CN109929863A - A method of isomaltoketose is produced using resting cell - Google Patents

Abstract

The invention discloses a kind of methods using resting cell production isomaltoketose, belong to gene engineering technology field.The present invention provides a kind of methods for producing isomaltoketose, the method is to produce isomaltoketose using the bacillus subtilis engineering bacteria resting cell for the gene (nucleotide sequence is as shown in SEQ ID NO.1) that can express encoding sucrose isomerase, production isomaltoketose is converted using the method, with conversion ratio, production intensity and the high advantage of yield；Utilize method conversion production isomaltoketose 9h of the invention, the isomaltoketose that can be 443g/L by the sucrose inversion of 500g/L in reaction system, Sucrose conversion, which is up to 94%, isomaltoketose yield and is up to 88.6%, isomaltoketose space-time yield, is up to 49.2g/Lh.

Description

A method of isomaltoketose is produced using resting cell

Technical field

The present invention relates to a kind of methods using resting cell production isomaltoketose, belong to technique for gene engineering neck Domain.

Background technique

Isomaltoketose (isomaltulose, α-D- glycopyranosyl -1,6-D- fructose), also referred to as palatinose, are sugarcanes A kind of isomer of sugar, has physical property similar with sucrose and mouthfeel, but unlike sucrose, sugariness only has The half of sucrose, without cariogenic tooth；Also, after isomaltoketose is eaten by human body, the speed of monosaccharide is discharged in blood of human body Degree is very slow, will not stimulate the secretion of insulin, be particularly suitable for diabetes patient and obese people；In addition, isomaltoketose It is the currently the only sweetener limited without dosage, therefore, isomaltoketose is as a kind of as a kind of reduction sexual function disaccharides Natural, novel functional sugar, is widely used in field of food.

Currently, the method for production isomaltoketose mainly has to sow: first is that microbe transformation method, second is that enzyme process biology closes At.Wherein, microbe transformation method be by by general city Serratia category, rhubarb horsetails Erwinia sp category, Klebsiella and point The some isomaltoketose main product wild-type strains for dissipating general Pseudomonas, which are seeded in the culture medium containing sucrose, to ferment to produce Isomaltoketose is obtained, the method usually makes thallus fermentation is synchronous with sucrose inversion to carry out, and still, produces different wheat using the method Bud ketose has the defects of cell concentration is low, conversion ratio is low, production intensity is weak, in addition, since these isomaltoketose main products are wild Raw type bacterial strain genetic background is unclear, may the potential different malt ketone that has virulence factor, will obtain using these strain fermentations Sugar is applied to food, and there are great security risks in the middle, therefore, comprehensively consider, and the microbe transformation method of wild-type strain is uncomfortable For large-scale industrial production.

Enzyme process biosynthesis is also classified into two kinds, and one is the engineering bacteria fermentations for first passing through building recombinant sucrose isomerase Sucrose isomerase is obtained, catalytic production is then carried out using sucrose as substrate by the sucrose isomerase that fermentation obtains and obtains different wheat Bud ketose；Another kind is the genetic engineering bacterium for first constructing recombinant sucrose isomerase, then directly using this genetic engineering bacterium with sugarcane Sugar carries out whole-cell catalytic as substrate and produces to obtain isomaltoketose.The method is due to that can express sucrose isomerase in building Select the aliment security level bacterial strain of green high-efficient as host cell when genetic engineering bacterium, advantage with high security, therefore, Enzyme process biosynthesis has become the research hotspot in food sucrose isomerase field.But to express sucrose different due to existing The equal yield of the genetic engineering bacterium of structure enzyme is not high, and the sucrose isomerase produced is also commonly present that specific enzyme activity is low, Sucrose conversion is low Defect, therefore, that there are still the substrate transformation rates is low, production intensity is weak using enzyme process biosynthesis isomaltoketose, low yield lacks It falls into.

For example, the sucrose isomerase in Enterobacter source is introduced streptococcus lactis by Jong-Yul Park etc. constructs weight Group streptococcus lactis MG1363, carries out the different malt of catalytic production using sucrose as substrate using this recombination lactic acid streptococcus m G1363 The substrate transformation rate of ketose only reaches 72% (specific visible document: Park J Y, Jung J H, Seo D H, et al.Microbial production of palatinose through extracellular expression of a sucrose isomerase from Enterobacter sp.FMB-1in Lactococcus lactis MG1363[J] .Bioresource Technology,2010,101(22):8828-8833.)；Gil-Yong Lee etc. is by Enterobacter source Sucrose isomerase introduce saccharomyces cerevisiae construct recombinant Saccharomyces cerevisiae, using this recombinant Saccharomyces cerevisiae using sucrose as substrate into The substrate transformation rate of row catalytic production isomaltoketose only reaches 6.4~7.4% (specific visible documents: Lee G Y, Jung J H,Seo D H,et al.Isomaltulose production via yeast surface display of sucrose isomerase from Enterobacter sp.FMB-1on Saccharomyces cerevisiae[J].Bioresour Technol,2011,102(19):9179-9184.)；Lingtian Wu etc. is by the sucrose isomerase in rhubarb horsetails Erwinia sp source Gene introduces bacillus subtilis and constructs recombined bacillus subtilis, by medium optimization, ferments after 35h, this recombination is withered Sucrose isomerase enzyme activity in careless fermentation of bacillus supernatant only reach 5.2U/mL (specific visible document: Wu L, Wu S, Qiu J,et al.Green synthesis of isomaltulose from cane molasses by,Bacillus subtilis,WB800-pHA01-pal I in a biologic membrane reactor[J].Food Chemistry, 2017,229:761-768.)；The sucrose isomerase gene for dispersing general bacterium source is introduced Yarrowia lipolytica by Peng Zhang etc. Recombination Yarrowia lipolytica is constructed, carries out catalysis 56h using sucrose as substrate using this recombination Yarrowia lipolytica, final Space-time yield only reaches 23.8g/Lh or so (specific visible document: Peng Z, Zhi-Peng W, Jun S, et al.High and efficient isomaltulose production using an engineered,Yarrowia lipolytica,strain[J].Bioresource Technology,2018:S0960852418308605-.)。

Therefore, be badly in need of finding it is a kind of can the high yield sucrose isomerase and sucrose isomerase specific enzyme activity that produces is high, sucrose inversion Enzyme process biosynthesis Sucrose conversion is low, production intensity is weak, low yield defect to solve for the high recombinant bacterium of rate.

Summary of the invention

[technical problem]

The technical problem to be solved in the present invention is to provide it is a kind of can high yield sucrose isomerase and the sucrose isomerase ratio that produces The recombinant bacterium that enzyme activity is high, Sucrose conversion is high, to improve the conversion ratio, yield and production of enzyme process biosynthesis isomaltoketose Intensity.

[technical solution]

The present invention provides the gene that one section can be used for encoding sucrose isomerase, the nucleotide sequence of the gene such as SEQ Shown in ID NO.1.

The present invention also provides the recombinant plasmids for carrying said gene.

In one embodiment of the invention, the carrier for constructing the recombinant plasmid is pMA5 carrier, pHT43 load Body, pET-20b (+) carrier, pXMJ19 carrier or pDXW-10 carrier.

In one embodiment of the invention, the carrier for constructing the recombinant plasmid is pMA5 carrier.

In one embodiment of the invention, the recombinant plasmid is by can be used for pMA5 carrier with above-mentioned one section The gene of encoding sucrose isomerase connects acquisition after restriction enzyme Nde I and Mlu I digestion.

The present invention also provides the host cells for carrying said gene or above-mentioned recombinant plasmid.

In one embodiment of the invention, the host cell is bacillus subtilis, Escherichia coli or glutamic acid Bar bacterium.

In one embodiment of the invention, the host cell is bacillus subtilis.

In one embodiment of the invention, the bacillus subtilis is bacillus subtilis (Bacillus subtilis)168。

The present invention also provides a kind of method for producing sucrose isomerase, the method is to cultivate above-mentioned host cell It is cultivated in base.

In one embodiment of the invention, the culture medium can be LB culture medium, TB culture medium or BHI culture medium.

In one embodiment of the invention, the temperature of the culture be 25~37 DEG C, the time be 20~for 24 hours.

The present invention also provides a kind of methods using resting cell production isomaltoketose, and the method is will be above-mentioned Host cell is added in the reaction system containing sucrose and carries out conversion reaction.

In one embodiment of the invention, the method is that above-mentioned host cell is resuspended with the buffer containing sucrose It is OD to cell concentration of the host cell in buffer₆₀₀=25~30, obtain reaction system；It is added in the reaction system thin Penetrating dose of progress conversion reaction of cell wall, obtains isomaltoketose.

In one embodiment of the invention, the temperature of the conversion is 25~35 DEG C, pH is 6.0~8.0, the time is 8~12h.

In one embodiment of the invention, the buffer is phosphate buffer.

In one embodiment of the invention, lead to for Qula for penetrating dose of the cell wall.

In one embodiment of the invention, the concentration of the sucrose in the reaction system is 500g/L.

The present invention also provides said genes or above-mentioned recombinant plasmid or above-mentioned host cell or the above method in production sugarcane Application in terms of sugared isomerase.

It is different in production that the present invention also provides said genes or above-mentioned recombinant plasmid or above-mentioned host cell or the above method Application in terms of maltulose.

[beneficial effect]

(1) the present invention provides gene (the nucleotide sequence such as SEQ ID NO.1 that one section can be used for encoding sucrose isomerase It is shown), this gene is expressed in bacillus subtilis, can enable bacillus subtilis efficiently synthesize specific enzyme activity it is high and The high sucrose isomerase of Sucrose conversion；

(2) for 24 hours by bacillus subtilis recombinant of the invention, the enzyme of the sucrose isomerase in crude enzyme liquid can be made It is living to be up to 125U/mL；

(3) the sucrose isomerase specific enzyme activity and sucrose produced using bacillus subtilis engineering bacteria of the invention is turned Rate is higher, wherein specific enzyme activity is up to 532U/mg, and Sucrose conversion is up to 94%；

(4) the present invention provides a kind of method for producing isomaltoketose, the method is different using that can express encoding sucrose The bacillus subtilis engineering bacteria resting cell of the gene (nucleotide sequence is as shown in SEQ ID NO.1) of structure enzyme produces different wheat Bud ketose converts production isomaltoketose using the method, with conversion ratio, yield and produces the high advantage of intensity；

It (5), can be by the sucrose of 500g/L in reaction system using method conversion production isomaltoketose 9h of the invention It is converted into the isomaltoketose of 443g/L, Sucrose conversion is up to 94%, isomaltoketose yield and is up to 88.6%, different malt ketone Sugared space-time yield is up to 49.2g/Lh.

Specific embodiment

The present invention will be further elaborated combined with specific embodiments below.

168, E. coli bacillus subtilis involved in following embodiments (Bacillus subtilis) JM109 is purchased from Promega company；Expression vector pMA5 involved in following embodiments is purchased from excellent precious biology；In following embodiments Sucrose, isomaltoketose, fructose, glucose, the trehalulose standard specimen being related to are purchased from SIGMA company, the U.S.；Following embodiments Involved in Na₂HPO₄Citrate buffer solution, phosphate buffer and Qula are logical to be purchased from Chinese Shanghai Chinese medicines group company.

Culture medium involved in following embodiments is as follows:

LB liquid medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L.

LB solid medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar powder 2% (m/v).

Fermentation medium: glucose 20g/L, peptone 10g/L, Angel Yeast powder 5g/L, KH₂PO₄ 0.75g/L、 K₂HPO₄·3H₂O 1.25g/L、MgSO₄·7H₂The NaoH tune pH to 7.0 of O 1g/L, 1mol/L.

Detection method involved in following embodiments is as follows:

The detection method of sucrose isomerase enzyme activity: the pure enzyme after taking 100 μ L suitably to dilute is added to containing 200g/L sucrose 50mM pH7.0 citrate-phosphate disodium hydrogen buffer 900 μ L reaction systems in；In 30 DEG C of reaction 20min, boiling water bath 5 For~10min to terminate enzymatic reaction, centrifuging and taking supernatant utilizes the content of isomaltoketose in HPLC detection reaction solution；

Wherein, the definition of sucrose isomerase enzyme activity: under conditions of 30 DEG C, 7.0 pH, every min catalysis sucrose reaction is generated Enzyme amount needed for 1 μm of ol product isomaltoketose is a unit of activity (U).

The detection method of sucrose isomerase specific enzyme activity: take the pure enzyme of 0.1mg that the 50mM pH7.0 containing 200g/L sucrose is added In the 1mL reaction system of citrate-phosphate disodium hydrogen buffer；20min is reacted at 30 DEG C, 5~10min of boiling water bath is to terminate Enzymatic reaction, centrifuging and taking supernatant utilize the content of isomaltoketose in HPLC detection reaction solution；

Wherein, the definition of sucrose isomerase specific enzyme activity: enzyme activity possessed by every milligram of zymoprotein, unit are U/mg.

The detection method of Sucrose conversion, isomaltoketose yield and isomaltoketose space-time yield: reaction is terminated Reaction solution sample afterwards carries out centrifuging and taking supernatant, and carries out dilution appropriate, and HPLC method measures the residual of sucrose in reaction solution The content of content and isomaltoketose；

Wherein, the calculation formula of Sucrose conversion are as follows: conversion ratio=(sucrose concentration/reaction before reacting in transformation system Sucrose concentration in transformation system afterwards)/react before sucrose concentration × 100% in reaction system；

The calculation formula of isomaltoketose yield are as follows: yield=(after reaction in conversion fluid isomaltoketose concentration/reaction The initial concentration of sucrose in preceding conversion fluid) × 100%；

The calculation formula of isomaltoketose space-time yield are as follows: after space-time yield=reaction in conversion fluid isomaltoketose it is dense Degree/reaction time；

The definition of isomaltoketose space-time yield: under 30 DEG C, the reaction condition of pH7.0, different wheat is generated in the unit time The yield of bud ketose.

(HPLC analysis: because sucrose and isomaltoketose can be detected in Composition distribution, using HPLC The concentration of method measurement substrate and product: where chromatographic condition: chromatographic column: Platisil NH2 (5 μm, 250mm × 4.6mm), stream Dynamic phase: acetonitrile-water (V/V=80:10), detector: RI Detector, column temperature: 40 DEG C, sample volume: 10 μ L, flow velocity: 0.9mL/ min。)

Embodiment 1: the bacillus subtilis engineering bacteria from the sucrose isomerase of Pantoea dispersa can be expressed Building

Specific step is as follows:

(1) sucrose for deriving from Pantoea dispersa UQ68J (GeneID:AY223549.1) is obtained from GENBANK The nucleotide sequence (as shown in SEQ ID NO.2) of isomerase (containing signal peptide) simultaneously obtains this sequence by artificial synthesized；It will This sequence and expression plasmid pMA5 are attached after restriction enzyme Nde I and Mlu I digestion, obtain recombinant plasmid pMA5-sim-1；Recombinant plasmid pMA5-sim-1 is converted into E. coli JM109, recombination bacillus coli is obtained E.coli JM109/pMA5-sim-1；

(2) sucrose for deriving from Pantoea dispersa UQ68J (GeneID:AY223549.1) is obtained from GENBANK The nucleotide sequence (as shown in SEQ ID NO.2) of isomerase (containing signal peptide), removes signal peptide institute on this nucleotide sequence Corresponding partial sequence simultaneously carries out codon optimization to this nucleotide sequence, obtains no signal peptide and optimized sucrose isomerase Nucleotide sequence (as shown in SEQ ID NO.1)；By no signal peptide and the nucleotide sequence of optimized sucrose isomerase with And expression plasmid pMA5 is attached after restriction enzyme Nde I and Mlu I digestion, obtains recombinant plasmid pMA5-sim- 2；Recombinant plasmid pMA5-sim-2 is converted into E. coli JM109, recombinant bacterium E.coli JM109/ is obtained pMA5-sim-2；

(3) by obtained recombinant bacterium E.coli JM109/pMA5-sim-1 and recombinant bacterium E.coli JM109/pMA5- It is 50 μ gmL that sim-2, which is coated on containing concentration,^-1Ampicillin LB solid medium on, in 37 DEG C of constant incubators It is inverted 8~12h of culture, obtains transformant；It is 50 μ gmL that picking transformant, which is seeded to 10mL to contain concentration,^-1Ampicillin LB liquid medium in, then 10~12h of shaking flask culture under conditions of 37 DEG C, 180rpm extracts plasmid and carries out single double enzymes Verifying is cut, obtains converting successful recombinant bacterium E.coli JM109/pMA5-sim-1 and recombinant bacterium E.coli JM109/ pMA5-sim-2。

(4) recombinant bacterium E.coli JM109/pMA5-sim-1 and recombinant bacterium E.coli JM109/pMA5-sim-2 is extracted In recombinant plasmid pMA5-sim-1 and pMA5-sim-2；Recombinant plasmid pMA5-sim-1 and pMA5-sim-2 are converted withered Careless bacillus (Bacillus subtilis) 168, obtains recombinant bacterium BS168/pMA5-sim-1 and recombinant bacterium BS168/ pMA5-sim-2；

(5) by obtained recombinant bacterium BS168/pMA5-sim-1 and recombinant bacterium BS168/pMA5-sim-2 be coated on containing Concentration is 100 μ gmL^-1Kanamycins LB solid medium on, in 37 DEG C of constant incubators be inverted culture 8~12h, Obtain transformant；Picking transformant is seeded in 10mL LB liquid medium, and final concentration of 100 μ gmL is added^-1Card that Mycin, under conditions of 37 DEG C, 180rpm, then shaking flask 10~12h of culture extracts plasmid and carries out single double digestion verifying, obtains Convert successful recombinant bacterium BS168/pMA5-sim-1 and recombinant bacterium BS168/pMA5-sim-2.

Embodiment 2: the structure of the bacillus subtilis engineering bacteria of the gene for expressing encoding sucrose isomerase in other sources It builds

Specific step is as follows:

(1) nucleotide sequence of the sucrose isomerase from Erwinia rhapontici NX-5 is obtained from GENBANK (as shown in SEQ ID NO.3), from Enterobacter sp.FMB-1 sucrose isomerase nucleotide sequence (such as SEQ Shown in ID NO.4) and the sucrose isomerase from Serratia plymuthica nucleotide sequence (such as SEQ ID Shown in NO.5) and these sequences are obtained by artificial synthesized；By these sequences respectively with by restriction enzyme Nde I and Expression plasmid pMA5 after Mlu I digestion is attached, and obtains recombinant plasmid pMA5-sim-3, pMA5-sim-4 and pMA5- sim-5；Recombinant plasmid pMA5-sim-3, pMA5-sim-4 and pMA5-sim-5 are converted respectively to E. coli In JM109, obtain recombination bacillus coli E.coli JM109/pMA5-sim-3, E.coli JM109/pMA5-sim-4 and E.coli JM109/pMA5-sim-5；

(2) by obtained recombinant bacterium E.coli JM109/pMA5-sim-3, E.coli JM109/pMA5-sim-4 and It is 100 μ gmL that E.coli JM109/pMA5-sim-5, which is coated on containing concentration,^-1Ampicillin LB solid medium it is flat On plate, it is inverted 8~12h of culture in 37 DEG C of constant incubators, obtains transformant；Picking transformant is seeded to 10mL and contains concentration For 100 μ gmL^-1Ampicillin LB liquid medium in, under conditions of 37 DEG C, 180rpm shaking flask culture 10~ Then 12h extracts plasmid and carries out single double digestion verifying, and is sent to sequencing company and carries out sequence verification, obtain converting successful weight Group bacterium E.coli JM109/pMA5-sim-3, E.coli JM109/pMA5-sim-4 and E.coli JM109/pMA5-sim- 5；

(3) extract recombinant bacterium E.coli JM109/pMA5-sim-3, recombinant bacterium E.coli JM109/pMA5-sim-4, with And recombinant plasmid pMA5-sim-3, pMA5-sim-4 and pMA5-sim- in recombinant bacterium E.coli JM109/pMA5-sim-5 5；Recombinant plasmid pMA5-sim-3, pMA5-sim-4 and pMA5-sim-5 are transformed into bacillus subtilis (Bacillus Subtilis) in 168, obtain converting successful recombinant bacterium BS168/pMA5-sim-3, recombinant bacterium BS168/pMA5-sim-4 with And recombinant bacterium BS168/pMA5-sim-5.

Embodiment 3: the expression of the sucrose isomerase of separate sources

Specific step is as follows:

(1) plasmid pMA5 is converted into bacillus subtilis (Bacillus subtilis) 168 and is expressed, recombinated Bacterial strain BS168/pMA5 is as blank control；

(2) picking embodiment 1 obtain recombinant bacterium BS168/pMA5-sim-1, recombinant bacterium BS168/pMA5-sim-2 and Recombinant bacterium BS168/pMA5-sim-3, recombinant bacterium BS168/pMA5-sim-4 and the recombinant bacterium BS168/ that embodiment 2 obtains The single colonie of pMA5-sim-5 is seeded to respectively in the LB culture medium of 10mL, is cultivated 12h under conditions of 37 DEG C, 180rpm, is obtained To seed liquor；

(3) seed liquor is seeded in 50mL fermentation medium with 1% inoculum concentration, under conditions of 25 DEG C, 180rpm Culture for 24 hours, respectively obtains recombinant bacterium BS168/pMA5-sim-1, recombinant bacterium BS168/pMA5-sim-2, recombinant bacterium BS168/ The culture solution of pMA5-sim-3, recombinant bacterium BS168/pMA5-sim-4 and recombinant bacterium BS168/pMA5-sim-5；

(4) culture solution is collected into thallus in 8000rpm, 4 DEG C of centrifugation 5min, first washes twice thallus, then benefit with buffer Thallus is resuspended to 5mL with buffer, ultrasonic cell disruption instrument smudge cells are then used, finally in 8000~10000rpm, 4 Supernatant is taken after carrying out 15~20min of centrifugation under conditions of DEG C, respectively obtains recombinant bacterium BS168/pMA5-sim-1, recombinant bacterium BS168/pMA5-sim-2, recombinant bacterium BS168/pMA5-sim-3, recombinant bacterium BS168/pMA5-sim-4 and recombinant bacterium The crude enzyme liquid of BS168/pMA5-sim-5 detects the sucrose isomerase enzyme activity in crude enzyme liquid.

Testing result is as follows: the sucrose isomerase enzyme activity in crude enzyme liquid that culture recombinant bacterium BS168/pMA5-sim-1 is obtained For 115U/mL, culture recombinant bacterium BS168/pMA5-sim-2 obtain crude enzyme liquid in sucrose isomerase enzyme activity be 125U/mL, The sucrose isomerase enzyme activity in the crude enzyme liquid that recombinant bacterium BS168/pMA5-sim-3 is obtained is cultivated to be 112U/mL, cultivate recombinant bacterium The sucrose isomerase enzyme activity in crude enzyme liquid that BS168/pMA5-sim-4 is obtained is 80U/mL, cultivates recombinant bacterium BS168/pMA5- The sucrose isomerase enzyme activity in crude enzyme liquid that sim-5 is obtained is 103U/mL.

As it can be seen that deriving from Pantoea dispersa, partial sequence corresponding to signal peptide on this nucleotide sequence is removed And the enzyme activity highest of the sucrose isomerase after codon optimization, higher than other sources and without the sucrose of codon optimization Therefore isomerase answers the gene of selected nucleotide sequence encoding sucrose isomerase as shown in SEQ ID NO.1 to be expressed.

Embodiment 4: the zymologic property of the sucrose isomerase of separate sources

Specific step is as follows:

Culture recombinant bacterium BS168/pMA5-sim-1, the recombinant bacterium BS168/pMA5-sim-2, recombination that embodiment 3 is obtained The thick enzyme that bacterium BS168/pMA5-sim-3, recombinant bacterium BS168/pMA5-sim-4 and recombinant bacterium BS168/pMA5-sim-5 are obtained Liquid respectively through affinity column after purification, obtain pure enzyme SIM-1, SIM-2, SIM-3, SIM-4 and SIM-5.

1, the specific enzyme activity of pure enzyme SIM-1, SIM-2, SIM-3, SIM-4 and SIM-5 are detected

Testing result are as follows: the specific enzyme activity of SIM-1, SIM-2, SIM-3, SIM-4 and SIM-5 be respectively as follows: 500U/mg, 532U/mg, 423U/mg, 120U/mg, 328U/mg, it is seen then that from Pantoea dispersa excision signal peptide and through overstocked The specific enzyme activity of sucrose isomerase enzyme after numeral optimization is different without signal peptide excision and other microbe-derived sucrose 1.06,1.25,4.43,1.62 times of structure enzyme, it is therefore, subsequently selected that other aspects zymology is carried out to pure enzyme SIM-1 and SIM-2 The comparison of matter.

2, influence of the detection temperature to pure enzyme SIM-1 and SIM-2

The pure enzyme SIM-1 and SIM-2 of 0.1mg is taken to be added separately to the pH that 1mL contains 200g/L sucrose as 7.0 50mM lemon It in lemon acid-disodium hydrogen phosphate buffer, is placed in the water-bath of different temperatures, temperature range is 20~45 DEG C, spacing gradient It is 5 DEG C, reacts after twenty minutes, 5~10min of boiling water bath termination enzymatic reaction, centrifuging and taking supernatant, after suitably dilution processing, HPLC method measures the enzyme activity in supernatant.

Testing result are as follows: SIM-1 and SIM-2 equal highest of enzyme activity under conditions of 30 DEG C, optimal reactive temperature are 30 DEG C, As it can be seen that the excision of signal peptide, optimal reactive temperature to enzyme is simultaneously had no significant effect.

3, influence of the detection temperature to pure enzyme SIM-1 and SIM-2

With reference to the detection method in 2, using the reaction system of 1mL, keeping reaction temperature is 30 DEG C, by the buffer of reaction It is substituted for citrate-phosphate disodium hydrogen buffer (pH range is 4.0~8.0), the spacing gradient 0.5 of different pH respectively, so After be separately added into SIM-2 and SIM-3 reaction 20min, 5~10min of boiling water bath terminates enzymatic reaction, centrifuging and taking supernatant, by suitable After dilution processing, HPLC method measures the enzyme activity in supernatant.

Testing result are as follows: SIM-1 and SIM-2 enzyme activity in the range of pH is 5.5~7.5 is relatively high, wherein pure enzyme The optimal reaction pH of SIM-1 is 6.5, and the optimal reaction pH of pure enzyme SIM-2 is 7.0, it is seen then that the excision of signal peptide, most to enzyme Suitable reaction pH influence is not very big.

4, the isomaltoketose yield of pure enzyme SIM-1 and SIM-2 is detected

Take the pure enzyme SIM-1 and SIM-2 of 0.1mg be added separately to the pH that 1mL contains 200g/L sucrose be respectively 6.5 with In 7.0 50mM citrate-phosphate disodium hydrogen buffer, after then reacting 20min in 30 DEG C of water-baths, boiling water bath 5~ 10min terminates enzymatic reaction, and centrifuging and taking supernatant, after suitably dilution processing, HPLC method measures the different malt ketone in supernatant Candy output.

Testing result are as follows: the isomaltoketose rate of SIM-1 and SIM-2 is respectively 82.5% and 90.6%, it is seen then that is derived from Pantoea dispersa and cut off signal peptide and codon optimization after sucrose isomerase isomaltoketose yield it is bright It is aobvious not cut off signal peptide and without the sucrose isomerase after codon optimization higher than from Pantoea dispersa.

Embodiment 5: the application of recombinant bacterium BS168/pMA5-sim-2

Specific step is as follows:

The recombinant bacterium BS168/pMA5-sim-2 that embodiment 3 is obtained carries out the system amplification culture of 5L fermentor, fermentation After producing enzyme, culture solution is centrifuged 5min in 4 DEG C, 8000rpm, discards supernatant and recycles thallus；Then it is with concentration The disodium hydrogen phosphate of 0.05mol/L-citrate buffer solution washing thalline suspends twice and then with buffer to thallus, Thallus in the phosphate buffer for being 7.0 to 0.05mol/L, pH containing 500g/L substrate sucrose after addition washing extremely buffers The concentration of thallus is OD in liquid₆₀₀=25~30, obtain the transformation system of 1L；The cell wall of 1mL is added in the reaction system simultaneously Penetrating dose of Qula is logical, and continuous transformation 9h under the conditions of 30 DEG C, 180rpm just obtains the conversion reaction of high concentration isomaltoketose Liquid；It needs to stablize by the way that ammonium hydroxide adjusting pH is added 6~7.5 in entire conversion process.

The Sucrose conversion of isomaltoketose, isomaltoketose yield and isomaltoketose space-time in reaction solution is detected to produce Rate.

Testing result are as follows:, can be by the sucrose of 500g/L in reaction system using the method conversion production isomaltoketose 9h It is converted into the isomaltoketose of 443g/L, isomaltoketose yield is up to 88.6%, Sucrose conversion and is up to 94%, space-time yield Up to 49.2g/Lh.

As it can be seen that preparing isomaltoketose with yield, conversion ratio and high excellent of Spatial-temporal Transformation rate yield using the method Gesture, the prospect of great large-scale industrial production.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

Sequence table

<110>Southern Yangtze University

<120>a kind of method using resting cell production isomaltoketose

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1737

<212> DNA

<213>artificial sequence

<400> 1

atggcaagcc cgctgaccaa gccgagtacc ccgatcgcag ccaccaacat ccagaagagc 60

gccgattttc cgatttggtg gaagcaagct gtgttctacc agatctaccc gcgcagtttc 120

aaggacagta acggcgacgg catcggtgat atcccgggca ttattgaaaa gctggattat 180

ttaaagatgc tgggtgtgga tgcaatctgg atcaacccgc actacgagag tccgaatacc 240

gacaacggct acgacatcag cgattatcgc aaaatcatga aagagtatgg cagcatggca 300

gactttgacc gtttagttgc cgaaatgaat aaacgcggca tgcgtctgat gatcgacatc 360

gttattaatc ataccagcga ccgccaccgt tggtttgttc agagccgtag cggcaaagac 420

aatccgtatc gtgactacta cttttggcgc gacggtaaac aaggtcaagc tccgaataat 480

tacccgagct tctttggcgg cagcgcatgg caactggata aacagaccga ccagtactat 540

ttacactact ttgccccgca acaaccggat ttaaattggg acaatccgaa agtgcgtgcc 600

gagctgtacg acatcttacg cttctggctg gataagggcg tgagcggttt acgttttgat 660

accgttgcca cattcagcaa aattccgggc ttcccggatc tgagcaaggc ccaactgaaa 720

aacttcgcag aggcatatac cgagggtccg aacatccaca agtacatcca cgagatgaac 780

cgtcaagttc tgagcaaata caatgtggcc accgctggtg agatctttgg tgttccggtg 840

agcgccatgc ccgattactt cgatcgtcgc cgcgaggagc tgaacattgc ctttaccttc 900

gatttaattc gcttagaccg ctaccccgat cagcgctggc gccgtaagcc gtggacttta 960

agtcaattcc gccaagttat cagccaaacc gatcgtgccg ccggtgagtt tggctggaat 1020

gccttctttt tagacaacca tgataaccct cgccaagtta gccattttgg cgatgatagc 1080

ccgcaatggc gtgaacgcag cgcaaaagct ttagccacac tgctgttaac ccagcgtgcc 1140

acccctttca tctttcaagg tgccgagctg ggcatgacca attatccttt taaaaatatt 1200

gaagagttcg atgacattga ggtgaagggc ttctggaacg actacgttgc aagtggcaag 1260

gttaacgccg ccgaatttct gcaagaagtt cgcatgacca gccgcgacaa tagccgtacc 1320

ccgatgcagt ggaacgatag cgttaatgcc ggcttcaccc aaggtaaacc gtggtttcat 1380

ctgaacccga actataagca aatcaacgcc gcccgcgaag tgaacaagcc ggacagcgtg 1440

tttagttact accgccagct gattaatctg cgccatcaga tcccggcact gaccagtggc 1500

gaataccgcg atttagatcc gcagaacaac caagtgtacg cctacacccg cattttagac 1560

aacgagaaat atttagttgt ggttaacttc aaaccggagc agctgcatta tgctttaccg 1620

gacaatttaa ccatcgccag ttctttactg gagaacgtgc atcagccgag tctgcaagaa 1680

aatgccagta ccttaacttt agccccgtgg caagccggca tttacaaatt aaattaa 1737

<210> 2

<211> 1797

<212> DNA

<213>artificial sequence

<400> 2

atgtttctta atggatttaa gacagttatt gctctgacta tggcaagctc gttttatctt 60

gccgccagcc cgttaactaa gccatcgacc cctattgccg caacgaatat acaaaagtcc 120

gctgattttc ccatttggtg gaaacaggca gtattttacc agatttatcc ccgctcattt 180

aaagatagca atggtgatgg tatcggcgat attcccggta tcattgagaa actggactat 240

ttaaaaatgc tgggagttga tgctatctgg ataaacccgc actatgagtc tcctaacacc 300

gacaatggtt acgatattag tgattatcgt aaaatcatga aggagtacgg cagcatggct 360

gactttgacc gtctggttgc cgaaatgaat aaacgtggta tgcgcctgat gattgatatt 420

gttatcaatc ataccagcga tcgtcaccgc tggtttgtgc agagccgttc aggtaaagat 480

aatccttacc gcgactatta tttctggcgt gatggtaaac agggacaggc tcccaataac 540

tatccctctt tctttggcgg ttcagcctgg caactggata aacagactga ccagtattat 600

ctgcactatt ttgcaccaca gcagccggat ctgaactggg ataacccaaa agttcgggct 660

gaactctacg atattctgcg tttctggctg gataaaggcg tatccggact acgttttgat 720

accgtggcta ctttctccaa aattcctggc ttcccggacc tgtcaaaagc gcagctgaag 780

aattttgccg aagcttatac tgaggggccg aatattcata aatatatcca tgaaatgaac 840

cgccaggtac tgtctaaata taatgttgcc accgctggtg aaatcttcgg tgtgccagtg 900

agtgctatgc cggattattt tgaccggcgg cgtgaagaac tcaatattgc tttcaccttt 960

gatttgatca ggctcgatcg ttatcccgat cagcgctggc gtcgtaaacc atggacatta 1020

agccagtttc gtcaagttat ctctcagact gaccgtgccg ccggtgaatt tggctggaac 1080

gcctttttcc ttgataacca tgataacccg cgccaggtct cacactttgg tgacgacagc 1140

ccacaatggc gcgaacgctc ggcaaaagca ctggcaacgc tgctgctgac gcagcgtgcc 1200

acgccgttta tctttcaggg ggcggagttg ggaatgacta attacccctt taaaaatata 1260

gaggaatttg atgatattga ggttaaaggc ttctggaacg actatgtagc cagcggaaaa 1320

gtaaacgctg ctgaattttt acaggaggtt cgcatgacca gccgcgataa cagccgaaca 1380

ccaatgcagt ggaacgactc tgttaatgcc ggattcaccc agggcaaacc ctggtttcac 1440

ctcaatccca actataagca aatcaatgcc gccagggagg tgaataaacc cgactcggta 1500

ttcagttact accgtcaact gatcaacctg cgtcaccaga tcccggcact gaccagtggt 1560

gaataccgtg atctcgatcc gcagaataac caggtctatg cctatacccg tatactggat 1620

aatgaaaaat atctggtggt agttaatttt aaacctgagc agctgcatta cgctctgcca 1680

gataatctga ctattgccag cagtctgctg gaaaatgtcc accaaccatc actgcaagaa 1740

aatgcctcca cgctgactct tgctccgtgg caagccggga tctataagct gaactga 1797

<210> 3

<211> 1803

<212> DNA

<213>artificial sequence

<400> 3

atggattctc aaggattgaa aacggctgtc gctatttttc ttgcaaccac tttttctgcc 60

acatcctatc aggcctgcag tgccgggcca gataccgccc cctcactcac cgttcagcaa 120

tcaaatgccc tgcccacatg gtggaagcag gctgtttttt atcaggtata tccacgctca 180

tttaaagata cgaatgggga tggcattggg gatttaaacg gtattattga gaatttagac 240

tatctgaaga aactgggtat tgatgcgatt tggatcaatc cacattacga ttcgcctaat 300

acggataatg gttatgacat ccgggattac cgtaagataa tgaaagaata cggtacgatg 360

gaagactttg accgtcttat ttcagaaatg aagaaacgca atatgcgttt gatgattgat 420

attgttatca accacaccag cgatcagcat gcctggtttg ttcagagcaa atcgggtaag 480

aacaacccct acagggacta ttacttctgg cgtgacggta aggatggcca tgcccccaat 540

aactatccct ccttcttcgg tggctcagcc tgggaaaaag acgataaatc aggccagtat 600

tacctccatt actttgccaa acagcaaccc gacctcaact gggacaatcc caaagtccgt 660

caagacctgt atgacatgct ccgcttctgg ttagataaag gcgtttctgg tttacgcttt 720

gataccgttg ccacctactc gaaaatcccg aacttccctg accttagcca acagcagtta 780

aaaaatttcg ccgaggaata tactaaaggt cctaaaattc acgactacgt gaatgaaatg 840

aacagagaag tattatccca ctatgatatc gccactgcgg gggaaatatt tggggttcct 900

ctggataaat cgattaagtt tttcgatcgc cgtagaaatg aattaaatat agcgtttacg 960

tttgatctga tcaggctcga tcgtgatgct gatgaaagat ggcggcgaaa agactggacc 1020

ctttcgcagt tccgaaaaat tgtcgataag gttgaccaaa cggcaggaga gtatgggtgg 1080

aatgcctttt tcttagacaa tcacgacaat ccccgcgcgg tttctcactt tggtgatgat 1140

cgaccacaat ggcgcgagca tgcggcgaaa gcactggcaa cattgacgct gacccagcgt 1200

gcaacgccgt ttatctatca gggttcagaa ctcggtatga ccaattatcc ctttaaaaaa 1260

atcgatgatt tcgatgatgt agaggtgaaa ggtttttggc aagactacgt tgaaacaggc 1320

aaagtgaaag ctgaggaatt ccttcaaaac gtacgccaaa ccagccgtga taacagcaga 1380

acccccttcc agtgggatgc aagcaaaaac gcgggcttta ccagtggaac cccctggtta 1440

aaaatcaatc ccaattataa agaaatcaac agcgcagatc agattaataa tccaaattcc 1500

gtatttaact attatagaaa gctgattaac attcgccatg acatccctgc cttgacctac 1560

ggcagttata ttgatttaga ccctgacaac aattcagtct atgcttacac ccgaacgctc 1620

ggcgctgaaa aatatcttgt ggtcattaat tttaaagaag aagtgatgca ctacaccctg 1680

cccggggatt tatccatcaa taaggtgatt actgaaaaca acagtcacac tattgtgaat 1740

aaaaatgaca ggcaactccg tcttgaaccc tggcagtcgg gcatttataa acttaatccg 1800

tag 1803

<210> 4

<211> 1800

<212> DNA

<213>artificial sequence

<400> 4

atgtcgtttt ttaaaaaatt aacaggggtt gccgtcacgt tttcatcctt gtttatgaca 60

ttagctcccg cagcgtatag tgcagaaact tccgtcactc agagtataca gactcagaag 120

gagagtacac ttccggcatg gtggaaagaa gctgtatttt atcaaattta tccccgctca 180

tttaaagata cgaacggaga tgggataggt gacattcgcg gcattataga aaaactggac 240

tatcttaaat cattagggat agatgcaatc tggattaatc cacattacga ctcacctaac 300

actgataatg gttatgatat cagagactac gaaaaaatta tgcaagagta cggaactatg 360

gaggattttg acacgttagt ttcagaaatg aaaaagcgca atatgcggtt aatgatagac 420

gtggtaatta atcatacaag cgatcaacac ccctggttta ttcaaagtaa aagcagtaaa 480

gaaaacccat atcgtgaata ttatttctgg cgtgacggta aggataatca gccacccaat 540

aattacccct cattctttgg cggctctgca tggcaaaaag atgataaaac aggacaatat 600

tatctccatt attttgccag acagcagcct gaccttaact gggataatcc gaaggtgcga 660

ggcgatcttt acgccatgct ccgcttctgg cttgataagg gcgtgtccgg gatgcggttt 720

gacaccgttg caacgtattc gaagattccg ggcttccccg atctgacgcc agagcagcag 780

aaaaattttg cagagcagta cacaacgggg ccgaatatcc atcgttatct acaagaaatg 840

aaacaggaag tcctttcgcg gtatgatgta gtgacggcag gcgaaatatt cggtgttcct 900

ctcgagcgct cgtctgattt ttttgaccgt cgtcgcaatg aactggatat gtcgtttatg 960

tttgacttaa tccgtctcga tcgcgacagc aatgaacgct ggcgtcataa aaaatggacg 1020

ctttctcagt ttcgtcagat tatcaacaaa atggattcta acgccggaga gtatggatgg 1080

aacaccttct ttttggacaa tcatgataac ccgcgggcag tgtctcattt tggggatgac 1140

agccctcagt ggatagagcc ttccgctaag gcattagcga caatcattct gacccagcgt 1200

gcgacacctt tcatttttca ggggtcagaa ctgggaatga ccaattatcc ctttaaaaag 1260

ctgaatgaat ttgacgatat tgaagtcaaa ggtttctggc aggattatgt ccagaccgga 1320

aaagtgtcgg ctgaagaatt tattgataac gtacgcctga caagccgtga taacagcagg 1380

accccttttc agtggaacga ccgtaagaac gctggtttta ccagtggaaa gccctggttc 1440

agaattaatc caaactatgt tgaaattaac gctgacaaag aattaatccg taatgactct 1500

gtgctcaatt attataagga aatgatcaag ttgcggcata aaacccctgc gctcatatac 1560

ggcacctata aagacatcag tcctgaagat gatagtgtct atgcttatac cagaaccctt 1620

ggaaaggaac gctatcttgt cgtaataaac tttaccgaaa agacagttcg ttatcctctg 1680

ccggaaaata atgttatcaa aagcatctta attgaggcca atcagaataa aaccgccgaa 1740

aaacagagta cagtcttaac attgagtccc tggcaggctg gggtttatga actccagtaa 1800

<210> 5

<211> 1803

<212> DNA

<213>artificial sequence

<400> 5

atgccccgtc aaggattgaa aactgcacta gcgatttttc taaccacatc attaagcgtc 60

tcatgccagc aagccttagg tacgcaacaa cccttgctta acgaaaagag tatcgaacag 120

tcgaaaacca tacctaaatg gtggaaggag gctgtttttt atcaggtgta tccgcgttcc 180

tttaaagaca ctaacgggga tggtatcggg gatattaaag gcatcataga aaaattagac 240

tatttaaaag ctttggggat tgatgccatt tggatcaacc cacattatga ctccccgaac 300

acggataatg gttacgatat acgtgattat cgaaaaatca tgaaagaata tggcacgatg 360

gaggattttg accgcctgat ttctgaaatg aaaaaacgta acatgcggtt gatgattgat 420

gtggtcatca accacaccag cgatcaaaac gaatggtttg ttaaaagtaa aagcagtaag 480

gataatcctt atcgtggcta ttacttctgg aaagatgcta aagaagggca ggcgcctaat 540

aattaccctt cattctttgg tggctcggcg tggcaaaaag atgaaaagac caatcaatac 600

tacctgcact attttgctaa acaacagcct gacctaaact gggataaccc caaagtccgt 660

caagatcttt atgcaatgtt gcgtttctgg ttagataaag gcgtgtctgg tttacgcttt 720

gatacggtag cgacctactc aaaaattccg gacttcccaa atctcaccca acaacagctg 780

aagaattttg cagctgagta taccaagggc cctaatattc atcgttacgt caatgaaatg 840

aatagagaag ttttgtctca ttacgacatt gccactgccg gtgaaatctt tggcgtaccc 900

ttggatcaat cgataaaatt cttcgatcgc cgtcgcgatg agctgaacat cgcatttacc 960

tttgacttaa tcagactcga tcgagactct gatcaaagat ggcgtcgaaa agagtggaaa 1020

ttgtcgcaat tccgacaggt catcgataac gttgaccgta ctgccggcga atatggttgg 1080

aatgccttct tcttggataa ccacgacaat ccgcgcgctg tctcccactt tggcgatgat 1140

cgcccacaat ggcgcgagcc atcggctaaa gcgcttgcaa ccttgacgct gactcaacga 1200

gcaacgcctt ttatttatca aggttcagaa ttgggcatga ccaattaccc cttcaaagct 1260

attgatgaat tcgatgatat tgaggtgaaa ggtttttggc atgactacgt tgagacagga 1320

aaggtgaaag ccgacgagtt cttgcaaaat gtacgcctga cgagcaggga taacagccgg 1380

acaccgttcc aatgggatac gagcaaaaat gcaggattca cgagcggaaa accttggttc 1440

aaggtcaatc caaactacca ggaaatcaat gcggtaagtc aagtcgcaca gcccgactcg 1500

gtatttaatt attatcgtca gttgatcaag ataaggcata acatcccggc actgacctat 1560

ggcacataca ccgatttgga tcctgcaaat gattcggtct acgcctatac acgcagcctt 1620

ggggcggaaa aatatcttgt tgtcgttaac ttccaggaac aagtgatgag atataaatta 1680

ccggataatc tatccatcga gaaagtgatt atagaaagca acagcaaaaa cgttgtgaaa 1740

aagaatgatt ccttactcga actaaaacca tggcagtcag gggtttataa actaaatcaa 1800

taa 1803

Claims (10)

1. one section of gene that can be used for encoding sucrose isomerase, which is characterized in that the nucleotide sequence of the gene such as SEQ Shown in IDNO.1.

2. carrying the recombinant plasmid of gene described in claim 1.

3. recombinant plasmid as claimed in claim 2, which is characterized in that for constructing the carrier of the recombinant plasmid as pMA5 load Body, pHT43 carrier, pET-20b (+) carrier, pXMJ19 carrier or pDXW-10 carrier.

4. carrying the host cell of recombinant plasmid described in gene described in claim 1 or Claims 2 or 3.

5. host cell as claimed in claim 4, which is characterized in that the host cell is bacillus subtilis, large intestine bar Bacterium or Corynebacterium glutamicum.

6. a kind of method for producing sucrose isomerase, which is characterized in that the method is by host described in claim 4 or 5 Cell is cultivated in the medium.

7. a kind of method using resting cell production isomaltoketose, which is characterized in that the method is by claim 4 Or host cell described in 5 is added in the reaction system containing sucrose and carries out conversion reaction.

8. a kind of method using resting cell production isomaltoketose as claimed in claim 7, which is characterized in that described Method is host cell described in claim 4 or 5 to be resuspended to host cell in buffer with the buffer containing sucrose Cell concentration is OD₆₀₀=25~30, obtain reaction system；Penetrating dose of progress conversion reaction of cell wall is added in the reaction system, Obtain isomaltoketose.

9. recombinant plasmid described in gene or Claims 2 or 3 described in claim 1 or the host cell of claim 4 or 5 or Application of claim 6 the method in terms of producing sucrose isomerase.

10. recombinant plasmid described in gene or Claims 2 or 3 described in claim 1 or the host cell of claim 4 or 5 Or the application of claim 7 or 8 the methods in terms of producing isomaltoketose.