Patent Application
20190270976
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.
Pullulanase (EC 3.2.1.41) specifically hydrolyzes α-1,6 glycosidic bonds in pullulan, soluble starch, amylopectin and corresponding oligosaccharides to generate a short linear dextrin. Combination of the pullulanase and other amylases can greatly improve the utilization ratio of the starch raw materials, so that the pullulanase is widely applied to the starch industry.
In the starch sugar industry, the pullulanase has the function of hydrolyzing α-1,6 glycosidic bonds, so that the pullulanase has wide application prospects, can be used for preparing amylose, maltose and branched cyclodextrin, and can be combined with CGTase to produce the cyclodextrin. In the production process of the cyclodextrin, by taking production of α-CD as an example, because α-CGTase can not hydrolyze α-1,6 bonds in starch substrates, when α-CGTase is used alone to produce α-CD, the conversion rate is only 40%-60%. The addition of the pullulanase can significantly increase the conversion rate of the cyclodextrin, but the pullulanase is easily inhibited by the cyclodextrin, so that the debranching reaction and the cyclization reaction need to be performed separately, resulting in problems such as long production cycle and resource waste. In order to shorten the production cycle, increase the utilization ratio of the raw material starch and increase the yield of the cyclodextrin, a one-pot method of compounding the pullulanase and cyclodextrin glycosyltransferase is adopted to produce the cyclodextrin, and therefore, reduction of inhibition on the pullulanase by the cyclodextrin becomes an urgent problem to be solved.
Marshall J J, Iwamoto H, Iwamoto H et al. study the inhibitory effect of the cyclodextrin on the pullulanase in terms of enzyme activity, reaction kinetics and the like, and confirm that the cyclodextrin is a competitive inhibitor of the pullulanase. Yu Bo et al. further study the inhibitory effect of the cyclodextrin on the pullulanase, and study the influence of the cyclodextrin on the endogenous fluorescence and the secondary structure of the pullulanase on the basis of conventional characterization of enzymatic properties, thereby proving that the inclusion effect between a hydrophobic cavity of the cyclodextrin and an aromatic amino acid of the pullulanase is the intrinsic driving force of the cyclodextrin for inhibition of pullulanase activity. Related studies at home and abroad only focus on the interaction between the cyclodextrin and the pullulanase and do not propose specific and effective solutions.
The inventors preliminarily analyze and compare the sequences of the pullulanase from different sources, and the crystal structure (PDB:2E8Z) combining the pullulanase (NCBI accession number: AF008220.1) derived from Bacillus subtilis str. 168 and the cyclodextrin is already published, so that the pullulanase derived from Bacillus subtilis is selected as a template for experimental design. After characterization of the wild type pullulanase and the mutant, the disclosure finds that the mutant can significantly reduce the inhibitory effect of the product cyclodextrin. The reduction of the inhibitory effect is favorable for improving the utilization ratio of the starch raw material, increasing the yield of the cyclodextrin, and providing greater industrial application value for cyclodextrin production.
In view of the above problems in the prior art, the disclosure provides a method for reducing the inhibitory effect of the cyclodextrin on 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 a pullulanase mutant having reduced sensibility to the cyclodextrin, thereby improving the utilization ratio of the starch raw material and the yield of the cyclodextrin.
The disclosure is directed to a pullulanase which is reduced in inhibitory effect of the cyclodextrin. 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 key amino acid is phenylalanine.
The amino acid sequence of the pullulanase comprises an amino acid obtained by mutating the last position F (phenylalanine) in FNDXXRDXXXGXXF (as shown in SEQ ID NO: 1) contained in a parent amino acid sequence based on the parent amino acid sequence, wherein X can be any naturally occurring amino acid.
In one example, the parent amino acid sequence is an amino acid sequence of the pullulanase derived from any one 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 pneumoniae, Thermotoga maritima, Escherichia coli, Enterobacter aerogenes, Bacillus tequilensis, Bacillus intestinalis, Bacillales, Bacillus licheniformis and Bacillus halotolerans.
In one example, the parent amino acid sequence is any one of the following amino acid sequences with accession numbers on NCBI: WP_003229246.1 (Bacillus subtilis), WP_024026701.1 (Bacillus vireti), WP_010789532.1 (Bacillus atrophaeus), KZE96788.1 (Geobacillus stearothermophilus), WP_032731297.1 (Bacillus mojavensis), CAA04522.1 (Thermotoga maritima), OEC77647.1 ([Brevibacterium] halotolerans), AAC15073.1 (Thermus sp. IM6501), AFQ12130.1 (Bacillus cereus FRI-35) and WP_106020274.1 (Bacillus halotolerans).
In one example, the amino acid sequence of the pullulanase comprises any segment of the following amino acid sequences:
(1) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 476 of the amino acid sequence of the pullulanase derived from Bacillus subtilis str. 168;
(2) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 476 of the amino acid sequence of the pullulanase derived from Bacillus vireti;
(3) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 476 of the amino acid sequence of the pullulanase derived from Bacillus atrophaeus;
(4) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 481 of the amino acid sequence of the pullulanase derived from Geobacillus stearothermophilus;
(5) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 476 of the amino acid sequence of the pullulanase derived from Bacillus mojavensis;
(6) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 601 of the amino acid sequence of the pullulanase derived from Thermotoga maritima;
(7) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 478 of the amino acid sequence of the pullulanase derived from [Brevibacterium] halotolerans;
(8) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 481 of the amino acid sequence of the pullulanase derived from Thermus sp. IM6501;
(9) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 613 of the amino acid sequence of the pullulanase derived from Bacillus cereus FRI-35; and
(10) an amino acid sequence obtained by mutating the amino acid phenylalanine at the position 476 of the amino acid sequence of the pullulanase derived from Bacillus halotolerans.
In one example, the parent amino acid sequence is an amino acid sequence of the pullulanase derived from Bacillus subtilis str. 168, and the sequence is shown in SEQ ID NO: 2.
In one example, the cyclodextrin is one or more of α-CD, β-CD and γ-CD.
In one example, the mutated amino acid phenylalanine is mutated into 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.
The disclosure is also directed to a nucleotide encoding the pullulanase.
The disclosure is further directed to a vector or cell containing the nucleotide.
In one example, the vector can be one of PMC series, pET series or pGEX series plasmids.
In one example, the cell can be a microbial cell constructed by taking gram-negative bacteria, gram-positive bacteria or fungi as host bacteria.
In one example, the host bacteria can be Escherichia coli, Bacillus subtilis or Saccharomyces.
The disclosure is further directed to a method for reducing the inhibitory effect of the cyclodextrin on the pullulanase. The method 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 method mutates the parent pullulanase; and the amino acid sequence of the mutated pullulanase comprises an amino acid obtained by mutating the last position F (phenylalanine) in FNDXXRDXXXGXXF contained in the parent amino acid sequence based on the parent amino acid sequence, wherein X can be any naturally occurring amino acid.
In one example, the mutation comprises the following specific processes:
(1) taking pullulanase genes as a template, selecting plasmids, constructing an expression vector, and preparing plasmids carrying the pullulanase genes, wherein the plasmids are selected from one of PMC series plasmids, pET series plasmids or pGEX series plasmids;
(2) analyzing a crystal structure to determine a key amino acid site for combination of the pullulanase and the cyclodextrin;
(3) designing a mutated primer of the key amino acid, and performing mutation by taking the plasmids carrying the pullulanase genes as a template to prepare mutated plasmids; and
(4) transferring the mutated plasmids into the host bacteria, performing positive monoclonal fermentation culture to obtain a pullulanase mutant crude enzyme, and performing separation and purification to obtain a pullulanase mutant purified enzyme.
In one example, the method of separation and purification in step (4) is affinity chromatography, hydrophobic chromatography, ultrafiltration chromatography or gel filtration chromatography.
The disclosure has the following beneficial technical effects:
According to sequence comparison of the pullulanase from different sources, the pullulanase with the known crystal structure is taken as a template, and the crystal structure is analyzed to obtain an amino acid related to the inhibitory effect, wherein the key amino acid is highly conserved. Based on the above, the expression vector is constructed, the mutated primer is designed, the site-directed mutation is utilized to obtain the pullulanase mutant, and the inhibitory effect of the cyclodextrin on the mutant is studied.
1. Nickel Affinity Chromatography Comprises the Following Steps:
(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;
(2) sample loading: loading a pretreated sample at a flow rate of 1 mL/min;
(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
(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.
2. Measurement of Enzyme Activity of the Pullulanase:
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.
3. Measurement of the Enzyme Activity Comprises the Following Steps:
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.;
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
C. measurement: measuring the light absorption value under the condition of 540 nm, and calculating the activity.
Reduction of the inhibitory effect of enzyme activity characterization:
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.;
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
C. measurement: measuring the light absorption value under the condition of 540 nm, and calculating the activity.
The disclosure is specifically described below in conjunction with accompanying drawings and examples.
A process for reasonable mutation and mutation effect analysis of the key amino acid of the pullulanase comprises the following steps:
1. Finding of the Key Amino Acid Related to the Inhibitory Effect:
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.
2. Acquisition of a Parent Amino Acid Sequence/Nucleotide Sequence
(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; 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.
(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 DH5a 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.
(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.
3. Construction, Expression and Purification of the Pullulanase Mutant
(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:
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 (1.25 μ/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.
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).
(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.
(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.
4. Performance Analysis of the Pullulanase Mutant
Inhibitory effects are compared, and experimental results are shown in
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.
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:
Inhibitory effects of three cyclodextrins on the wild enzyme and the mutant F476D are compared and analyzed, and experimental results are shown in
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.
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.
The accession numbers of the sequences of the pullulanase from 40 different sources on NCBI are respectively: WP_003229246.1 (Bacillus subtilis), WP_024026701.1 (Bacillus vireti), WP_010789532.1 (Bacillus atrophaeus), KZE96788.1 (Geobacillus stearothermophilus), WP_032731297.1 (Bacillus mojavensis), CAA04522.1 (Thermotoga maritima), OEC77647.1 ([Brevibacterium] halotolerans), AAC15073.1 (Thermus sp. IM6501), AFQ12130.1 (Bacillus cereus FRI-35), WP_106020274.1 (Bacillus halotolerans), AMJ80567.1 (Alteromonas mediterranea), AFU74026.1 (Klebsiella pneumoniae), CAA04522.1 (Thermotoga maritima MSBB), OWC08995.1 (Escherichia coli), AAA25124.1 (Enterobacter aerogenes), WP_010789532.1 (Bacillus atrophaeus), WP_032731297.1 (Bacillus mojavensis), WP_095714534.1 (Bacillus sp. 7705b), WP_024714639.1 (Bacillus tequilensis), WP_079288107.1 (Bacillus intestinalis), WP_014114809.1 (Bacillales), KFI03882.1 (Bacillus sp. BSC154), WP_075747083.1 (Bacillus licheniformis), WP_069840139.1 (Bacillus sp. F56), WP_014665024.1 (Bacillus sp. IS), WP_071577010.1 (Bacillus sp. FMQ74), WP_103749897.1 (Bacillus sp. MBGLi97), WP_095432444.1 (Bacillus sp. X2(2017)), OTQ82147.1 (Bacillus subtilis subsp. subtilis), WP_046160744.1 (Bacillus sp. CMAA 1185), WP_103803268.1 (Bacillus sp. Ru63), WP_059291821.1 (Bacillus halotolerans), WP_059335607.1 (Bacillus halotolerans), WP_069150087.1 (Bacillus subtilis), KFF55698.1 (Bacillus subtilis), PTU26911.1 (Bacillus subtilis), WP_061670344.1 (Bacillus atrophaeus), WP_106293419.1 (Bacillus halotolerans), WP_099043342.1 (Bacillus halotolerans) and WP_103748328.1 (Bacillus subtilis).
Specific amino acid fragments (FNDXXRDXXXGXXF) corresponding to the sequences of the pullulanase from 10 different sources are listed below:
Bacillus subtilis
Bacillus vireti
Bacillus atrophaeus
Geobacillus
stearothermophilus
Bacillus mojavensis
Thermotoga maritima
halotolerans
Thermus sp. IM6501
Bacillus cereus FRI-35
Bacillus halotolerans
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.
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.
Geobacillus
stearothermophilus
halotolerans
Bacillus
halotolerans
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018101701346 | Mar 2018 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2018/094612 | Jul 2018 | US |
| Child | 16288804 | US |
