CN107201373B

CN107201373B - A kind of maltogenic amylase and its gene, the engineering bacteria containing the gene and its application

Info

Publication number: CN107201373B
Application number: CN201610158814.7A
Authority: CN
Inventors: 崔中利; 周杰; 李周坤; 吴佳乐; 黄彦
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2016-03-18
Filing date: 2016-03-18
Publication date: 2019-09-24
Anticipated expiration: 2036-03-18
Also published as: CN107201373A

Abstract

The invention discloses a kind of maltogenic amylase and its genes, the engineering bacteria containing the gene and its application.The present invention provides a kind of maltogenic amylase gene for producing ultra-high purity maltose, nucleotide sequences are as follows: the encoded maltogenic amylase enzyme amino acid sequence of SEQ ID NO.1 are as follows: SEQ ID NO.2.Using the gene constructed pichia pastoris engineered strain energy high efficient expression maltogenic amylase, which can efficiently hydrolyze starch and generate ultra-high purity maltose, and maltose content hydrolyzes up to 90% or more and is free of glucose in final product.Its Rate activity is up to 980U/mg when the enzyme is using soluble starch as substrate.It can be used for the industries such as food, frozen food, baking, brewing, beverage industry and medicine using the enzyme preparation that gene produces, considerable economic benefit can also be obtained while solving practical problems.

Description

A kind of maltogenic amylase and its gene, the engineering bacteria containing the gene and its application

Technical field

The invention belongs to applicable industry microorganism field, discloses a kind of maltose alpha-amylase gene, contains the gene Engineering bacteria and its application.

Background technique

Starch is by glucose monomer unit with glucoside bond as one of polymer the most abundant of content on the earth It is formed by connecting, two kinds of important components: amylose (linear α-Isosorbide-5-Nitrae key connection glucan) is divided into according to the difference of glycosidic bond With amylopectin (being connected with α -1, the branch of 6 keys on linear α-Isosorbide-5-Nitrae key connection glucan), 6% of total glycosidic linkages or so is accounted for α -1,6 key makes starch sugar chain form branched structure.Starch Hydrolysis can provide energy, while its water for different life entities Solution product is also widely used in the various aspects of human lives.It is formed into the hydrolysate of starch by one kind is common Point-maltose (Maltose), maltose is the reducing disaccharides that two glucose units are combined by α-Isosorbide-5-Nitrae glycosidic bond, point Minor C₁₂H₂₂O₁₁, chemical name is 4-O- α-D- glycopyranosyl-D-Glucose, molecular weight 342.30, because of C_lThe position of hydroxyl Different and have α-and β-two kinds of isomers, structural formula is as follows:

Free maltose is not present in nature, can only be generated by Starch Hydrolysis.Maltose syrups are due to some Outstanding advantages, such as nutritive value height, sweet taste is mild, sugariness only has the 1/3 of sucrose, and do not participate in insulin metabolism, and viscosity is low, inhales Tide is low, and crystallinity is low, high temperature resistant, clear and transparent.It has in the application aspect of frozen food, baking, brewing and beverage industry There is the maltose of very high commercial value, especially high-purity to have important purposes in medical industry.Maltose syrups press it Middle maltose content can be divided into three classes: first is that maltose content < 50% is common maltose syrups；Second is that maltose content It is high malt sugar syrup between 50-75%；Third is that maltose content > 80% is then superelevation maltose syrups.Domestic market Malt sugar product also have a superhigh maltose syrup product based on maltose, high maltose syrup, but ultra-high purity maltose and Crystal malt sugar product is seldom.

Maltogenic alpha-amylase (EC3.2.1.133, maltogenic α-amylase), the full name Portugal Isosorbide-5-Nitrae-α-D- are poly- Sugar-α-malt-base hydrolase, is commonly called as maltogenic amylase, be acted in alpha-amylase family starch and related polysaccharide with Maltose unit is hydrolyzed in the direction of the clock from the non-reducing end of starch chain, and principal product is α-maltose one kind Enzyme belongs to glycoside hydrolase Families GH13.Maltogenic amylase enzymatic property is very unique, cuts at random different from typical amylase α-Isosorbide-5-Nitrae key glycosidic bond in starch, it not only can fast hydrolyzing utilize amylose or cyclodextrine, can also utilize branch on a small quantity Starch has more substrate specificities；But also there is a degree of transglycosylation.

The maltogenic amylase of separate sources produce maltose ability and in terms of there is also differences.Source In Penicillium expansum high yield maltogenic amylase, molecular weight 69kDa, optimal pH 4.5, in pH 3.6-6 model It encloses interior holding to stablize, optimum temperature is 60 DEG C, and maltose content is up to 74% in hydrolyzed starch product, but has 21% Portugal simultaneously Grape sugar generates.Amylase optimal pH from the high yield maltose of Streptomyces sp.IMD 2679 is 5.5, most thermophilic Degree is 60-65 DEG C, is 3042U/mg by the Rate activity of substrate of soluble starch, maltose content is up in hydrolysate 79%, but the glucose of simultaneous 13% generates.Maltose is produced from Rhizopus oryzae CICIM 0071 to form sediment Powder enzyme molecular weight is 48kDa, optimal pH 4.0-6.0, keeps stablizing in 4.5-6.5 range, and optimum temperature is 60 DEG C, is lower than 50 DEG C keep stablize.It is 1123U/mg by the Rate activity of substrate of soluble starch, the maltose that hydrolyse potato starch produces contains Amount up to 74%, wherein glucose content is 14% in hydrolysate.

Maltogenic amylase from Thermomonospora curvata NCIMB 10081 is with soluble starch Rate activity when substrate is 5400U/mg, and the molecular weight of albumen about 62kDa, optimal pH 6.0, optimum temperature is 65 DEG C, malt Sugared content is up to 73%, and glucose is not generated in hydrolytic process, but has a large amount of maltotrioses to remain.The proteolysis starch generates Maltose content need 20h when reaching highest, while when using 20mM maltotriose as substrate, reacting maltose after 30min Content only has 10%, illustrates that the albumen shows lower catalytic efficiency while high yield maltose.

It is such a to maltogenic amylase that there is important work as people further increase maltose quality requirement The research of the novel enzyme preparation of industry application value has become hot spot.Foreign countries have carried out a large amount of research to maltogenic amylase, several Large-scale enzyme preparation company such as Novi's letter, Danisco, family etc., existing maltogenic amylase emerges, but expensive.The country is to wheat The research of bud saccharogenic amylase is still in the primary stage, therefore domestic maltogenic amylase used at present relies on import substantially.Mesh Preceding maltose production is to handle starch by fungal amylase and beta amylase, is generated by glucose, maltose, malt three The common malt syrup of the malto-oligosaccharides such as sugar, tetrose and polysaccharide composition.High maltose syrup is obtained, also to add de- branch again Enzyme and beta amylase collective effect, or common malt syrup is post-processed with glucoamylase etc..Due to these productions All there are the by-products such as the oligosaccharide malts oligosaccharides such as glucose, maltotriose, tetrose in the process, seriously affect its maltose quality, So that the maltose purity of enzyme process preparation is difficult to reach the requirement of superhigh maltose syrup, it is therefore desirable to which subsequent technique further removes Glucose, this be just production process it is additional increase cost, limit the application range of albumen.

Summary of the invention

The purpose of the present invention is to provide a kind of new maltogenic amylase genes, and its protein of coding.

It is a further object of the present invention to provide the engineering strains containing the maltogenic amylase gene.

It is yet another object of the invention to provide the applications of the gene and its coding protein.

A kind of maltogenic amylase gene, nucleotide sequence are as follows: SEQ ID NO.1, overall length is (from initiation codon to end Only codon) it is 1665bp, G+C content is 69.19%, encodes 554 amino acid.

The maltogenic amylase zymoprotein of maltogenic amylase gene coding of the present invention, amino acid sequence are as follows: SEQ ID NO.2。

Maltogenic amylase optimal reaction pH of the present invention be 7.0, optimal reactive temperature be 50 DEG C, and 20 DEG C- Keep activity stabilized between 40 DEG C (5h) and pH 6.0-9.0 (for 24 hours), and enzyme activity does not depend on Ca²⁺Ion.Using starch as substrate When generate superelevation maltose, maltose content can be up to 90% or more, and hydrolyze final product and do not contain glucose.

Recombinant plasmid containing maltogenic amylase gene of the present invention.

The maltogenic amylase gene is preferably cloned into gained in pEFaA by the recombinant plasmid.

Recombinant microorganism containing recombinant plasmid of the present invention.

The recombinant microorganism, preferably with Pichia pastoris (Pichia pastoris GS115) for host strain.

Maltogenic amylase zymoprotein of the present invention is in Starch Hydrolysis or superelevation maltose industrial production and food processing The application of aspect.

Maltogenic amylase zymoprotein joint α -1,4 amylase restriction endonuclease of the present invention is producing answering in superelevation maltose With.

The preferred number of patent application of α -1,4 amylase restriction endonuclease is that " 201310043628.5 " (international application no is PCT/CN2013/078680), in entitled " a kind of alpha-amylase and its gene, the engineering bacteria containing the gene and its application " α -1,4 amylase the restriction endonuclease of protection.The encoding gene of the α -1,4 amylase restriction endonuclease such as proprietary sequence table SEQ ID Shown in NO.1, amino acid sequence is as shown in proprietary sequence table SEQ ID NO.2.Expression, zymologic property, function are at this It is disclosed in patent, has been detailed in the disclosure of the patent.

Beneficial effect

1. the present invention using slime bacteria bacterial strain EGB as material, with reference to EGB Genomic sequence information and combines PCR amplification, success Obtain maltogenic amylase gene sequence.The full length gene (from initiation codon to terminator codon) is 1665bp, G+C content It is 69.19%, encodes 554 amino acid.

2. expanding the complete maltogenic amylase gene piece of end I containing Xho and Xba I restriction enzyme site by round pcr Section connects it to the Xho I of Pichia pastoris P.pastoris GS115 high-expression vector pEFaA (purchased from Novegen company) On Xba I restriction enzyme site, conversion expression host strain P.pastoris GS115 (being purchased from Invitrogen company) carries out methanol Inducing expression, inducing expression amount are 64mg/L.

3. the present invention carries out enzyme to maltogenic amylase to the product of maltogenic amylase gene expression, by DNS method Determination of activity, the maltogenic amylase can effectively act on soluble starch, cornstarch, potato starch, corn dextrin, directly Chain starch, amylopectin and various Fructus Hordei Germinatus oligoses generate ultra-high purity maltose, and maltose content is produced up to 90% or more, and eventually Do not occur glucose in object.Rate activity when using soluble starch as substrate is up to 980U/mg.

4. the gene constructed engineered strain energy high efficient expression maltogenic amylase is utilized, using starch as substrate hydrolysis mistake Cheng Zhong, maltogenic amylase joint, which produces maltose amylase, can efficiently improve malt sugar yield, wherein maltose content Improve nearly 5 times.

Detailed description of the invention

The PCR amplification electropherogram of Fig. 1 maltogenic amylase gene

1:DL5000 nucleic acid Marker；2: maltogenic amylase gene PCR amplification

The policy map of Fig. 2 maltogenic amylase gene clone

Fig. 3 maltogenic amylase gene high efficient expression experimental program figure in P.pastoris GS115 (pEFaA)

The SDS-PAGE electrophoresis of Fig. 4 recombination maltogenic amylase

M: standard protein Marker；1: the pure enzyme of recombinant protein；2: with the enzyme spectrum analysis of soluble starch plate

Fig. 5 maltogenic amylase zymologic property

A: maltogenic amylase optimal pH；B: optimum temperature；C: maltogenic amylase pH stability；D: temperature stability

The Ca of Fig. 6 maltogenic amylase²⁺Ion is not dependent

Fig. 7 maltogenic amylase catalysis characteristics

A: thin-layer chromatography TLC analysis chart of the maltogenic amylase to starch substrate hydrolysis product

1-7 malto-oligosaccharide standard items (G1-G7)；

8-13: being glycogen, amylose, amylopectin, soluble starch enzyme, potato starch, the not enzyme place of dextrin respectively Reason control；

14-19: being different malto-oligosaccharide standard items G2, G3, G4, G5, G6 respectively, and G7 adds maltogenic amylase enzymatic treatment Product；

20-25: glycogen, amylose, amylopectin, soluble starch, potato starch, dextrin add at maltogenic amylase Manage product；

B: the thin-layer chromatography TLC analysis chart of maltogenic amylase enzymatic maltotriose

1-6: malto-oligosaccharide standard items (G1-G6)；

7-29: being maltogenic amylase hydrolysis respectively in 20mM maltotriose 5min, 10min, 20min, 30min, 40min, 50min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, for 24 hours, the production after 36h Object；

Fig. 8 maltogenic amylase combines the application that α -1,4 amylase restriction endonuclease produces superelevation maltose in hydrolysis starch

A: standard sample control；

The independent processing soluble starch products of B: α -1,4 amylase restriction endonuclease；

C: the independent processing soluble starch products of maltogenic amylase；

D: α -1,4 amylase restriction endonuclease and maltogenic amylase enzyme Combined Treatment product

Biomaterial preservation information

Corallococcus coralloldes EGB, is preserved in China typical culture collection center, and preservation address is Wuhan, China, Wuhan University, the deposit date is on December 17th, 2012, deposit number was CCTCC NO:M2012528.

Specific embodiment

The clone of 1 maltogenic amylase gene of embodiment and the building of expression vector

The PCR amplification of 1.1 maltogenic amylase genes

With reference to slime bacteria EGB (CCTCC NO:M2012528) genome sequence and NCBI genomic information is combined to carry out ORF Prediction designs maltogenic amylase gene primer with full length sequence, using the genomic DNA of EGB bacterium as template, carries out maltose shallow lake The PCR amplification of powder enzyme gene overall length obtains the full length sequence of maltogenic amylase gene, and the primer is F and R, as a result sees figure 1.Detailed process is referring to Fig. 2.

F:ctcgagAAAAGAGAGCGCCCCCTCCGCGGACTCTC(Xho I)(SEQ ID NO.3)

R:tctagaTTAATGATGATGATGATGATGGCGCAGCCGCCAGATGCC(Xba I)(SEQ ID NO.4)

PCR amplification system are as follows:

PCR amplification condition:

Pcr amplification product product carries out 0.75% agarose gel electrophoresis gel extraction.

The preparation of the 1.2 common competence of E.coli DH5 α

Standardized ring thallus E.coli DH5 α scribing line is connected on SOB plate in the strain saved from -80 DEG C, 37 DEG C of cultures 16 ~20h chooses the 250mL triangular flask for being seeded to the culture medium of SOB containing 25mL with sterile toothpick picking diameter in the bacterium colony of 2mm or so In, 37 DEG C with 250rmin^-1Speed oscillation 6~8h of culture is inoculated with 2mL respectively, and 4mL, 10mL culture solution is in equipped with 250mL In the 1L triangular flask of SOB fluid nutrient medium, with 200rpm speed oscillation overnight incubation in 18 DEG C of shaking tables.Pass through spectrophotometer Measure the density (OD of thallus₆₀₀Nm), the culture solution that OD is approximately equal to 0.45 is taken, culture is first placed in 10min on ice, 4 DEG C, 4,000rpm centrifugation 10min, abandon liquid, centrifuge tube are tipped upside down on 2min on blotting paper and is drained off remaining culture medium, adds 80mL pre- Cold 0.1mM CaCl₂Conversion buffer has gently hanged thallus in ice-water bath, and in 4 DEG C, 4,000rpm centrifugation 10min are abandoned Upper layer buffer simultaneously buckles dry, to be pre-chilled again with 20mL 0.1mM CaCl on blotting paper₂It converts buffer and bacterial precipitation is resuspended, Add 1.5mL DMSO.Bacterial suspension is mixed gently, 10min on ice is placed in, is sub-packed in 1.5mL sterile centrifugation tube by every 200 μ L of pipe In, quick-frozen in liquid nitrogen, as Efficiency Competent Cells are set, it is stand-by that -80 DEG C of refrigerators can be transferred to.

1.3 enzymes even convert

3:1 is mixed in molar ratio for the maltogenic amylase DN Α segment and pMD19-T Vector (TaKaRa) generated through PCR It closes, under connection enzyme solution effect, 16 DEG C of water-baths are stayed overnight.Enzyme disjunctor system is as follows:

10 μ L enzyme-linked products are added in the E.coli DH5 α competent cell after 200 μ l melt on ice, ice bath 30min, in 42 DEG C of water-baths after heat shock 90s.It is quickly transferred in ice bath cooling 3min, 800 μ L liquid are added into every pipe LB culture medium, 37 DEG C of shaking table 80-90rpm incubate 45min, recovery cell.4000rpm is centrifuged 3min, and remaining 200 μ L competence are thin Born of the same parents are coated on the LB agar plate of the ampicillin containing 100mg/L, and plate is inverted in 37 DEG C of incubator cultures.

The extraction and sequencing of 1.4 target gene plasmids

Overnight incubation in single colonie LB culture medium with ampicillin in picking 1.3,6000rpm are centrifuged 1min and collect Thallus extracts plasmid using plasmid extraction kit, serves the sequencing of extra large Invitrogen biology Co., Ltd.As a result the full length gene (from initiation codon to terminator codon) is 1665bp, and G+C content is 69.19%, and sequence is SEQ ID NO.1；The gene 554 amino acid are encoded, amino acid sequence is SEQ ID NO.2.

1.5 by the recombinant plasmid extracted in 1.4 Xho I and Xba I double digestion

System is as follows:

Reaction system is placed in reaction overnight in 37 DEG C of water-baths, the rear 1% Ago-Gel nucleic acid electrophoresis that runs recycles enzyme Cut product.

1.6 by expression vector pEF α A (being purchased from Novegen) with Xho I and Xba I double digestion (refer to 1.5)

1.7 enzymes even convert

System is as follows:

System is placed in reaction overnight in 16 DEG C of water-baths and obtains the recombinant plasmid pEF α A-amyZ containing target gene.It will After enzyme-linked product is transformed into E.coli DH5 α, the LLB culture medium for being coated on the Zeocin containing final concentration of 25 μ g/mL is flat On plate, picking transformant plasmid and digestion verification are stored in -80 DEG C of low temperature refrigerators of final concentration of 15% glycerol after sequencing is correct In.

The linearisation of 1.8 recombinant plasmid pEF α A-amyZ

Correct recombinant plasmid is linearized using restriction enzyme Nde I, system is placed in 37 DEG C of water-baths Under the conditions of react 8h after recycle digestion products.

Reaction system is as follows:

This experiment linearizes product using ethanol precipitation purification and recovery, and the specific method is as follows:

1. the dehydrated alcohol of 3M sodium acetate (pH 5.2) and 2 times of volumes of 1/10 volume of pre-cooling is added, after mixing gently It is placed in 20min at -20 DEG C；

2. the then 12000rpm under the conditions of 4 DEG C, is centrifuged 10min, supernatant is abandoned；

3. 70% ethyl alcohol of pre-cooling of 2 times of volumes is added into centrifuge tube, 12000rpm is centrifuged 3min, is repeated once, in abandoning Clearly；

4. the dissolution of 30 μ L sterile waters is added after ethyl alcohol volatilization is clean.

The preparation of 1.9 P.pastoris GS115 competent cells

From the P.pastoris GS115 being seeded on the fresh inclined-plane YPD (purchased from China typical culture collection center) Picking single colonie；Inoculation single bacterium drops down onto the 250mL triangular flask of 50mL YPD culture medium, and 28 DEG C, 200rpm, overnight incubation is extremely OD₆₀₀Value is between 1.0-1.5；Culture solution is drawn to be seeded to by 5% inoculum concentration in the 250mL triangular flask of 50mL YPD culture medium, 28 DEG C, 200rpm, culture to OD₆₀₀Value is between 0.3-0.5；4500rpm is centrifuged 5min, collects thallus；By thallus with it is fresh After the 8mL yeast cells competence mother liquor of preparation mixes gently, it is placed at room temperature for 30min, every 10min gently shakes up once； 4500rpm is centrifuged 5min, collects thallus；It is resuspended with the 1M sorbierite of the pre-cooling of 2mL, then under the conditions of 4 DEG C, 4000rpm, It is centrifuged 5min, collects thallus, in triplicate；Then it is resuspended, is dispensed into 1.5mL centrifuge tube with the 1M sorbierite of 100 μ L pre-cooling, Every 80 μ L of pipe, saves at -80 DEG C.

1.10 electrotransformation

0.2cm electrotransformation cup is placed in 5min on ice, opens electric converter, electrotransformation parameter needed for adjusting: voltage 1.5kV, 250 Ω of resistance, 25 μ F of capacitor；The DNA linearized in 1-3 μ g 1.6 is added into the competent yeast cells prepared Segment is transferred in electric revolving cup after being placed in 5min on ice after mixing gently；Electricity, which is carried out, according to instrument operation instructions turns operation； The 1M sorbierite of 1mL pre-cooling is added after electrotransformation into electric revolving cup at once, is transferred in 1.5mL centrifuge tube, is placed on 30 DEG C of cultures Stationary culture 1h in case；3min is centrifuged under 4000rpm at room temperature, abandons supernatant, is resuspended after collecting thallus with 200 μ L YPDS；It will 100 μ L cell suspending liquids are coated on the YPD plate containing 100 μ g/mL Zeocin；Plate is placed at 30 DEG C and cultivates 3-4 It, grows to thallus.

The single colonie grown on plate is chosen to the YPD plate containing 100 μ g/mL Zeocin, mesh is used after growing Gene characteristic primer carry out bacterium colony PCR verifying, whether be integrated into yeast chromosomal with testing goal gene；25 μ L reaction System, 60 DEG C of annealing temperature, extension of time 1min 45s.PCR product is big with 0.75% agarose gel electrophoresis detection DNA fragmentation It is small.It is engineering strain P.pastoris GS115 (pEF α A-amyZ) integrated therein with maltogenic amylase gene.

High efficient expression of 2. maltogenic amylase gene of embodiment in P.pastoris GS115 (pEFaA)

The expression of 2.1 maltogenic amylase AmyZ

By recombinant microorganism bacterial strain P.pastoris GS115 (pEF α A-amyZ) streak plate culture, picking single bacterium is dropped down onto In 50mL liquid YPD triangular flask, 28 DEG C, 200rpm is cultivated for 24 hours；Then 4500rpm, centrifugation 10min are discarded supernatant at room temperature Thallus 25mL BMMY culture medium is resuspended liquid, starts the expression for inducing yeast cells；Under the conditions of continuing 28 DEG C, 200rpm training It supports, every adding methanol for 24 hours to final concentration of 0.5% (v/v), co-cultures 96h；After cultivating, it is collected by centrifugation in fermentation Clear liquid is maltogenic amylase crude enzyme liquid.Appropriate maltogenic amylase crude enzyme liquid is taken to add to the soluble shallow lake that 1ml contains 0.5% In the Tris-HCl buffer of powder, after 50 DEG C of reaction 10min, the generation situation of reduced sugar is detected by DNS.Testing goal egg White enzyme activity, as a result, it has been found that the maltogenic amylase realizes high expression in Pichia pastoris.

The purifying of 2.2 maltogenic amylase AmyZ

Fermentation supernatant of the P.pastoris GS115 (pEF α A-amyZ) Jing Guo 96 hours inducing expressions is taken, using salting out method First fermentation enzyme solution is concentrated.Fractional precipitation is carried out by the ammonium sulfate saturated gradient of 0-80%.Operating procedure: by the enzyme solution that ferments Volume has been measured, has been poured into beaker, magnet rotor is placed in beaker, has placed the beaker on ice, opens magnetic stirring apparatus.By crude enzyme liquid Volume be slowly added to ammonium sulfate, so that saturation degree is reached 80%, 4 DEG C, 13,000rpm centrifugation 20min, precipitating is with not sulfur-bearing on a small quantity The buffer of the 50mM Tris-HCl pH7.0 of sour ammonium is resuspended, and measures protein content and enzyme activity.Enzyme solution after resuspension is at 4 DEG C The dialysed overnight in 50mM Tris-HCl pH7.0 buffer.By the enzyme solution loading after dialysis in advance with twice column volume The Ni of 50mM Tris-HCl pH7.0 balance²⁺- NTA affinity column, 4 DEG C of incubation 1h, with the 50mM Tris- of 5 times of column volumes HCl (pH7.0,50mM imidazoles) washes away foreign protein, then with 50mM Tris-HCl (pH7.0,100mM, 300mM imidazoles) progress Gradient elution collects the highest eluent of merger and reorganization enzyme purity through enzyme activity determination and SDS-PAGE electrophoresis, and 4 DEG C, with retention The bag filter of molecular weight 10KDa dialysed overnight in 50mM Tris-HCl (pH 7.0) buffer removes imidazoles, obtains maltose The pure enzyme solution of amylase.When taking the pure enzyme solution of appropriate maltogenic amylase using 0.5% soluble starch as substrate, maltogenic amylase The Rate activity of enzyme is 980U/mg.

2.3 protein electrophorese SDS-PAGE identification and renaturation zymogram

Polyacrylamide gel electrophoresis (SDS-PAGE) of the protein sample 12%, after the completion of electrophoresis, to containing enzyme sample Swimming lane carry out rubber tapping processing, be placed in containing pre-cooling 2.5%Triton-X 100 plate in, after 4 DEG C of placement 30min, Triton-X 100 is replaced, 30min is placed；Then band is transferred to the slow of the 0.1M pH 7.0Tris-HCl containing pre-cooling In fliud flushing, 4 DEG C, after 30min, buffer is replaced, continues to place 30min；Same operation is placed on 50mM pH 7.0Tris- In HCl buffer, time interval 15min；The good band of renaturation is spread over and is dissolved in 50mM pH 7.0Tris-HCl buffering On the soluble starch solid plate of liquid, after 37 DEG C of reaction 20min, iodine solution is added dropwise and develops the color.Due to maltogenic amylase AmyZ can hydrolyze starch so that it can not color reaction occur with iodine solution, so item takes active maltogenic amylase AmyZ Place will not generate royal purple colour response and form transparent circle, whether have work by the enzyme that this phenomenon can detect electrophoresis postcondition Property.Maltogenic amylase band after Fig. 4 display expression is more single, and the single band is maltogenic amylase to zymogram as the result is shown Expression of enzymes purpose band.

3. maltogenic amylase AmyZ zymology Quality Research of embodiment

Influence of 3.1 temperature to enzyme activity

The measurement of optimal reactive temperature: in different temperatures (20 DEG C, 30 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 70 DEG C), the activity of measurement recombination enzyme purification enzyme, is set as 100% (Fig. 5 B and D) for highest enzyme activity under conditions of pH7.0.It is hot steady Qualitatively measurement: recombination enzyme purification enzyme solution is kept the temperature under 20 DEG C, 30 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, pH7.0 1h is sampled every 10min, cooling rapidly on ice, respectively measures residual enzyme activity, is 100% (figure with uninsulated enzyme activity 5D).The amylase optimal reactive temperature is 50 DEG C after measured, and keeps stablizing between 20 DEG C -40 DEG C.

Influence of 3.2 pH to enzyme activity

The measurement of optimal reaction pH: in different pH value (3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0), 50 The activity of measurement recombination enzyme purification enzyme solution, is set as 100% (Fig. 5 A and C) for highest vigor at DEG C.The measurement of pH stability: will Enzyme purification enzyme solution is recombinated at pH 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0,4 DEG C keep for 24 hours, rear respective Its remaining vigor is measured, with the enzyme activity of pH 7.0 for 100% (Fig. 5 C).Maltogenic amylase optimal reaction pH is after measured 7.0, and keep stablizing in pH 6.0-9.0.

The Ca of 3.3 maltogenic amylases²⁺Ion is not dependent

(50mM Tirs-HCl, pH 7.0 contains 0.5% soluble starch), addition was whole respectively in amylase live body system Concentration is 1mM and 5mM Ca²⁺Ion measures various concentration Ca²⁺The influence of ions enzyme vigor.Not add any Ca²⁺Ion Reaction system compare.Enzyme activity determination is 50 DEG C of reaction 10min, detects remaining starch enzyme activity.1mM Ca after measured²⁺Ion The maltogenic amylase enzyme activity is influenced less, however in 5mM Ca²⁺The maltogenic amylase enzyme activity remnants are after ion processing 78% (such as table 1).

Table 1

It takes appropriate recombination maltogenic amylase AmyZ to be added in optimal reaction system, adds 1mM Ca²⁺Ion, then It is placed at 30 DEG C, 40 DEG C and keeps the temperature, timing sampling measures enzyme activity.Not add any Ca²⁺Ion detects Ca as control²⁺Ion Influence to enzyme stability.Show after measured in addition 1mM Ca²⁺After ion, which declines (Fig. 6), Show that the enzyme is one and does not depend on Ca²⁺The maltogenic amylase of ion.

3.4 maltogenic amylase substrate specificities

With soluble starch, potato starch, corn dextrin, amylopectin, amylose, pulullan polysaccharide, glycogen, A Ka Wave sugar and alpha-cyclodextrin, gamma-cyclodextrin are substrate, carry out substrate specificity to the maltogenic amylase of expression and analyze (such as table 2), as a result, it has been found that the maltogenic amylase forms sediment to soluble starch, potato starch, corn dextrin, amylopectin, straight chain The substrates such as powder, glycogen, pulullan polysaccharide, gamma-cyclodextrin all have activity, to acarbose and alpha-cyclodextrin without activity. Activity highest when wherein using soluble starch as substrate, the Rate activity of maltogenic amylase are 980U/mg.

Table 2

3.5 maltogenic amylase catalysis characteristics

Using the polysaccharide such as different Fructus Hordei Germinatus oligose and starch, dextrin, amylose, amylopectin, glycogen as substrate, take It is different to maltogenic amylase enzyme hydrolysis by thin-layer chromatography TLC after suitable maltogenic amylase is sufficiently reacted from different substrates The final product of substrate is tested and analyzed (Fig. 7 a).The result shows that maltogenic amylase has efficient conversion maltose ability, nothing By being Fructus Hordei Germinatus oligose or polysaccharide as substrate, final product is based on maltose, and glucose does not occur in product.

By the method for thin-layer chromatography (TLC) to maltogenic amylase with soluble starch, cornstarch, potato starch, Corn dextrin, amylose, the polysaccharide such as amylopectin and different Fructus Hordei Germinatus oligoses are as the sugared ingredient during substrate hydrolysis It is analyzed.For this sentences the hydrolytic process of hydrolysis maltotriose, takes suitable maltogenic amylase and concentration is 20mM's The mixing of maltotriose substrate is placed in 37 DEG C of reactions, respectively in 5min, 10min, 20min, 30min, 40min, 50min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, for 24 hours, 36h sampling, boiling water bath 3min carry out enzyme solution Inactivation treatment.Centrifuging and taking supernatant carries out TLC analysis.It will form various oligosaccharide malt oligosaccharides, including malt two at its hydrolysis initial stage Sugar, trisaccharide, tetrose, pentasaccharides, six sugar etc. have a small amount of glucose in hydrolysis mid-term and generate, but the final wheat for generating high-purity Bud sugar, and be free of glucose (Fig. 7 b).Show that the enzyme also has while cutting hydrolyzes starch glycosidic bond and turns very much glucosides work by force Property, so as to form high-purity malt sugar, maltose content reaches 90% or more, and is free of glucose.

4. maltogenic amylase AmyZ of embodiment combines related amylolytic enzyme and produces in superelevation maltose in hydrolysis starch Using

Due to, containing a large amount of amylopectin, causing amylorrhexis incomplete in starch, influencing the content of hydrolysate, And α-Isosorbide-5-Nitrae amylase restriction endonuclease Degree of Liquefaction is high, generates a large amount of oligosaccharide malt oligosaccharides, and the enzymatic hydrolysis effect of maltose can be improved Rate.Therefore maltogenic amylase cooperates the amylase of other liquefaction types that can significantly improve starch in maltose production application Utilization efficiency improves the output of maltose product.

It is quoted from according to 201310043628.5 patents, α-Isosorbide-5-Nitrae amylase restriction endonuclease is the starch of a high yield maltose Hydrolase.This patent is used in combination using the α-Isosorbide-5-Nitrae amylase restriction endonuclease with maltogenic amylase of the present invention, to improve starch water Solve the maltose content in product.Using soluble starch as substrate, it is added in every g soluble starch in α-Isosorbide-5-Nitrae amylase of 9U Enzyme cutting, 45 DEG C of processing 1h, rear maltogenic amylase/g starch that 10U is added, 37 DEG C handle r for 24 hours, high performance liquid chromatography detection wheat The content of bud sugar.Liquid phase chromatogram condition: Agilent liquid chromatogram, Cosmosil Sugar-D nh 2 column, ELSD ES2000 evaporation Light scattering detector, mobile phase are acetonitrile/water=65/35 (v/v), and flow velocity is 1mL/min, 30 DEG C of column temperature.Shown in Fig. 8, malt Saccharogenic amylase and α-Isosorbide-5-Nitrae amylase restriction endonuclease Combined Treatment (Fig. 8 D), compared with single maltogenic amylase enzymatic treatment (Fig. 8 C), 5 times of the output increased of maltose.

Claims

1. maltogenic amylase gene, which is characterized in that the nucleotide sequence of the gene are as follows: SEQ ID NO.1.

2. the maltogenic amylase of maltogenic amylase gene coding described in claim 1, which is characterized in that the maltose The amino acid sequence of amylase are as follows: SEQ ID NO.2.

3. the recombinant plasmid containing maltogenic amylase gene described in claim 1.

4. recombinant plasmid according to claim 3, which is characterized in that the recombinant plasmid is will be described in claim 1 Maltogenic amylase gene is cloned into gained in pEFaA.

5. the recombinant microorganism containing recombinant plasmid as claimed in claim 3.

6. recombinant microorganism according to claim 5, which is characterized in that using Pichia pastoris GS115 as host strain.

7. application of the maltogenic amylase gene described in claim 1 in terms of genetic engineering produces superelevation maltose syrups；Institute The superelevation maltose syrups stated refer to the maltose syrups of maltose mass content > 80%.

8. maltogenic amylase as claimed in claim 2 is in Starch Hydrolysis, industrial production superelevation maltose syrups or food processing The application of aspect；The superelevation maltose syrups refer to the maltose syrups of maltose mass content > 80%.

9. maltogenic amylase as claimed in claim 2 combines other liquefying amylases in Starch Hydrolysis or superelevation maltose sugar Application in terms of slurry industrial production or food processing；The superelevation maltose syrups refer to the wheat of maltose mass content > 80% Bud sugar syrup.

10. according to application described in right 9, which is characterized in that other described liquefying amylases are α-Isosorbide-5-Nitrae amylase inscribe Enzyme.

11. application according to claim 10, it is characterised in that the amino acid sequence of other liquefying amylases As shown in SEQ ID NO.5.