CN102277308B - Immobilized catalyst for use in production of maltose and preparation method thereof - Google Patents

Abstract

The invention relates to an immobilized catalyst for use in the production of maltose and a preparation method thereof and belongs to the technical field of enzyme engineering and agricultural and sideline product deep processing. The preparation method comprises: extracting a beta-amylase gene from sweet potatoes by reverse transcription technology; fusing the beta-amylase gene with an alpha-lectin coding gene from a wine brewing yeast in a reading frame; transforming a wine brewing yeast W303-1A by the fusion gene to obtain a wine brewing yeast on the surface of which the beta-amylase is anchored; further performing the large-scale culture the recombinant yeast cells and obtaining cells; and stabilizing the recombinant yeast by chemical and physical processes and crosslinking the recombinant yeast to obtain immobilized beta-amylase, wherein the enzyme activity is 50 to 250DPo/g. When the immobilized beta-amylase is used, high maltose syrup at a concentration of 55 to 65 percent can be directly produced from liquefied starch; and the immobilized beta-amylase can be used repeatedly for 10 times with the enzyme activity loss rate less than 20 percent.

Description

A kind of immobilized catalyst of producing for maltose and preparation method thereof

Technical field

The present invention relates to the special fixation Catalysts and its preparation method that a kind of starch is processed as maltose, belong to enzyme engineering and processing of agriculture product technical field.

Background technology

Maltose has higher nutritive value, and its sugariness only has 1/3 of sucrose, and is more more agreeable to the taste than sucrose.Mainly contain following application characteristic in foodstuffs industry: 1) maltose is not participated in insulin metabolism, is conducive to the control of diabetes; 2) crystallinity is low, and the effect that prevents the starch cohesion is arranged; 3) thermostability is high, and acid-resistant stability is high, and cokeability is low, is heated to be difficult for producing coloring matter; 4) have less water-absorbent, generally absorb 6% ~ 12% moisture after, just no longer suction also no longer discharges moisture, helps to suppress the dehydration of candy, makes candy keep soft and prolongs the shelf-lives of candy; 5) be suitable for the continous vacuum film and stir off and water mold forming, especially to making the hard candy successful, and can reduce the viscosity of candy, improve the local flavor of candy.Pharmaceutically, because maltose does not need Regular Insulin just can be absorbed in body metabolism, therefore be that agent is treated in diabetic subject's nutrition agent and auxiliary smelting, with pure maltose transfusion instillation vein.In addition, the raw material of maltose or multiple great product (such as trehalose, isomaltose).

The production of maltose is to make by hydrolyzed starch, usually with the malt syrup form as product form, high-purity crystallized maltose product form is also arranged.Malt syrup is take starch as raw material, and that makes through enzymatic hydrolysis is a kind of take maltose as main syrup, by method for making be divided into (tables 1) such as maltose, high maltose syrup and superhigh maltose syrups different from maltose content.

The main sugared moiety of all kinds of malt syrups of table 1

Classification	The DE value	Glucose (%)	Maltose (%)	Trisaccharide maltose (%)	Other (%)
						Maltose	35-50	<10	20-30	10-20	30-40
High maltose syrup	35-50	0.5-3	45-60	10-25	--
						Superhigh maltose syrup	45-60	1.5-2	70-85	8-21	--

Enzyme in being applied to take maltose production as main starch processing mainly contains: a-amylase, b-amylase, fungi a-amylase, Pullulanase, isoamylase etc.Use or various combination use and can be used for producing variant production separately.

Maltose

Maltose is China's a kind of sweet food product since ancient times, with starchy material---rice, corn, jowar, potato class are produced through the saccharifying agent effect, and the sugar composition is mainly maltose, dextrin and oligose, and nutritive value is higher, sweet taste is soft, tasty and refreshing, is infant's good food.China's special product " sesame candy ", " crisp sweets ", Fructus Hordei Germinatus sugar, peanut brittle etc. all are the reproduced goods of maltose.

Maltose is produced according to the raw material form different, and solid saccharogenic method and liquid enzyme process are arranged, and the former is saccharifying agent with Fructus Hordei Germinatus, and equipment is simple, and labour intensity is large, and production efficiency is low; The latter is liquefied to starch slurry with α-amylase first, carries out saccharification with wheat bran or Fructus Hordei Germinatus (beta-amylase wherein) again.Maltose content accounts for 20% ~ 30% of total reducing sugar usually.

High maltose syrup

High maltose syrup is similar to the method for making of maltose, but maltose content requires more than 45%, and product should be through decolouring, the refined syrup of ion-exchange, its outward appearance is clean and clear such as water, protein and ash oontent are atomic, and syrup infusion temperature generally reaches more than 140 ℃ far above maltose.

Make the saccharifying agent of high maltose syrup except the Fructus Hordei Germinatus beta-amylase, also the beta-amylase of being produced by sweet potato, barley, wheat bran, soybean commonly used.For guaranteeing that the maltose growing amount is not less than 45%, debranching factor commonly used during saccharification.Also available fungal alpha-amylase is made high maltose syrup.Fungal alpha-amylase can from the inner α-Isosorbide-5-Nitrae key that cuts of starch molecule, generate maltose and α-limit dextrin of being with α-1,6 key.The latter's relative molecular mass is the low and good fluidity of little, made high maltose syrup viscosity more than β-limit dextrin.The high maltose syrup of European and American countries is produced as saccharifying agent with fungi a-amylase mostly, maltose accounted for 45% ~ 60% during it formed, trisaccharide maltose approximately 20%, glucose 2% ~ 7% and other oligose and dextrin etc., in the product glucose content higher be its weak point.

Superhigh maltose syrup or liquid maltose

The maltose content of superhigh maltose syrup surpasses 70%, and its purposes is different from general high maltose syrup, mainly is for the manufacture of pure maltose, makes maltitol powder after the drying, makes maltose alcohol etc. after the hydrogenation.Must simultaneously or in succession use beta-amylase and debranching factor when producing superhigh maltose syrup.Sometimes in order to improve the content of maltose, often use more than one debranching factor and saccharification enzyme, and strictly control Degree of Liquefaction, liquefaction DE value should be no more than 10% or lower, and the concentration of substrate general control is below 30% or lower.

As above can find out, beta-amylase is one of most crucial zymin during maltose is made.

Beta-amylase (EC 3.2.1.2), it is a kind of circumscribed-type saccharifying enzyme, it can cut off one by one from non reducing end α-1 take maltose as unit, the 4-dextran chain, hydrolysate is maltose, trisaccharide maltose and β-limit dextrin, is mainly used in the industries such as maltose in the foodstuffs industry, high maltose syrup, superhigh maltose syrup, beer.Beta-amylase extensively is present in the high plants such as barley, wheat, sweet potato, soybean.The beta-amylase enzyme activity of plant origin is high, good heat resistance, action pH scope are wide, but at present in all techniques, from plant extract obtain high-purity beta-diastatic cost of manufacture is higher.

The present invention is by extracting the beta-amylase gene of Ipomoea batatas, merge in frame with the α lectin encoding gene that derives from yeast saccharomyces cerevisiae again, fusion gene is expressed in yeast saccharomyces cerevisiae again, and expressed beta-amylase is fixed at the brewing yeast cell surface anchoring through yeast α lectin.Further this recombinant yeast cell of large scale culturing, harvested cell is stablized crosslinked this recombination yeast with chemistry, physical method, obtains the immobilization beta-amylase, and enzyme work is 50 ~ 250 DP ^o/ g.Use this immobilization beta-amylase can be by Starch Production high maltose syrup and superhigh maltose syrup.

Summary of the invention

One of the object of the invention provides a kind of for from Starch Production high maltose syrup and the required immobilization beta-amylase of superhigh maltose syrup.

Two of the object of the invention provides a kind of immobilization beta-diastatic preparation method.

Technical scheme of the present invention is:

A kind of recombination yeast, the called after yeast saccharomyces cerevisiae ( Saccharomyces cerevisiae) HH20110602, expressed beta-amylase is fixed in yeast saccharomyces cerevisiae W303-1A cell surface grappling through yeast α lectin, obtain recombination yeast, be preserved in Wuhan, China Wuhan University Chinese Typical Representative culture collection center, deposit number CCTCC NO:M 2011220.

The immobilized catalyst that is used for high maltose syrup production of described recombination yeast preparation is in the nature beta-amylase, by the beta-amylase molecule is realized that in the grappling of yeast saccharomyces cerevisiae W303-1A cell surface it is immobilized;

Described immobilized catalyst is the immobilization beta-amylase;

Expressed beta-amylase is fixed in yeast saccharomyces cerevisiae W303-1A cell surface grappling through yeast α lectin, obtain recombination yeast CCTCC NO:M 2011220, further this recombinant yeast cell of large scale culturing, centrifugal cell harvesting, suspend and lower this recombination yeast of irradiation of ultraviolet ray with glutaraldehyde solution, obtain the immobilization beta-amylase;

Enzyme work is 50 ~ 250 DP ^o/ g; PH is 3.0 ~ 10.5, and temperature is 55 ~ 75 ℃, and immobilized catalyst is stable.

1. the immobilization beta-amylase produces the structure of bacterium recombination yeast

1) preparation beta-amylase gene Amy-β: take Ipomoea batatas as raw material, extract Ipomoea batatas RNA with the cetyl trimethylammonium bromide method, obtain its cDNA with the reverse transcription technology, take cDNA as template, design primer amplification beta-amylase gene Amy-β, the pcr amplification condition is: 95 ℃ of denaturation 5 min; Circulate with 94 ℃ of 1 min, 54 ℃ of 1 min, 72 ℃ of 2 min and 30 to take turns; Last 72 ℃ are extended 10 min.

Described PCR the primer is:

BB-F: 5’-ATGGCTCCAATCCCCGGT-3’

BB-R:5’-ATAAAAGAGCTTTTGGCGCTAATCAAACGGGTTTGAGCCAT-3’

2) preparation α lectin encoding gene Ag-Sc: with yeast saccharomyces cerevisiae W303-1A(Thomas BJ, Rothstein RJ. Cell. 1989.) genomic dna is raw material, derives from the α lectin encoding gene of yeast saccharomyces cerevisiae karyomit(e) W303-1A with the amplification of polymerase chain reaction,PCR (PCR) technology Ag-Sc, reaction conditions is: 95 ℃ of denaturation 5 min, and with 94 ℃ of 30 s, 56 ℃ of 1 min, 72 ℃ of 2 min circulate and 30 take turns, and last 72 ℃ are extended 10 min.

Described pcr amplification Ag-Sc, the primer is:

AF:5’-TGGCTCAAACCCGTTTGATTAGCGCCAAAAGCTCTTTTATC-3’

AR:5’- TTTGATTATGTTCTTTCTAT-3’

Described yeast saccharomyces cerevisiae W303-1A derives from Chinese Typical Representative culture collection center;

3) preparation fusion gene Amy-β- Ag-Sc: utilize the method for overlapping PCR, with above-mentioned beta-amylase gene Amy-β and α lectin encoding gene Ag-ScIn frame, merge, obtain fusion gene Amy-β- Ag-ScDescribed overlapping PCR, the primer is:

Upstream primer BB-F:5 '-ATGGCTCCAA TCCCCGGT-3 ',

Downstream primer AR:5 '-TTTGATTATGTTCTTTCTAT-3 '.

The PCR program is as follows: 95 ℃ of denaturation 5 min, and with 94 ℃ of 30 s, 55 ℃ of 1 min, 72 ℃ of 3 min carries out 30 circulations, and 72 ℃ are extended 10 min.

4) preparation recombination yeast CCTCC NO:M 2011220: with pMGK (Zhong-Peng Guo, Gui-Yang Shi. Yeast.2010) plasmid warp EcoThe RI enzyme is cut purifying, pfu polysaccharase and is filled behind the purifying and above-mentioned fusion gene Amy-β- Ag-ScConnect, obtain recombinant plasmid pBA-AG, with this recombinant plasmid warp SacThe II linearizing, electricity is transformed among the yeast saccharomyces cerevisiae W303-1A, conversion fluid is coated the YPD flat board that G418 concentration is 300 μ g/mL, cultivate 3 ~ 5d for 30 ℃, the dibbling of picking transformant in YPGS dull and stereotyped (YNB0.34%, ammonium sulfate 1%, starch 1%, glucose 0.5%) use iodine staining behind 30 ℃ of cultivation 2 d, the screening transformant has obtained thus beta-amylase and has been anchored to its surperficial recombination yeast CCTCC NO:M 2011220.

2. immobilization beta-diastatic production

1) fermentation harvested cell: add respectively the substratum that adds respectively 70% volume in the fermentor tank in 15 ~ 1000 L fermentor tanks, its mass concentration is 1% ~ 3% glucose, 0.5% ~ 2.5% yeast extract paste, 0.5% ~ 3.0% NaCl, all the other are the substratum of ultrapure water, pH 3.5 ~ 5.5.With recombination yeast CCTCC NO:M 2011220 take volume ratio in 0.5% ~ 10% access substratum, the control temperature is that 30 ℃ ± 2 ℃, dissolved oxygen are not less than 20%, 48 ~ 72h time is cultivated in pH4.0 ~ 5.0.Harvested cell.

2) with 0.1% ~ 1.5% glutaraldehyde solution suspension cell of 0.1 ~ 1 times of this cell volume, under 10 ~ 30 cm ultraviolet rays, shine 30 ~ 120 min again, oven drying at low temperature below 40 ℃ to moisture less than 5%, obtain product immobilization beta-amylase, i.e. immobilized catalyst.

3. immobilization beta-diastatic use

Above-mentioned immobilization beta-catalyzed by amylase Starch Hydrolysis is maltose, and enzyme work is 50 ~ 250 DP ^o/ g.Analyze immobilization beta-diastatic enzyme is alive under different temperature and pH respectively, the result shows that pH is 3.0 ~ 10.5, and temperature is 55 ~ 75 ℃, and immobilized catalyst is stable.

Above-mentioned immobilization beta-amylase uses in batches or in fluidized-bed continuously, can be used for the preparation of high maltose syrup, and massfraction is 30% ~ 33% starch material, obtains maltose behind this immobilization beta-diastatic action, its transformation efficiency 48.5% ~ 55.0%.

This immobilized catalyst stable performance gets final product Reusability more than 10 times through simple separated and collected thalline, and enzyme is lived loss less than 20%.

Beneficial effect of the present invention:

The present invention is anchored to the brewing yeast cell surface with beta-amylase, with this as the immobilized enzyme preparation, its catalysis activity obviously improves than resolvase, and its thermotolerance, stability, reaction pH width etc. all obviously promote, further, the immobilized enzyme that the present invention obtains can reuse by simple separated and collected thalline in reaction, thus, industrial application attribute and the Effective Raise maltose of beta-amylase be can significantly improve and efficient and the added value of industry made, but the decrease production cost.A kind of new industrial source mode of beta-amylase also is provided in addition.

Biological material specimens preservation: with gene Ag-Sc, AmyThe recombinant plasmid pBA-AG transformed saccharomyces cerevisiae W303-1A that-β and cloning vector pMGK form obtains transformant, Classification And Nomenclature be yeast saccharomyces cerevisiae ( Saccharomyces cerevisiae) HH20110602, be deposited in Wuhan, China Wuhan University Chinese Typical Representative culture collection center, preservation date: on June 26th, 2011, deposit number CCTCC NO:M 2011220.

Description of drawings

Fig. 1 Ipomoea batatas beta-amylase encoding gene nucleic acid electrophoresis collection of illustrative plates.

The physical map of Fig. 2 recombinant plasmid pBA-AG.

Fig. 3 synthesizes immobilization beta-diastatic recombination yeast CCTCC NO:M 2011220.

Fig. 4 immobilization beta-diastatic production process route.

Fig. 5 hydrolyzed starch prepares the operational path of maltose.

The HPLC of Fig. 6 malt syrup analyzes collection of illustrative plates.

The optimal reactive temperature of Fig. 7 immobilized catalyst.

The optimal reaction pH of Fig. 8 immobilized catalyst.

The temperature stability of Fig. 9 immobilized catalyst.

The pH stability of Figure 10 immobilized catalyst.

Embodiment

The structure of embodiment 1 immobilization beta-diastatic recombination yeast

At first, the nucleotide sequence according to Ipomoea batatas beta-amylase gene designs primer

Upstream primer: 5 '-ATGGC TCCAATCCCCGGT-3 ',

Downstream primer: 5 '-ATAAAAGAGCTTTTGGCGCTATCAAACGGGTTTGAGCCAT-3 '

Carry out the pcr amplification total length take Ipomoea batatas cDNA as template Amy-β, the PCR program is as follows: 95 ℃ of denaturation 5 min, with 94 ℃ of 1 min, 54 ℃ of 1 min, 72 ℃ of 2 min carries out 30 circulations, and 72 ℃ are extended 10 min.

Secondly, the nucleotide sequence according to the α lectin designs primer:

Upstream primer 5 '-TGGCTCAAACCCGTTTGATTAGCGCCAAAAGCTCTTTTATC-3 '

Downstream primer: 5 '-TTTGATTATGTT CTTTCTAT-3 '.

Pcr amplification adopts 50 μ L systems: polysaccharase pfu 0.5 μ L, polymerase buffer 5 μ L, dNTP 4 μ L, primer BB-F and BB-R(25 μ mol) each 0.5 μ L, template cDNA 0.2 μ L is with two H that steam ₂O complements to 50 μ L.

Take yeast saccharomyces cerevisiae karyomit(e) W303-1A as template, carry out the pcr amplification total length Ag-Sc, the PCR program is as follows: 95 ℃ of denaturation 5 min, and with 94 ℃ of 30s, 56 ℃ of 1 min, 72 ℃ of 2 min carries out 30 circulations, and 72 ℃ are extended 10 min.

Pcr amplification adopts 50 μ L systems: polysaccharase pfu 0.5 μ L, polymerase buffer 5 μ L, dNTP 4 μ L, primer AF and AR(25 μ mol) each 0.5 μ L, template karyomit(e) 0.2 μ L is with two H that steam ₂O complements to 50 μ L.

Difference purifying beta-amylase gene Amy-β and α lectin encoding gene Ag-Sc, respectively get 1 μ L and add overlapping PCR system as template, amplification Amy-β- Ag-Sc(upstream primer adopts amplification to the fusion gene fragment AmyThe upstream primer of-β: 5 '-ATGGCTCCAA TCCCCGGT-3 ', downstream primer adopts amplification Ag-ScDownstream primer: 5 '-TTTGATTATGTTCTTTCTAT-3 ', the PCR program is as follows: 95 ℃ of denaturation 5 min, with 94 ℃ of 30 s, 55 ℃ of 1 min, 72 ℃ of 3 min carries out 30 circulations, 72 ℃ are extended 10 min.The fusion gene product purification is simultaneously plasmid pMGK warp EcoThe RI enzyme is cut rear purifying, and 72 ℃ of 10 min fills with the pfu polysaccharase, uses T behind the product purification ₄Dna ligase connects, and transforms the e. coli jm109 competence, obtains recombinant plasmid pBA-AG.

Recombinant plasmid pBA-AG warp SacThe method that II linearizing electricity consumption transforms changes yeast saccharomyces cerevisiae W303-1A over to.Conversion fluid is coated the YPD flat board that G418 concentration is 300 μ g/mL, cultivates 3 ~ 5d for 30 ℃, and the dibbling of picking transformant is in the dull and stereotyped YPGS(YNB 0.34% of starch, ammonium sulfate 1%, starch 1%, glucose 0.5%), use iodine staining behind 30 ℃ of cultivation 2 d, observe transparent circle (seeing accompanying drawing 3).

The transformant that obtains is inoculated in 5 mLYPD nutrient solutions, and 30 ℃, 200 r/min overnight incubation are collected thalline, the saccharogenic power (DP of Application standard ^o) measuring method detection beta-amylase vigor, the beta-amylase of different recombinant bacterium surface anchorings is slightly different, and enzyme work is 50 ~ 250 DP ^o/ g.

Above-mentioned electric method for transformation is as follows:

(1) connects yeast list bacterium colony to 30 mL liquid YPD substratum (2% peptone, 1% yeast extract paste, 2% glucose), cultivate 12 h for 30 ℃;

(2) get culture and be transferred in the fresh YPD liquid nutrient medium of 30 mL with 1% inoculum size, cultivate 8 h, make thalline reach the synchronous growth state for 30 ℃;

(3) yeast culture is placed place 30 min on ice, centrifugal 5 min of 6000 r/min collect thalline;

(4) abandon supernatant, add the 15 mL ultrapure waters washing thalline of precooling once;

Centrifugal 5 min of (5) 6000 r/min collect thalline, with 15 mL, 1 mol/L Sorbitol Solution USP repeated washing thalline three times;

(6) centrifugal collection thalline adds 100 μ L, 1 mol/L Sorbitol Solution USP suspension thalline, gets in bacteria suspension to the 1.5 mL centrifuge tube of 100 μ L;

(7) in this 100 μ L bacteria suspension, add 20 μ L (≤5 μ g) warp SacThe linearizing plasmid pBA-AG of II, gently ice bath 10 min behind the mixing;

(8) bacteria suspension behind the ice bath is changed in the electric revolving cup of precooling over to 1500 V, 5 ms that shock by electricity;

(9) bacteria suspension after 1 mol/L Sorbitol Solution USP of an amount of volume of adding turns electricity washes out from electric revolving cup, getting 200 μ L coats conversion fluid to coat G418 concentration is that the YPD of 300 μ g/mL is dull and stereotyped, cultivate 3 ~ 5 d for 30 ℃, the dibbling of picking transformant in dull and stereotyped 30 ℃ of YPGS cultivate 2 d, iodine staining, the screening transformant.

Embodiment 2. immobilization betas-diastatic production

1) adding the 700L mass concentration in 1000 L fermentor tanks is 2% glucose, 2% yeast extract paste, and 2% NaCl, all the other are ultrapure water, the substratum of pH4.5 ~ 5.5.With recombination yeast CCTCC NO:M 2011220 take volume ratio in 5% access substratum, the control dissolved oxygen is not less than and cultivates 60 ~ 72 h under 20% pH4.0.Harvested cell.

2) with 1% glutaraldehyde solution suspension cell of 0.5 times of volume, under 20 cm ultraviolet rays, shine again 100 min.Obtained thus that a collection of enzyme activity is high, the immobilization beta-amylase of good stability.

Embodiment 3. immobilization beta-amylases are used for the production of high maltose syrup

1) batching: taking by weighing 33 g over dry W-Gums is raw material, adds the damping fluid of 57 mL water, 10 mL pH 6.0, adds the Calcium Chloride Powder Anhydrous of 100 μ L5 M, stirs.

2) liquefaction: add by every gram raw material (dry weight) that warm amylase feeds intake among 6 ~ 8 U, be warming up to 75 ℃ ~ 80 ℃, keep 40 ~ 80 min, the high temperature enzyme that goes out controls that DE is 16 ~ 22 behind the enzyme that goes out.

3) in batches saccharification: the starch fluid by liquefaction adds 0.2 ~ 0.3 DP by every gram raw material (dry weight) ^oAdd the immobilization beta-amylase, uniform stirring is controlled 55 ± 5 ℃, and saccharification 20 ~ 30 h obtain the high density malt syrup of transformation efficiency 48.5% ~ 63.0%.

Continuous conversion: the starch fluid by liquefaction adds 0.2 ~ 0.3 DP by every gram raw material (dry weight) ^oAdd the immobilization beta-amylase in fluidized-bed, control 55 ± 5 ℃, feed liquid circulating reaction 20 ~ 30 h obtain the high density malt syrup of transformation efficiency 50.0% ~ 65.0%.

The malt syrup that a copy of it is made carries out HPLC analysis, nh 2 column, moving phase 65% acetonitrile solution after trichoroacetic acid(TCA) is processed.The HPLC collection of illustrative plates of gained as shown in Figure 6.Obtain maltose 175.7 g by the HPLC analytical calculation, the maltose transformation efficiency is 53.2%

The preparation of embodiment 4. maltose

Take by weighing 330 g over dry W-Gums, add the damping fluid of 570 mL water, 100 mL pH 6.0, add Calcium Chloride Powder Anhydrous, making final concentration is 5 mM, stirs.The middle temperature amylase 62 μ L that add 2000 U/mL, heating is warming up to 80 ℃ with the speed of 2 ℃ of per minutes, is incubated 120 min, continues to be warming up to 115 ℃ with the speed of 3 ℃ of per minutes, keeps the 10 min enzyme that goes out.Recording the rear DE value of liquefaction is 15.23.

In the starch liquefacation mash behind the above-mentioned enzyme that goes out, add the Pullulanase of 1 ~ 10 U or the isoamylase of 125 ~ 750 U according to the every gram of solid content, 45 ~ 60 ℃ of lower insulation 1 ~ 4 h add 0.2 ~ 0.3 DP again ^oImmobilization beta-amylase of the present invention, 55 ~ 65 ℃ of lower insulation 20 ~ 30 h and agitation as appropriate obtain 330.2 g maltose, starch is 91.5% to the transformation efficiency of maltose.

The Best of embodiment 5. immobilized catalysts

1) optimal reactive temperature

Recombinant yeast cell suspension is respectively at 20,30,40,45,50,55,60,70 ℃, pH 5.0(0.04M Na ₂HPO ₄-citrate buffer solution) under the condition, react 0.5 h with 1% starch solution, measurement result as shown in Figure 7, this immobilized enzyme has the highest amylase activity about 65 ℃.

2) optimal pH condition

Recombinant yeast cell suspension is respectively at pH 3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,9.0,10.0,10.5 and 11.0, under 65 ℃ the condition, react 0.5 h with 1% starch solution, the result shows that this immobilized enzyme has the highest amylase activity at pH about 6.0.

3) temperature stability

Recombinant yeast cell suspension is respectively under 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃ and 70 ℃, pH6.0 temperature, insulation 10,20,30,40,50,60 min, measure its enzyme and live, its result as shown in Figure 9, this immobilized enzyme is lived stable at 55 ℃ ~ enzyme below 75 ℃.

4) pH stability

Recombinant yeast cell suspension is incubated 10,30,60 min respectively under pH 3.0,3.5,4.0,5.0,6.0,7.0,8.0,9.0,10.0,10.5 and 11.0,30 ℃ of conditions, measuring its enzyme lives, its result as shown in figure 10, this immobilized enzyme is lived stable at pH3.0 ~ 10.5 enzymes.

Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other are not anyly deviating from the change done under spirit of the present invention and the principle, modification, are substituting, combination, are simplifying; bacterium should be the substitute mode of equivalence, is included within protection scope of the present invention.

＜110〉Southern Yangtze University

＜120〉a kind of immobilized catalyst of producing for maltose and preparation method thereof

<160> 6

<170> PatentIn version 3.3

<210> SEQ ID NO: 1

<211> 1498

<212> DNA

＜213〉the beta-amylase gene ( Amy-β)

<400> 1

atggctccaa tccccggt gt aatgccaatt ggtaactacg tctctctgta tgtaatgctc 60

ccgttgggag ttgtgaatgc ggacaatgtt tttccggaca aagagaaggt ggaagatgag 120

ctgaagcagg tgaaagcagg ggggtgcgat ggggtgatgg tggatgtgtg gtgggggatc 180

atcgaggcca aagggccaaa gcagtacgat tggtctgctt acagggagtt attccagttg 240

gttaagaaat gtgggctcaa aatccaggcg atcatgtctt ttcaccaatg cggcggcaat 300

gtcggcgacg ccgtcttcat ccccatccct caatggattc tccaaatcgg cgacaaaaac 360

cccgatatct tctacaccaa ccgggccggt aaccgaaacc aggagtacct ctctctcggc 420

gtcgacaatc aacgtctctt ccagggccgc accgctctcg agatgtacag ggatttcatg 480

gaaagtttca gggataatat ggcagacttt ttgaaggctg gagatattgt agacattgaa 540

gtagggtgtg gggctgccgg agagcttcgg tatccctcgt atcccgagac tcaaggatgg 600

gtttttcccg gcatcggaga atttcagtgc tatgacaagt acatggtggc agactggaag 660

gaggctgtga agcaagctgg gaatgcagat tgggagatgc cgggaaaagg caccgggact 720

tacaacgaca cgccggacaa gacggaattc ttccgcccaa acgggactta caagacggat 780

atgggcaagt ttttcttgac ctggtactcc aacaagctca tcatccatgg cgatcaagtc 840

ctcgaagaag ccaacaaagt tttcgttggg ctccgcgtca acatagctgc caaagtttct 900

ggaattcact ggtggtacaa ccatgtgagc catgcggcgg agctcaccgc cggattctac 960

aatgtggcgg gaagagacgg ttatcggcct attgccagga tgctggcaag gcaccatgcc 1020

actctgaatt tcacttgcct tgagatgaga gactccgaac agcctgccga ggccaagagt 1080

gctcctcaag aactcgttca acaggtgttg agcagcggat ggaaagagta tatcgatgtg 1140

gcaggtgaga atgcactgcc aagatatgat gccacggcat acaaccaaat acttctgaac 1200

gtcaggccaa acggcgtcaa ccttaacggc cctcccaagc tcaagatgtc cggcttgaca 1260

tatctccgtt tgtcggacga tctgttacag acagacaact tcgaactctt caagaaattc 1320

gtcaagaaga tgcacgctga tctggatcca tccccgaacg ctatctctcc ggcggtgttg 1380

gagagatcaa actcggcgat caccattgat gaactgatgg aagccaccaa aggtagcagg 1440

ccattcccat ggtatgacgt cacagacatg ccggtggatg gctcaaaccc gtttgatt 1498

<210> 2

<211> 18

＜212〉artificial sequence

＜213〉primer BB-F

<400> 2

5’-ATGGCTCCAA TCCCCGGT-3’

<210> 3

<211> 41

＜212〉artificial sequence

＜213〉primer BB-R

<400> 3

5’-ATAAAAGAGCTTTTGGCGCTAATCAAACGGGTTTGAGCCAT-3’

<210> SEQ ID NO: 4

<211> 1409

<212> DNA

＜213〉α lectin encoding gene ( Ag-Sc)

<400> 4

agcgccaaaa gctcttttat ctcaaccact actactgatt taacaagtat aaacactagt 60

gcgtattcca ctggatccat ttccacagta gaaacaggca atcgaactac atcagaagtg 120

atcagtcatg tggtgactac cagcacaaaa ctgtctccaa ctgctactac cagcctgaca 180

attgcacaaa ccagtatcta ttctactgac tcaaatatca cagtaggaac agatattcac 240

accacatcag aagtgattag tgatgtggaa accattagca gagaaacagc ttcgaccgtt 300

gtagccgctc caacctcaac aactggatgg acaggcgcta tgaatactta catcccgcaa 360

tttacatcct cttctttcgc aacaatcaac agcacaccaa taatctcttc atcagcagta 420

tttgaaacct cagatgcttc aattgtcaat gtgcacactg aaaatatcac gaatactgct 480

gctgttccat ctgaagagcc cacttttgta aatgccacga gaaactcctt aaattccttc 540

tgcagcagca aacagccatc cagtccctca tcttatacgt cttccccact cgtatcgtcc 600

ctctccgtaa gcaaaacatt actaagcacc agttttacgc cttctgtgcc aacatctaat 660

acatatatca aaacggaaaa tacgggttac tttgagcaca cggctttgac aacatcttca 720

gttggcctta attcttttag tgaaacagca ctctcatctc agggaacgaa aattgacacc 780

tttttagtgt catccttgat cgcatatcct tcttctgcat caggaagcca attgtccggt 840

atccaacaga atttcacatc aacttctctc atgatttcaa cctatgaagg taaagcgtct 900

atatttttct cagctgagct cggttcgatc atttttctgc ttttgtcgta cctgctattc 960

taaaacgggt actgtacagt tagtacattg agtcgaaata tacgaaatta ttgttcataa 1020

ttttcatcct ggctcttttt ttcttcaacc atagttaaat ggacagttca tatcttaaac 1080

tctaataata cttttctagt tcttatcctt ttccgtctca ccgcagattt tatcatagta 1140

ttaaatttat attttgttcg taaaaagaaa aatttgtgag cgttaccgct cgtttcatta 1200

cccgaaggct gtttcagtag accactgatt aagtaagtag atgaaaaaat ttcatcacca 1260

tgaaagagtt cgatgagagc tactttttca aatgcttaac agctaaccgc cattcaataa 1320

tgttacgttc tcttcattct gcggctacgt tatctaacaa gaggttttac tctctcatat 1380

ctcattcaaa tagaaagaac ataatcaaa 1409

<210> 5

<211> 41

＜212〉artificial sequence

<213> AF

<400> 5

5’-TGGCTCAAACCCGTTTGATTAGCGCCAAAAGCTCTTTTATC-3’

<210> 6

<211> 20

＜212〉artificial sequence

<213> AR

<400> 6

5’-TTTGATTATGTTCTTTCTAT-3’

Claims (3)

1. immobilized catalyst that high maltose syrup is produced that is used for recombination yeast CCTCC NO:M 2011220 preparation, it is characterized in that being in the nature beta-amylase, by the beta-amylase molecule is realized that in the grappling of yeast saccharomyces cerevisiae W303-1A cell surface it is immobilized;

Described immobilized catalyst is the immobilization beta-amylase;

Expressed beta-amylase is fixed in yeast saccharomyces cerevisiae W303-1A cell surface grappling through yeast α lectin, obtain recombination yeast CCTCC NO:M 2011220, further this recombinant yeast cell of large scale culturing, centrifugal cell harvesting, suspend and lower this recombination yeast of irradiation of ultraviolet ray with glutaraldehyde solution, obtain the immobilization beta-amylase;

Enzyme work is 50 ~ 250 DP ^o/ g; PH is 3.0 ~ 10.5, and temperature is 55 ~ 75 ℃, and immobilized catalyst is stable.

2. the preparation method of immobilized catalyst claimed in claim 1 is characterized in that step is:

1) preparation beta-amylase gene Amy-β: take Ipomoea batatas as raw material, extract Ipomoea batatas RNA with the cetyl trimethylammonium bromide method, obtain its cDNA with the reverse transcription technology, take cDNA as template, design primer amplification beta-amylase gene Amy-β, the pcr amplification condition is: 95 ℃ of denaturation 5 min; Circulate with 94 ℃ of 1 min, 54 ℃ of 1 min, 72 ℃ of 2 min and 30 to take turns; Last 72 ℃ are extended 10 min;

Described PCR the primer is:

BB-F:5’-ATGGCTCCAATCCCCGGT-3’，

BB-R:5’-ATAAAAGAGCTTTTGGCGCTAATCAAACGGGTTTGAGCCAT-3’；

2) preparation α lectin encoding gene Ag-Sc: take yeast saccharomyces cerevisiae W303-1A genomic dna as raw material, derive from the α lectin encoding gene of yeast saccharomyces cerevisiae karyomit(e) W303-1A with the amplification of polymerase chain reaction,PCR round pcr Ag-Sc, reaction conditions is: 95 ℃ of denaturation 5 min; Circulate with 94 ℃ of 30 s, 56 ℃ of 1 min, 72 ℃ of 2 min and 30 to take turns; Last 72 ℃ are extended 10 min;

Described pcr amplification Ag-Sc, the primer is:

AF:5’-TGGCTCAAACCCGTTTGATTAGCGCCAAAAGCTCTTTTATC-3’，

AR:5’- TTTGATTATGTTCTTTCTAT-3’；

Described yeast saccharomyces cerevisiae W303-1A derives from Chinese Typical Representative culture collection center;

3) preparation fusion gene Amy-β- Ag-Sc: utilize the method for overlapping PCR, with above-mentioned beta-amylase gene Amy-β and α lectin encoding gene Ag-ScIn frame, merge, obtain fusion gene Amy-β- Ag-Sc

Described overlapping PCR, the primer is:

Upstream primer BB-F:5 '-ATGGCTCCAA TCCCCGGT-3 ',

Downstream primer AR:5 '-TTTGATTATGTTCTTTCTAT-3 ';

The PCR program is as follows: 95 ℃ of denaturation 5 min; Carry out 30 circulations with 94 ℃ of 30 s, 55 ℃ of 1 min, 72 ℃ of 3 min; 72 ℃ are extended 10 min;

4) preparation recombination yeast CCTCC NO:M 2011220: with plasmid pMGK warp EcoThe RI enzyme is cut purifying, pfu polysaccharase and is filled behind the purifying and above-mentioned fusion gene Amy-β- Ag-ScConnect, obtain recombinant plasmid pBA-AG, with this recombinant plasmid pBA-AG warp SacThe II linearizing, electricity is transformed among the yeast saccharomyces cerevisiae W303-1A, conversion fluid is coated the YPD flat board that G418 concentration is 300 μ g/mL, cultivates 3 ~ 5d for 30 ℃, and the dibbling of picking transformant is dull and stereotyped in YPGS, contain YNB 0.34% in this YPGS flat board, ammonium sulfate 1% is used iodine staining behind 0.5%, 30 ℃ of cultivation of starch 1% and glucose, 2 d, the screening transformant has obtained thus beta-amylase and has been anchored to its surperficial recombination yeast CCTCC NO:M 2011220;

5) fermentation harvested cell: add respectively the substratum of 70% volume in 15 ~ 1000 L fermentor tanks, its mass concentration is 1% ~ 3% glucose, 0.5% ~ 2.5% yeast extract paste, 0.5% ~ 3.0% NaCl, all the other are the substratum of ultrapure water, pH 3.5 ~ 5.5; With recombination yeast CCTCC NO:M 2011220 take volume ratio in 0.5% ~ 10% access substratum, the control temperature is that 30 ℃ ± 2 ℃, dissolved oxygen are not less than 20%, 48 ~ 72 h, harvested cell time are cultivated in pH4.0 ~ 5.0;

6) with 0.1% ~ 1.5% glutaraldehyde solution suspension cell of 0.1 ~ 1 times of this cell volume, under 10 ~ 30 cm ultraviolet rays, shine 30 ~ 120 min again, oven drying at low temperature below 40 ℃ to moisture less than 5%, obtain product immobilization beta-amylase, i.e. immobilized catalyst.

3. the application of immobilized catalyst claimed in claim 1 is characterized in that this immobilized catalyst is used for high maltose syrup production, uses in batches or continuously; Reusability is more than 10 times.

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