CN107345223A - Alpha-amylase variants and its application - Google Patents
Alpha-amylase variants and its application Download PDFInfo
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- CN107345223A CN107345223A CN201610816934.1A CN201610816934A CN107345223A CN 107345223 A CN107345223 A CN 107345223A CN 201610816934 A CN201610816934 A CN 201610816934A CN 107345223 A CN107345223 A CN 107345223A
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
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Abstract
The invention discloses alpha-amylase variants and its application.Described alpha-amylase variants increase by five extra amino acid KTTVS in C-terminal and obtained by increasing by two extra amino acid A and N in B.licheniformis alpha amylase N-terminal, while have remained in that the ability of parental generation hydrolyzing alpha Isosorbide-5-Nitrae glycosidic bond；It is preferred that the alpha-amylase variants as shown in SEQ ID NO.4.A series of alpha-amylase variants provided by the invention, under the acid conditions of pH 5.0 5.8 and there is higher catalytic activity under the conditions of 100 DEG C of high temperature above.The acidproof and heat endurance of these alpha-amylase variants, suitable for starch liquefacation.
Description
Technical field
The invention belongs to enzyme engineering field, is related to alpha-amylase variants and its application.
Background technology
In industrial quarters, mainly since alpha amylase come, these derive from the alpha amylase of microorganism for the hydrolysis of starch
With the synergistic application of other enzymes, such as Pullulanase, carbohydrase and glucose isomerase, can effectively starch-splitting divide greatly
Son, these caused micromolecular polysaccharides or monose have very in industries such as food manufacturing, cereal processing, beer processing, Alcohol Productions
More applications, it is most important.Alpha amylase is under the jurisdiction of one kind of saccharification enzyme, and its key structural feature is (α/β)8Fold, its
In contain special starch substrates binding site, length is generally no more than 10 sugar monomers, but much combinations of amylase
Site acts on together, it is possible to multi-point combination is carried out, so as to successfully shear starch polymer.
Alpha amylase can effectively shear α-Isosorbide-5-Nitrae glycosidic bond in starch substrates, so as to quickly reduce the molecule of starch substrates
Amount and viscosity, product are mainly the dextrin of different length.Alpha amylase has different species, the application of these species industrially
Condition is very different according to the sex character of required product.
Alpha amylase (α -1,4-glucan-4-glucanohydrolases, E.C.3.2.1.1) can effectively hydrolysis starch and
α-Isosorbide-5-Nitrae glycosidic bond in other polysaccharide.In view of in raising of the starch in hydrolytic process to enzyme efficiency and reduce production cost
Demand, seek to support the alpha amylase of effective starch liquefacation to turn into the one of academia and industrial quarters in different application field
Individual important R＆D direction.Currently with the focus of the technique improvement of the enzyme engineering enzyme mainly in heat resistance, degrees energy
Improvement and liquefaction effect lifting.
It is commercially valuable that many alpha amylases are found and defined from plant and microorganism, are mainly included
B.licheniformis alpha amylases, B.amyloliquefaciens alpha amylases and G.stearothermophilus α starch
Enzyme, wherein being that template has derivative variant quantity to be most using B.licheniformis α-amylase (L-type), using also most wide
It is general.
In the present invention, to adapt to the needs of industrialized production, we utilize B.licheniformis α-amylase (L
Type) construct a series of new alpha-amylase variants for template, improve the application efficiency of the enzyme, particularly in low pH and
Liquefaction efficiency can match with the mainstream product in the market in the case of reducing addition.
The content of the invention
It is an object of the invention to provide a series of B.licheniformis α-amylase (L-type) variants, the serial variant
Liquefaction efficiency can be improved, and adapts to the demand of industrialized production.Temperature and pH particularly more than 100 DEG C are 5.0-
Under conditions of 5.8, the enzyme activity of alpha-amylase variants of the invention and other properties can match with the mainstream product in the market.
It is an object of the invention to provide the gene for encoding the alpha-amylase variants.
It is yet another object of the invention to provide the production method of the alpha-amylase variants and application.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of alpha-amylase variants, described alpha-amylase variants are increased by the alpha amylase N-terminal in B.licheniformis
Add two extra amino acid A and N, and increase by five extra amino acid KTTVS in C-terminal and obtain, while remain in that
The ability of the glycosidic bond of parental generation hydrolyzing alpha -1,4；The two amino acid sequence homology reaches more than 95%.
The preferably natural alpha amylase of described parental generation alpha amylase, i.e. bacterial alpha amylase, further preferred Bacillus
Subtilis, B.licheniformis, B.amyloliquefaciens, G.stearothermophilus or Bacillus
The alpha amylase of any one in cereus, still more preferably B.licheniformis or G.stearothermophilus
Alpha amylase, most preferably B.licheniformis alpha amylase.
The total length coding gene sequence of described B.licheniformis alpha amylase is shown in SEQ ID NO.1；It is corresponding
Amino acid sequence is shown in SEQ ID NO.2.
The amino acid sequence of described alpha-amylase variants is further preferably as shown in SEQ ID NO.4.
The nucleotide coding sequence of described alpha-amylase variants is preferably freely shown in SEQ ID NO.3.
Encode the gene of alpha-amylase variants of the present invention.
Described gene is preferably as shown in SEQ ID NO.3.
For expressing the expression vector of alpha-amylase variants of the present invention, wherein being formed sediment containing coding for alpha of the present invention
The gene of powder enzyme variants.
Described expression vector is included mainly by a promoter sequence that is natural or synthesizing, and one natural or synthetic
Ribosome bind site, a natural or synthetic terminator sequence, and the base of coding alpha-amylase variants of the present invention
Because sequence together constitutes an expression component.
A kind of recombinant cell for being used to express alpha-amylase variants of the present invention, wherein including this one or more hair
The gene of bright described coding alpha-amylase variants.
The host cell of recombinant cell preferably is selected from Bacillus bacterial strains, further preferred B.licheniformis or process
Genetic engineering transforms the Bacillus bacterial strains for having inactivated some endogenous proteins；Most preferably inactivated by genetic engineering transformation
AprE and/or Blase B.licheniformis.
A kind of production method of alpha-amylase variants of the present invention, it is included in the condition of suitable alpha-amylase variants expression
Under the recombinant cell containing coding alpha-amylase variants gene order is cultivated, and from recombinant cell or its culture supernatant
Alpha-amylase variants are obtained in liquid.
Application of the alpha-amylase variants of the present invention in α -1,4 glycosidic bonds of Polysaccharides；It is preferred that high temperature and/
Or the application under low ph condition in α -1,4 glycosidic bonds of Polysaccharides；Preferably 80 DEG C~110 DEG C of described high temperature, it is further excellent
Select 100 DEG C~110 DEG C；Described low pH preferable ph is 5.0~5.8.
Beneficial effect
A series of alpha-amylase variants provided by the invention, under pH 5.0-5.8 acid conditions and 100 DEG C of high temperature above bars
There is higher catalytic activity under part.The acidproof and heat endurance of these alpha-amylase variants, suitable for starch liquefacation.
Brief description of the drawings
Fig. 1 is pYF-tsDE carriers, including a temperature sensitivity original paper (having replication activity at 30 DEG C), an erythromycin are determined
Determine gene (ErmC) -- 300 μ g/mL erythromycin can be resistant in E.coli and can be resistant in B.licheniformis
5 μ g/mL erythromycin.The recombinant host cell containing coding alpha-amylase variants nucleotide sequence is filtered out with erythromycin.
Fig. 2 is the schematic diagram of pUC57-KS-erm carriers, and the pYF-tsDE that can be obtained from the carrier in the present invention is carried
Body.
Fig. 3 is the schematic diagram of pYF-tsINT-amy carriers.
Fig. 4 is under the conditions of different injection temperations, amylase liquefaction application compares.
Fig. 5 is under different starch slurry concentration, and amylase liquefaction application is compared.
Fig. 6 is under pH5.0, and the liquefaction application of amylase difference enzyme concentration is compared.
Fig. 7 is under the conditions of different concentration of substrate, amylase liquefaction application compares.
Fig. 8 is under condition of different pH, and amylase liquefaction application is compared.
Fig. 9 is under the conditions of different concentration of substrate, amylase liquefaction application compares.
Figure 10 is under condition of different pH, and amylase liquefaction application is compared.
Figure 11 is that application of the amylase in corn alcohol liquefaction is compared.
The detailed description of the present invention
Unless otherwise prescribed, all technologies and scientific terminology in the present invention there is identical implication, be generally understood as
In general professional technique.In this application, some explicans are the same as described in specification.It must be noted that used herein and institute
Attached claim, singulative "one" " this " includes plural form, unless otherwise expressly specified in context.
In the present invention, alpha amylase refers to being capable of the enzyme of α-Isosorbide-5-Nitrae glycosidic bond of Polysaccharides.For example, alpha amylase can incite somebody to action
Starch Hydrolysis is into dextrin.
In the present invention, parental generation alpha amylase refers to natural alpha amylase.Natural alpha amylase is bacterial alpha amylase, source
Including but not limited to Bacillus subtilis, B.licheniformis, B.amyloliquefaciens,
G.stearothermophilus and Bacillus cereus.
According to a preferred embodiment of the invention, natural alpha amylase is derived from Bacillus strain ----especially
B.licheniformis and G.stearothermophilus.B.licheniformis complete encoding sequence is shown in SEQ ID
NO.1；Corresponding amino acid sequence is shown in SEQ ID NO.2.
In the present invention, term " alpha-amylase variants " refer to it is non-naturally occurring, in the alpha amylase amino acid sequence of parental generation
Available point position carry out increase, deletion and/or the replacement of one or several amino acid residues, while remained in that parental generation hydrolysis
The alpha amylase of the ability of α -1,4 glycosidic bonds.
" liquefaction " generally refers to the process for the polysaccharide of small molecule by carbohydrate breakdown in the present invention.Formed sediment when adding α
When powder enzyme or alpha-amylase variants, " liquefaction " refers in particular to hydrolyze α-Isosorbide-5-Nitrae glycosidic bond of carbohydrate.
In the present invention, " α-Isosorbide-5-Nitrae glycosidic bond ", which refers to, connects the C1 of previous glucose and the latter glucose C4
Key, as α-Isosorbide-5-Nitrae glycosidic bond.
The present invention relates to " alpha-amylase variants " that parental generation alpha amylase is done to sequence alterations acquisition.Parental generation alpha amylase is day
Right alpha amylase, especially derive from the natural alpha amylase of bacterium.According to an embodiment of the invention, alpha-amylase variants refer to parent
The available point position of the alpha amylase amino acid sequence in generation carries out increase, deletion and/or the replacement of one or several amino acid residues,
The alpha amylase of the ability of the glycosidic bond of parental generation hydrolyzing alpha -1,4 has been remained in that simultaneously.
The present invention includes a series of alpha-amylase variants.According to an embodiment of the invention, this series of alpha amylase becomes
The homology of the amino acid sequence of body is at least up to 95%, respectively reaches 95%, 96%, 97%, 98%, 99% or 100%.
As illustrative and non-limiting example of the present invention, alpha-amylase variants are the parental generations by B.licheniformis
Alpha amylase increases by two amino acid residue AN in its N- end, and its C- end increases by 5 amino acid residues on this basis
KTTVS and obtain, see SEQ ID NO.4.
Alpha-amylase variants in the present invention maintain the ability of the glycosidic bond of hydrolyzing alpha -1,4.In addition, the property of these alpha amylases
It can use and above meet demand of industrial production, such as, the raising of liquefaction efficiency, catalytic activity is stable under acid pH or hot conditions.
According to an embodiment of the invention, an alpha-amylase variants are higher than 100 DEG C in pH 5.0 acid condition or temperature
Condition (especially temperature 100 DEG C -108 DEG C between) under, catalytic activity is stable.The performance of these raisings of alpha-amylase variants
More adapt to the liquefaction reaction of starch industry.Because liquefaction process is usually carried out under low pH and hot conditions on starch industry.
All alpha-amylase variants of the present invention may be used to liquefaction reaction.In a preferred embodiment, alpha amylase becomes
Body derives from parental generation alpha amylase, the especially alpha amylase from B.licheniformis parental generations.Specifically it is being preferable to carry out
In example, alpha-amylase variants amino acid sequence is as shown in SEQ ID NO.4 in sequence table.
According to the present invention, any carbohydrate containing α-Isosorbide-5-Nitrae glycosidic bond may be used to liquefaction reaction.Contain one
Or the carbohydrate of multiple α-Isosorbide-5-Nitrae glycosidic bond includes but are not limited to starch, amylopectin, amylose and dextran.
Many carbohydrate contain α -1,6- glycosidic bonds and α-Isosorbide-5-Nitrae-glycosidic bond, such as, amylopectin." α -1,4- sugar
Glycosidic bond " refers to the key that the C1 of the previous glucose and C4 of the latter glucose is connected, as α-Isosorbide-5-Nitrae glycosidic bond.Therefore,
The alpha-amylase variants of the present invention can be used cooperatively with being capable of the Pullulanase of the glycosidic bond of hydrolyzing alpha -1,6 in saccharifying.Energy
The enzyme of enough glycosidic bonds of hydrolyzing alpha -1,4 includes but are not limited to alpha amylase.In a preferred embodiment of the invention, catalytically hydrolyzing alpha-
The enzyme of 1,4 glycosidic bonds is alpha amylase.
Therefore, according to an embodiment of the invention, it is with the use of general that further catalysis saccharification reaction, which puies forward efficient method,
Shandong orchid enzyme.In the present invention, " Pullulanase " is the hydrolase for referring to hydrolyzing alpha -1,6 glycosidic bond.
Alpha amylase in the present invention and Pullulanase coordinate in the saccharifying of starch use can improve glucose and
The purity of maltose.In addition, can effectively reduce concentration of substrate using above-mentioned complex enzyme in saccharification reaction, transformation efficiency is improved,
Can also there is higher catalytic activity at acid pH or higher temperature, be suitable for the condition of industrial hydrolysis starch.
The invention provides a kind of alpha-amylase variants can under the conditions of the temperature of any suitable industrialized production and pH water
The method that solution α-Isosorbide-5-Nitrae glycosidic bond carries out saccharification reaction.According to the present invention, liquefaction reaction can be at a high temperature of 80 DEG C to 110 DEG C
Reaction, such as 80 DEG C, 90 DEG C, 100 DEG C, 105 DEG C and 110 DEG C.Saccharification reaction also can be in pH5.0 to pH5.8 condition of acidic pH
Lower progress, such as pH5.0,5.2,5.4,5.6, and 5.8.
According to an embodiment of the invention, the liquefaction reaction of alpha-amylase variants catalysis is in acid pH and 100 DEG C of temperatures above bars
Catalytic activity is stable under part.
On the other hand, the expression vector in the present invention includes the nucleotide sequence of the coding alpha-amylase variants of a synthesis,
And recombinant host cell contains above-mentioned expression vector.Expression vector contains the nucleosides of the coding alpha-amylase variants of a synthesis
Acid sequence.Expression vector can be integrated on the genome of host cell.For example, expression vector contains the nucleotide sequence of synthesis
SEQ ID NO.3。
Expression vector in the present invention preferably includes natural or synthesis a promoter sequence, and one natural or synthetic
Ribosome bind site, a natural or synthetic terminator sequence.These hereditary original papers and the alpha-amylase variants of synthesis are compiled
Code sequence together constitutes an expression component, and expression component constitutes expression vector with carrier framework.For example, expression vector bag
An expression component is included, and expressing component includes following element：One promoter sequence, the ribosome binding site of a synthesis
Point, the nucleotide sequence and a terminator sequence that encode alpha-amylase variants in the present invention of a synthesis.Signal sequence can
The secretion of alpha-amylase variants is instructed, signal sequence is introduced into expression vector or expression component, especially introduced signal sequence
The upstream of initiation codon is more beneficial for the secretion of alpha-amylase variants.
According to the preferred embodiment of the present invention, expression vector is suitably expressed in bacterium, especially Bacillus bacterial strains, more suitable
Preferably expressed in B.licheniformis.In a particularly preferred embodiment, expression vector can be incorporated into Bacillus gene
In group, especially on B.licheniformis genome.Host available for the polynucleotide sequence being incorporated into chromosome
The expression vector of cell, and the construction method of this expression vector, it is that well-known one of Contemporary Biology field is common
Technical ability.
According to embodiments of the present invention, recombinant host cell can be transformed with genetic engineering and become comprising one or more alpha amylases
The nucleotide sequence of body gene expression.Any technology is used equally for genetic engineering engineered host cell to include one or more present invention
In alpha-amylase variants coding synthesis nucleotide sequence, for example, chromosomal integration.Contain the sensitive origin of temperature and resistance screening
The carrier of mark can be used for integration step.These carriers are integrated by the specific region of Campbell's mechanism and genome, are passed through
Resistance screening obtains recombinant bacterium, and recombinant bacterium removes resistance screening by homologous recombination in subsequent incubation and marked.
According to embodiments of the present invention, recombinant host cell has inactivated some endogenous proteins by genetic engineering transformation.Energy
The endogenous being deactivated includes but is not limited to extracellular protease.The transformation of host cells of restructuring contains alpha-amylase variants expression
Some endogenous proteins are inactivated before or after the nucleotide sequence of gene.Method preferably is become being transferred to alpha amylase
The inactivation of Host Strains external source extracellular proteinase is carried out before the carrier of body expressing gene.
First, B.licheniformis has inactivated some extrinsic protein enzyme genes by transformation.Particularly
B.licheniformis bacterial strains can inactivate some extracellular proteases, such as subtilisin (AprE), glutamic acid-
specific protease(Blase).These genetic engineerings, which are transformed, causes B.licheniformis bacterial strains alpha amylase preferably
The expression and secretion of variant.
The invention provides a kind of production method of alpha-amylase variants.According to an embodiment of the invention, this method is included in
The recombinant host cell containing coding alpha-amylase variants nucleotide sequence is carried out under conditions of suitable alpha-amylase variants expression
Culture, and obtain alpha-amylase variants from recombinant host cell or its supernatant.
All recombinant host cells of the present invention can produce alpha-amylase variants.Recombinant host cell contains at least
The nucleotide sequence of the coding alpha-amylase variants of one copy.The nucleotide sequence of these coding alpha-amylase variants can be suitable
Alpha-amylase variants are expressed under conditions of preferably.The alpha-amylase variants secreted from recombinant host cell can from recombinant cell or on
It is collected into clear liquid.The method of collection includes but are not limited to filter, centrifugation etc..
According to an embodiment of the invention, transforming B.licheniformis by genetic engineering by fermentation being capable of high yield α shallow lake
Powder enzyme variants.B.licheniformis has imported the nucleotide sequence of coding alpha-amylase variants by genetic engineering transformation.More
Good, the B.licheniformis in the present invention has eliminated resistance screening gene, environmental sound and the α produced
Amylase variant is particularly suited for food industry.
Following example further illustrates the essence of the present invention in the present invention.It should be appreciated that following example does not limit
The present invention, the scope of the present invention are determined by appended claim.
Embodiment
Embodiment 1:The structure of pYF-tsDE plasmids
PYF-tsDE (Fig. 1) is the E.coli/B.licheniformis shuttle plasmids of a temperature sensitive type.The plasmid is by one
The replication orgin (active at 30 DEG C) of individual responsive to temperature type and erythromycin resistance gene (ErmC) composition, the resistant gene
Resistance in E.coli is 300ug/ml, and the resistance in B.licheniformis is 5ug/ml.At 37 DEG C, on plasmid
Replication orgin inactivation, plasmid is integrated into the specified site of Host Strains genome, screened with ErmC.
The building process of pYF-tsDE plasmids is：By plasmid pUC57-KS-erm, (commission Genscript synthesis, sequence are shown in
CN104073458A, Fig. 2) use BglII double digestions, recovery purifying 3.8kbp fragment, with T4 ligases (New England
Biolabs) connect certainly, the plasmid cloned is exactly pYF-tsDE.Transformant is bred in E.coli TOP10, and conduct
The skeleton of all genetic manipulations below.
Embodiment 2:The structure of protease-deficient B.licheniformis bacterial strains
As restructuring enzyme product host cell genetic engineering bacterial strain had document report (Widner et al.,
Journal of Industrial Microbiology&Biotechnology,25,204-212,2000).These restructuring
Host cell generally comprises the nucleic acid structure of one or more encoding target sequences to the expression of enzyme.In the present invention,
B.licheniformis is used as the recipient bacterium of genetic manipulation.Bacillus conversion can pass through highly developed hand at present
Section reaches, as competent cell converts, electricity conversion and protoplast transformation (Young et al., J Bacteriology,
81,823-829,1961；Shigekawa et al.,Biotechniques,6,742-751,1988；Chang et al.,
Molecular General Genetics,168,111-115,1979)。
In the present invention, a single alpha-amylase variants expression cassette, comprising naturally or synthesis promoter sequence,
One signal peptide sequence screened from bacillus, the ribosome bind site of a synthesis, one comes from
B.licheniformis alpha-amylase variants encoding gene, and a transcription terminator.Such design will greatly enhance place
The secretory volume of the expression of gene and alpha-amylase variants in main bacterial strain.Alpha-amylase variants encoding gene is replaced
Specific site in B.licheniformis cellular genomes is recombinated by plasmid-mediated single cross-over homologous to realize.
In B.licheniformis, the active secretion to heterologous enzyme of extracellular protease is unfavorable.Confirm
2 kinds of main extracellular proteases：Subtilisin (AprE), glutamic acid-specific protease (Blase),
The extracellular protease activity of big paces is all derived from both protease in B.licheniformis.
In the present invention, in order to obtain the structural integrity of alpha-amylase variants gene expression, above-mentioned two gene is gone out
It is living, using continuity mode Characteristics for Single Staggered Campbell's type mechanism.Concrete operations are as follows：
2.1pYF-tsDE suppresses to connect certainly via after BglII digestions with CIP processing；
2.2 gene knockout
(1) in order to obtain each gene delection fragment, the method using B.lieheniformis genomic DNA as template PCR
About 500bp homologous sequence is respectively expanded from the gene both sides to be lacked.The monoclonal of bacillus subtilis passes through 98 DEG C, 5 points
After clock pre-degeneration genomic DNA template can be used as directly to be used in PCR reactions.
Primer for PCR reactions is synthesized by Genscript companies.Primer sequence is as follows：
Amplification Apr gene upstream sequences primer be：
lichApr_F1 TTATTGAGCGGCAGCTTCGACATTGATCAGACCTT
lichApr_R1 CCTTACGGCATTCCTCTCAACAGCGGATCTTCAG
Amplification Apr downstream of gene sequences primer be：
lichApr_F2 CCTGAAGATCCGCTGTTGAGAGGAATGCCGTAAGG
lichApr_R2 ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGAC
Amplification Blase gene upstream sequences primer be：
blalich_F1 TTATTGTGCGCTGTTTTTCCAGTTGGTCAAATTGTCG
blalich_cR1 CGGACAAGGGTCACCAACGGGACAACTGTTACCATC
Amplification Blase downstream of gene sequences primer be：
blalich_cF2 GATGGTAACAGTTGTCCCGTTGGTGACCCTTGTCC
blalich_R2 CGGCGTTGGTTAGTAAAAAGAGTGTTAAACGAGGTTTGAT
PCR amplification system is 50ul, and response procedures are as follows：
(1) 98 DEG C, 8 minutes of 14580 monoclonal pre-degenerations of bacillus subtilis B.licheniformis；
(2) 96 DEG C, 15 seconds；
(3) 58 DEG C, 15 seconds；
(4) 72 DEG C, 30 seconds；Repeat 2-4 steps 25-30 times；
(5) 72 DEG C, 2 minutes are extended eventually.
PCR primer is pursued progress kits after being detected with 0.8% agarose gel electrophoresis with love.
The target gene of the internal about 400-500bp sequence deletions of 2.3 overlapping PCR methods amplification
Gene internal deletion fragment is obtained with overlapping PCR method (overlap extension PCR, SOE), specifically
Operation is as follows：
(1) each upstream region of gene, downstream PCR fragment and purified in being separately recovered 2.2；
(2) with the upstream and downstream homologous sequence fragment 1 of each target gene:Template is used as after 1 mixed in molar ratio, with primer XX-
CZ-F1 and XX-CZ-R2 (" XX " represents Apr or Blase) PCR amplifications obtain the AprE genes or Blase bases of internal deletion fragment
Cause.
Above-mentioned fragment is then recombinated into by BglII lines with Clone-EZ Cloning Kits (offer of Genscript companies)
In the pYF-tsDE carriers of property, the recombinant plasmid of acquisition is respectively designated as：PYF-tsDE-Apr and pYF-tsDE-Blase.This
A little recombinant plasmids are temperature sensitive type plasmid, wherein the Apr genes or Blase genes that include lacked for complete genome it is interior
Portion about 400-500bp sequence.
The replacement of iso-allele can not realized by homologous recombination.Method is referring to CN102124112A, it is possible to use
The method of other known homologous recombinations of this area.
2.4 plasmids convert
Using that will knock out plasmid, to be transformed into method and screening process in B.lieheniformis competent cell as follows for this experiment：
(1) by temperature sensitive type plasmid pYF-tsDE-Apr or pYF-tsDE-Blase convert B.lieheniformis (CICC 22794, in
State microorganism fungus kind storehouse is bought) competent cell；
(2) under conditions of 30 DEG C, used on LB (every liter of 10g containing peptone, yeast extract 5g, sodium chloride 10g) culture medium
Erythromycin (5ug/ml) resistance carrys out screening positive clone bacterial strain；
(2) positive colony bacterial strain is transferred under conditions of 37 DEG C again and cultivated, the temperature-sensitive plasmid is fused to host
On genome.In order that gene is replaced in the site of setting, select several clones while be inoculated in 2 × YT culture mediums and connect
After continuous culture 24 hours again subculture once, whole process subculture 4-5 times (generally requiring 5-7 days).
(3) bacillus subtilis cell of screening erythromycin-sensitive enters performing PCR identification and can put down simultaneously with 1% skim milk LB
Plate observes hydrolysis, and the strain after knockout should show the hydrolysis circle being reduced significantly.
PCR primer used in identification：
AprE：Apr-seqF1/Apr-seqR3
Blase：Blase-seqF1/Blase-seqR3
Apr-seqF1：GCCAGGTTGAAGCGGTCTATTCAT
Apr-seqR3：TACGGCCATCCGACCATAATGGAAC
Blase-seqF1：GAAGAGCCGGTCACAATTGC
Blase-seqR3：GGCCGTTAGATGTGACAGCC
Embodiment 3:The construction and integration of alpha-amylase variants bacterial strain
3.1 amylase express framework establishment
The structure of integrated plasmid is using the same method of above-mentioned pYF-tsDE plasmids.In order to which expression cassette is incorporated into design
The homology region in the AmyE sites on genome, the upstream and downstream design 800bp in the AmyE sites on genome or so, is connected to
One alpha-amylase variants expression cassette both sides.Simultaneously assemble some from first to last the bacterial chromosomal dna fragment of natural selection and
The composition sequence of function, these be all control table reach alpha-amylase variants gene necessary to.
One typical amylase expression cassette has following components composition：One typical alpha-amylase variants expression cassette
It is made up of elements below：One natural or synthesis promoter sequence (SEQ ID NO.5), the ribosomes of a synthesis combine
Site aaaggagg, an alpha-amylase variants encoding gene from B.licheniformis (is respectively SEQ ID
NO.3) and one synthesis terminator sequence (SEQ ID NO.6).One strong natural signals screened from bacillus subtilis
Sequence (SEQ ID:NO.7 the upstream of alpha-amylase variants encoding gene promoters) is inserted into, to the secretion of Enhanced expressing enzyme
Efficiency.Complete alpha-amylase variants expression cassette is inserted to the pYF- of linearisation with Clone-EZ Cloning Kits (Genscript)
BglII sites in tsDE, the temperature sensitive type integrated plasmid finally given are named as pYF-tsINT-amy (Fig. 3).Above-mentioned sequence
Synthesis completed by Genscript companies, by above-mentioned sequence successively it is seamless series connection obtain alpha amylase expression of enzymes frame.This framework
Middle signal peptide sequence filters out from bacillus subtilis, can effectively improve the secretion of alpha amylase.
3.2 plasmids convert
Above-mentioned whole alpha amylase expression cassette (including amyE gene upstream and downstream homologous fragment) is cyclized using recombinant technique
The pYF-tsDE plasmids (recombination kit is provided by Genscript companies) of BglII linearisations, the temperature sensitive type plasmid life built
Entitled pYF-tsINT-amy.The plasmid is used to convert into the bacillus licheniformis of AprE and Blase protease genes missing
(CICC22794, Chinese microorganism strain storehouse are bought), the alpha-amylase variants expression cassette of non-resistant mark will replace AmyE.Using
Above-mentioned method, the bacterial strain successfully integrated on alpha-amylase variants encoding gene to B.licheniformis chromosomes form sediment in blueness
Transparent circle is produced on powder flat board, PCR further verifies that expression cassette is incorporated into the AmyE sites of F-strain.
The B.licheniformis engineered strains of alpha-amylase variants are produced in -80 DEG C of preservations.
Embodiment 4:The shake flask fermentation of alpha-amylase variants production
The bacterial colony (containing alpha-amylase variants expression cassette) of an activation is taken, is inoculated into 20ml culture mediums and (contains
Malt syrup 4.0%, peptone 2.0%, dusty yeast 0.1%KH2PO40.6% and corresponding antibiotic) culture arrive logarithmic phase.
1.2ml nutrient solutions are taken to be inoculated into 30ml culture mediums (containing malt syrup 12.0%, peptone 1.0%, dusty yeast 1%KH2PO4
0.2%, MnCl20.003%), 120rpm shaken cultivations 3 days in reciprocal shaker.Respectively at 24 hours, 48 hours and 72 small
When sample 1ml, 1000rpm centrifugations 1min.Supernatant is preserved, does SDS-PAGE analyses.Alpha-amylase variants molecular weight is about
53kD。
Alpha-amylase variants activity is determined, method is the same as embodiment 6.
Embodiment 5:Alpha-amylase variants substep fed-batch fermentation technique
The line of genetic engineering B.licheniformis bacterial strains and agar by -80 DEG C of freezen protectives obtained in embodiment 3
On inclined-plane, 37 DEG C of overnight renewal cultivations.Agar slant formula is as follows：Peptone 1%, yeast extract 0.5%, NaCl 1%,
Agar powder 2%.
First, several fresh colonies are chosen to cultivate 16 hours in 37 DEG C in the seed flask for filling 50ml culture mediums.Kind
Sub- shaking flask formula：Malt syrup 4.0%, peptone 2.0%, yeast extract 0.1%, KH2PO40.6%., will after 16 hours
All seed fermentation liquid is fully transferred to fill in the 7L stainless steel fermentation tanks of 4L culture mediums in 37 DEG C, mixing speed
350rpm, continuing fermentation 12 hours under conditions of Ventilation Rate is 650L/H.Fermentation tank formula：Malt syrup 6.0%, peptone
1.0%, yeast extract 1%, KH2PO40.2%, MnCl20.003%.Then with 5% phosphoric acid control fermentation pH 5.7 ±
0.2 or so, and first 18 hours with speed 1L/18hrs after constantly mended into fermentation tank with speed 0.5L/18hrs within 110 hours
Material.Feed supplement formula is as follows：Malt syrup 48%, peptone 6%, yeast extract 8%.Whole fermentation process continues 140-150
Hour.Collect and all culture mediums in 4 DEG C, 1010krpm, 30 minutes centrifugation fermentation tanks, the supernatant after centrifugation are used for α starch
Enzyme variants enzyme activity is analyzed.
Embodiment 6:Amylase activity determines
Amylase activity measure is hundred this outstanding amylase activities (BAU).The definition of 1 BAU unit：In pH6.0,70
Under the conditions of DEG C, the enzyme amount required for 1 minute liquefaction 1mg soluble starch.
Briefly, enzyme activity determination operates as follows：20ml 20g/L soluble starches solution and 5ml pH6.0 phosphoric acid buffers
Liquid mixes, and first preheats 8min at 70 DEG C, the enzyme liquid added after 1.0ml dilutions, accurate response 5 minutes, then takes 1ml reaction solutions,
Addition is filled in 0.5ml 0.1mol/L hydrochloric acid solutions and the test tube of the dilute iodine solutions of 5ml in advance, is shaken up, and with 0.5ml 0.1mol/L
Hydrochloric acid solution and the dilute iodine solutions of 5ml are blank, under 660nm wavelength, determine its light absorption value rapidly, are tabled look-up according to absorbance, are obtained
The enzyme activity of test sample.
Embodiment 7:The application of amylase
Except what is illustrated in addition, 1BAU:The vitality test of amylase is hundred this outstanding amylase units (BAU).One BAU
It is defined as under the conditions of pH6.0,70 DEG C, the enzyme amount required for 1 minute liquefaction 1mg soluble starch.
tDS:Dry per ton
The amylase variant obtained is expressed and separated from Bacillus licheniformis cell carries out first with cornstarch first
Wheel liquefaction test.Test condition：18 Baume degrees (° B é), fully mixing, pH are adjusted to 5.2 with hydrochloric acid.Add 0.4kg/tDS's
Amylase, be respectively adopted injection temperation 100,105,108,112,115 degree, maintain 5-8min after flash, 95 degree maintain 120min.
DE and iodine examination test are carried out after liquefaction, while pays attention to observing albumen flocculation and viscosity situation, using Wild type as control, as a result
It is shown in Table lattice 1 and Fig. 4.
Table 1:Under the conditions of different injection temperations, amylase liquefaction application is compared
| Temperature (DEG C) | Wild type DE (%) | 8008 mutant 2DE (%) |
| 100 | 18.02 | 19.80 |
| 105 | 17.79 | 19.59 |
| 108 | 15.00 | 19.17 |
| 112 | 13.85 | 17.75 |
| 115 | 7.05 | 15.81 |
As a result showing, 8008 mutant 2 (alpha-amylase variants of the present invention of above-mentioned preparation) are substantially better than wild type,
8008 mutant 2 are in the case of different injection temperations, and 100,105,108 DEG C of liquefaction are excessive, and 112 DEG C of liquefaction are just right, and egg
White cotton fiber coagulates, and at 115 DEG C, liquefaction effect is still preferable, and albumen flocculation is normal, illustrates that alpha-amylase variants of the present invention have very
Good heat resistance, and wild type are not resistant to 115 DEG C of high temperature.
Secondly, we are tested by the liquefaction under different starch slurry concentration conditions, and it is dense to high substrate to determine amylase
The tolerance of degree.Liquefaction reaction condition is same as above, and injection temperation is 108 DEG C, using Wild type as control, the results are shown in Table 2 Hes
Fig. 5.
Table 2:Under different starch slurry concentration, amylase liquefaction application is compared
| Baume degrees (° B é) | Wild type DE (%) | 8008 mutant 2DE (%) |
| 15 | 15.27 | 18.56 |
| 18 | 15.04 | 18.47 |
| 20 | 14.98 | 18.40 |
| 22 | 12.49 | 16.89 |
As shown in table 2,8008 mutant 2 are substantially better than wild type, and 8008 mutant 2 are in different starch slurry concentration bars
Under part, under the conditions of at concentrations up to 22 ° B é of starch slurry, L-type amylase remains to normally liquefy, and illustrates that alpha-amylase variants of the present invention can
To carry out underflow liquefaction, effectively save factory cost.
Then, we determine the acid resistance of amylase, while carry out the liquefaction performance under the conditions of different enzyme concentrations.Liquefaction
Reaction condition is same as above, pH 5.0, enzyme concentration is respectively 0.2,0.3,0.4,0.5,0.6kg/tDS, using wild type as
Control, the results are shown in Table 3 and Fig. 6.
Table 3:Under pH5.0, the liquefaction application of amylase difference enzyme concentration is compared
| Enzyme concentration (kg/tDS) | Wild type DE (%) | 8008 mutant 2DE (%) |
| 0.2 | 8.24 | 11.47 |
| 0.3 | 11.61 | 15.62 |
| 0.4 | 14.98 | 18.58 |
| 0.5 | 15.16 | 18.65 |
| 0.6 | 16.77 | 20.17 |
As shown in table 3,8008 mutant 2 are substantially better than wild type, and 8008 mutant 2 are in low pH, 0.2-0.3kg/
Under the conditions of tDS additions, alpha-amylase variants of the present invention remain to normally liquefy, and illustrate that alpha-amylase variants of the present invention have to low pH
Stronger tolerance, while under the conditions of the low enzyme concentrations of 0.2kg/tDS, alpha-amylase variants of the present invention remain to normally liquefy, and this can
Effectively reduce factory's enzyme cost.
In addition, We conducted influence test of the amylase to saccharification, while with Wild type amylase, Liquozyme
Liquefier made from Supra (being purchased from Novozymes) liquefaction is contrasted, test condition：32% dry (DS), fully mix
Even, pH is adjusted to 4.3 with hydrochloric acid.0.45kg/tDS compounded saccharifying enzymes are added, 200ml reaction reacts 24 and 48 respectively at 60 DEG C
Hour.Sample is used for HPLC after 0.22um membrane filtrations and 100 DEG C of inactivations and analyzed.It the results are shown in Table 4.
Table 4:Influence of the amylase to saccharification
As shown in table 4, using alpha-amylase variants liquefier of the present invention and Wild type liquefiers, alpha amylase of the present invention
Variant saccharification result is substantially better than Wild type, while uses alpha-amylase variants liquefier of the present invention and Liquozyme
Supra liquefiers, both saccharification results are just the same, illustrate that alpha-amylase variants of the present invention can apply to starch sugar industry.
In addition, We conducted influence test of the amylase to wheaten starch, test condition：22nd, 25,28,30% (W/W)
Different concentration of substrate, fully mixing, pH are adjusted to 5.6 with hydrochloric acid.0.4kg/tDS amylase is added, using 91-95 DEG C of maintenance
120min.After liquefaction carry out DE and iodine examination test, while pay attention to observe albumen flocculation and viscosity situation, using wild type as pair
According to the results are shown in Table 5 and Fig. 7.
Table 5:Under the conditions of different concentration of substrate, amylase liquefaction application is compared
| Concentration of substrate (%) | Wild type DE (%) | 8008 mutant 2DE (%) |
| 22 | 20.85 | 21.21 |
| 25 | 20.21 | 20.47 |
| 28 | 19.64 | 19.82 |
| 30 | 18.14 | 19.02 |
As a result showing, 8008 mutant 2 are similar to wild type results, in the case of different concentration of substrate, 22-25%
Liquefy just right, and albumen flocculates, and 28 and 30%, liquefaction effect is still preferable, and albumen flocculation is normal, illustrates the present invention
Alpha-amylase variants can carry out underflow liquefaction, effectively save factory cost.
Secondly, we determine the acid resistance of amylase, while carry out the liquefaction performance under condition of different pH.Liquefaction reaction
Condition is same as above, and pH is respectively 4.8,5.2,5.6,6.0, enzyme concentration 0.4kg/tDS, using wild type as control, as a result
It is shown in Table 6 and Fig. 8.
Table 6:Under different pH, amylase liquefaction application is compared
| pH | Wild type DE (%) | 8008 mutant 2DE (%) |
| 4.8 | 8.11 | 13.30 |
| 5.2 | 20.43 | 19.07 |
| 5.6 | 21.77 | 20.98 |
| 6.0 | 21.97 | 21.05 |
As shown in table 6, under the conditions of pH4.8, alpha-amylase variants of the present invention remain to normally liquefy, and illustrate α starch of the present invention
Enzyme variants have stronger tolerance to low pH, and wild type are not resistant to low pH.
Then, We conducted influence test of the amylase to rice, test condition：12nd, 15,18,20 Baume degrees (° B é)
Different concentration of substrate, fully mixing, pH are adjusted to 5.2 with hydrochloric acid.Addition 0.4kg/tDS amylase, 108 DEG C of injection temperation,
Flashed after maintaining 5-8min, 95 degree of maintenance 120min.After liquefaction carry out DE and iodine examination test, while pay attention to observe albumen flocculation and
Viscosity situation, using wild type as control, it the results are shown in Table lattice 7 and Fig. 9.
Table 7:Under the conditions of different concentration of substrate, amylase liquefaction application is compared
| Concentration of substrate (° B é) | Wild type DE (%) | 8008 mutant 2DE (%) |
| 12 | 19.53 | 22.33 |
| 15 | 18.87 | 21.59 |
| 18 | 17.06 | 21.17 |
| 20 | 15.72 | 20.46 |
As a result show, 8008 mutant 2 are substantially better than wild type, and 8008 mutant 2 are in different concentration of substrate situations
Under, 12-18 ° of B é liquefaction is just right, and albumen flocculates, and still preferable in 20 ° of B é, liquefaction effect, and albumen flocculation is normal, says
Bright alpha-amylase variants of the present invention can carry out underflow liquefaction, effectively save factory cost.
Secondly, we are tested by the liquefaction under the conditions of different starch pH (4.8,5.2,5.4,5.6,5.8), are determined
Tolerance of the amylase to high concentration of substrate.Liquefaction reaction condition is same as above, and injection temperation is 108 DEG C, and enzyme concentration is
0.4kg/tDS, using wild type as control, it the results are shown in Table 8 and Figure 10.
Table 8:Under different pH, amylase liquefaction application is compared
As shown in table 8, under the conditions of pH4.8-5.8, alpha-amylase variants of the present invention remain to normally liquefy, and illustrate α of the present invention
Amylase variant has stronger tolerance to low pH, and wild type acid resistances are poor.
Finally, because amylase has important application in alcohol industry production, we are also tested for amylase and given birth in alcohol
Liquefaction effect in production, the corn flour (40 mesh) of different material-water ratios is prepared, with salt acid for adjusting pH to 5.8, add 0.145kg/tDS
Amylase.In 95 DEG C of cooking and liquefaction 120min.Reaction terminate after, determination sample DE and viscosity, at the same with Liquozyme Supra
(being purchased from Novozymes) carries out contrast test,.It the results are shown in Table 9 and Figure 11.
Table 9:Application of the amylase in corn alcohol liquefaction is compared
| The corn flour of different material-water ratios | DE (%) | Viscosity (mPas) |
| Amylase -1:2 | 11.88 | 1562 |
| Amylase -1:2.3 | 12.51 | 1109 |
| Amylase -1:2.5 | 13.15 | 586 |
| Amylase -1:2.7 | 13.30 | 429 |
| Liquozyme Supra-1:2 | 12.19 | 1702 |
| Liquozyme Supra-1:2.3 | 12.78 | 1114 |
| Liquozyme Supra-1:2,5 | 12.88 | 506 |
| Liquozyme Supra-1:2.7 | 13.32 | 352 |
As shown in table 9, alpha-amylase variants of the present invention can reach similar application effect to Liquzoyme Supra.Say
Bright its can apply to corn alcohol industry.
In summary, according to the experimental result in the present invention, alpha-amylase variants of the present invention have preferable heat resistance, pH resistance to
By property, the liquefaction of high concentration starch slurry is can apply to, therefore can apply to starch sugar industry and alcohol industry.
Embodiments of the invention are in addition to applying technical method in the art, the guidance of more too busy to get away inventive concept.
Therefore, the present invention is not limited only to disclosed specific embodiment, more to cover the revised provision in spirit and scope of the invention, in detail
See claim.
Claims (13)
1. a kind of alpha-amylase variants, it is characterised in that described alpha-amylase variants pass through the α starch in B.licheniformis
Enzyme N-terminal increases by two extra amino acid A and N, and increases by five extra amino acid KTTVS in C-terminal and obtain, while still
Maintain the ability of the glycosidic bond of parental generation hydrolyzing alpha -1,4.
2. alpha-amylase variants according to claim 1, it is characterised in that the amino acid sequence of described alpha-amylase variants
As shown in SEQ ID NO.4 in sequence table.
3. alpha-amylase variants according to claim 2, it is characterised in that the nucleotide coding of described alpha-amylase variants
Sequence is as shown in SEQ ID NO.3 in sequence table.
4. the gene of coding alpha-amylase variants according to any one of claims 1 to 3.
5. gene according to claim 4, it is characterised in that nucleotide sequence is as shown in SEQ ID NO.3 in sequence table.
6. the expression vector for expressing alpha-amylase variants according to any one of claims 1 to 3, it is characterised in that contain
The gene of coding alpha-amylase variants described in claim 4 or 5.
7. expression vector according to claim 6, it is characterised in that described expression vector includes mainly natural by one
Or the promoter sequence of synthesis, a natural or synthetic ribosome bind site, a natural or synthetic terminator sequence
Row, and the gene order of the coding alpha-amylase variants described in claim 4 together constitute an expression component.
A kind of 8. recombinant cell for being used to express alpha-amylase variants according to any one of claims 1 to 3, it is characterised in that
Include the gene of the coding alpha-amylase variants described in claim 4.
9. recombinant cell according to claim 8, it is characterised in that the host cell of recombinant cell is selected from Bacillus bacterium
Strain, preferably B.licheniformis or the Bacillus bacterial strains that some endogenous proteins have been inactivated by genetic engineering transformation.
10. recombinant cell according to claim 9, it is characterised in that the host cell of recombinant cell, which is selected from, passes through gene work
Journey transforms the B.licheniformis for having inactivated AprE and/or Blase.
11. a kind of production method of alpha-amylase variants according to any one of claims 1 to 3, it is characterised in that be included in suitable
The recombinant cell containing coding alpha-amylase variants gene order is cultivated under conditions of suitable alpha-amylase variants expression, and from
Alpha-amylase variants are obtained in recombinant cell or its culture supernatant.
12. application of the alpha-amylase variants according to any one of claims 1 to 3 in α -1,4 glycosidic bonds of Polysaccharides.
13. application according to claim 12, it is characterised in that described alpha-amylase variants are in high temperature and/or low pH bars
Application under part in α -1,4 glycosidic bonds of Polysaccharides；Preferably 80 DEG C~110 DEG C of described high temperature, further preferred 100 DEG C~
110℃；Described low pH preferable ph is 5.0~5.8.
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| CN101668855A (en) * | 2007-03-23 | 2010-03-10 | 丹尼斯科美国公司 | Enhanced amylase production by n-terminal addition to mature amylase protein |
| CN101815783A (en) * | 2007-05-30 | 2010-08-25 | 丹尼斯科美国公司 | The improvement variant of bacillus licheniformis alpha-amylase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101668855A (en) * | 2007-03-23 | 2010-03-10 | 丹尼斯科美国公司 | Enhanced amylase production by n-terminal addition to mature amylase protein |
| CN101815783A (en) * | 2007-05-30 | 2010-08-25 | 丹尼斯科美国公司 | The improvement variant of bacillus licheniformis alpha-amylase |
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| Title |
|---|
| LEE S等: ""Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis alpha-amylases and insights into engineering alpha-amylase variants active under acidic conditions."", 《J BIOCHEM.》 * |
| YAMABHAI，M.等: ""Genbank序列登录号：FJ556804.1"", 《GENBANK》 * |
| YUUKI T.等: ""UniProt：P06278"", 《CHEMBI》 * |
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