OLIGOSACCHARIDE DEBRANCHING ENZYME MUTANT AND USE THEREOF IN GLUCOSE MOTHER LIQUOR

Abstract

The invention provides an oligosaccharide debranching enzyme mutant and use thereof in a glucose mother liquor. The mutant is obtained by mutating valine at position 219 in SEQ ID NO: 1 into alanine. According to oligosaccharide debranching enzyme mutant V219A, a primary mother liquor, a secondary mother liquor, or a tail liquid after chromatographic separation is used as a substrate, the percentage contents of glucose in the products are 99.21% (primary mother liquor), 98.89% (secondary mother liquor) and 97.97% (tail liquid after chromatographic separation) respectively, which are 2.86%, 8.64%, and 28.67% higher than that of glucose obtained with the wild-type oligosaccharide debranching enzyme. Therefore, the mutant V219A obviously improves the percentage content of glucose in the glucose mother liquor, and the scope of application of the mother liquor can be expanded by the high product purity and substrate conversion rate, so the mutant V219A has higher industrial application value.

Claims

1. An oligosaccharide debranching enzyme mutant, obtained by mutating valine at position 219 in an amino acid sequence as shown in SEQ ID NO: 1 into alanine.

2. A gene encoding the oligosaccharide debranching enzyme mutant according to claim 1.

3. The gene according to claim 2, having a nucleotide sequence as shown in SEQ ID NO: 2.

4. A recombinant plasmid carrying the gene according to claim 2.

5. A host cell expressing the oligosaccharide debranching enzyme mutant according to claim 1.

6. The cell according to claim 5, wherein the host cell is a bacterial, fungal, plant or animal cell.

7. Use of the oligosaccharide debranching enzyme mutant according to claim 1 in the hydrolysis of an oligosaccharide or the production of glucose.

8. The use according to claim 7, wherein glucose is produced with the oligosaccharide as a substrate, and the oligosaccharide debranching enzyme mutant, or a whole cell or a preparation comprising the mutant as a catalyst.

9. The use according to claim 7, wherein the oligosaccharide comprises linear maltooligosaccharide or isomaltooligosaccharide.

10. A method for regenerating glucose with a glucose mother liquor, comprising: treating the glucose mother liquor with the oligosaccharide debranching enzyme mutant according to claim 1, or a whole cell or a preparation comprising the mutant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the SDS-PAGE analysis of the oligosaccharide debranching enzyme.

[0024] FIG. 2 shows a HPAEC-PAD curve obtained when the oligosaccharide debranching enzyme acts on 1 mg/mL panose.

[0025] FIG. 3 is a HPLC curve obtained when the oligosaccharide debranching enzyme acts on 5% (w/w) primary mother liquor.

[0026] FIG. 4 is a HPLC curve obtained when the oligosaccharide debranching enzyme acts on 5% (w/w) secondary mother liquor.

[0027] FIG. 5 is a HPLC curve obtained when the oligosaccharide debranching enzyme acts on 5% (w/w) tail liquid after chromatographic separation.

[0028] FIG. 6 shows the enzyme activities of various oligosaccharide debranching enzyme mutants.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention will be further described below with reference to the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention; however, the present invention is not limited thereto.

[0030] Detection methods involved:

[0031] p-nitrobenzene--d-glucopyranoside (pNPG) is used as the substrate. The reaction mixture includes 150 L of a 500 mM phosphate buffer (pH 6.0), 800 L of 10 mM pNPG and 50 L of enzyme. After incubation at 50 C. for 5 min, 1 mL of 1 M sodium carbonate is added, to stop the reaction. The standard curve is measured and calibrated by measuring the increase of absorbance at 410 nm of the released p-nitrobenzene (pNP). One unit (U) of hydrolysis activity is defined as the amount of enzyme required to produce 1 mol pNP per minute under the experimental conditions.

[0032] The contents of various components in the product from enzymatic catalysis of panose are analyzed by high performance anion exchange chromatography (HPAEC-PAD). Analytical conditions: chromatographic column CarboPac PA 200, mobile phase: 0.25 M NaOH, 1 M NaAc and ultrapure water, flow rate: 0.5 mL/min, column temperature: 35 C., and injection volume: 10 L.

[0033] The contents of various components in the product from enzymatic catalysis of the glucose mother liquor are analyzed by high performance anion exchange chromatography (HPLC). Analytical conditions: chromatographic column: Hypersil APS2, mobile phase: 70% (V/V) acetonitrile and ultrapure water, flow rate: 1.0 mL/min, column temperature: 30 C., and injection volume: 10 L.

Example 1. Production of Gene Sequence Encoding Oligosaccharide Debranching Enzyme Mutant

[0034] The expression vector oga/pET-28a was used as a template, the complementary primer chains needed for the experiment were designed (see Table 1), and the primers were synthesized by Genewiz Biotech Corp. Site-directed mutation was carried out according to the method in the instructions for use of STAR Primer GXL kit of TaKaRa company. The PCR reaction system were based on the conditions set in the instructions for use of STAR Primer kit: 5PrimeSTAR Buffer (Mg.sup.2+ Plus) 10 L, template DNA 1 L, forward and reverse primers (10 M) each 1 L, PrimeSTAR HS DNA Polymerase (2.5 U/L) 0.5 L, dNTPs (2.5 mM) 4 L, and ultrapure water 32.5 L. PCR amplification conditions: predenaturation at 98 C. for 3 min, 35 cycles of denaturation at 98 C. for 30 s, annealing at 60 C. for 30 s, and extension at 68 C. for 3 min, and incubation at 68 C. for 5 min.

TABLE-US-00003 TABLE1 Introductionofmutationsitesto oligosaccharidedebranchingenzyme Primers: Primersequence(5-3).sup.1 V219A- GACGGCTTCCGTATG For GACGCCATCAACCT V219A- CTTGGAGATCAGGTT Rev GATGGCGTCCATAC Note: .sup.1The underlined bases correspond to the correspondingly mutated amino acids.

Example 2. Construction of Genetically Engineered Strain

[0035] (1) At 37 C., the PCR product obtained in Example 1 was treated with Dpn I for 2 h, and then the treated PCR product was transformed into E. coli JM109. The transformed E. coli JM109 was coated onto LB agar medium containing 100 g/mL kanamycin, and cultured overnight for 12 h in an incubator at 37 C. Single colonies were picked up and inoculated into LB liquid medium containing 100 g/mL kanamycin, and cultured overnight at 37 C. and 200 r/min. The plasmids were extracted, identified and sequenced according to the instruction for use of the plasmid extraction kit.

[0036] (2) The gene encoding the oligosaccharide debranching enzyme mutant obtained in Example 1 was ligated to the pET-28a plasmid vector by homologous recombination. Ligation system: purified PCR fragments of gene encoding oligosaccharide debranching enzyme mutant (50 ng/L) 1 L, PCR fragments of purified pET-28a plasmid vector (50 ng/L) 2 L, 5CE II Buffer 4 L, Exnase II 2 L, ddH.sub.2O 11 L. The conditions for homologous recombination include incubation at 37 C. for 30 min. Then, the recombination product was transformed into E. coli JM109, and the cells were coated onto LB agar medium containing 100 g/mL kanamycin, and cultured overnight for 12 h in an incubator at 37 C. Single colonies were picked up and inoculated into LB liquid medium containing 100 g/mL kanamycin, and cultured overnight at 37 C. and 200 r/min. The plasmids were extracted, identified and sequenced according to the instruction for use of the plasmid extraction kit. The plasmid sequenced to be correct and containing the gene encoding the oligosaccharide debranching enzyme mutant was transformed into the expression host competent E. coli BL21 (DE 3) cells. Finally, the genetically engineered strain E. coli BL21 (DE 3) (pET-28a/oga) was obtained.

Example 3. Expression of Oligosaccharide Debranching Enzyme Mutant

[0037] (1) Activation and culture of host strain: The genetically engineered strain E. coli BL21 containing the expression plasmid pET-28a/oga obtained in Example 2 was inoculated onto LB solid medium by streak plate method, and then cultured overnight in a constant-temperature incubator at 37 C. The positive single colony was picked up and inoculated in a 250 ml conical flask containing 50 mL LB liquid culture medium. Kanamycin with a final concentration of 100 g/mL was added before inoculation. The conical flask was placed in a rotary shaker at 200 r/min and cultured at 37 C. for 12 h.

[0038] (2) Fermentation culture: The activated seed culture was inoculated into a 250 mL conical flask with 50 ml fermentation medium in an inoculation amount of 4% (v/v), and cultured in a rotary shaker for 48 h (at a rotation speed of 200 r/min). Kanamycin with a final concentration of 100 g/mL L was added before inoculation. After fermentation, the fermentation broth was centrifuged at 4 C. and 10,000g for 15 min, and the cells were collected. The cells were suspended in 50 mM sodium acetate buffer, ultrasonically homogenized for 30 min, and then centrifuged at 10,000g for 15 min to obtain a supernatant that is the crude enzyme solution.

Example 4. Analysis of Products Obtained with Oligosaccharide Debranching Enzyme Mutant

[0039] 1 mg/mL panose (pH 6.0) was prepared, the wide type (having an amino acid sequence as shown in SEQ ID NO: 1) or the oligosaccharide debranching enzyme mutant V219A was added in an amount of 5 U/g, and the reaction was continued for 24 hrs at 50 C. After the reaction, the enzyme was inactivated by standing in a boiling water bath. The reaction solution was centrifuged at 10,000g for 5 min, diluted by a certain factor and filtered through a 0.22 m needle filter. Qualitative and quantitative analysis were carried out with G1-G7 over a certain gradient of concentrations mixed with the standard as a control.

[0040] The results of product analysis are shown in FIG. 2. The wild type catalyzes the hydrolysis of panose to glucose and maltose, and the oligosaccharide debranching enzyme mutant catalyzes the hydrolysis of panose to glucose only. The rate of conversion of panose to glucose by the oligosaccharide mutant is 100%. Compared with the wild type, the conversion ability of the oligosaccharide mutant is significantly improved.

TABLE-US-00004 TABLE 2 Analysis of products obtained with with oligosaccharide debranching enzyme Components in the product (%) Wild type V219A G1 60 100 G2 33 0 Note: G1 and G2 represent glucose and maltose respectively.

Example 5. Production of Glucose Through Hydrolysis of Primary Mother Liquor by Oligosaccharide Debranching Enzyme

[0041] 5% (w/w) primary mother liquor was prepared, adjusted to pH 6.0, and allowed to stand in a water bath at 50 C. for 15 min. The stirring speed was 300 r/min. The oligosaccharide debranching enzyme or the mutant V219A was added at a dosage of 10 U/g dry starch, and the reaction was timed. 24 hrs after reaction, the product was determined by HPLC after centrifugation, membrane filtration and dilution. The percentage contents of various sugar components are shown in Table 3. The percentage content of glucose in the hydrolysate of wild-type oligosaccharide debranching enzyme is 96.35%. The percentage content of glucose in the hydrolysate of the variant V219A is 99.21%, and increased by 2.86%.

TABLE-US-00005 TABLE 3 Analysis of products obtained by treatment of primary mother liquor with oligosaccharide debranching enzyme in Primary Components mother liquor Wide type V219A product (%) (%) (%) G1 93.97 96.35 99.21 G2 3.08 3.26 0.28 IG2 2.50 0.39 0.51 G3 P 0.45 IG3 Note: G1 to G4 represents glucose, maltose, linear maltotriose, and linear maltotetraose respectively; IG2 to IG3 represents isomaltose, and isomaltotriose respectively; and P represents panose.

Example 6. Production of Glucose Through Hydrolysis of Secondary Mother Liquor by Oligosaccharide Debranching Enzyme

[0042] 20% (w/w) secondary mother liquor was prepared, adjusted to pH 6.0, and allowed to stand in a water bath at 50 C. for 15 min. The stirring speed was 300 r/min. The oligosaccharide debranching enzyme or the mutant V219A was added at a dosage of 10 U/g dry starch, and the reaction was timed. 24 hrs after reaction, the product was determined by HPLC after centrifugation, membrane filtration and dilution. The percentage contents of various sugar components are shown in Table 4. The percentage content of glucose in the hydrolysate of wild-type oligosaccharide debranching enzyme is 90.25%. The percentage content of glucose in the hydrolysate of the variant V219A is 98.89%, and increased by 8.64%.

TABLE-US-00006 TABLE 4 Analysis of products obtained by treatment of secondary mother liquor with oligosaccharide debranching enzyme Components in Secondary Wide type V219A product mother liquor (%) (%) (%) G1 82.72 90.25 98.89 G2 7.99 8.83 0.36 IG2 7.20 0.92 0.75 G3 P 1.84 IG3 0.25 Note: G1 to G3 represents glucose, maltose, and linear maltotriose respectively; IG2 to IG3 represents isomaltose, and isomaltotriose respectively; and P represents panose.

Example 7. Production of Glucose Through Hydrolysis of Tertiary Mother Liquor by Oligosaccharide Debranching Enzyme

[0043] 20% (w/w) tail liquid after chromatographic separation was prepared, adjusted to pH 6.0, and allowed to stand in a water bath at 50 C. for 15 min. The stirring speed was 300 r/min. The oligosaccharide debranching enzyme or the mutant V219A was added at a dosage of 10 U/g dry starch, and the reaction was timed. 24 hrs after reaction, the product was determined by HPLC after centrifugation, membrane filtration and dilution. The percentage contents of various sugar components are shown in Table 5. The percentage content of glucose in the hydrolysate of wild-type oligosaccharide debranching enzyme is 69.30%. The percentage content of glucose in the hydrolysate of the variant V219A is 97.97%, and increased by 28.67%.

TABLE-US-00007 TABLE 5 Analysis of products obtained by treatment of tertiary mother liquor with oligosaccharide debranching enzyme Components Tertiary mother liquor Wide type V219A in product (%) (%) (%) G1 52.07 69.30 97.97 G2 23.94 29.62 1.35 IG2 13.35 0.48 G3 0.24 0.73 0.21 P 8.87 0.36 IG3 0.25 G4 and higher 1.28 Note: G1 to G4 represents glucose, maltose, linear maltotriose, and linear maltotetraose respectively; IG2 to IG3 represents isomaltose, and isomaltotriose respectively; and P represents panose.

Comparative Example. Analysis of Products Obtained with Oligosaccharide Debranching Enzyme Mutant

[0044] 1 mg/mL panose (pH 6.0) was prepared, the wide type (having an amino acid sequence as shown in SEQ ID NO: 1) or the oligosaccharide debranching enzyme mutant V219A, V219D, V219L, V219T, or V219M was added in an amount of 5 U/g, and the reaction was continued for 24 hrs at 50 C. After the reaction, the enzyme was inactivated by standing in a boiling water bath. The reaction solution was centrifuged at 10,000g for 5 min, diluted by a certain factor and filtered through a 0.22 m needle filter. Qualitative and quantitative analysis were carried out with G1-G7 over a certain gradient of concentrations mixed with the standard as a control.

[0045] The results of product analysis are shown in FIG. 2. The oligosaccharide debranching enzyme mutant V219A catalyzes the hydrolysis of panose to glucose only. The rate of conversion of panose to glucose by the oligosaccharide mutant is 100%. The wild type and other variants catalyze the hydrolysis of panose to glucose and maltose. These suggest that the wide type and V219D, V219L, V219T and V219M mutant cannot hydrolyze the -1,4-glycosidic bonds of panose, and they can't hydrolyze maltooligosaccharide.

[0046] The enzyme activities of oligosaccharide debranching enzyme mutants V219A, V219D, V219L, V219T, and V219M are shown in FIG. 6.

[0047] Apparently, the above-described embodiments are merely examples provided for clarity of description, and are not intended to limit the implementations of the present invention. Other variations or changes can be made by those skilled in the art based on the above description. The embodiments are not exhaustive herein. Obvious variations or changes derived therefrom also fall within the protection scope of the present invention.