amino acid sequence of a maltooligosyl trehalose trehalohydrolase derived from Sulfolobus acidocaldarius set forth in SEQ ID NO.2.

2. The mutant of claim 1, comprising comprises an amino acid sequence with mutations selected from the group consisting of: mutation of methionine (Met) at position 140 to leucine (Leu), and the mutant is named M140L; mutation of leucine (Leu) at position 202 to proline (Pro), and the mutant is named L202P; mutation of leucine (Leu) at position 218 to aspartic acid (Asp), and the mutant is named L218D; mutation of tyrosine (Tyr) at position 323 to glycine (Gly), and the mutant is named Y323G; mutation of phenylalanine (Phe) at position 338 to serine (Ser), and the mutant is named F338S; mutation of isoleucine (Ile) at position 404 to threonine (Thr), and the mutant is named I404T; mutation of leucine (Leu) at position 202 to proline (Pro), and mutation of tyrosine (Tyr) at position 323 to glycine (Gly), and the mutant is named L202P/Y323G; mutation of leucine (Leu) at position 202 to proline (Pro), mutation of leucine (Leu) at position 218 to aspartic acid (Asp), and mutation of tyrosine (Tyr) at position 323 to glycine (Gly), and the mutant is named L202P/L218D/Y323G; mutation of methionine (Met) at position 140 to leucine (Leu), mutation of leucine (Leu) at position 202 to proline (Pro), mutation of leucine (Leu) at position 218 to aspartic acid (Asp), and mutation of tyrosine (Tyr) at position 323 to glycine (Gly), and the mutant is named M140L/L202P/L218D/Y323G; mutation of methionine (Met) at position 140 to leucine (Leu), mutation of leucine (Leu) at position 202 to proline (Pro), mutation of leucine (Leu) at position 218 to aspartic acid (Asp), mutation of tyrosine (Tyr) at position 323 to glycine (Gly), and mutation of phenylalanine (Phe) at position 338 to serine (Ser), and the mutant is named M140L/L202P/L218D/Y323G/F338S; and mutation of methionine (Met) at position 140 to leucine (Leu), mutation of leucine (Leu) at position 202 to proline (Pro), mutation of leucine (Leu) at position 218 to aspartic acid (Asp), mutation of tyrosine (Tyr) at position 323 to glycine (Gly), mutation of phenylalanine (Phe) at position 338 to serine (Ser), and mutation of isoleucine (Ile) at position 404 to threonine (Thr), and the mutant is named M140L/L202P/L218D/Y323G/F338S/I404T.

3. Genes encoding the mutant of claim 1.

4. A vector or recombinant cell carrying the genes of claim 3.

5. A method for preparing the mutant of claim 1, comprising the following steps: (1) designing a site-directed mutagenic primer based on a determined mutation site, and carrying out site-directed mutagenesis by using a vector carrying a maltooligosyl trehalose trehalohydrolase gene as a template; constructing a plasmid vector containing the gene encoding the mutant; (2) transforming a mutant plasmid into a host cell; and (3) picking out a positive clone for fermentation, and collecting cells by centrifugation, wherein cell wall breaking supernatant is a crude enzyme solution of the mutant maltooligosyl trehalose trehalohydrolase.

6. A method for producing the mutant of claim 1, comprising: inoculating a fermentation medium with a recombinant Escherichia coli expressing the mutant of claim 1, culturing at 37 C. for 8 to 14 hours, adding lactose at a flow rate of 0.1 g L.sup.1 h.sup.1, and performing induction at 30 C. for 10 to 20 hours.

7. The method of claim 6, wherein the fermentation medium per liter contains 4.0 g of diammonium phosphate, 13.5 g of potassium dihydrogen phosphate, 1.7 g of citric acid, 1.4 g of magnesium sulfate heptahydrate, 1.2 g of peptone, 2.4 g of yeast extract, 8.0 g of glycerol, and 10 mL of metal ion liquid, and the pH is adjusted to 6.5 to 7.5.

8. A method of use of the mutant of claim 1 in producing trehalose.

9. A method of use of the genes of claim 3 or the vector or recombinant cell of claim 4 in producing trehalose.

10. An enzyme preparation for producing trehalose, comprising the mutant of claim 1.

Description

BRIEF DESCRIPTION OF FIGURES

[0033] FIG. 1 shows the growth conditions of a mutant MTHase;

[0034] FIG. 2 shows the enzyme activity of a mutant MTHase.

DETAILED DESCRIPTION

Example 1: Expression of Wild Type MTHase

[0035] An LB liquid medium (containing 100 mg/L kanamycin) is inoculated with Tre Z/pET24a(+)/BL21 (DE3) stored in a glycerol tube in the laboratory for growth for 8 h, and a TB liquid fermentation medium (containing 100 mg/L kanamycin) are inoculated with seeds according to 5% of inoculation amount. After Escherichia coli is cultured in a shaker at 25 C. for 24 h, a certain volume of fermentation broth is centrifuged at 4 C. and 12,000 rpm for 10 min. The supernatant is discarded to collect the bacteria, then the bacterial precipitate is resuspended with 20 mmol.Math.L.sup.1 Na.sub.2HPO.sub.4NaH.sub.2PO.sub.4 buffer of pH 8.0, and mixed well. The cell wall of the bacterial suspension is broken by an ultrasonic cell disruptor (the operating conditions of the ultrasonic cell disruptor: 6 operating probe, operating time 10 min, operating for 2 s and stopping for 3 s, and the operating power 20%), and then centrifuging is performed at 12,000 rpm for 10 min. After centrifugation, the supernatant is a fermented intracellular crude enzyme solution.

Example 2: Preparation and Expression of MTHase Single Mutant

[0036] (1) Preparation of Mutant

[0037] Primers into which M140L, L202P, L218D, Y323G, F338S, and I404T mutations are introduced are designed and synthesized according to the gene sequence of Sulfolobus acidocaldarius MTHase, and site-directed mutagenesis of the MTHase gene Tre Z is carried out, and sequencing is carried out respectively to confirm whether the MTHase mutant encoding genes are correct; a vector carrying the mutant gene is introduced into Escherichia coli for expression, to obtain a single mutant MTHase.

[0038] PCR amplification of encoding genes of site-directed mutant comprises: a rapid PCR technique is adopted, and the expression vector Tre Z/pET-24a(+) carrying the genes encoding the wild type MTHase as a template.

[0039] The site-directed mutagenic primers into which M140L mutation is introduced are as follows:

TABLE-US-00001 Forwardprimer: 5-GTATTGAACTGCTGCCGGTGGCGCA-3 (underlinedas mutantbases); and Reverseprimer: 5-TGCGCCACCGGCAGCAGTTCAATAC-3 (underlinedas mutantbases).

[0040] The site-directed mutagenic primers into which L202P mutation is introduced are as follows:

TABLE-US-00002 Forwardprimer: 5-CAATTATCTGCCGGGCCTGGGCCCGTATTTC-3 (underlined asmutantbases); and Reverseprimer: 5-GAAATACGGGCCCAGGCCCGGCAGATAATTG-3 (underlined asmutantbases).

[0041] The site-directed mutagenic primers into which L218D mutation is introduced are as follows:

TABLE-US-00003 Forwardprimer: 5-CGCCGTGGGGCGACACCTTCAATTTTGACGAC-3 (underlinedasmutantbases); and Reverseprimer: 5-GTCGTCAAAATTGAAGGTGTCGCCCCACGGCG-3 (underlinedasmutantbases).

[0042] The site-directed mutagenic primers into which Y323G mutation is introduced are as follows:

TABLE-US-00004 Forwardprimer: 5-CAAGGAAAAGGACGGCTATTATCAGGACTTTTGGTC-3 (underlinedasmutantbases); and Reverseprimer: 5-GACCAAAGTCCTGATAATAGCCGTCCTTTTCCTTG-3 (underlinedasmutantbases).

[0043] The site-directed mutagenic primers into which F338S mutation is introduced are as follows:

TABLE-US-00005 Forwardprimer: 5-GACATTGAGAAGACGTCTAAGGATGTGTTTGTG-3 (underlinedasmutantbases); and Reverseprimer: 5-CACAAACACATCCTTAGACGTCTTCTCAATGTC-3 (underlinedasmutantbases).

[0044] The site-directed mutagenic primers into which I404T mutation is introduced are as follows:

TABLE-US-00006 Forwardprimer: 5-GCGACCCTGTATACTCTGAGCCCGT-3 (underlinedas mutantbases); and Reverseprimer: 5-ACGGGCTCAGAGTATACAGGGTCGC-3 (underlinedas mutantbases).

[0045] PCR reaction system are all as follows: 5PS buffer 10 L, dNTPs Mix (2.5 mmol.Math.L.sup.1) 4 L, forward primer (10 mol.Math.L.sup.1) 1 L, reverse primer (10 mol.Math.L.sup.1) 1 L, template DNA 1 L, and PrimeStar HS (5 U/L) 0.5 L; distilled water is added to achieve 50 L.

[0046] PCR procedure comprises the steps: first, performing denaturation at 94 C. for 4 min; then starting 30 cycles: denaturation at 98 C. for 10 s, annealing at 55 C. for 5 s, and extension at 72 C. for 7 min 20 s; finally performing extension at 72 C. for 10 min, performing heat preservation at 4 C. The PCR product is detected by 1% agarose gel electrophoresis.

[0047] After the correct PCR product is verified, Dpn I is added to the PCR product, water bath is performed at 37 C. for 2 h to degrade the template. Afterwards, Escherichia coli JM109 competent cells are transformed and the transformed product is used for coating an LB solid medium containing 100 mg/L kanamycin, and culturing is performed at 37 C. for 10 to 12 h. Positive clones are selected and cultured in an LB liquid medium for 8 to 10 h. The correct mutants are sequenced, and an LB medium is inoculated from a glycerol tube, and culturing is performed overnight. Plasmids are extracted and transformed to express host Escherichia coli BL21 (DE3) competent cells, so as to obtain recombinant strains that can express mutants M140L, L202P, L218D, Y323G, F338S and I404T.

[0048] (2) Expression of Mutants

[0049] The mutant expression process is described as that in Example 1.

Example 3: Analysis of MTHase Enzyme Activity

[0050] Enzyme activity is defined as the amount of enzyme required to produce one micromole of maltooligosaccharides per minute.

[0051] Determination of enzyme activity comprises the steps: preheating: taking 0.45 mL of 2% maltodextrin solution (DE 9 to 13 pH 6.0 phosphate buffer) in a test tube and placing in a 60 C. water bath to preheat for 10 min; reaction: adding 0.05 mL of diluted fermented intracellular crude enzyme solution, shaking well, for 10 min (accurate timing), then adding 0.1 mL of NaOH solution (4 mol/L) to terminate the reaction, and taking 0.3 mL of reaction solution in a test tube with a stopper, then adding 0.7 mL of distilled water and 4 mL of DNS, shaking well to stop the reaction, then boiling for 7 min and cooling; measurement: adding distilled water to the above reaction system to a constant volume of 15 mL, mixing well, measuring the absorbance at the wavelength of 540 nm and calculating the enzyme activity.

[0052] OD.sub.600nm of shake flask culture for 24 h and enzyme activity of the wild type MTHase (WT) and mutants are listed in Table 1. The results show that all the mutants have higher enzyme activity than the wild type.

TABLE-US-00007 TABLE 1 OD.sub.600 nm of shake flask and enzyme activity of wild type MTHase and single mutant enzyme Enzyme activity Enzyme OD.sub.600 nm (U/mL) WT 12.9 31.2 M140L 12.7 39.9 L202P 12.1 41.9 L218D 12.9 40.9 Y323G 12.2 42.9 F338S 11.5 47.7 I404T 10.5 30.2

Example 4: Preparation, Expression and Enzyme Activity Analysis of MTHase Double Mutant

[0053] By using the plasmid of the mutant L202P constructed in Example 2 as a template for double mutation and according to site-directed mutagenic primers designed in Example 2, site-directed mutagenesis is performed on the plasmid carrying the genes encoding the mutant L202P through a rapid PCR technique, so as to construct a double mutant L202P/Y323G. Sequencing is performed to identify whether the encoding genes of MTHase double mutant are correct, and the plasmid with correct sequencing result is introduced into Escherichia coli for expression, so as to obtain double mutant MTHase.

[0054] OD.sub.600nm of shake flask culture for 24 h and enzyme activity of the wild type MTHase (WT) and the double mutant are listed in Table 2. The results show that the mutant L202P/Y323G has higher enzyme activity than the wild type.

TABLE-US-00008 TABLE 2 OD.sub.600 nm of shake flask and enzyme activity of wild type MTHase and double mutant enzyme Enzyme activity Enzyme OD.sub.600 nm (U/mL) WT 12.9 31.2 L202P/Y323G 13.1 47.7

Example 5: Preparation, Expression and Enzyme Activity Analysis of MTHase Triple Mutant

[0055] By using the plasmid of the mutant L202P/Y323G constructed in Example 2 and Example 4 as a template for triple mutation and according to site-directed mutagenic primers designed in embodiment 2, site-directed mutagenesis is performed on the plasmid carrying the genes encoding the mutant L202P/Y323G through a rapid PCR technique, so as to construct a triple mutant L202P/L218D/Y323G. Sequencing is performed to identify whether the encoding genes of MTHase triple mutant are correct, and the plasmid with correct sequencing result is introduced into Escherichia coli for expression, so as to obtain triple mutant MTHase.

[0056] OD.sub.600nm of shake flask culture for 24 h and enzyme activity of the wild type MTHase (WT) and the triple mutant are listed in Table 3. The results show that the mutant has higher enzyme activity than the wild type.

TABLE-US-00009 TABLE 3 OD.sub.600 nm of shake flask and enzyme activity of wild type MTHase and triple mutant enzyme Enzyme activity Enzyme OD.sub.600 nm (U/mL) WT 12.9 31.2 L202P/L218D/Y323G 12.9 59.4

Example 6: Preparation, Expression and Enzyme Activity Analysis of MTHase Quadruple Mutant

[0057] By using the plasmid of the triple mutant L202P/L218D/Y323G constructed in Example 5 as a template for quadruple mutant and according to site-directed mutagenic primers designed in Example 2, site-directed mutagenesis is performed on the plasmid carrying the genes encoding the mutant L202P/L218D/Y323G through a rapid PCR technique, so as to construct a quadruple mutant M140L/L202P/L218D/Y323G. Sequencing is performed to identify whether the encoding genes of MTHase quadruple mutant are correct, and the plasmid with correct sequencing result is introduced into Escherichia coli for expression, so as to obtain quadruple mutant MTHase.

[0058] OD.sub.600nm of shake flask culture for 24 h and enzyme activity of the wild type MTHase (WT) and the quadruple mutant are listed in Table 4. The results show that the mutant has higher enzyme activity than the wild type.

TABLE-US-00010 TABLE 4 OD.sub.600 nm of shake flask and enzyme activity of wild type MTHase and quadruple mutant enzyme Enzyme Enzyme OD.sub.600 nm activity (U/mL) WT 12.9 31.2 M140L/L202P/L218D/Y323G 12.9 64.3

Example 7: Preparation, Expression and Enzyme Activity Analysis of MTHase Quintuple Mutant

[0059] By using the plasmid of the mutant M140L/L202P/L218D/Y323G constructed in Example 2 as a template for quintuple mutant and according to site-directed mutagenic primers designed in Example 2, site-directed mutagenesis is performed on the plasmid carrying the genes encoding the mutant M140L/L202P/L218D/Y323G through a rapid PCR technique, so as to construct a quintuple mutant M140L/L202P/L218D/Y323G/F338S. Sequencing is performed to identify whether the encoding genes of MTHase quintuple mutant are correct, and the plasmid with correct sequencing result is introduced into Escherichia coli for expression, so as to obtain quintuple mutant MTHase.

[0060] OD.sub.600nm of shake flask culture for 24 h and enzyme activity of the wild type MTHase (WT) and the quintuple mutant are listed in Table 5. The results show that the mutant has higher enzyme activity than the wild type.

TABLE-US-00011 TABLE 5 OD.sub.600 nm of shake flask and enzyme activity of wild type MTHase and quintuple mutant enzyme Enzyme activity Enzyme OD.sub.600 nm (U/mL) WT 12.9 31.2 M140L/L202P/L218D/Y323G/F338S 11.6 60.4

Example 8: Preparation, Expression and Enzyme Activity Analysis of MTHase Sextuple Mutant

[0061] By using the plasmid of the mutant M140L/L202P/L218D/Y323G/F338S constructed in Example 2 as a template for sextuple mutant and according to site-directed mutagenic primers designed in Example 2, site-directed mutagenesis is performed on the plasmid carrying the genes encoding the mutant M140L/L202P/L218D/Y323G/F338S through a rapid PCR technique, so as to construct a sextuple mutant M140L/L202P/L218D/Y323G/F338S/I404T. Sequencing is performed to identify whether the encoding genes of MTHase sextuple mutant are correct, and the plasmid with correct sequencing result is introduced into Escherichia coli for expression, so as to obtain sextuple mutant MTHase.

[0062] OD.sub.600nm of shake flask culture for 24 h and enzyme activity of the wild type MTHase (WT) and the sextuple mutant are listed in Table 6. The results show that the enzyme activity of the sextuple mutant is 2.4 times that of the wild type enzyme.

TABLE-US-00012 TABLE 6 OD.sub.600 nm of shake flask and enzyme activity of wild type MTHase and sextuple mutant enzyme Enzyme activity Enzyme OD.sub.600 nm (U/mL) WT 12.9 31.2 M140L/L202P/L218D/Y323G/F338S/ 12.6 74.9 I404T

Example 9: Affinity to Substrate and Trehalose Conversion Rate Analysis of MTHase Mutant M140L/L202P/L218D/Y323G/F338S/I404T

[0063] The affinity of the wild type enzyme and the mutant M140L/L202P/L218D/Y323G/F338S/I404T to a maltohexose substrate at 60 C. is determined. Results are shown in Table 7.

[0064] The results show that, compared with the wild type enzyme (WT), the K.sub.m value of the mutant M140L/L202P/L218D/Y323G/F338S/I404T decreases to some extent, and the decrease of the K.sub.m value indicates that the affinity of the mutant to the substrate is enhanced.

TABLE-US-00013 TABLE 7 K.sub.m values of MTHase mutant Enzyme K.sub.m (mM) WT 3.89 0.34 M140L/L202P/L218D/Y323G/F338S/I404T 3.4 0.94

[0065] In the trehalose synthesis process, 150 g/L maltodextrin (DE value of 5-7) is used as a substrate, the initial pH is adjusted to 5.5, then the pullulanase (5 U/g starch) is added, and MTHase (20 U/g starch) and MTSase (80 U/g starch) at a ratio of 4 to 1 are added separately. The reaction conditions are 60 C. and 150 r/min. After reaction for 30 to 40 h, samples are boiled to terminate the reaction and undergo treatment. After the product is diluted and precipitated, the content of trehalose is determined by high performance liquid chromatography (HPLC) and the yield is calculated. HPLC detection conditions are as follows: mobile phase (acetonitrile:water=80:20), flow rate: 0.8 mL min.sup.1; column temperature: 40 C.; NH.sub.2 column (APS-2 HYPERSIL, Thermo Scientific), and refractive index detector (RID). After calculation and analysis, it is shown that the conversion rate of wild type MTHase has reached 76.1%, while the conversion rate of mutant MTHase has reached 73.8%, which can be considered as no significant effect on the conversion rate of trehalose.

Example 10: Fermentation Enzyme Production Experiment of Mutant M140L/L202P/L218D/Y323G/F338S/I404T in 3 L Fermenters

[0066] 1) Seed activation: The preserved glycerol tubes is taken out from a 80 C. freezer, and an LB solid medium is inoculated with a small amount of bacteria solution by streak inoculation with inoculation needles, then incubation is performed in a 37 C. thermostatic incubator for 10-12 h. A single colony is selected, an LB liquid medium is inoculated with the single colony, and culture is performed at 37 C., and 200 r.Math.min.sup.1 for 8-10 h.

[0067] 2) Seed culture: 100 L of the above activated bacterial solution is drawn, a 50 mL seed fermentation medium is inoculated with the bacterial solution, and culture is performed at 37 C., and 200 r.Math.min.sup.1 for 8 h.

[0068] 3) Culture in 3 L fermenter: NBS 3 L full-automatic fermenters are used, with initial liquid volume of 1.2 L. The seed liquid obtained in the previous step is fed into a fermenter with an inoculation amount of 8%, and antibiotics are added at the same time. The initial dissolved oxygen in the fermenter is set to 100%, the initial rotation speed is set to 300 r min.sup.1, and pH is set to 7.0. After seeding, the dissolved oxygen in the fermenter is coupled with the rotation speed to maintain the dissolved oxygen in the fermenter at about 30%. After inoculation for 7 to 8 hours, the carbon source in the fermenter is used up and the dissolved oxygen rebounds, at this time, feed solution is added for feeding at a specific growth rate p=0.2 h.sup.1. Samples are taken every 3 hours. The pH is adjusted by adding 25% aqueous ammonia to maintain the pH in the fermenter at 6.8 to 7.2 during the whole fermentation process. When the OD.sub.600 reaches 50, the induction temperature is controlled at 30 C. and lactose is added for induction at a flow rate of 0.1 g.Math.L.sup.1.Math.h.sup.1.

Example 11: Fermentation Growth and Enzyme Production Effect of Mutant M140L/L202P/L218D/Y323G/F338S/I404T

[0069] The bacteria are induced to produce target protein in a 3 L fermenter under the conditions of induction temperature of 30 C., cell concentration OD.sub.600 of 50, and inducer concentration (flow rate of lactose of 0.1 g.Math.L.sup.1.Math.h.sup.1). Results are shown in FIG. 1. The growth rate of bacteria is good at OD.sub.600 of 50, and the flow rate of lactose has no significant effect on the bacteria growth. The maximum value of bacteria OD.sub.600 is 181.5. The corresponding enzyme activity assay results are shown in FIG. 2. The maximum enzyme activity is 444.8 U.Math.mL.sup.1. The maximum enzyme activity of wild type MTHase is 82 U.Math.mL.sup.1 under the above condition, and the modification effect of the mutant MTHase is significant, and is increased by about 5 times.