Pullulanase mutant

Abstract

The disclosure herein relates to a method for reducing the inhibitory effect of a cyclodextrin on a pullulanase and belongs to the technical field of gene engineering, enzyme engineering or food science. The method of the disclosure prepares a pullulanase mutant by reasonably mutating the key amino acid of the pullulanase interactive with the cyclodextrin to reduce the inhibitory effect of the cyclodextrin on the pullulanase, thereby improving the hydrolysis activity of the pullulanase. The disclosure finds the interactive sites of the pullulanase and the cyclodextrin based on analysis of crystal structures of enzymes and inhibitors and sequence comparison of enzymes from different sources, and utilizes site-directed mutation to obtain the pullulanase mutant having reduced sensibility to the cyclodextrin, thereby improving the utilization ratio of the starch raw material and the yield of the cyclodextrin.

Claims

1. A pullulanase mutant, wherein the pullulanase mutant comprises a mutation of a phenylalanine (F) at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1), wherein FNDXXRDXXXGXXF (SEQ ID NO: 1) is a part of a parent pullulanase amino acid sequence, wherein X is any naturally occurring amino acid; wherein an inhibitory effect of a cyclodextrin on the pullulanase mutant is significantly reduced relative to an inhibitory effect on the parent pullulanase, and wherein the cyclodextrin is one or more of α-CD, β-CD and γ-CD, with a reduction rate of 5.6 for the β-CD.

2. The pullulanase mutant of claim 1, wherein the parent pullulanase is obtained from a microorganism selected from the group consisting of: Bacillus subtilis, Bacillus vireti, Bacillus atrophaeus; Geobacillus stearothermophilus, Bacillus mojavensis, Thermotoga maritima, Brevibacterium halotolerans, Thermus sp. IM6501, Bacillus cereus FRI-35, Bacillus halotolerans, Alteromonas mediterranea, Klebsiella pneumonia, Thermotoga maritima, Escherichia coli, Enterobacter aerogenes, Bacillus teguilensis, Bacillus intestinalis, Bacillus licheniformis and Bacillus halotolerans.

3. The pullulanase mutant of claim 1, wherein the amino acid sequence of the pullulanase mutant comprises any one of the following: (1) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 476 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 17) obtained from Bacillus subtilis str. 168; (2) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 476 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 18) obtained from Bacillus vireti; (3) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 476 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 19) obtained from Bacillus atrophaeus; (4) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 481 relative to a parentamino acid sequence of a parent pullulanase (SEQ ID NO: 20) obtained from Geobacillus stearothermophilus; (5) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 476 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 21) obtained from Bacillus mojavensis; (6) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 601 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 22) obtained from Thermotoga maritima; (7) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 478 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 23) obtained from Brevibacterium halotolerans; (8) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 481 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 24) obtained from Thermus sp. IM6501; (9) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 613 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 25) obtained from Bacillus cereus FRI-35; and (10) a mutation of phenylalanine at the last position in FNDXXRDXXXGXXF (SEQ ID NO: 1) which is position 476 relative to a parent amino acid sequence of a parent pullulanase (SEQ ID NO: 26) obtained from Bacillus halotolerans.

4. The pullulanase mutant of claim 1, wherein the parent amino acid sequence is set forth in SEQ ID NO: 1.

5. The pullulanase mutant of claim 1, wherein the mutation of the phenylalanine comprises mutation from phenylalanine to any one of the following amino acids: glycine, alanine, leucine, isoleucine, valine, proline, methionine, serine, glutamine, threonine, cysteine, aspartic acid, asparagine, glutamic acid, lysine, arginine or histidine.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is a partial schematic diagram of Bacillus subtilis pullulanase and cyclodextrin crystal structure;

(2) FIG. 2 is a schematic diagram of a plasmid construction process;

(3) FIG. 3 is enzyme activity characterization of the inhibitory effect of a cyclodextrin on a wild type and a Bacillus subtilis pullulanase F476G mutant; and

(4) FIG. 4 is enzyme activity characterization of the inhibitory effect of the cyclodextrin on a wild type and a Bacillus subtilis pullulanase F476D mutant.

DETAILED DESCRIPTION

(5) 1. Nickel Affinity Chromatography Comprises the Following Steps:

(6) (1) balancing: balancing a nickel column with 20 mM Tris-HCl, 500 mM NaCl and a buffer solution of which the pH value is 7.5;

(7) (2) sample loading: loading a pretreated sample at a flow rate of 1 mL/min;

(8) (3) cleaning: cleaning impure protein with 20 mM Tris-HCl, 500 mM NaCl and the buffer solution of which the pH value is 7.5; and

(9) (4) elution: eluting target protein with 20 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole and the buffer solution of which the pH value is 7.5, enabling the detection wavelength to be 280 nm, and collecting eluent with pullulanase activity, thereby obtaining the purified enzyme of the mutant F476G.

(10) 2. Measurement of Enzyme Activity of the Pullulanase:

(11) A 3,5-dinitrosalicylic acid (DNS) method is adopted. The pullulanase catalyzes the hydrolysis of pullulan to produce reducing sugar under certain conditions, 3,5-dinitrosalicylic acid and the reducing sugar are reduced under thermal conditions to produce a brown-red amino complex, the color depth is directly proportional to the amount of the reducing sugar within a certain range, and the measurement can be performed at the wavelength of 540 nm so as to calculate the enzyme activity. Enzyme activity unit definition: the amount of the enzyme that catalyzes the production of the reducing sugar equivalent to 1 μmol of glucose reducing power per minute is defined as one activity unit.

(12) 3. Measurement of the Enzyme Activity Comprises the Following Steps:

(13) A. preheating: putting 1 mL of a 1% pullulan solution of which the pH value is 6.0 and 0.1 mL of a buffer solution of which the pH value is 6.0 in a centrifuge tube, and performing thermal insulation for 10 min on a water bath of 40° C.;

(14) B. reaction: adding 0.1 mL (10 U) of an enzyme solution, performing shaking and mixing uniformly, performing accurate timing for 30 min, adding 1.5 mL of DNS for stopping reaction, performing treatment in a boiling water bath for 10 min, and performing instant cooling; and

(15) C. measurement: measuring the light absorption value under the condition of 540 nm, and calculating the activity.

(16) Reduction of the Inhibitory Effect of Enzyme Activity Characterization:

(17) A. preheating: putting 1 mL of the 1% pullulan solution of which the pH value is 6.0 and 0.1 mL of 10 mM cyclodextrin in the centrifuge tube, and performing thermal insulation for 10 min on the water bath of 40° C.;

(18) B. reaction: adding 0.1 mL (10 U) of the enzyme solution, performing shaking and mixing uniformly, performing accurate timing for 30 min, adding 1.5 mL of DNS for stopping reaction, performing treatment in the boiling water bath for 10 min, and performing instant cooling; and

(19) C. measurement: measuring the light absorption value under the condition of 540 nm, and calculating the activity.

(20) The disclosure is specifically described below in conjunction with accompanying drawings and examples.

Example 1

(21) A process for reasonable mutation and mutation effect analysis of the key amino acid of the pullulanase comprises the following steps:

(22) 1. Finding of the Key Amino Acid Related to the Inhibitory Effect:

(23) The crystal structure of the pullulanase derived from Bacillus subtilis is analyzed, and the key amino acid F476 related to the inhibitory effect is found, wherein the amino acid has an inclusion relationship with the cyclodextrin structurally; and the gene sequences of the pullulanase from different sources are compared to find that the key amino acid is highly conserved in the pullulanase sequence.

(24) FIG. 1 is a partial schematic diagram of the Bacillus subtilis pullulanase and cyclodextrin compound crystal structure. Seen from the figure, a similar inclusion effect exists between the phenylalanine at the position 476 in the pullulanase and the cavity of the cyclodextrin, and the inclusion effect is based on the hydrophobic interaction between the benzene ring of the phenylalanine and the hydrophobic cavity of the cyclodextrin.

(25) 2. Acquisition of a Parent Amino Acid Sequence/Nucleotide Sequence

(26) (1) The pullulanase (PDB:2E8Z) derived from Bacillus subtilis str. 168 in a crystal structure in which the cyclodextrin interacts with the pullulanase is taken as a template, wherein the accession number of the pullulanase on NCBI is CAB14971.2 (SEQ ID NO:2); and a pullulanase gene sequence AmyX is synthesized by adopting a chemical total synthesis method. The plasmids used for constructing an Escherichia coli expression vector are pET20b (+) with a T7 promoter.

(27) (2) NcoI and BamI double-enzyme digestion are respectively performed on the pET20b(+) plasmids and the plasmids containing the AmyX gene, the enzyme digestion product is recovered by tapping, then connection is performed with T4 ligase, the connecting product is transformed to E. coli DH5α competent cells, culture is performed for 8-12 h at 37° C., transformants are picked into a liquid LB culture medium containing 100 μg/mL ampicillin for shaking culture, the plasmids are extracted, and enzyme digestion verification is performed to obtain an AmyX/Pet20b(+) expression vector.

(28) (3) The plasmids AmyX/Pet20b(+) are transformed into E. coli BL21(DE3) host bacteria, an LB panel containing ampicillin (100 mg/mL) is coated with the plasmids, and culture is performed for 8 h at 37° C. to obtain AmyX/Pet20b(+)/BL21(DE3); and a single colony is picked into the liquid LB culture medium, overnight culture is performed at 37° C., and a strain is performed with glycerol.

(29) FIG. 2 is a schematic diagram of the plasmid construction process. pET-20b(+) plasmids are selected, BamHI and NcoI enzyme digestion sites are selected, and the pullulanase is connected to the plasmids to construct the AmyX/pET-20b(+) expression vector.

(30) 3. Construction, Expression and Purification of the Pullulanase Mutant

(31) (1) A mutated primer is designed, and the phenylalanine (F) at the position 476 is mutated into glycine (G), wherein mutated primers are as shown in table 1 below:

(32) TABLE-US-00001 TABLE 1 F476G-For 5′-cgctgtaaaagggaacaccggtcaccttaaggcaacaggg-3′ Shown in SEQ ID NO: 3 F476G-Rev 5′-ccctgttgccttaaggtgaccggtgttcccttttacagcg-3′ Shown in SEQ ID NO: 4 Note: Underlines represent mutated basic groups.

(33) Site-directed mutation is performed by utilizing PCR, wherein a PCR reaction system comprises 10 μL of 5×Primer STAR GXL Buffer, 4 μL of dNTPMixture (2.5 mM), 1.5 μL of a forward primer F476G-For (10 μM), 1.5 μL of a reverse primer F476G-Rev (10 μM), 1 μL of template DNA (10 ng/μL) and 1 μL of Prime STAR GXL DNA Polymerase (1.25μ/μL); double distilled water is added to 50 μL; and PCR amplification conditions are as follows: pre-degeneration is performed for 3 min at 98° C., then 30 cycles (98° C. 20 s, 60° C. 30 s, 68° C. 6 min) are performed, and extension is continued for 10 min at 68° C.

(34) The PCR product is digested through Dpn I (Thermo Fisher), the digestion product is transformed into Escherichia coli DH5α competent cells, overnight culture is performed on the competent cells in an LB solid culture medium (containing 100 μg/mL ampicillin), a single colony is picked into an LB liquid culture medium (containing 100 μg/mL ampicillin) to be cultured, plasmids are extracted, and correctly sequenced plasmids are transformed into expression host Escherichia coli BL21(DE3).

(35) (2) A positive single colony transformed into expression host Escherichia coli BL21 (DE3) is picked into the LB liquid culture medium (containing 100 μg/mL) to be cultured for 8-12 h at 37° C. and 200 rpm, and inoculation is performed to a TB culture medium (containing 100 μg/mL ampicillin) under the inoculation volume of 5%; and culture is performed at 37° C. and 200 rpm until the OD is equal to 0.6, IPTG of which the final concentration is 0.2 mM is added, induced expression is performed for 96 h at 25° C. and 160 rpm, fermentation liquid is centrifuged at 10000 g for 10 min at 4° C., and the supernatant liquid is collected.

(36) (3) Fermented supernatant liquid is centrifuged at 4000 g with a 10 kDa ultrafiltration centrifuge tube for 10 min, and preliminary concentration and separation are performed; and the concentrated fermentation liquid passes through a 0.22 μm filter membrane, and purification is performed by nickel affinity chromatography, wherein the whole purification process is performed under low temperature conditions.

(37) 4. Performance Analysis of the Pullulanase Mutant

(38) Inhibitory effects are compared, and experimental results are shown in FIG. 3. Comparison of a mutant F476G with a wild type pure enzyme finds that the inhibitory effects of three cyclodextrins on the pullulanase mutant are significantly lower than that of the wild type pullulanase, thereby achieving the purpose of reducing the inhibitory effect of the cyclodextrin on the pullulanase. When an inhibitor α-CD exists, the reduction rate of the specific enzyme activity of the wild type pure enzyme is 14.3%, and the reduction rate of the specific enzyme activity of the mutant F476G is 10.6%; when an inhibitor β-CD exists, the reduction rate of the specific enzyme activity of the wild type pure enzyme is 42.8%, and the reduction rate of the specific enzyme activity of the mutant F476G is 17.1%; and when an inhibitor γ-CD exists, the reduction rate of the specific enzyme activity of the wild type pure enzyme is 7.1%, and the reduction rate of the specific enzyme activity of the mutant F476G is 17.6%.

Example 2

(39) The mutated primer is redesigned by adopting a method similar to example 1, the amino acid (F) at the position 476 is mutated into other amino acids based on the parent of the pullulanase derived from Bacillus subtilis str. 168, and expression and purification are performed.

(40) The mutated primer is designed, the phenylalanine (F) at the position 476 is mutated into aspartic acid (D), and a new mutant F476D is constructed, wherein mutated primers are shown in table 2 below:

(41) TABLE-US-00002 TABLE 2 F476D-For 5′-ccctgttgccttaaggtgatcggtgttcccttttacagcg-3′ Shown in SEQ ID NO: 5 F476D-Rev 5′-cgctgtaaaagggaacaccgatcaccttaaggcaacaggg-3′ Shown in SEQ ID NO: 6 Note: Underlines represent mutated basic groups.

(42) Inhibitory effects of three cyclodextrins on the wild enzyme and the mutant F476D are compared and analyzed, and experimental results are shown in FIG. 4. Comparison of the mutant with the wild type pure enzyme finds that the inhibitory effects of three cyclodextrins on the pullulanase mutant are significantly lower than that of the wild type pullulanase, thereby achieving the purpose of reducing the inhibitory effect of the cyclodextrin on the pullulanase. When the inhibitor α-CD exists, the reduction rate of the specific enzyme activity of the wild type pure enzyme is 14.3%, and the reduction rate of the specific enzyme activity of the mutant F476D is 5.6%; when the inhibitor β-CD exists, the reduction rate of the specific enzyme activity of the wild type pure enzyme is 42.8%, and the reduction rate of the specific enzyme activity of the mutant F476D is 11.1%; and when the inhibitor γ-CD exists, the reduction rate of the specific enzyme activity of the wild type pure enzyme is 17.1%, and the reduction rate of the specific enzyme activity of the mutant F476D is 11.0%.

(43) Based on the method similar to example 1, the inventors select the pullulanase (PDB:2E8Z) derived from Bacillus subtilis str. 168 as a template to mutate the amino acid at the position 476 and also construct mutants F4761, F476M, F476V, F476C and F476A, and the results are shown in table 3.

(44) TABLE-US-00003 TABLE 3 Inhibitory conditions of different mutants by cyclodextrin Reduction rate of specific Reduction rate of Reduction rate of enzyme specific enzyme specific enzyme activity in activity in existence activity in existence of existence of α-CD β-CD of γ-CD Wild enzyme 14.3% 42.8% 17.1% Mutant F476I 8.2% 21.7% 15.2% Mutant 6.5% 20.6% 16.1% F476M Mutant F476V 7.2% 16.7% 12.3% Mutant F476C 4.3% 17.2% 5.6% Mutant F476A 3.5% 5.6% 5.8%

Example 3

(45) The inventors compare the sequences of the pullulanase from 40 different sources and find that non-X amino acids (thickened amino acids) in FNDXXRDXXXGXXF in the sequence (shown in SEQ ID NO:1, wherein X can be any naturally occurring amino acid) are highly conserved, wherein the last position F (phenylalanine) in FNDXXRDXXXGXXF corresponds to the phenylalanine at the position 476 of the amino acid sequence of the pullulanase derived from Bacillus subtilis str. 168.

(46) The accession numbers of the sequences of the pullulanase from 40 different sources on NCBI are respectively: WP_003229246.1 (Bacillus subtilis)(as shown in SEQ ID NO: 17), WP_024026701.1 (Bacillus vireti)(as shown in SEQ ID NO: 18), WP_010789532.1 (Bacillus atrophaeus)(as shown in SEQ ID NO: 19), KZE96788.1 (Geobacillus stearothermophilus) as shown in SEQ ID NO: 20), WP_032731297.1 (Bacillus mojavensis)(as shown in SEQ ID NO: 21), CAA04522.1 (Thermotoga maritima)(as shown in SEQ ID NO: 22), OEC77647.1 ([Brevibacterium] halotolerans)(as shown in SEQ ID NO: 23), AAC15073.1 (Thermus sp. IM6501)(as shown in SEQ ID NO: 24), AFQ12130.1 (Bacillus cereus FRI-35)(as shown in SEQ ID NO: 25), WP_106020274.1 (Bacillus halotolerans)(as shown in SEQ ID NO: 26), AMJ80567.1 (Alteromonas mediterranea) as shown in SEQ ID NO: 27), AFU74026.1 (Klebsiella pneumoniae)(as shown in SEQ ID NO: 28), CAA04522.1 (Thermotoga maritima MSB8)(as shown in SEQ ID NO: 29), OWC08995.1 (Escherichia coli)(as shown in SEQ ID NO: 30), AAA25124.1 (Enterobacter aerogenes) as shown in SEQ ID NO: 31), WP_010789532.1 (Bacillus atrophaeus)(as shown in SEQ ID NO: 32), WP_032731297.1 (Bacillus mojavensis)(as shown in SEQ ID NO: 33), WP_095714534.1 (Bacillus sp. 7705b)(as shown in SEQ ID NO: 34), WP_024714639.1 (Bacillus tequilensis)(as shown in SEQ ID NO: 35), WP_079288107.1 (Bacillus intestinalis)(as shown in SEQ ID NO: 36), WP_014114809.1 (Bacillales)(as shown in SEQ ID NO: 37), KF103882.1 (Bacillus sp. BSC154) as shown in SEQ ID NO: 38), WP_075747083.1 (Bacillus licheniformis)(as shown in SEQ ID NO: 39), WP_069840139.1 (Bacillus sp. F56)(as shown in SEQ ID NO: 40), WP_014665024.1 (Bacillus sp. JS)(as shown in SEQ ID NO: 41), WP_071577010.1 (Bacillus sp. FMQ74)(as shown in SEQ ID NO: 41, WP_103749897.1 (Bacillus sp. MBGLi97)(as shown in SEQ ID NO: 43), WP_095432444.1 (Bacillus sp. X2 (2017))(as shown in SEQ ID NO: 44), OTQ82147.1 (Bacillus subtilis subsp. subtilis)(as shown in SEQ ID NO: 45), WP_046160744.1 (Bacillus sp. CMAA 1185)(as shown in SEQ ID NO: 46), WP_103803268.1 (Bacillus sp. Ru63)(as shown in SEQ ID NO: 47), WP_059291821.1 (Bacillus halotolerans)(as shown in SEQ ID NO: 48), WP_059335607.1 (Bacillus halotolerans)(as shown in SEQ ID NO: 49), WP_069150087.1 (Bacillus subtilis) as shown in SEQ ID NO: 50), KFF55698.1 (Bacillus subtilis)(as shown in SEQ ID NO: 51), PTU26911.1 (Bacillus subtilis)(as shown in SEQ ID NO: 52), WP_061670344.1 (Bacillus atrophaeus) as shown in SEQ ID NO: 53), WP_106293419.1 (Bacillus halotolerans)(as shown in SEQ ID NO: 54), WP_099043342.1 (Bacillus halotolerans) (as shown in SEQ ID NO: 55) and WP_103748328.1 (Bacillus subtilis)(as shown in SEQ ID NO: 56).

(47) Specific amino acid fragments (FNDXXRDXXXGXXF) corresponding to the sequences of the pullulanase from 10 different sources are listed below:

(48) TABLE-US-00004 TABLE 4 NCBI SEQ accession Sequence Specific amino acid ID Source strain number position fragment sequence NO. Bacillus subtilis  WP_003229246.1 463-476 FNDMFRDAVKGNTF  7 str. 168 Bacillus vireti WP_024026701.1 463-476 FNDKFRDTIKGSTF  8 Bacillus atrophaeus WP_010789532.1 463-476 FNDSFRDAVKGSTF  9 Geobacillus KZE96788.1 468-481 FNDRFRDAVKGSTF 10 stearothermophilus Bacillus mojavensis WP_032731297.1 463-476 FNDSFRDAVKGNTF 11 Thermotoga maritima CAA04522.1 588-601 FNDEFRDAIRGSVF 12 MSB8 [Brevibacterium] OEC77647.1 465-478 FNDSFRDAVKGNTF 13 halotolerans Thermus sp. IM6501 AAC15073.1 468-481 FNDRFRDAVKGSTF 14 Bacillus cereus FRI-35 AFQ12130.1 600-613 FNDNIRDGLKGSVF 15 Bacillus halotolerans WP_106020274.1 463-476 FNDSFRDAVKGNTF 16

(49) Comparison shows that the site corresponding to the phenylalanine at the position 476 of the pullulanase (PDB:2E8Z) of Bacillus subtilis str. 168 used in the disclosure is highly conserved in the pullulanase from different sources.

(50) On the basis, the phenylalanine at the position 481 of the Geobacillus stearothermophilus, the phenylalanine at the position 478 of the [Brevibacterium] halotolerans and the phenylalanine at the position 476 of the Bacillus halotolerans are mutated, obtained mutants and the mutation effects are shown in the table below. The key amino acid site is applicable to the pullulanase from different sources.

(51) TABLE-US-00005 TABLE 5 Reduction rate of Reduction rate of Reduction rate of specific enzyme specific enzyme specific enzyme activity in activity in activity in Source strain existence of α-CD existence of β-CD existence of γ-CD Geobacillus Wild 16.9% 51.2% 7.7% stearothermophilus enzyme F481A 7.1% 21.5% 7.1% F481G 5.2% 18.2% 6.9% [Brevibacterium] Wild 15.3% 47.2% 6.1% halotolerans enzyme F478M 10.1% 23.1% 5.5% F478G 6.2% 16.5% 4.7% Bacillus Wild 17.2% 35.9% 6.7% halotolerans enzyme F476A 15.2% 12.5% 5.5% F476S 10.7% 15.2% 6.1%