CN107189998B - A kind of sucrose isomerase enzyme mutant and its application - Google Patents

A kind of sucrose isomerase enzyme mutant and its application Download PDF

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CN107189998B
CN107189998B CN201710257308.8A CN201710257308A CN107189998B CN 107189998 B CN107189998 B CN 107189998B CN 201710257308 A CN201710257308 A CN 201710257308A CN 107189998 B CN107189998 B CN 107189998B
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罗科
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Luo Ke
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Wilkin LLC
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99011—Isomaltulose synthase (5.4.99.11)

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Abstract

The invention discloses a kind of sucrose isomerase enzyme mutant and its applications.The present invention provides a kind of protein, are named as PRSi-QRY albumen, as shown in the sequence 3 of sequence table.The gene of coding PRSi-QRY albumen also belongs to protection scope of the present invention.The present invention also protects PRSi-QRY albumen as the application of sucrose isomerase.The present invention also protects a kind of recombinant bacterium, is the recombinant bacterium for obtaining recombinant plasmid pPicZa-PRSi-QRY importing Pichia pastoris X33；The recombinant plasmid pPicZa-PRSi-QRY is the recombinant plasmid for obtaining gene insertion pPICZA carrier.The present invention also protects a kind of method for producing sucrose isomerase, includes the following steps: the recombinant bacterium that ferments, obtains sucrose isomerase.The present invention has very huge application prospect and industrial value.

Description

A kind of sucrose isomerase enzyme mutant and its application
Technical field
The present invention relates to a kind of sucrose isomerase enzyme mutant and its applications.
Background technique
Isomaltoketose is naturally present in honey, and content can be up to 1%, in sugar-cane juice there is also.Different malt Ketose is a kind of functional sweetener, and natural taste is pure, non-hygroscopic, is not deliquesced, not cariogenic, and there are many health-care effects.Different wheat The health characteristic of bud ketose is mainly in the following aspects: (1) securely and reliably, can large amounts of food；The safety of isomaltoketose The abundant approval of Japanese health ministry, food safety department, European Union, U.S. FDA etc. is obtained；(2) it not will form plaque, more It can inhibit the formation of decayed tooth；Food and drug administration (FDA) ratifies isomaltoketose as non-cariogenicity sugar source；(3) Lasting, slow, balanced offer energy improves anti-fatigue ability；(4) disaccharides such as sucrose, glucose are stabilized to blood glucose fluctuation It influences, blood glucose is inhibited to rise；(5) inhibit Fat Accumulation, promote the oxygenolysis of body fat；(6) improve cerebral function, keep Brain attention is concentrated；(7) sweet taste is pure, can cover peculiar smell (e.g., DHA fish oil fishy smell, beany, ginseng, blueberry, apricot The peculiar smell of benevolence), it can also balance mouthfeel, improve food flavor.Due to these features of isomaltoketose, it is widely used in functional sugar Fruit, energy slow-release sports products, generation meal based food and sugarfree foods etc. exploitation, become most popular sucrose succedaneum, have Vast market prospect.Currently, isomaltoketose is in global marketing amount, oneself is reached 300,000 tons, and the output value is more than 1,000,000,000 dollars.
Sucrose isomerase (sucrose isomerase, EC5.4.99.11) is the key enzyme for producing isomaltoketose.Mesh Before, the method for producing isomaltoketose mainly passes through sucrose isomerase enzymatic conversion sucrose.Currently used for production isomaltoketose Wild strain or expression of recombinant e. coli sucrose isomerase ability be not high, and production of enzyme is very low, and is all desmoenzyme, and sucrose is molten Liquid needs to enter by cell membrane into the cell could be with sucrose isomerase enzyme reaction.This process affects isomaltoketose yield, Increase the cost of production.It is unfavorable for large-scale production.In addition, microbe-derived sucrose isomerase enzymatic forms different malt ketone When sugared, the by-products such as trehalulose are also generated.Therefore, the specificity and table for improving sucrose isomerase are transformed by protein molecule Up to amount, isomaltoketose production efficiency can be improved, reduce industrial production cost.
Summary of the invention
The object of the present invention is to provide a kind of sucrose isomerase enzyme mutant and its applications.
The present invention provides a kind of protein, are named as PRSi-QRY albumen, for following (a) or (b) or (c):
(a) protein that the amino acid sequence shown in sequence 3 in sequence table forms；
(b) fused protein obtained in N-terminal or/and C-terminal the connection label of (a)；
(c) protein that the amino acid sequence shown in sequence 5 in sequence table forms.
The gene of coding PRSi-QRY albumen also belongs to protection scope of the present invention.
The gene is following (d1) or (d2) or (d3) or (d4):
(d1) code area DNA molecular as shown in the sequence 4 of sequence table；
(d2) DNA molecular shown in the sequence 4 of sequence table；
(d3) code area DNA molecular as shown in the sequence 6 of sequence table；
(d4) DNA molecular shown in the sequence 6 of sequence table.
Expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing the gene belong to protection of the invention Range.
The present invention also protects PRSi-QRY albumen as the application of sucrose isomerase.
The present invention also protects application of the PRSi-QRY albumen in production isomaltoketose.
The present invention also protects PRSi-QRY albumen producing the application in isomaltoketose using sucrose as raw material.
The present invention also protects a kind of recombinant bacterium, is to obtain recombinant plasmid pPicZa-PRSi-QRY importing Pichia pastoris X33 Recombinant bacterium；The recombinant plasmid pPicZa-PRSi-QRY is the recombinant plasmid for obtaining gene insertion pPICZA carrier. Recombinant plasmid pPicZa-PRSi-QRY is concretely by XhoI the and NotI digestion position of gene forward direction insertion pPICZA carrier The recombinant plasmid obtained between point.
The present invention also protects application of the recombinant bacterium in production sucrose isomerase.
The present invention also protects a kind of method for producing sucrose isomerase, includes the following steps: the recombinant bacterium that ferments, obtains Sucrose isomerase.
The method is specially method first or method second.
In the method first, the design parameter of the fermentation is as follows:
Seed liquor is seeded to basal medium to ferment；
The control parameter of entire fermentation process: 25-30 DEG C of temperature, pH 4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5-1vvm, pressure 0.03-0.05MPa, DO >=20%；
Glucose solution is added as first time DO >=80% with the flow velocity of 10mL/h/L in lasting culture, until thallus is wet Stop that glucose solution is added when weighing to 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol solution is added with the flow velocity of 1-3mL/h/L after 1h (initial flow rate 1mL/h/L, flow velocity are incremented by with the speed linearity of 0.1mL/h/L, until flow velocity keeps being somebody's turn to do after reaching 3mL/h/L Flow velocity is constant)；
Entire fermented incubation time is 72h.
In the method first, the parameter of the fermentation is as follows:
Seed liquor is seeded to basal medium to ferment；
The control parameter of entire fermentation process: 25-30 DEG C of temperature, pH4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5- 1vvm, pressure 0.03-0.05MPa, DO >=20%；
As first time DO >=80% glucose solution is added with the flow velocity of 5-20mL/h/L, until thallus in lasting culture Weight in wet base stops that glucose solution is added when reaching 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol is added with the flow velocity of 1-3mL/h/L after 1-1.5h Solution (according to the flow velocity of dissolved oxygen and thallus culture situation control methanol solution with the speed increase of 0.1-0.2ml/h/L, works as flow velocity It is not further added by when reaching 3mL/h/L).
In the method first, seed culture medium concretely: the PBS buffer solution of solvent pH6.0,0.1M；Solute and its Concentration is as follows: yeast extract 1g/100mL, peptone 2g/100mL, glycerol 1g/100mL, YNB1.34g/100mL, biotin 4 ×10-5g/100mL。
In the method first, basal medium (pH5.0) concretely: solvent is water；Solute and its concentration are as follows: grape Sugared 2g/100mL, 85% phosphatase 24 .5g/100mL, calcium sulfate 0.93g/100mL, potassium sulfate 1.82g/100mL, epsom salt 1.5g/100mL, potassium hydroxide 0.4g/100mL, PTM1 solution 0.44mL/100mL.
In the method first, glucose solution concretely: solvent is water；Solute and its concentration are as follows: glucose 50g/ 100mL, PTM1 solution 1.2mL/100mL.
In the method first, methanol solution concretely: solvent is methanol；Solute and its concentration are as follows: PTM1 solution 1.2mL/100mL。
In the method second, the design parameter of the fermentation is as follows:
Seed liquor is seeded to basal medium to ferment；
The control parameter of entire fermentation process: 29-31 DEG C of temperature, pH4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5- 1vvm, pressure 0.03-0.05MPa, DO >=20%；
As first time DO >=80% glycerite is added with the flow velocity of 10mL/h/L, until thallus weight in wet base in lasting culture Stop that glycerite is added when reaching 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol solution is added with the flow velocity of 1-3mL/h/L after 1h (initial flow rate 1mL/h/L, flow velocity are incremented by with the speed linearity of 0.1mL/h/L, until flow velocity keeps being somebody's turn to do after reaching 3mL/h/L Flow velocity is constant), start when the flow velocity of methanol solution reaches 3mL/h/L molten with the flow velocity addition sorbierite of 0.8mL/h/L simultaneously Liquid；
Entire fermented incubation time is 72h.
In the method second, the parameter of the fermentation is as follows:
Seed liquor is seeded to basal medium to ferment；
The control parameter of entire fermentation process: 29-31 DEG C of temperature, pH4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5- 1vvm, pressure 0.03-0.05MPa, DO >=20%；
Glycerite is added as first time DO >=80% with the flow velocity of 5-20mL/h/L in lasting culture, until thallus is wet Stop that glycerite is added when weighing to 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol is added with the flow velocity of 1-3mL/h/L after 1-1.5h Solution (according to the flow velocity of dissolved oxygen and thallus culture situation control methanol solution with the speed increase of 0.1-0.2ml/h/L, works as methanol Solution flow velocity is not further added by when reaching 3mL/h/L)；Start when the flow velocity of methanol solution reaches 3mL/h/L simultaneously with 0.5- Sorbitol solution is added in the flow velocity of 1mL/h/L.
In the method second, seed culture medium concretely: the PBS buffer solution of solvent pH6.0,0.1M；Solute and its Concentration is as follows: yeast extract 1g/100mL, peptone 2g/100mL, glycerol 1g/100mL, YNB1.34g/100mL, biotin 4 ×10-5g/100mL。
In the method second, basal medium (pH5.0) concretely: solvent is water；Solute and its concentration are as follows: glycerol 2g/100mL, 85% phosphoric acid 5%g/100mL, calcium sulfate 0.1g/100mL, potassium sulfate 2g/100mL, epsom salt 1.5g/ 100mL, potassium hydroxide 0.4g/100mL, PTM1 solution 0.44mL/100mL.
In the method second, glycerite concretely: solvent is water；Solute and its concentration are as follows: glycerol 50g/ 100mL, PTM1 solution 1.2mL/100mL.
In the method second, methanol solution concretely: solvent is methanol；Solute and its concentration are as follows: PTM1 solution 1.2mL/100mL。
In the method second, sorbitol solution concretely: solvent is water；Solute and its concentration are as follows: sorbierite 1g/ 100mL, PTM1 solution 1.2mL/100mL.
In any description above method, the L in flow rate is directed to the volume of fermentation system.
In any description above method, specifically, 1155L OD is added in every 10 tons of basal mediums600nmValue=8-12 Seed liquor.
In any description above method, the preparation method of seed liquor is specific as follows:
1. recombination yeast is seeded in 300mL seed culture medium, 30 DEG C, 250rpm shaken cultivation to OD600nmValue is 4- 6；
2. all systems of step 1. are added in 105L basal medium, in " 29-31 DEG C, 200rpm, ventilatory capacity 0.5- It cultivates under conditions of 1vvm, pressure 0.03-0.05MPa, pH4.5-5.5, DO >=20% " to OD600nmValue is 8-12；
3. all systems of step 2. are added in 1050L basal medium, in " 29-31 DEG C, 200rpm, ventilatory capacity It is cultivated under conditions of 0.5-1vvm, pressure 0.03-0.05MPa, pH4.5-5.5, DO >=20% ", obtains OD600nmValue is 8-12's Seed liquor.
Compared with PRSi albumen (wild-type protein), PRSi-QRY albumen provided by the invention has the advantages that expression Amount significantly increases；Specificity significantly increases；Enzyme activity as sucrose isomerase dramatically increases.For sucrose isomerase production field For, PRSi-QRY albumen provided by the invention has very huge application prospect and industrial value.
Further, the present inventor is weighed the channel genes Pichia pastoris of coding PRSi-QRY albumen Group bacterium.
One step, the present inventor has carried out the technique using the recombinant bacterium industrialized production sucrose isomerase excellent Change, generated good effect is as follows:
(1) in production using glycerol as carbon source, with using glucose as carbon source compared with, can substantially reduce metabolism repressor second The generation of alcohol acetic acid, to increase the yield of the sucrose isomerase of final production；
(2) it is produced using glycerol as carbon source, because glycerol will not generate in autoclaving process as carbon source Caramelization, it is possible to the color of fermentation liquid is substantially improved, to make saccharomycete can be with normal growth；
(3) current adding substrate sorbierite can play the role of stimulation induction, to improve the final production of sucrose isomerase Amount；
(4) sucrose isomerase is produced using this method, the final amount ratio for producing sucrose isomerase is using glucose as carbon source and not The production technology of current adding substrate sorbierite increases 55-60%, can significantly reduce sucrose isomerase production cost.
The present invention is with a wide range of applications and huge economic benefit.
Detailed description of the invention
Fig. 1 is the protein electrophoresis figure in embodiment 3.
Fig. 2 is the result of embodiment 4.
Fig. 3 is the result of embodiment 5.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.
Pichia pastoris (Pichia postoris) X33:Cat#C180-00；Life Technologies,Grand Island,NY.USA.PPICZA carrier: Cat#V190-20；Life Technologies,Grand Island,NY.USA. PTM1 solution: Cat#97064-804；VWR.Atlanta,USA.
The discovery of embodiment 1, mutain
On the basis of the sucrose isomerase shown in the sequence 1 of sequence table, building mutain library (including it is single mutation, more The forms such as mutation, are made of the protein of hundreds of thousands particular sequence).It is different as sucrose to each albumen in mutain library The enzyme activity of structure enzyme is detected, and the mutain that enzyme activity significantly increases, the sequence 3 of one of mutain such as sequence table are excavated It is shown.
Protein shown in sequence 1 by sequence table is named as PRSi albumen, 2 institute of sequence of encoding gene such as sequence table Show.Protein shown in sequence 3 by sequence table is named as PRSi-QRY albumen, 4 institute of sequence of encoding gene such as sequence table Show.Compared with PRSi albumen, there are three point mutation for PRSi-QRY albumen tool, are followed successively by K132Q, K171R and D329Y.
The acquisition of embodiment 2, recombination yeast
One, the acquisition of recombination yeast PRSi
1, by the sequence of sequence table 2, the double chain DNA molecule forward direction shown in the nucleotide of 5 ' end the 1st to 1800 is inserted into Between XhoI the and NotI restriction enzyme site of pPICZA carrier, recombinant plasmid pPicZa-PRSi is obtained.
2, recombinant plasmid pPicZa-PRSi is subjected to single endonuclease digestion with restriction enzyme SacI, obtains linearization plasmid.
3, the linearization plasmid for obtaining step 2 imports Pichia pastoris X33, obtains recombination yeast PRSi.
Two, the acquisition of recombination yeast PRSi-QRY
1, by the sequence of sequence table 4, the double chain DNA molecule forward direction shown in the nucleotide of 5 ' end the 1st to 1800 is inserted into Between XhoI the and NotI restriction enzyme site of pPICZA carrier, recombinant plasmid pPicZa-PRSi-QRY is obtained.In recombinant plasmid, shape Fusion shown in sequence 6 at sequence table, fusion protein shown in the sequence 5 of expressed sequence table.
2, recombinant plasmid pPicZa-PRSi-QRY is subjected to single endonuclease digestion with restriction enzyme SacI, obtains linearisation matter Grain.
3, the linearization plasmid for obtaining step 2 imports Pichia pastoris X33, obtains recombination yeast PRSi-QRY.
The zymologic property of embodiment 3, the preparation of albumen and albumen as sucrose isomerase
One, the preparation of albumen
BMGY fluid nutrient medium: the PBS buffer solution of solvent pH6.0,0.1M；Solute and its concentration are as follows: yeast extracts Object 10g/L, tryptone 20g/L, glycerol 10g/L, YNB 13.4g/L, biotin 4 × 10-4g/L。
BMMY fluid nutrient medium: the PBS buffer solution of solvent pH6.0,0.1M；Solute and its concentration are as follows: yeast extracts Object 10g/L, tryptone 20g/L, methanol 0.5% (volumn concentration), YNB 13.4g/L, biotin 4 × 10-4g/L。
Solution I: the Tris-HCl buffer of solvent pH8.0,20mM；Solute and its concentration are as follows: 20mM imidazoles, 300mM NaCl。
Solution II: the Tris-HCl buffer of solvent pH8.0,20mM；Solute and its concentration are as follows: 250mM imidazoles, 300mM NaCl。
Recombination yeast are as follows: recombination yeast PRSi or recombination yeast PRSi-QRY.
1, recombination yeast is seeded in 3mL BMGY fluid nutrient medium, 30 DEG C, 250rpm shaken cultivation 18 hours obtain OD600nm=3 seed liquor.
2, the seed liquor for taking 1mL step 1 to obtain is seeded in 10mL BMMY fluid nutrient medium, 30 DEG C, 250rpm oscillation Culture 96 hours.In incubation, 100 μ L methanol respectively are added to cultivating system after culture 24,48,72 hours.
3, after completing step 2, it is rounded a cultivating system, 4 DEG C, 8000rpm centrifugation 15 minutes collect supernatant.
4, it takes nickel ion column (1mL, Qiagen Products), the supernatant that loading step 3 obtains, then uses 10mL solution I washing pillar, then elutes destination protein with 2mL solution II, collects the solution after crossing column when being eluted with solution II.
5, by step 4 obtain cross column after solution be transferred in bag filter, in deionized water 4 DEG C dialyse 48 hours (every 6 The deionized water that hour more renews), the liquid phase in bag filter is then taken, is freeze-dried, obtains dry powder.
Recombination yeast PRSi carries out the dry powder i.e. PRSi albumen that above-mentioned steps obtain.Every 10mL is completed in the system of step 2, The yield of PRSi albumen is 2.0mg.
Recombination yeast PRSi-QRY carries out the dry powder i.e. PRSi-QRY albumen that above-mentioned steps obtain.Every 10mL completes step 2 System in, the yield of PRSi-QRY albumen is 4.5mg.
Use the citrate-phosphate disodium hydrogen buffer of pH6.0,100mM molten respectively in PRSi albumen and PRSi-QRY albumen Polyacrylamide gel electrophoresis, the result is shown in Figure 1 are carried out after solution.In Fig. 1,1 is molecular weight protein marker, and 2 be PRSi albumen, 3 For PRSi-QRY albumen.
Two, enzymatic activity of the albumen as sucrose isomerase
The measurement of sucrose isomerase enzymatic activity uses 3,5- dinitrosalicylic acid system.Sucrose isomerase under certain condition, Catalysis sucrose generates a large amount of isomaltoketoses and a small amount of trehalulose, glucose and fructose.Isomaltoketose is reduced sugar, 3, The amido complex compound of aobvious brownish red is reduced to after 5- dinitrosalicylic acid and reduction sugar juice heat together, in a certain range its face The depth of color and the amount of reduced sugar are directly proportional, therefore colorimetric can be carried out under the wavelength of 540nm, calculate enzyme activity.Enzyme-activity unit is fixed Justice: the enzyme amount of 1 μm of ol isomaltoketose of release is as a unit of activity per minute.
DNS solution: sodium potassium tartrate tetrahydrate 182g/L, NaOH 10g/L, dinitrosalicylic acid 10g/L, phenol 2g/L, sodium sulphate 2g/L, surplus are water.
Solution to be measured: PRSi albumen and PRSi-QRY albumen are used to the citrate-phosphate disodium hydrogen of pH6.0,100mM respectively Buffer solution obtains the solution that protein concentration is 1mg/mL.
1, using the citrate-phosphate disodium hydrogen buffer of pH6.0,100mM as solvent, preparing sucrose concentration is The sucrose solution of 730mM.
2, test tube is taken, the sucrose solution of addition 0.9mL step 1 preparation and O.1mL solution to be measured simultaneously mix, and 30 DEG C of water-baths are anti- It answers 10 minutes, 2mL DNS solution is then added and mixes, be subsequently placed in ice water environment and terminate reaction, be subsequently placed in boiling water bath Middle 5min measures light absorption value at 540nm after natural cooling.
The calibration curve equation of light absorption value and reduced sugar (isomaltoketose) concentration is Y=1.003X-0.2576, R2= 0.9995；Wherein, Y is OD540nm light absorption value, and X is isomaltoketose concentration (mM).
The Rate activity of PRSi albumen is 512U/mg, and the Rate activity of PRSi-QRY albumen is 638U/mg.With PRSi albumen phase Than the Rate activity of PRSi-QRY albumen improves 24.6%.
Three, specificity of the albumen as sucrose isomerase
Solution to be measured: PRSi albumen and PRSi-QRY albumen are used to the citrate-phosphate disodium hydrogen of pH6.0,100mM respectively Buffer solution obtains the solution that protein concentration is 1mg/mL.
1, using the citrate-phosphate disodium hydrogen buffer of pH6.0,100mM as solvent, preparing sucrose concentration is The sucrose solution of 730mM.
2, test tube is taken, the sucrose solution of addition 0.9mL step 1 preparation and O.1mL solution to be measured simultaneously mix, and 30 DEG C of water-baths are anti- It answers 120 minutes.
3, after completing step 2, it is rounded a reaction system, is diluted to 10 times of volumes with deionized water, then use efficient liquid The content of phase chromatographic determination isomaltoketose in the product.
HPLC condition: LC-3000HPLC system, differential refraction detector；
Chromatographic column: Rezex RCM-Monosaccharide Ca2+ column；80 DEG C of column temperature；
Mobile phase is pure water, and flow velocity is O.6mL/min.
Isomaltoketose standard items are purchased from Sigma (St Louis, USA), catalog number P2007, and peak position is out Third peak (retention time 7.8min, under identical chromatographic conditions, retention time ± 0.1min be can determine whether as same substance).
Trehalulose standard items are purchased from Carbosynth (San Diego USA), catalog number OT09774, appearance Position is that (retention time 8.6min, under identical chromatographic conditions, retention time ± 0.1min be can determine whether as same object at the 4th peak Matter).
PRSi albumen carries out above-mentioned steps, and shared mass percentage is 81.9% to isomaltoketose in the product, sea Shared mass percentage is 9.2% to algae ketose in the product.PRSi-QRY albumen carries out above-mentioned steps, and isomaltoketose exists Shared mass percentage is 96.2% in product, and shared mass percentage is 2.1% to trehalulose in the product.Knot Fruit shows that compared with PRSi albumen, PRSi-QRY albumen is greatly improved as the specificity of sucrose isomerase, and isomaltoketose contains Amount improves 14.3%, and trehalulose content decline 7.1% has and is preferably applied to industrial potentiality.
The large scale fermentation of embodiment 4, recombination yeast PRSi-QRY
One, culture medium and related solution are prepared
Seed culture medium: the PBS buffer solution of solvent pH6.0,0.1M；Solute and its concentration are as follows: yeast extract 1g/ 100mL, peptone 2g/100mL, glycerol 1g/100mL, YNB 1.34g/100mL, biotin 4 × 10-5g/100mL。
Basal medium (pH5.0): solvent is water；Solute and its concentration are as follows: glucose 2g/100mL, 85% phosphoric acid 4.5g/100mL, calcium sulfate 0.93g/100mL, potassium sulfate 1.82g/100mL, epsom salt 1.5g/100mL, potassium hydroxide 0.4g/100mL, PTM1 solution 0.44mL/100mL.
Glucose solution: solvent is water；Solute and its concentration are as follows: glucose 50g/100mL, PTM1 solution 1.2mL/ 100mL。
Methanol solution: solvent is methanol；Solute and its concentration are as follows: PTM1 solution 1.2mL/100mL.
Two, it ferments
1, seed liquor is prepared
Recombination yeast PRSi-QRY is seeded in 300mL seed culture medium, 30 DEG C, 250rpm shaken cultivation 20 hours, Obtain seed liquor (OD600nm=5).
In practical application, incubation time can be 18-24 hours, the OD of seed liquor600nmValue can be 4-6.
2, primary seed solution is prepared
It is packed into 105L basal medium in 150L first class seed pot, all seed liquors that step 1 obtains then are added, It is trained under conditions of " 29-31 DEG C, 200rpm, ventilatory capacity 0.5-1vvm, pressure 0.03-0.05MPa, pH4.5-5.5, DO >=20% " It supports 20 hours, obtains primary seed solution (OD600nm=10).The degerming ammonium hydroxide of use >=20% controls pH during the cultivation process.
In practical application, incubation time can be 18-24 hours, the OD of seed liquor600nmValue can be 8-12.
3, secondary seed solution is prepared
It is packed into 1050L basal medium in 1500L secondary seed tank, all level-one kinds that step 2 obtains then are added Sub- liquid, at " 29-31 DEG C, 200rpm, ventilatory capacity 0.5-1vvm, pressure 0.03-0.05MPa, pH4.5-5.5, DO >=20% " Under the conditions of cultivate 16 hours, obtain secondary seed solution (OD600nm=10).>=20% degerming ammonium hydroxide control is used during the cultivation process PH processed.
In practical application, incubation time can be 12-18 hours, the OD of seed liquor600nmValue can be 8-12.
4, production fermentation
The L in flow rate in this step is directed to the volume of fermentation system.
It is packed into 10 tons of basal mediums in 20 tons of fermentors, all secondary seed solutions that step 3 obtains then are added.
The control parameter of entire fermentation process: 25-30 DEG C of temperature, pH 4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5-1vvm, pressure 0.03-0.05MPa, DO >=20%；The degerming ammonium hydroxide of use >=20% controls pH during the cultivation process；
Grape is added as first time DO >=80% (glucose consumption is complete) with the flow velocity of 10mL/h/L in lasting culture Sugar juice, until thallus weight in wet base stops that glucose solution is added when reaching 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol solution is added with the flow velocity of 1-3mL/h/L after 1h (initial flow rate 1mL/h/L, flow velocity are incremented by with the speed linearity of 0.1mL/h/L, until flow velocity keeps being somebody's turn to do after reaching 3mL/h/L Flow velocity is constant)；
Entire fermented incubation time is 72h.
In fermentation process, interval time sampling is detected in the thallus weight in wet base and every milliliter of fermentation in every liter of fermentation system Enzyme activity clearly as sucrose isomerase (Enzyme activity assay method is with the step of embodiment 3 two)
As a result see Fig. 2.
After fermentation, the sucrose isomerase enzyme activity of fermentation supernatant is 515U/mL.
In practical application, production fermentation parameter can be as follows:
The control parameter of entire fermentation process: 25-30 DEG C of temperature, pH4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5- 1vvm, pressure 0.03-0.05MPa, DO >=20%；The degerming ammonium hydroxide of use >=20% controls pH during the cultivation process；
Portugal is added as first time DO >=80% (glucose consumption is complete) with the flow velocity of 5-20mL/h/L in lasting culture Grape sugar juice, until thallus weight in wet base stops that glucose solution is added when reaching 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol is added with the flow velocity of 1-3mL/h/L after 1-1.5h Solution works as methanol according to the flow velocity of dissolved oxygen and thallus culture situation control methanol solution with the speed increase of 0.1-0.2ml/h/L Flow velocity is not further added by when solution flow velocity reaches 3mL/h/L, until fermentation ends.
The large scale fermentation (advanced optimizing technique) of embodiment 5, recombination yeast PRSi-QRY
One, culture medium and related solution are prepared
Seed culture medium: the PBS buffer solution of solvent pH6.0,0.1M；Solute and its concentration are as follows: yeast extract 1g/ 100mL, peptone 2g/100mL, glycerol 1g/100mL, YNB 1.34g/100mL, biotin 4 × 10-5g/100mL。
Basal medium (pH5.0): solvent is water；Solute and its concentration are as follows: glycerol 2g/100mL, 85% phosphoric acid 5% G/100mL, calcium sulfate 0.1g/100mL, potassium sulfate 2g/100mL, epsom salt 1.5g/100mL, potassium hydroxide 0.4g/ 100mL, PTM1 solution 0.44mL/100mL.
Glycerite: solvent is water；Solute and its concentration are as follows: glycerol 50g/100mL, PTM1 solution 1.2mL/100mL.
Methanol solution: solvent is methanol；Solute and its concentration are as follows: PTM1 solution 1.2mL/100mL.
Sorbitol solution: solvent is water；Solute and its concentration are as follows: sorbierite 1g/100mL, PTM1 solution 1.2mL/ 100mL。
Two, it ferments
1, seed liquor is prepared
With the step of embodiment 4 two 1.
2, primary seed solution is prepared
With the step of embodiment 4 two 2.
3, secondary seed solution is prepared
With the step of embodiment 4 two 3.
4, production fermentation
The L in flow rate in this step is directed to the volume of fermentation system.
It is packed into 10 tons of basal mediums in 20 tons of fermentors, all secondary seed solutions that step 3 obtains then are added.
The control parameter of entire fermentation process: 29-31 DEG C of temperature, pH4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5- 1vvm, pressure 0.03-0.05MPa, DO >=20%；The degerming ammonium hydroxide of use >=20% controls pH during the cultivation process；
It is molten that glycerol is added with the flow velocity of 10mL/h/L as first time DO >=80% (glycerol consumption is complete) in lasting culture Liquid, until thallus weight in wet base stops that glycerite is added when reaching 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol solution is added with the flow velocity of 1-3mL/h/L after 1h (initial flow rate 1mL/h/L, flow velocity are incremented by with the speed linearity of 0.1mL/h/L, until flow velocity keeps being somebody's turn to do after reaching 3mL/h/L Flow velocity is constant), start when the flow velocity of methanol solution reaches 3mL/h/L molten with the flow velocity addition sorbierite of 0.8mL/h/L simultaneously Liquid；
Entire fermented incubation time is 72h.
In fermentation process, interval time sampling is detected in the thallus weight in wet base and every milliliter of fermentation in every liter of fermentation system Enzyme activity clearly as sucrose isomerase (Enzyme activity assay method is with the step of embodiment 3 two)
As a result see Fig. 3.
After fermentation, the sucrose isomerase enzyme activity of fermentation supernatant is 1219U/ml.
In practical application, production fermentation parameter can be as follows:
The control parameter of entire fermentation process: 29-31 DEG C of temperature, pH4.5-5.5, revolving speed 90-150rpm, ventilatory capacity 0.5- 1vvm, pressure 0.03-0.05MPa, DO >=20%；The degerming ammonium hydroxide of use >=20% controls pH during the cultivation process；
Glycerol is added as first time DO >=80% (glycerol consumption is complete) with the flow velocity of 5-20mL/h/L in lasting culture Solution, until thallus weight in wet base stops that glycerite is added when reaching 180g/L；
Lasting culture, starts timing as second of DO >=80%, and methanol is added with the flow velocity of 1-3mL/h/L after 1-1.5h Solution works as methanol according to the flow velocity of dissolved oxygen and thallus culture situation control methanol solution with the speed increase of 0.1-0.2ml/h/L Flow velocity is not further added by when solution flow velocity reaches 3mL/h/L, until fermentation ends；When the flow velocity of methanol solution reaches 3mL/h/L Start that sorbitol solution is added with the flow velocity of 0.5-1mL/h/L simultaneously.
SEQUENCE LISTING
<110>Weir gold Co., Ltd
<120>a kind of sucrose isomerase enzyme mutant and its application
<130> GNCYX170835
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 600
<212> PRT
<213>protaminobacter ruber
<400> 1
Met Pro Arg Gln Gly Leu Lys Thr Ala Leu Ala Ile Phe Leu Thr Thr
1 5 10 15
Ser Leu Cys Ile Ser Cys Gln Gln Ala Phe Gly Thr Gln Gln Pro Leu
20 25 30
Leu Asn Glu Lys Ser Ile Glu Gln Ser Lys Thr Ile Pro Lys Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Ile Asn Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ala Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln Asn Glu Trp Phe Val Lys Ser Lys Ser Ser Lys
145 150 155 160
Asp Asn Pro Tyr Arg Gly Tyr Tyr Phe Trp Lys Asp Ala Lys Glu Gly
165 170 175
Gln Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Glu Lys Thr Asn Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asp Phe Pro Asn Leu Thr
245 250 255
Gln Gln Gln Leu Lys Asn Phe Ala Ala Glu Tyr Thr Lys Gly Pro Asn
260 265 270
Ile His Arg Tyr Val Asn Glu Met Asn Lys Glu Val Leu Ser His Tyr
275 280 285
Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Gln Ser
290 295 300
Ile Lys Phe Phe Asp Arg Arg Arg Asp Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asp Gln Arg Trp Arg Arg
325 330 335
Lys Asp Trp Lys Leu Ser Gln Phe Arg Gln Ile Ile Asp Asn Val Asp
340 345 350
Arg Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu Pro Ser Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Ala Ile Asp Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp His Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Asp Glu Phe Leu
435 440 445
Gln Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Gly Ser Lys Asn Ala Gly Phe Thr Ser Gly Lys Pro Trp Phe
465 470 475 480
Lys Val Asn Pro Asn Tyr Gln Glu Ile Asn Ala Val Ser Gln Val Thr
485 490 495
Gln Pro Asp Ser Val Phe Asn Tyr Tyr Arg Gln Leu Ile Lys Ile Arg
500 505 510
His Asp Ile Pro Ala Leu Thr Tyr Gly Thr Tyr Thr Asp Leu Asp Pro
515 520 525
Ala Asn Asp Ser Val Tyr Ala Tyr Thr Arg Ser Leu Gly Ala Glu Lys
530 535 540
Tyr Leu Val Val Val Asn Phe Lys Glu Gln Met Met Arg Tyr Lys Leu
545 550 555 560
Pro Asp Asn Leu Ser Ile Glu Lys Val Ile Ile Asp Ser Asn Ser Lys
565 570 575
Asn Val Val Lys Lys Asn Asp Ser Leu Leu Glu Leu Lys Pro Trp Gln
580 585 590
Ser Gly Val Tyr Lys Leu Asn Gln
595 600
<210> 2
<211> 1803
<212> DNA
<213>protaminobacter ruber
<400> 2
atgccccgtc aaggattgaa aactgcacta gcgatttttc taaccacatc attatgcatc 60
tcatgccagc aagccttcgg tacgcaacaa cccttgctta acgaaaagag tatcgaacag 120
tcgaaaacca tacctaaatg gtggaaggag gctgtttttt atcaggtgta tccgcgctcc 180
tttaaagaca ccaacggaga tggcatcggg gatattaacg gcatcataga aaaattagac 240
tatctaaaag ccttggggat tgatgccatt tggatcaacc cacattatga ttctccgaac 300
acggataatg gttacgatat acgtgattat cgaaaaatca tgaaagaata tggcacgatg 360
gaggattttg accgcctgat ttctgaaatg aaaaaacgga atatgcggtt gatgattgat 420
gtggtcatca accacaccag cgatcaaaac gaatggtttg ttaaaagtaa aagcagtaag 480
gataatcctt atcgcggcta ttatttctgg aaagatgcta aagaagggca ggcgcctaat 540
aattaccctt cattctttgg tggctcggcg tggcaaaaag atgaaaagac caatcaatac 600
tacctgcact attttgctaa acaacagcct gacctaaact gggataatcc caaagtccgt 660
caagatcttt atgcaatgtt acgtttctgg ttagataaag gcgtgtctgg tttacgtttt 720
gatacggtag cgacctactc aaaaattccg gatttcccaa atctcaccca acaacagctg 780
aagaattttg cagcggagta taccaagggc cctaatattc atcgttacgt caatgaaatg 840
aataaagagg tcttgtctca ttacgacatt gcgactgccg gtgaaatctt tggcgtaccc 900
ttggatcaat cgataaagtt cttcgatcgc cgccgtgatg agctgaacat tgcatttacc 960
tttgacttaa tcagactcga tcgagactct gatcaaagat ggcgtcgaaa agattggaaa 1020
ttgtcgcaat tccggcagat catcgataac gttgaccgta ctgcaggaga atatggttgg 1080
aatgccttct tcttggataa ccacgacaat ccgcgcgctg tctcgcactt tggcgatgat 1140
cgcccacaat ggcgtgagcc atcggctaaa gcgcttgcaa ccttgacgct gactcaacga 1200
gcaacacctt ttatttatca aggttcagaa ttgggcatga ccaattaccc gtttaaagct 1260
attgatgaat tcgatgatat tgaggtgaaa ggtttttggc atgactacgt tgagacagga 1320
aaggtcaaag ccgacgagtt cttgcaaaat gtacgcctga cgagcaggga taacagccgg 1380
acgccgttcc aatgggatgg gagcaaaaat gcaggattca cgagcggaaa accttggttc 1440
aaggtcaacc caaactacca ggaaatcaat gcagtaagtc aagtcacaca acccgactca 1500
gtatttaact attatcgtca gttgatcaag ataaggcatg acatcccggc actgacctat 1560
ggtacataca ccgatttgga tcctgcaaat gattcggtct acgcctatac acgcagcctt 1620
ggggcggaaa aatatcttgt tgttgttaac ttcaaggagc aaatgatgag atataaatta 1680
ccggataatt tatccattga gaaagtgatt atagacagca acagcaaaaa cgtggtgaaa 1740
aagaatgatt cattactcga gctaaaacca tggcagtcag gggtttataa actaaatcaa 1800
taa 1803
<210> 3
<211> 600
<212> PRT
<213>artificial sequence
<400> 3
Met Pro Arg Gln Gly Leu Lys Thr Ala Leu Ala Ile Phe Leu Thr Thr
1 5 10 15
Ser Leu Cys Ile Ser Cys Gln Gln Ala Phe Gly Thr Gln Gln Pro Leu
20 25 30
Leu Asn Glu Lys Ser Ile Glu Gln Ser Lys Thr Ile Pro Lys Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Ile Asn Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ala Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser
115 120 125
Glu Met Lys Gln Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln Asn Glu Trp Phe Val Lys Ser Lys Ser Ser Lys
145 150 155 160
Asp Asn Pro Tyr Arg Gly Tyr Tyr Phe Trp Arg Asp Ala Lys Glu Gly
165 170 175
Gln Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Glu Lys Thr Asn Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asp Phe Pro Asn Leu Thr
245 250 255
Gln Gln Gln Leu Lys Asn Phe Ala Ala Glu Tyr Thr Lys Gly Pro Asn
260 265 270
Ile His Arg Tyr Val Asn Glu Met Asn Lys Glu Val Leu Ser His Tyr
275 280 285
Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Gln Ser
290 295 300
Ile Lys Phe Phe Asp Arg Arg Arg Asp Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Tyr Ser Asp Gln Arg Trp Arg Arg
325 330 335
Lys Asp Trp Lys Leu Ser Gln Phe Arg Gln Ile Ile Asp Asn Val Asp
340 345 350
Arg Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu Pro Ser Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Ala Ile Asp Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp His Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Asp Glu Phe Leu
435 440 445
Gln Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Gly Ser Lys Asn Ala Gly Phe Thr Ser Gly Lys Pro Trp Phe
465 470 475 480
Lys Val Asn Pro Asn Tyr Gln Glu Ile Asn Ala Val Ser Gln Val Thr
485 490 495
Gln Pro Asp Ser Val Phe Asn Tyr Tyr Arg Gln Leu Ile Lys Ile Arg
500 505 510
His Asp Ile Pro Ala Leu Thr Tyr Gly Thr Tyr Thr Asp Leu Asp Pro
515 520 525
Ala Asn Asp Ser Val Tyr Ala Tyr Thr Arg Ser Leu Gly Ala Glu Lys
530 535 540
Tyr Leu Val Val Val Asn Phe Lys Glu Gln Met Met Arg Tyr Lys Leu
545 550 555 560
Pro Asp Asn Leu Ser Ile Glu Lys Val Ile Ile Asp Ser Asn Ser Lys
565 570 575
Asn Val Val Lys Lys Asn Asp Ser Leu Leu Glu Leu Lys Pro Trp Gln
580 585 590
Ser Gly Val Tyr Lys Leu Asn Gln
595 600
<210> 4
<211> 1803
<212> DNA
<213>artificial sequence
<400> 4
atgccccgtc aaggattgaa aactgcacta gcgatttttc taaccacatc attatgcatc 60
tcatgccagc aagccttcgg tacgcaacaa cccttgctta acgaaaagag tatcgaacag 120
tcgaaaacca tacctaaatg gtggaaggag gctgtttttt atcaggtgta tccgcgctcc 180
tttaaagaca ccaacggaga tggcatcggg gatattaacg gcatcataga aaaattagac 240
tatctaaaag ccttggggat tgatgccatt tggatcaacc cacattatga ttctccgaac 300
acggataatg gttacgatat acgtgattat cgaaaaatca tgaaagaata tggcacgatg 360
gaggattttg accgcctgat ttctgaaatg aaacaacgga atatgcggtt gatgattgat 420
gtggtcatca accacaccag cgatcaaaac gaatggtttg ttaaaagtaa aagcagtaag 480
gataatcctt atcgcggcta ttatttctgg agggatgcta aagaagggca ggcgcctaat 540
aattaccctt cattctttgg tggctcggcg tggcaaaaag atgaaaagac caatcaatac 600
tacctgcact attttgctaa acaacagcct gacctaaact gggataatcc caaagtccgt 660
caagatcttt atgcaatgtt acgtttctgg ttagataaag gcgtgtctgg tttacgtttt 720
gatacggtag cgacctactc aaaaattccg gatttcccaa atctcaccca acaacagctg 780
aagaattttg cagcggagta taccaagggc cctaatattc atcgttacgt caatgaaatg 840
aataaagagg tcttgtctca ttacgacatt gcgactgccg gtgaaatctt tggcgtaccc 900
ttggatcaat cgataaagtt cttcgatcgc cgccgtgatg agctgaacat tgcatttacc 960
tttgacttaa tcagactcga tcgatactct gatcaaagat ggcgtcgaaa agattggaaa 1020
ttgtcgcaat tccggcagat catcgataac gttgaccgta ctgcaggaga atatggttgg 1080
aatgccttct tcttggataa ccacgacaat ccgcgcgctg tctcgcactt tggcgatgat 1140
cgcccacaat ggcgtgagcc atcggctaaa gcgcttgcaa ccttgacgct gactcaacga 1200
gcaacacctt ttatttatca aggttcagaa ttgggcatga ccaattaccc gtttaaagct 1260
attgatgaat tcgatgatat tgaggtgaaa ggtttttggc atgactacgt tgagacagga 1320
aaggtcaaag ccgacgagtt cttgcaaaat gtacgcctga cgagcaggga taacagccgg 1380
acgccgttcc aatgggatgg gagcaaaaat gcaggattca cgagcggaaa accttggttc 1440
aaggtcaacc caaactacca ggaaatcaat gcagtaagtc aagtcacaca acccgactca 1500
gtatttaact attatcgtca gttgatcaag ataaggcatg acatcccggc actgacctat 1560
ggtacataca ccgatttgga tcctgcaaat gattcggtct acgcctatac acgcagcctt 1620
ggggcggaaa aatatcttgt tgttgttaac ttcaaggagc aaatgatgag atataaatta 1680
ccggataatt tatccattga gaaagtgatt atagacagca acagcaaaaa cgtggtgaaa 1740
aagaatgatt cattactcga gctaaaacca tggcagtcag gggtttataa actaaatcaa 1800
taa 1803
<210> 5
<211> 630
<212> PRT
<213>artificial sequence
<400> 5
Pro Arg Met Pro Arg Gln Gly Leu Lys Thr Ala Leu Ala Ile Phe Leu
1 5 10 15
Thr Thr Ser Leu Cys Ile Ser Cys Gln Gln Ala Phe Gly Thr Gln Gln
20 25 30
Pro Leu Leu Asn Glu Lys Ser Ile Glu Gln Ser Lys Thr Ile Pro Lys
35 40 45
Trp Trp Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys
50 55 60
Asp Thr Asn Gly Asp Gly Ile Gly Asp Ile Asn Gly Ile Ile Glu Lys
65 70 75 80
Leu Asp Tyr Leu Lys Ala Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro
85 90 95
His Tyr Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr
100 105 110
Arg Lys Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu
115 120 125
Ile Ser Glu Met Lys Gln Arg Asn Met Arg Leu Met Ile Asp Val Val
130 135 140
Ile Asn His Thr Ser Asp Gln Asn Glu Trp Phe Val Lys Ser Lys Ser
145 150 155 160
Ser Lys Asp Asn Pro Tyr Arg Gly Tyr Tyr Phe Trp Arg Asp Ala Lys
165 170 175
Glu Gly Gln Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala
180 185 190
Trp Gln Lys Asp Glu Lys Thr Asn Gln Tyr Tyr Leu His Tyr Phe Ala
195 200 205
Lys Gln Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp
210 215 220
Leu Tyr Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu
225 230 235 240
Arg Phe Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asp Phe Pro Asn
245 250 255
Leu Thr Gln Gln Gln Leu Lys Asn Phe Ala Ala Glu Tyr Thr Lys Gly
260 265 270
Pro Asn Ile His Arg Tyr Val Asn Glu Met Asn Lys Glu Val Leu Ser
275 280 285
His Tyr Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp
290 295 300
Gln Ser Ile Lys Phe Phe Asp Arg Arg Arg Asp Glu Leu Asn Ile Ala
305 310 315 320
Phe Thr Phe Asp Leu Ile Arg Leu Asp Arg Tyr Ser Asp Gln Arg Trp
325 330 335
Arg Arg Lys Asp Trp Lys Leu Ser Gln Phe Arg Gln Ile Ile Asp Asn
340 345 350
Val Asp Arg Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp
355 360 365
Asn His Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro
370 375 380
Gln Trp Arg Glu Pro Ser Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr
385 390 395 400
Gln Arg Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr
405 410 415
Asn Tyr Pro Phe Lys Ala Ile Asp Glu Phe Asp Asp Ile Glu Val Lys
420 425 430
Gly Phe Trp His Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Asp Glu
435 440 445
Phe Leu Gln Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro
450 455 460
Phe Gln Trp Asp Gly Ser Lys Asn Ala Gly Phe Thr Ser Gly Lys Pro
465 470 475 480
Trp Phe Lys Val Asn Pro Asn Tyr Gln Glu Ile Asn Ala Val Ser Gln
485 490 495
Val Thr Gln Pro Asp Ser Val Phe Asn Tyr Tyr Arg Gln Leu Ile Lys
500 505 510
Ile Arg His Asp Ile Pro Ala Leu Thr Tyr Gly Thr Tyr Thr Asp Leu
515 520 525
Asp Pro Ala Asn Asp Ser Val Tyr Ala Tyr Thr Arg Ser Leu Gly Ala
530 535 540
Glu Lys Tyr Leu Val Val Val Asn Phe Lys Glu Gln Met Met Arg Tyr
545 550 555 560
Lys Leu Pro Asp Asn Leu Ser Ile Glu Lys Val Ile Ile Asp Ser Asn
565 570 575
Ser Lys Asn Val Val Lys Lys Asn Asp Ser Leu Leu Glu Leu Lys Pro
580 585 590
Trp Gln Ser Gly Val Tyr Lys Leu Asn Gln Arg Gly Arg Gln Leu Gly
595 600 605
Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp
610 615 620
His His His His His His
625 630
<210> 6
<211> 1893
<212> DNA
<213>artificial sequence
<400> 6
ccgcgcatgc cccgtcaagg attgaaaact gcactagcga tttttctaac cacatcatta 60
tgcatctcat gccagcaagc cttcggtacg caacaaccct tgcttaacga aaagagtatc 120
gaacagtcga aaaccatacc taaatggtgg aaggaggctg ttttttatca ggtgtatccg 180
cgctccttta aagacaccaa cggagatggc atcggggata ttaacggcat catagaaaaa 240
ttagactatc taaaagcctt ggggattgat gccatttgga tcaacccaca ttatgattct 300
ccgaacacgg ataatggtta cgatatacgt gattatcgaa aaatcatgaa agaatatggc 360
acgatggagg attttgaccg cctgatttct gaaatgaaac aacggaatat gcggttgatg 420
attgatgtgg tcatcaacca caccagcgat caaaacgaat ggtttgttaa aagtaaaagc 480
agtaaggata atccttatcg cggctattat ttctggaggg atgctaaaga agggcaggcg 540
cctaataatt acccttcatt ctttggtggc tcggcgtggc aaaaagatga aaagaccaat 600
caatactacc tgcactattt tgctaaacaa cagcctgacc taaactggga taatcccaaa 660
gtccgtcaag atctttatgc aatgttacgt ttctggttag ataaaggcgt gtctggttta 720
cgttttgata cggtagcgac ctactcaaaa attccggatt tcccaaatct cacccaacaa 780
cagctgaaga attttgcagc ggagtatacc aagggcccta atattcatcg ttacgtcaat 840
gaaatgaata aagaggtctt gtctcattac gacattgcga ctgccggtga aatctttggc 900
gtacccttgg atcaatcgat aaagttcttc gatcgccgcc gtgatgagct gaacattgca 960
tttacctttg acttaatcag actcgatcga tactctgatc aaagatggcg tcgaaaagat 1020
tggaaattgt cgcaattccg gcagatcatc gataacgttg accgtactgc aggagaatat 1080
ggttggaatg ccttcttctt ggataaccac gacaatccgc gcgctgtctc gcactttggc 1140
gatgatcgcc cacaatggcg tgagccatcg gctaaagcgc ttgcaacctt gacgctgact 1200
caacgagcaa caccttttat ttatcaaggt tcagaattgg gcatgaccaa ttacccgttt 1260
aaagctattg atgaattcga tgatattgag gtgaaaggtt tttggcatga ctacgttgag 1320
acaggaaagg tcaaagccga cgagttcttg caaaatgtac gcctgacgag cagggataac 1380
agccggacgc cgttccaatg ggatgggagc aaaaatgcag gattcacgag cggaaaacct 1440
tggttcaagg tcaacccaaa ctaccaggaa atcaatgcag taagtcaagt cacacaaccc 1500
gactcagtat ttaactatta tcgtcagttg atcaagataa ggcatgacat cccggcactg 1560
acctatggta catacaccga tttggatcct gcaaatgatt cggtctacgc ctatacacgc 1620
agccttgggg cggaaaaata tcttgttgtt gttaacttca aggagcaaat gatgagatat 1680
aaattaccgg ataatttatc cattgagaaa gtgattatag acagcaacag caaaaacgtg 1740
gtgaaaaaga atgattcatt actcgagcta aaaccatggc agtcaggggt ttataaacta 1800
aatcaacgcg gccgccagct tgggcccgaa caaaaactca tctcagaaga ggatctgaat 1860
agcgccgtcg accatcatca tcatcatcat tga 1893

Claims (15)

1. a kind of protein, the protein formed for the amino acid sequence shown in sequence 3 in sequence table.
2. a kind of fused protein, the protein formed for the amino acid sequence shown in sequence 5 in sequence table.
3. encoding the gene of protein described in claim 1.
4. gene as claimed in claim 3, it is characterised in that: the gene is code area as shown in the sequence 4 of sequence table DNA molecular.
5. gene as claimed in claim 4, it is characterised in that: the gene is DNA molecular shown in the sequence 4 of sequence table.
6. encoding the gene of fused protein described in claim 2.
7. gene as claimed in claim 6, it is characterised in that: the gene is code area as shown in the sequence 6 of sequence table DNA molecular.
8. gene as claimed in claim 7, it is characterised in that: the gene is DNA molecular shown in the sequence 6 of sequence table.
9. expression cassette, recombinant vector or recombinant bacterium containing claim 3 or 4 or 5 or 6 or 7 or 8 genes.
10. application of the fused protein described in protein or claim 2 described in claim 1 as sucrose isomerase.
11. application of the fused protein described in protein or claim 2 described in claim 1 in production isomaltoketose.
12. fused protein described in protein or claim 2 described in claim 1 is producing different malt ketone by raw material of sucrose Application in sugar.
13. a kind of recombinant bacterium is the recombinant bacterium for obtaining recombinant plasmid pPicZa-PRSi-QRY importing Pichia pastoris X33；It is described Recombinant plasmid pPicZa-PRSi-QRY is to obtain claim 3 or 4 or the 5 or 6 or 7 or 8 gene insertion pPICZA carriers Recombinant plasmid.
14. application of the recombinant bacterium described in claim 13 in production sucrose isomerase.
15. a kind of method for producing sucrose isomerase includes the following steps: the recombinant bacterium described in claim 13 that ferments, obtains sugarcane Sugared isomerase.
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CN113174385B (en) * 2021-04-13 2024-04-12 苏州朗邦营养科技有限公司 Sucrose isomerase mutant with high activity and high conversion rate and application thereof
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Citations (1)

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CN105483107A (en) * 2015-12-31 2016-04-13 天津科技大学 Sucrose isomerase mutant and method for producing isomaltulose

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Publication number Priority date Publication date Assignee Title
CN105483107A (en) * 2015-12-31 2016-04-13 天津科技大学 Sucrose isomerase mutant and method for producing isomaltulose

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Title
蔗糖异构酶突变菌株的构建及其应用研究;藤菲等;《生物工程》;20150930;第143-147页

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