CN112481247A - Method for producing F42 and F55 high fructose corn syrup on same line - Google Patents

Method for producing F42 and F55 high fructose corn syrup on same line Download PDF

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CN112481247A
CN112481247A CN202011521801.4A CN202011521801A CN112481247A CN 112481247 A CN112481247 A CN 112481247A CN 202011521801 A CN202011521801 A CN 202011521801A CN 112481247 A CN112481247 A CN 112481247A
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fructose
syrup
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CN112481247B (en
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李林海
董得平
黄继红
闫佳佳
董瑜琪
杨秋霞
侯银臣
廖爱美
冯军伟
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Henan Feitian Agricultural Development Co ltd
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Abstract

The invention belongs to the technical field of high fructose corn syrup production, and particularly relates to a technical process for producing F42 and F55 high fructose corn syrup in the same line. The method takes starch as a raw material, and obtains F42 and F55 syrup by the same-line production through liquefaction, saccharification, ion exchange, evaporation, isomerization, chromatographic separation and blending of products at different stages, so that the frequent syrup switching can be reduced, the production efficiency is improved, and the single-day yield of fructose (480 tons to 520 tons) is improved. Meanwhile, the novel glucose isomerase is adopted, so that the isomerization efficiency is improved, the process cost is reduced, and the novel glucose isomerase has a good application prospect.

Description

Method for producing F42 and F55 high fructose corn syrup on same line
The technical field is as follows:
the invention belongs to the technical field of high fructose corn syrup production, and particularly relates to a technical process for producing F42 and F55 high fructose corn syrup in the same line.
Background art:
the high fructose corn syrup is prepared by hydrolyzing corn starch and belongs to starch sugar. The starch sugar industry conforms to the agricultural industrialization policy, under the influence of the national three-agriculture policy, under the condition that the production technology of the starch sugar industry is continuously improved and the scale is continuously enlarged, and along with the continuous improvement of the application technology of the fructose-glucose syrup in food, more and more food enterprises can select the fructose-glucose syrup, so that the replacement of most of sucrose by the fructose-glucose syrup in the food industry is inevitable.
42 fructose (F42) has a fructose content of 42-44% and a DS of 63% or 71% (71% as an example in this project), 55 fructose (F55) has a fructose content of 55-57% and a DS of 77%; the products are of two different specifications. The existing process can only produce one product independently, a system needs to be switched and cleaned when the product is replaced, another product is produced again, a collinear production process of F42 and F55 is developed, and the production of products with two specifications can be adjusted at any time according to the requirements of customers.
The invention content is as follows:
in order to solve the technical problems, the invention provides an online production method of F42 and F55 high fructose corn syrup, which comprises the following steps:
(1) liquefaction: adjusting the concentration Be' 17-25 of starch slurry, adding high-temperature resistant amylase, and liquefying under the condition of pH5.0-7.0 to DE12-18, wherein the iodine test is brown;
preferably, a secondary injection liquefaction method may be employed;
(2) saccharification: adding saccharifying enzyme into the liquefied solution at pH of 3.8-4.5 and temperature of 58-62 deg.C for saccharifying until DX (fructose + glucose) is greater than or equal to 95%;
further, the saccharification time is 30-40 h;
further, the adding amount of the saccharifying enzyme is 0.4-0.45 kg/tds;
(3) removing slag by using a plate frame: at the temperature of 62-65 ℃, the saccharified liquid is filtered by a plate and frame filter to remove protein in the saccharified liquid, so as to obtain clear sugar liquid;
(4) plate frame decoloring: adding active carbon, and decolorizing the sugar solution at 70-75 deg.C;
further, the adding amount of the active carbon is 0.8-1.1 kg/tds;
(5) aga filtration (secondary decolorization): adding activated carbon into the filtrate transfer tank, and carrying out secondary decolorization on the sugar liquid at the decolorization temperature of 65-70 ℃;
further, the adding amount of the active carbon is 0.8-1.1 kg/tds;
(6) ceramic membrane: filtering the decolorized solution with ceramic membrane to remove part of macromolecular protein and dextrin in the sugar solution, and retaining various microorganisms;
(7) f00 ion exchange: passing the sugar solution with the temperature of 52-56 ℃ from bottom to top through a cation exchange column, discharging the sugar solution from the cation exchange column, and introducing the sugar solution into an anion exchange column, so that the pH of the discharged sugar solution is 4.0-6.5, and the electric conductivity is less than or equal to 20 mu s/cm;
the method removes anions and cations in the sugar solution, reduces ash content, purifies the sugar solution, and prevents impurities in the sugar solution from damaging isomerase;
(8) f00 evaporation: concentrating under vacuum by an evaporator to obtain a syrup with DS of 50%;
(9) isomerization: after flash evaporation and deoxidation, the pH value of the sugar solution is adjusted to 7.6-7.9, the sulfur dioxide content is adjusted to 80-120ppm, the magnesium ion content is adjusted to 30-80ppm, and then the sugar solution enters an isomerase fixed column (the feeding flow is 1-6 m)3H), isomerizing by glucose isomerase at 55-60 ℃ to obtain sugar liquid F42 which is converted from glucose into fructose with the content of 42-44%;
further, the isomerase added into the fixed column is glucose isomerase GIM, and the amino acid sequence is shown as SEQ ID NO: 3 is shown in the specification;
(10) f42 ion exchange/mixed bed: passing the obtained sugar solution through a cation exchange column and an anion exchange column in sequence, and then through special resin for a mixed bed to ensure that the pH of the discharged material is more than or equal to 4.5 and the conductivity is less than or equal to 20 mus/cm;
the method removes anions and cations in the sugar, reduces ash content, purifies sugar liquid, prevents impurities in the sugar liquid from causing harm to chromatographic resin, and ensures the quality of a finished 42 fructose product;
(11) f42 evaporation: evaporating and concentrating a part of F42 fructose passing through an F42 ion exchange/mixing bed, controlling the discharge concentration to be 71.2-71.4%, and controlling the pH to be 3.3-4.5, namely F42 finished syrup, so that the storage and the transportation are convenient;
(12) and (3) chromatography: separating the other part of F42 fructose subjected to the F42 ion exchange/mixed bed into syrup AD with the fructose content of more than 85 percent and raffinate BD (the fructose content is less than or equal to 10 percent);
(13) f55 blending and decoloring: mixing the syrup AD and a part of F42 fructose which passes through an F42 ion exchange/mixed bed into syrup with the fructose content of 55-57% according to a certain proportion, and then adsorbing organic substances such as flocculent precipitates, pigments and the like in the sugar solution at the temperature of 62-65 ℃ by utilizing the porosity and microporosity of activated carbon to obtain F55 fructose with the chroma of less than 10;
further, the adding amount of the active carbon is 1.0-1.5 kg/tds;
(14) f55 deodorization: f55 fructose passes through the deodorizing column from top to bottom, part of protein and colored and odorous substances in the sugar, such as HMF, are removed, the quality of the syrup is improved, and the storage time is prolonged.
(16) F55 mixed bed: the sugar solution is processed by special resin for mixed bed, the feeding temperature is 32-37 ℃, the discharging pH is more than or equal to 4.5, anions and cations in the sugar are removed, the ash content is reduced, the sugar solution is purified, and the storage life of the syrup is prolonged;
(17) f55 evaporation: and (3) concentrating the sugar liquid from the F55 mixed bed to ensure that the pH value of the discharged material is 3.3-4.5, and the concentration is 77.2% -77.4%, namely F55 finished syrup, so that the storage and the transportation are convenient.
Has the advantages that:
(1) the invention provides an online production process of F42 and F55 high fructose corn syrup, which can reduce frequent syrup switching, improve the production efficiency and improve the single-day yield of fructose (480 tons are improved to 520 tons);
(2) by adopting the novel glucose isomerase, the improvement of the enzyme activity can greatly shorten the isomerization time, improve the efficiency and reduce the process cost.
Description of the drawings:
FIG. 1 error-prone PCR electrophoresis results.
FIG. 2 shows the restriction map of pGAPZ. alpha.C-gim.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
The invention provides an on-line production method of F42 and F55 high fructose corn syrup, which comprises the following process steps:
(1) liquefaction: adjusting the concentration Be' 17-25 of starch slurry, adding high-temperature resistant amylase, and liquefying under the condition of pH5.0-7.0 to DE12-18, wherein the iodine test is brown;
in some embodiments of the invention, this may be achieved by:
slurry mixing: adjusting the concentration Be' 17-25 of starch slurry; pH5.0-7.0; high temperature resistant amylase: 0.08-0.11kg/tds (properly adjusted according to the liquefied DE value, the high temperature resistant amylase used in the embodiment of the invention is Novoxil NWS 10098;
secondly, liquefying by adopting a double-injection method:
primary spraying: preheating an ejector and a laminar flow tank to 100 ℃, then carrying out ejection liquefaction, controlling the temperature of the ejector to be 102-110 ℃, carrying out vacuum primary flash evaporation cooling, and reducing the temperature of a system to 95-98 ℃ to enter the laminar flow tank for maintaining for 60-90 min;
secondary spraying: heating the feed liquid to the temperature of 120-145 ℃ by a secondary ejector, maintaining the temperature for 3-5min by a pipeline, then carrying out vacuum secondary flash evaporation cooling, reducing the system temperature to 95-98 ℃, entering a secondary laminar flow tank, maintaining the temperature for 20-30min, carrying out vacuum tertiary flash evaporation cooling, reducing the system temperature to 78-72 ℃, reducing the temperature to 58-62 ℃ by a plate heat exchanger, entering a pH tank, adjusting the pH value, and then entering a saccharification tank;
(2) saccharification: saccharifying at pH of 3.8-4.5 and temperature of 58-62 deg.C for 30-40h to ensure that DX (fructose + glucose) is not less than 95%;
in some embodiments of the invention, the saccharifying enzyme is added at a rate of 0.4-0.45kg/tds (the saccharifying enzyme used in embodiments of the invention is Nanjing Pasteur
Figure BDA0002849238570000041
Ultra 3.0)；
(3) Removing slag by using a plate frame: at the temperature of 62-65 ℃, the saccharified liquid is filtered by a plate and frame filter to remove protein in the saccharified liquid, so as to obtain clear sugar liquid;
(4) plate frame decoloring: adding active carbon, and decolorizing the sugar solution at 70-75 deg.C (with active carbon addition of 0.8-1.1 kg/tds);
(5) aga filtration (secondary decolorization): transferring the filtrate into a tank, adding active carbon (the addition amount of the active carbon is 0.8-1.1kg/tds), and performing secondary decolorization on the sugar solution at the decolorization temperature of 65-70 ℃;
(6) ceramic membrane: filtering the decolorized solution with ceramic membrane to remove part of macromolecular protein and dextrin in the sugar solution, and retaining various microorganisms;
(7) f00 ion exchange: sugar liquor with the temperature of 52-56 ℃ is discharged from a positive ion exchange column from bottom to top through the positive ion exchange column (the type of the positive resin adopted in the embodiment of the invention: dispute D001FD), enters an negative ion exchange column (the type of the negative resin adopted in the embodiment of the invention: D354FD) to remove negative and positive ions in the sugar liquor, reduces ash content, purifies the sugar liquor, prevents impurities in the sugar liquor from damaging isomerase, and has the discharge pH of 4.0-6.5 and the conductance less than or equal to 20 mu s/cm;
(8) f00 evaporation: obtaining syrup with DS of 50 percent by vacuum concentration of a five-effect evaporator (five effect: vacuum degree of 50-150mbar, temperature of 35-45 ℃, four effect: vacuum degree of 100-;
(9) isomerization: after flash evaporation and deoxidation, the pH value of the sugar solution is adjusted to 7.6-7.9, the sulfur dioxide content is adjusted to 80-120ppm, the magnesium ion content is adjusted to 30-80ppm, and then the sugar solution enters an isomerase fixed column (the feeding flow is 1-6 m)3H), converting glucose by glucose isomerase isomerization at 55-60 deg.CSugar solution F42 containing 42% -44% of fructose;
in some embodiments of the present invention, the isomerase added to the fixed column is glucose isomerase GIM, and the amino acid sequence is as shown in SEQ ID NO: 3 is shown in the specification;
(10) f42 ion exchange/mixed bed: the obtained sugar solution sequentially passes through a cation exchange column (bleached positive resin: PPC150S is used in the embodiment of the invention) and an anion exchange column (bleached negative resin: PPA103S is used in the embodiment of the invention), and then passes through a mixed bed special resin (competing light mixed bed positive resin: D001MB and mixed bed negative resin: D202MB are used in the embodiment of the invention), so that negative and positive ions in the sugar are removed, ash content is reduced, the sugar solution is purified, the harm of impurities in the sugar solution to chromatographic resin is prevented, the finished product quality of 42 fructose is ensured, the discharging pH is more than or equal to 4.5, and the conductance is less than or equal to 20 mus/cm;
(11) f42 evaporation: concentrating a part of F42 fructose which passes through an F42 ion exchange/mixing bed, controlling the discharge concentration to be 71.2-71.4% and the pH to be 3.3-4.5, namely F42 finished syrup, and facilitating storage and transportation;
in some embodiments of the invention, five-effect evaporators are used for vacuum concentration: five effects are as follows: vacuum degree of 50-150mbar, and temperature of 35-45 deg.C; four effects are as follows: the vacuum degree is 100-; three effects are as follows: the vacuum degree is 150-; two effects are as follows: the vacuum degree is 200-: the vacuum degree is 250-;
(12) and (3) chromatography: separating the other part of F42 fructose subjected to the F42 ion exchange/mixed bed into syrup AD with the fructose content of more than 85 percent and raffinate BD (the fructose content is less than or equal to 10 percent);
in some embodiments of the invention, the dow: separating with 99ca/310 chromatographic separation resin at 58-65 ℃;
(13) f55 blending and decoloring: mixing the syrup AD and a part of F42 fructose which passes through an F42 ion exchange/mixed bed into syrup with the fructose content of 55-57% according to a certain proportion, and adsorbing organic substances such as flocculent precipitates, pigments and the like in sugar solution at the temperature of 62-65 ℃ by utilizing the porosity and microporosity of active carbon (the adding amount of the active carbon is 1.0-1.5kg/tds) to obtain F55 fructose with the chroma of 10;
(14) f55 deodorization: removing partial protein and colored and odorous substances such as HMF (HMF) less than or equal to 40ppm from top to bottom through a deodorizing column, improving the quality of the syrup and prolonging the storage time;
(15) f55 mixed bed: the hypoglycemic liquid passes through the special resin for the mixed bed, the feeding temperature is 32-37 ℃, the discharging pH is more than or equal to 4.5, anions and cations in the sugar are removed, the ash content is reduced, the sugar liquid is purified, and the storage life of the syrup is prolonged;
(16) f55 evaporation: concentrating the sugar liquid from the F55 mixed bed, discharging the sugar liquid with pH of 3.3-4.5 and concentration of 77.2% -77.4%, namely F55 finished syrup, and facilitating storage and transportation;
in some embodiments of the invention, ineffective vacuum evaporation is employed: five effects are as follows: vacuum degree of 50-150mbar, and temperature of 40-55 deg.C; four effects are as follows: the vacuum degree is 100-; three effects are as follows: the vacuum degree is 150-; two effects are as follows: the vacuum degree is 200-: the vacuum degree is 250-.
The invention will be further explained below by means of specific embodiments.
Example 1A Glucose Isomerase (GI) mutant
The invention provides a Glucose Isomerase (GI) mutant and a gene thereof, the invention uses an error-prone PCR technology to randomly mutate a Glucose isomerase coding Gene (GI) from Streptomyces rubiginosus to obtain a Glucose isomerase mutant gene (gim), the enzyme activity of the mutant is improved by 57% compared with the original gene, and the mutant is expressed in pichia pastoris to obtain the high-activity Glucose isomerase.
The following definitions are used in the present invention:
(1) nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
(2) Identification of glucose isomerase mutants
"amino acid substituted at original amino acid position" is used to indicate a mutated amino acid in a glucose isomerase mutant. Such as Met88Lys, the amino acid at position 88 is replaced by Met of the original glucose isomerase to Lys, and the numbering of the position corresponds to SEQ ID NO: 1, amino acid sequence number of wild-type glucose isomerase.
In the present invention, GI represents the original glucose isomerase (amino acid sequence shown in SEQ ID NO: 1), GIM represents the mutated glucose isomerase (amino acid sequence shown in SEQ ID NO: 3); gi represents the coding gene of the original glucose isomerase (shown as SEQ ID NO: 2), and gim represents the coding gene of the mutated glucose isomerase (shown as SEQ ID NO: 4).
Figure BDA0002849238570000061
The host cell for expressing the glucose isomerase mutant is Pichia pastoris SMD1168, and the expression vector is pGAPZ alpha C.
1. Obtaining of wild-type glucose isomerase DNA
The amino acid sequence (SEQ ID NO.1) of wild-type glucose isomerase of Streptomyces rubiginosis was searched using NCBI database, and codon optimization was performed for Escherichia coli (E.coli) to obtain a DNA sequence (SEQ ID NO. 2). Synthesizing the whole gene of SEQ ID NO.2, connecting to pUC57 vector, transferring into E.coli DH5 alpha, preparing into glycerol strain, and storing at-80 deg.C for a long time.
1 glycerol strain was inoculated into a test tube containing 5ml of Amp-resistant LB medium, cultured overnight at 37 ℃ and subjected to Plasmid mini-extraction using High Pure Plasmid Isolation Kit from Roche to obtain a wild-type glucose isomerase DNA fragment as a template for subsequent random mutagenesis.
2. Obtaining of glucose isomerase mutant Gene
(1) Random mutagenesis
Carrying out random mutation by using TaKaRa Taq PCR amplification enzyme of TaKaRa company based on an error-prone PCR technology to obtain a high-activity glucose isomerase gene;
primers were designed as follows:
upstream P1(SEQ ID No. 5):
5’-TAAGAAGGAGATATACCATGGATGAACTACCAGCCGACCCCGGA-3’
downstream P2(SEQ ID No. 6):
5’-GTGGTGGTGGTGGTGCTCGAG TTA gccacgtgcgcccagca-3’
the reaction system for amplification is as follows:
10×PCR buffer 5μL
dNTPs(2.5mmol/L each) 5μL
upstream primer P1 (10. mu. mol/L) 1.5μL
Downstream primer P2 (10. mu. mol/L) 1.5μL
25mmol/L MgCl2 11μL
5mmol/L MnCl2 5μL
Form panel 20pmol
Taq DNA polymerase 1μL
ddH2O Make up to 50 μ L
The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 60 s; annealing at 61 ℃ for 60s, extending at 72 ℃ for 180s, and reacting for 30 cycles; keeping the temperature at 72 ℃ for 10 min; storing at 4 ℃. The PCR amplification product was detected by 1.0% agarose gel electrophoresis, and a band of about 1200bp was observed (FIG. 1). The PCR amplification product does not need to be processed, can be immediately used for vector construction, and can also be stored for a long time at the temperature of 20 ℃ below zero.
(2) Expression vector linearization
The pET-28a plasmid was linearized using the conventional restriction enzyme TaKaRa, as follows:
Nco I 5μL
Xho I 5μL
10*K buffer 10μL
0.1％BSA 10μL
pET-28a 5μg
ddH2O make up to 100 mu L
Linearization conditions are as follows: preserving the heat for 3 hours at 37 ℃; keeping the temperature at 65 ℃ for 20 min; storing at 4 ℃. The linearized product can be used for vector construction immediately and can also be stored for a long time at the temperature of 20 ℃ below zero.
(3) Vector construction
The gim mutant expression vector library was constructed by ligating the error-prone PCR product with linearized pET-28a using Clonexpress II one-step ligase from Vazyme. To ensure sufficient storage capacity, 5 ligation reactions were performed simultaneously, totaling 100. mu.L of ligation system.
The linking system is as follows:
5*CE II buffer 4μL
PCR product 112ng
Linearized vector 110ng
Exnase II 2μL
ddH2O Make up to 20 mu L
Note that: the reaction system was prepared in an ice bath.
Reaction conditions are as follows: keeping the temperature at 37 ℃ for 30 min; keeping the temperature at 4 ℃ for 5 min.
After the reaction is finished, the product can be stored for a short time at 4 ℃ or for a long time at-20 ℃.
(4) gim mutant expression library construction
Connecting the mutant expression vector of the step (3) with products (20. mu.L/cell), and transforming the expression strain E.coli BL21 as follows:
e.coli BL21 competent cells (100. mu.L/cell) were taken out from-80 ℃, dissolved on ice, 20. mu.L of the ligation product was added to 100. mu.L of E.coli competent cells BL21 immediately after dissolution in a sterile environment, placed on ice for 30min, heat-shocked in a water bath at 42 ℃ for 90s, cooled on ice for 1.5min, added to 900. mu.L of LB medium, and pre-cultured at 37 ℃ and 200r/min for 30 min. Centrifuging at 3000rpm for 2min, discarding 600 μ L of supernatant, mixing thallus precipitate and the rest supernatant by pipette, coating each 100 μ L of concentrated bacterial liquid on LB plate containing Kan resistance, making 4 parallel groups, and culturing at 37 deg.C in constant temperature incubator upside down overnight.
Finally, 20 blocks of gim mutant expression strain libraries are obtained and coated on a flat plate, and after sealing by a sealing film, the flat plate is stored for a short time at 4 ℃.
(5) Wild type expression Strain construction
Wild type gi gene was directly amplified by general PCR, and by referring to the steps (2)/(3)/(4) in this example, wild type expression strain was constructed as a control sample for screening enzyme activity.
(6) Enzyme activity screening sample preparation
Preparing an induction culture medium:
consists of the following components: 12.0 g of Tryptone, 24.0 g of Yeast extract, 2.0g of alpha-lactose, 0.5g of glucose, 17.05g of Studier salt and pH7.0 +/-0.2.
Weighing 55.55g of dehydrated culture medium powder, and heating and dissolving with 1L of deionized water; boiling for 1 min; sterilizing at 115 deg.C for 20 min.
Positive transformants were selected by plating plates from 20 pools of gim mutant expression strains. At least 2000 positive transformants (containing 1 wild-type expression strain) were picked and each positive transformant was inoculated into a new kanamycin-resistant plate for strain preservation and simultaneously inoculated into a 96-well shallow well plate containing 200. mu.L of LB liquid medium (containing 50. mu.g/mL of Kan) per well.
After the strain preservation plate is cultured overnight at 37 ℃, the strain preservation plate is sealed by a sealing film and preserved at 4 ℃.
Inoculated into a 96-well shallow plate (each plate contains 1 wild-type expression strain as a control), and cultured at 37 ℃ for 12h at 400 r/min. 50. mu.l of overnight-cultured bacterial suspension was transferred to a 96-well deep-well plate (1ml of auto-induction medium/well, Kan concentration 50. mu.g/ml), cultured at 37 ℃ and 400rpm for 4 hours, and then induced at 25 ℃ for expression for 20 hours. Centrifuging at 4000r/min at 4 deg.C for 30min, collecting thallus, standing at-80 deg.C for 2h, standing at room temperature for 1h, and repeating the above operation for 2 times. Add 70. mu.l of bacteriolysis buffer (0.5mg/ml lysozyme, 0.7U/ml Dnasel, 50mmol/L PBS, pH7.5) to each well to resuspend the thallus, transfer to a new 96-well shallow-well plate, and place in a 37 ℃ incubator for 90min to fully break the thallus. The mutant was centrifuged at 4000rpm for 30min at 4 ℃ and 10. mu.l of the supernatant enzyme solution was carefully pipetted into a new 96-well ELISA plate in preparation for the measurement of the specific activity of the mutant.
3. Glucose isomerase preliminary screening
The enzyme activity is measured by cysteine-carbazole method, each sample well of ELISA plate contains 10 μ l enzyme solution, 0.6mol D-glucose 25 μ l, 0.025mol triethanolamine-hydrochloric acid (containing 10mmol MgSO)4·7H2O) 20. mu.l of a buffer solution of pH8, and reacted at 35 ℃ for 15 minutes. The reaction was stopped by adding 5. mu.l of 50% trichloroacetic acid. Immediately adding 300 mul of 70% sulfuric acid which is cooled in an ice way, 10 mul of 2.4% cysteine-hydrochloride and 10 mul of 0.12% ethanol-carbazole bath solution, uniformly mixing, reacting for 30 minutes at 25 ℃, and detecting the absorption value A of each hole by using a 560nm wavelength in an enzyme-labeling instrument.
And (3) recording the strain number of the strain with the absorption value A larger than that of the wild type control well, re-inoculating the strain into a new photocopy plate (convenient for centralized storage) and a 96-well shallow-hole plate from the photocopy storage plate, repeating the step (6) of the step (2) to prepare the sample, re-testing the enzyme activity, and finally, performing the next round of screening on the remaining 28 mutant strains.
4. Specific activity detection of glucose isomerase mutant
28 mutant strains and 1 wild-type strain were inoculated into a test tube containing 5ml of Kan-resistant LB medium and cultured overnight at 37 ℃ with shaking at 160 rpm. Inoculating a 250mL shake flask containing 50mL Kan resistant LB culture medium according to the inoculation amount of 1%, carrying out shake culture at 37 ℃ and 200rpm until OD600 reaches 0.6, adding IPTG (final concentration is 1mmol/L), inducing for 16h at 16 ℃, centrifuging for 15min at 4 ℃ and 4000r/min, collecting thalli, suspending in 15mL precooled PBS buffer solution with pH of 7.4, crushing cells by using a low-temperature ultrahigh-pressure continuous flow cell crusher, centrifuging for 45min at 4 ℃ and 12000r/min, collecting supernatant to obtain crude enzyme solution, and then carrying out specific activity detection.
The glucose isomerase concentration was estimated by SDS-PAGE electrophoresis.
The enzyme activity is measured by cysteine-carbazole method, adding 0.6mol D-glucose 250 μ l glucose isomerase 100 μ l (equivalent to 0.2-0.8 activity unit), and 0.025mol triethanolamine-hydrochloric acid (containing 10mmol MgSO4·7H2O) 200. mu.l of a buffer solution of pH8, and reacted at 35 ℃ for 15 minutes. The reaction was stopped by adding 50. mu.l of 50% trichloroacetic acid. 3ml of 70% sulfuric acid, 100. mu.l of 2.4% cysteine-hydrochloride and 100. mu.l of 0.12% ethanol-carbazole bath solution, which are cooled in the ice, are immediately added, and after uniform mixing, the mixture is reacted at 25 ℃ for 30 minutes, and the absorption value A is measured on the quartz cup side with the wavelength of 560nm and the optical path length of 1cm on a spectrophotometer.
The amount of enzyme required to produce 1. mu.g fructose per minute in a standard reaction mixture is defined as 1 activity unit ((U), and the specific activity is expressed in terms of enzyme activity per mg glucose isomerase, i.e., U/mg.
Numbering Specific activity Numbering Specific activity Numbering Specific activity Numbering Specific activity
Wild type 100 281 132 889 83 1593 86
147 91 409 93 950 112 1747 107
170 80 473 116 996 108 1794 131
217 98 488 114 1015 142 1835 146
222 141 725 88 1063 130 1977 121
226 151 822 101 1240 129
244 148 862 156 1309 103
271 126 872 123 1507 140
The detection result shows that the specific activity of the mutant 862 is the highest, and is improved by 56% compared with the wild type enzyme activity. The mutant is sequenced by using a universal primer T7/T7 ter, and the sequencing result shows that the mutant is a high-activity glucose isomerase gene gim with Met88Lys, Ala131Pro, Ala136Gly, Gly248Cys, the nucleotide sequence is shown in SEQ ID No.4, and the corresponding amino acid sequence is shown in SEQ ID No. 3.
5. Construction of glucose isomerase mutant free expression pichia pastoris recombinant bacteria
Constructing a pichia pastoris high-activity glucose isomerase free expression recombinant strain. The high-activity glucose isomerase (gim) is optimized by a codon and added with a stop codon and an EcoRI/XbaI enzyme cutting site (SEQ ID NO:7) to carry out whole gene synthesis to be connected with a pichia pastoris secretion expression vector pGAPZ alpha C, so that a high-activity glucose isomerase pichia pastoris expression vector pGAPZ alpha C-gim is constructed (enzyme cutting verification is shown in figure 2), and pichia pastoris is transformed.
(1) Preparation of linearized plasmid DNA
Before transformation of Pichia pastoris, the constructed recombinant expression plasmid pGAPZ alpha C-gim is linearized to improve the integration efficiency of the plasmid on Pichia pastoris chromosome. The linearized digestion was carried out with the restriction enzyme BspH I.
(2) Electrically transforming pichia pastoris with linearized plasmid pGAPZ alpha C-gim, identifying positive transformants and screening high-yield strains of glucose isomerase
Adding 80 mu L of pichia pastoris SMD1168 competent cells and 10 mu g of linearized DNA into a 1.5mL precooled centrifuge tube, uniformly mixing, and transferring the reaction solution into a conversion cup in an ice bath in advance;
and secondly, carrying out ice bath on a transformation cup filled with transformation liquid for 5min, and carrying out pichia pastoris electrotransformation according to parameters recommended by an electrotransformation device:
③ immediately adding 1mL of precooled 1mol/L sorbitol solution into the transformation cup after pulse, and transferring the transformation solution into a new 1.5mL centrifuge tube;
standing and culturing at 30 ℃ for 1.5h, and sucking 200 mu L of pichia pastoris SMD1168 electrotransfer liquid and coating the liquid on an MD culture medium;
fifthly, culturing at 30 ℃ until transformants appear;
sixthly, selecting a single colony of the transformant, dissolving the single colony in 10 mu L of deionized water, taking 2 mu L of bacterial liquid, adding Lyticase wall-breaking enzyme, reacting at 30 ℃ for L0min, immediately placing the reaction liquid in a refrigerator at-80 ℃ for freezing for L0min, cracking the cell wall of the yeast, and taking the released genome as a template for PCR. Positive transformants were determined using empty plasmid-transferred Pichia pastoris SMD 1168/pGAPZ. alpha.C as a control.
Seventhly, on the basis of determining positive transformants, firstly, using resistance plates containing geneticin with different concentrations to screen the transformants with high geneticin resistance, and then respectively measuring the enzyme activity of glucose isomerase of the transformants with high geneticin resistance so as to obtain the production strain SMD1168/pGAPZ alpha C-palm of the glucose isomerase.
6. Expression and preparation of glucose isomerase mutant in pichia free expression recombinant bacteria
Inoculating the recombinant strain SMD1168/pPIC9K-gim of the Pichia pastoris free expression glucose isomerase mutant into a YPD liquid culture medium, and culturing at 30 ℃ and 250r/min for 24 h. Transferring the strain into a fresh BMGY culture medium with the inoculation amount of 1%, culturing at 30 ℃ and 250r/min for 24h, centrifuging at 6000r/min for 5min to obtain thalli, and transferring the thalli into a BMMY culture medium. Culturing at 30 deg.C and 250r/min for 120 hr to obtain crude glucose isomerase enzyme solution, precipitating high activity glucose isomerase by fractional salting-out method, collecting protein precipitate, dissolving, dialyzing to remove salt, performing ion exchange chromatography and gel chromatography, and freeze drying to obtain high activity pure glucose isomerase enzyme powder.
Approximately 211mg of pure enzyme powder of glucose isomerase mutant per liter of medium was obtained.
In the same manner, about 180mg of pure enzyme powder of wild-type glucose isomerase per liter of the medium was obtained.
And (4) determining the specific enzyme activities of the original (wild type) glucose isomerase GI and the mutated glucose isomerase GIM to be 110U/mg and 173.14U/mg respectively by adopting the enzyme activity determination method in the step 4, wherein the specific enzyme activity of the mutated glucose isomerase is improved by 57 percent compared with that before mutation.
In the following examples of the present invention, F42 and F55 syrups were produced using the mutated glucose isomerase GIM obtained in example 1 as an isomerase.
Example 2 an in-line production method of F42, F55 high fructose corn syrup
An in-line production method of F42 and F55 high fructose corn syrup comprises the following process steps:
(1) liquefaction: adjusting the concentration of starch slurry Be' 17, adding high-temperature resistant amylase 0.08kg/tds, and liquefying under the condition of pH6.0 to DE12, wherein the iodine test is brown;
liquefying by twice spraying
Primary spraying: preheating an ejector and a laminar flow tank to 100 ℃, then carrying out ejection liquefaction, controlling the temperature of the ejector to be 102 ℃, carrying out vacuum primary flash evaporation cooling, and reducing the temperature of a system to 95 ℃ to enter the laminar flow tank for maintaining for 60 min;
secondary spraying: heating the feed liquid to 120 ℃ through a secondary ejector, maintaining the temperature for 3min through a pipeline, then feeding the feed liquid into a vacuum secondary flash evaporation cooling system, cooling the system temperature to 95 ℃, feeding the system temperature into a secondary laminar flow tank, maintaining the system temperature for 20min, performing vacuum tertiary flash evaporation cooling, cooling the system temperature to 72 ℃, cooling the system temperature to 58 ℃ through a plate heat exchanger, feeding the system temperature into a pH tank, adjusting the pH value into a saccharification tank;
(2) saccharification: adding saccharifying enzyme 0.4kg/tds at pH3.8 and temperature 58 deg.C, and saccharifying for 30 hr until DX (fructose + glucose) reaches 95.2%;
(3) removing slag by using a plate frame: at the temperature of 62 ℃, the saccharified liquid is filtered by a plate and frame filter to remove protein in the saccharified liquid, and clear sugar liquid is obtained;
(4) plate frame decoloring: adding active carbon, and decolorizing the sugar solution at 70 deg.C (with active carbon addition of 0.8 kg/tds);
(5) aga filtration (secondary decolorization): adding active carbon (the adding amount of the active carbon is 0.8kg/tds) into the filtrate in a transferring tank, and carrying out secondary decolorization on the sugar solution at the decolorization temperature of 65 ℃;
(6) ceramic membrane: filtering the decolorized solution with ceramic membrane to remove part of macromolecular protein and dextrin in the sugar solution, and retaining various microorganisms;
(7) f00 ion exchange: feeding sugar liquor with the temperature of 52 ℃ from bottom to top through a cation exchange column (cation resin model: dispute D001FD) from the discharge of the cation exchange column to an anion exchange column (anion resin model: D354FD) to remove anions and cations in the sugar liquor, reduce ash content, purify the sugar liquor, prevent impurities in the sugar liquor from damaging isomerase, ensure that the discharge pH is 5.0 and the electrical conductance is less than or equal to 20 mu s/cm;
(8) f00 evaporation: concentrating under vacuum by five-effect evaporator (five-effect: vacuum degree 50mbar, temperature 35 deg.C; four-effect: vacuum degree 100mbar, temperature 40 deg.C; three-effect: vacuum degree 150mbar, temperature 45 deg.C; two-effect: vacuum degree 200mbar, temperature 50 deg.C; one-effect: vacuum degree 250mbar, temperature 60 deg.C) to obtain syrup with DS of 50%;
(9) isomerization: after flash evaporation and deoxidation, the pH value of the sugar solution is required to be adjusted to 7.6, the sulfur dioxide content is 80ppm, the magnesium ion content is 30ppm, and then the sugar solution enters an isomerase fixed column (the feeding flow is 6 m)3H), isomerizing the sugar solution by glucose isomerase for 60min at 55 ℃ to obtain sugar solution F42 containing 42% of fructose;
(10) f42 ion exchange/mixed bed: passing the obtained sugar solution through a cation exchange column (bleaching positive resin: PPC150S) and an anion exchange column (bleaching negative resin: PPA103S) in sequence, and then passing through a special mixed bed resin (competing light mixed bed positive resin: D001MB and mixed bed negative resin: D202MB) to remove negative and positive ions in sugar, reduce ash content, purify sugar solution, prevent impurities in the sugar solution from causing harm to chromatographic resin and ensure the quality of 42 fructose finished product, wherein the discharging pH is 5.0, and the electric conductance is less than or equal to 20 mu s/cm;
(11) f42 evaporation: concentrating a part of F42 fructose which passes through an F42 ion exchange/mixed bed to obtain F42 finished syrup with the discharge concentration of 71.2 percent and the pH value of 4.0, and being convenient for storage and transportation;
vacuum concentration by using a five-effect evaporator: five effects are as follows: vacuum degree of 50mbar, temperature of 35 ℃; four effects are as follows: vacuum degree of 100mbar and temperature of 40 ℃; three effects are as follows: vacuum degree of 150mbar and temperature of 50 ℃; two effects are as follows: vacuum degree of 200mbar and temperature of 55 ℃; the method has the following effects: vacuum degree of 250mbar and temperature of 60 ℃;
(12) and (3) chromatography: another portion of F42 fructose that passed through F42 ion/mixed bed was passed through dow: 99ca/310 chromatographic separation resin, and chromatographically separating at 58 ℃ into syrup AD with fructose content of 86% and raffinate BD with fructose content of 9.5%;
(13) f55 blending and decoloring: mixing the syrup AD (fructose content 86%) and a part of F42 fructose which passes through an F42 ion exchange/mixed bed into syrup with the fructose content of 55% according to the proportion of 0.42:1, and adsorbing organic substances such as flocculent precipitates, pigments and the like in sugar solution at the temperature of 62 ℃ by utilizing the porosity and microporosity of activated carbon (the adding amount of the activated carbon is 1.0kg/tds) to obtain F55 fructose with the chroma of 10;
(14) f55 deodorization: part of protein and colored and odorous substances in F55 fructose are removed from top to bottom through a deodorizing column, so that HMF is 30ppm, the quality of the syrup is improved, and the storage time is prolonged;
(15) f55 mixed bed: the hypoglycemic liquid passes through the special resin for the mixed bed, the feeding temperature is 32 ℃, the discharging pH is 4.6, anions and cations in the sugar are removed, the ash content is reduced, the sugar liquid is purified, and the storage life of the syrup is prolonged;
(16) f55 evaporation: concentrating the sugar liquid from the F55 mixed bed, discharging the sugar liquid with pH4.0 and 77.2 percent concentration, namely F55 finished syrup, and facilitating storage and transportation;
five-effect vacuum evaporation is adopted: five effects are as follows: vacuum degree of 50mbar, temperature of 40 deg.C; four effects are as follows: the vacuum degree is 100mbar, and the temperature is 50 ℃; three effects are as follows: vacuum degree of 150mbar, temperature of 55 ℃; two effects are as follows: vacuum degree of 200mbar and temperature of 60 ℃; the method has the following effects: vacuum 250mbar and temperature 65 ℃.
Example 3 an in-line production method of F42, F55 high fructose corn syrup
An in-line production method of F42 and F55 high fructose corn syrup comprises the following process steps:
(1) liquefaction: adjusting the concentration Be' 20 of starch slurry, adding 0.1kg/tds of high-temperature resistant amylase, and liquefying under the condition of pH6.5 until DE15, wherein the iodine test is brown;
liquefaction by double injection
Primary spraying: preheating an ejector and a laminar flow tank to 100 ℃, then carrying out jet liquefaction, controlling the temperature of the ejector to be 105 ℃, carrying out vacuum primary flash evaporation cooling, and reducing the temperature of a system to 96 ℃, and then, entering the laminar flow tank for maintaining for 70 min;
secondary spraying: heating the feed liquid to 130 ℃ through a secondary ejector, maintaining the temperature for 4min through a pipeline, entering a vacuum secondary flash evaporation tank for cooling, cooling the system temperature to 96 ℃, entering a secondary laminar flow tank for maintaining the temperature for 25min, performing vacuum tertiary flash evaporation cooling, cooling the system temperature to 75 ℃, reducing the temperature to 60 ℃ through a plate heat exchanger, entering a pH tank, adjusting the pH, and entering a saccharification tank;
(2) saccharification: adding saccharifying enzyme 0.42kg/tds at 60 deg.C and pH4.2, and saccharifying for 35 hr to ensure that DX (fructose + glucose) reaches 95.5%;
(3) removing slag by using a plate frame: at the temperature of 64 ℃, the saccharified liquid is filtered by a plate and frame filter to remove protein in the saccharified liquid, so as to obtain clear sugar liquid;
(4) plate frame decoloring: adding activated carbon, and decolorizing the sugar solution at 72 deg.C (with the addition of activated carbon of 1.0 kg/tds);
(5) aga filtration (secondary decolorization): adding activated carbon (the adding amount of the activated carbon is 1.0kg/tds) into the filtrate in a transferring tank, and carrying out secondary decolorization on the sugar solution at the decolorization temperature of 68 ℃;
(6) ceramic membrane: filtering the decolorized solution with ceramic membrane to remove part of macromolecular protein and dextrin in the sugar solution, and retaining various microorganisms;
(7) f00 ion exchange: feeding sugar liquor with the temperature of 55 ℃ from bottom to top through a cation exchange column (cation resin model: dispute D001FD) from the discharge of the cation exchange column to an anion exchange column (anion resin model: D354FD) to remove anions and cations in the sugar liquor, reduce ash content, purify the sugar liquor, prevent impurities in the sugar liquor from damaging isomerase, ensure that the discharge pH is 5.5 and the electrical conductance is less than or equal to 20 mu s/cm;
(8) f00 evaporation: concentrating under vacuum by five-effect evaporator (five-effect: vacuum degree of 100mbar, temperature of 40 deg.C; four-effect: vacuum degree of 150mbar, temperature of 50 deg.C; three-effect: vacuum degree of 200mbar, temperature of 55 deg.C; two-effect: vacuum degree of 250mbar, temperature of 60 deg.C; one-effect: vacuum degree of 300mbar, temperature of 65 deg.C) to obtain syrup with DS of 50%;
(9) isomerization: after flash evaporation and deoxidation, the pH value of the sugar solution is adjusted to 7.8, the sulfur dioxide content is 100ppm, the magnesium ion content is 50ppm, and then the sugar solution enters an isomerase fixed column (the feeding flow is 5.5 m)3H) at 58 ℃ byGlucose isomerase is used for isomerization for 65min to obtain sugar liquid F42 containing 43% of fructose;
(10) f42 ion exchange/mixed bed: passing the obtained sugar solution through a cation exchange column (bleaching positive resin: PPC150S) and an anion exchange column (bleaching negative resin: PPA103S) in sequence, and then passing through a special mixed bed resin (competing light mixed bed positive resin: D001MB and mixed bed negative resin: D202MB) to remove negative and positive ions in sugar, reduce ash content, purify sugar solution, prevent impurities in the sugar solution from causing harm to chromatographic resin and ensure the quality of 42 fructose finished product, wherein the discharging pH is 4.7, and the electric conductivity is less than or equal to 20 mu s/cm;
(11) f42 evaporation: concentrating a part of F42 fructose which passes through an F42 ion exchange/mixing bed, controlling the discharge concentration to be 71.3 percent and controlling the discharge concentration to be pH4.2, namely F42 finished syrup, and facilitating the storage and transportation;
vacuum concentration by using a five-effect evaporator: five effects are as follows: vacuum degree of 100mbar and temperature of 40 ℃; four effects are as follows: vacuum degree of 150mbar and temperature of 50 ℃; three effects are as follows: vacuum degree of 200mbar and temperature of 55 ℃; two effects are as follows: vacuum degree of 250mbar and temperature of 60 ℃; the method has the following effects: vacuum degree of 300mbar and temperature of 65 ℃;
(12) and (3) chromatography: another portion of F42 fructose that was subjected to F42 ion/mixed bed was used as dow: separating with 99ca/310 chromatographic resin at 62 deg.C to obtain syrup AD with fructose content of 87% and residue BD with fructose content of 8.6%;
(13) f55 blending and decoloring: mixing the syrup AD and a part of F42 fructose which passes through an F42 ion exchange/mixed bed according to the ratio of 2:5 to obtain syrup with the fructose content of 56 percent, and adsorbing organic substances such as flocculent precipitates, pigments and the like in the sugar solution at 63 ℃ by utilizing the porosity and microporosity of activated carbon (the adding amount of the activated carbon is 1.2kg/tds) to obtain F55 fructose with the chromaticity of 10;
(14) f55 deodorization: part of protein and colored and odorous substances in the sugar are removed from top to bottom through a deodorizing column, so that the HMF content reaches 30ppm, the quality of the syrup is improved, and the storage time is prolonged;
(15) f55 mixed bed: the hypoglycemic liquid passes through the special resin for the mixed bed, the feeding temperature is 35 ℃, the discharging pH is 5.0, anions and cations in the sugar are removed, the ash content is reduced, the sugar liquid is purified, and the storage life of the syrup is prolonged;
(16) f55 evaporation: the sugar liquid from the F55 mixed bed is concentrated to ensure that the discharged material has pH of 4.2 and the concentration of 77.3 percent, namely F55 finished syrup, and the purpose is convenient for storage and transportation;
five-effect vacuum evaporation is adopted: five effects are as follows: vacuum degree of 100mbar and temperature of 45 ℃; four effects are as follows: vacuum degree of 150mbar, temperature of 55 ℃; three effects are as follows: vacuum degree of 200mbar and temperature of 60 ℃; two effects are as follows: vacuum degree of 250mbar and temperature of 65 ℃; the method has the following effects: vacuum 300mbar and temperature 70 ℃.
Example 4 an in-line production method of F42, F55 high fructose corn syrup
An in-line production method of F42 and F55 high fructose corn syrup comprises the following process steps:
(1) liquefaction: adjusting the concentration Be' 25 of starch slurry, adding 0.11kg/tds of high-temperature resistant amylase, and liquefying under the condition of pH7.0 to obtain DE18, wherein the iodine test is brown;
the liquefaction is carried out by adopting a double-injection method:
primary spraying: preheating an ejector and a laminar flow tank to 100 ℃, then carrying out ejection liquefaction, controlling the temperature of the ejector to be 110 ℃, carrying out vacuum primary flash evaporation cooling, and reducing the temperature of a system to 98 ℃ to enter the laminar flow tank for maintaining for 90 min;
secondary spraying: heating the feed liquid to 145 ℃ through a secondary ejector, maintaining the temperature for 5min through a pipeline, then feeding the feed liquid into a vacuum secondary flash evaporation cooling tank, feeding the feed liquid into a secondary laminar flow tank at the system temperature of 98 ℃ for 30min, performing vacuum tertiary flash evaporation cooling, reducing the system temperature to 78 ℃, then reducing the temperature to 62 ℃ through a plate heat exchanger, feeding the feed liquid into a pH tank, adjusting the pH value, and feeding the feed liquid into a saccharification tank;
(2) saccharification: adding saccharifying enzyme 0.45kg/tds at pH4.5 and 62 deg.C, and saccharifying for 40 hr until DX (fructose + glucose) reaches 96%;
(3) removing slag by using a plate frame: at the temperature of 65 ℃, the saccharified liquid is filtered by a plate and frame filter to remove protein in the saccharified liquid, so as to obtain clear sugar liquid;
(4) plate frame decoloring: adding activated carbon, and decolorizing the sugar solution at 75 deg.C (with the addition of activated carbon of 1.1 kg/tds);
(5) aga filtration (secondary decolorization): adding activated carbon (the adding amount of the activated carbon is 1.1kg/tds) into the filtrate in a transferring tank, and carrying out secondary decolorization on the sugar solution at the decolorization temperature of 70 ℃;
(6) ceramic membrane: filtering the decolorized solution with ceramic membrane to remove part of macromolecular protein and dextrin in the sugar solution, and retaining various microorganisms;
(7) f00 ion exchange: feeding 56 ℃ sugar solution from bottom to top through a cation exchange column (cation resin model: dispute D001FD) from the discharge of the cation exchange column to an anion exchange column (anion resin model: D354FD) to remove anions and cations in the sugar solution, reduce ash content, purify the sugar solution, prevent impurities in the sugar solution from damaging isomerase, wherein the discharge pH is 6.5, and the electrical conductance is less than or equal to 20 mus/cm;
(8) f00 evaporation: concentrating under vacuum by five-effect evaporator (five-effect: vacuum degree 150mbar, temperature 45 deg.C; four-effect: vacuum degree 250mbar, temperature 55 deg.C; three-effect: vacuum degree 300mbar, temperature 60 deg.C; two-effect: vacuum degree 350mbar, temperature 65 deg.C; one-effect: vacuum degree 450mbar, temperature 75 deg.C) to obtain syrup with DS of 50%;
(9) isomerization: after flash evaporation and deoxidation, the pH value of the sugar solution is adjusted to 7.9, the sulfur dioxide content is adjusted to 120ppm, the magnesium ion content is adjusted to 80ppm, and then the sugar solution enters an isomerase fixed column (the feeding flow is 5 m)3H), isomerizing by glucose isomerase for 75min at 60 ℃ to obtain sugar liquid F42 containing 44% of fructose;
(10) f42 ion exchange/mixed bed: passing the obtained sugar solution through a cation exchange column (bleaching positive resin: PPC150S) and an anion exchange column (bleaching negative resin: PPA103S) in sequence, and then passing through a special mixed bed resin (competing light mixed bed positive resin: D001MB and mixed bed negative resin: D202MB) to remove negative and positive ions in sugar, reduce ash content, purify sugar solution, prevent impurities in the sugar solution from causing harm to chromatographic resin and ensure the quality of 42 fructose finished product, wherein the discharging pH is 5.0, and the electric conductance is less than or equal to 20 mu s/cm;
(11) f42 evaporation: concentrating a part of F42 fructose which passes through an F42 ion exchange/mixing bed, controlling the discharge concentration to be 71.4 percent and controlling the discharge concentration to be pH4.5, namely F42 finished syrup, and facilitating the storage and transportation;
vacuum concentration by using a five-effect evaporator: five effects are as follows: vacuum degree of 150mbar and temperature of 45 ℃; four effects are as follows: vacuum degree of 250mbar and temperature of 55 ℃; three effects are as follows: vacuum degree of 300mbar and temperature of 60 ℃; two effects are as follows: the vacuum degree is 350mbar, and the temperature is 65 ℃; the method has the following effects: vacuum degree of 450mbar and temperature of 75 ℃;
(12) and (3) chromatography: the other part of F42 fructose which passes through the F42 ion mixed bed adopts the Dow: 99ca/310 chromatographic separation resin, and chromatographically separating at 65 ℃ into syrup AD with fructose content of 87% and raffinate BD with fructose content of 8%;
(13) f55 blending and decoloring: mixing the syrup AD and a part of F42 fructose which passes through an F42 ion exchange/mixed bed according to the proportion of 1:2 to form syrup with the fructose content of 57 percent, and adsorbing organic substances such as flocculent precipitates, pigments and the like in the sugar solution at the temperature of 65 ℃ by utilizing the porosity and microporosity of activated carbon (the adding amount of the activated carbon is 1.5kg/tds) to obtain F55 fructose with the chroma of 10;
(14) f55 deodorization: part of protein and colored and odorous substances in the sugar are removed from top to bottom through a deodorizing column, so that the HMF content reaches 30ppm, the quality of the syrup is improved, and the storage time is prolonged;
(15) f55 mixed bed: the hypoglycemic liquid passes through the special resin for the mixed bed, the feeding temperature is 37 ℃, the discharging pH is 5.3, anions and cations in the sugar are removed, the ash content is reduced, the sugar liquid is purified, and the storage life of the syrup is prolonged;
(16) f55 evaporation: concentrating the sugar liquid from the F55 mixed bed, discharging the sugar liquid with pH of 4.5 and concentration of 77.4 percent, namely F55 finished syrup, and facilitating storage and transportation;
five-effect vacuum evaporation is adopted: five effects are as follows: vacuum degree of 150mbar, temperature of 55 ℃; four effects are as follows: vacuum degree of 250mbar and temperature of 60 ℃; three effects are as follows: vacuum degree of 300mbar and temperature of 70 ℃; two effects are as follows: the vacuum degree is 350mbar, and the temperature is 75 ℃; the method has the following effects: vacuum 450mbar and temperature 80 ℃.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
SEQUENCE LISTING
<110> Henan flying agricultural development Co., Ltd
<120> same-line production method of F42 and F55 high fructose corn syrup
<130> 1
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 388
<212> PRT
<213> Streptomyces rubiginosus)
<400> 1
Met Asn Tyr Gln Pro Thr Pro Glu Asp Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
Thr Val Gly Trp Gln Gly Arg Asp Pro Phe Gly Asp Ala Thr Arg Arg
20 25 30
Ala Leu Asp Pro Val Glu Ser Val Gln Arg Leu Ala Glu Leu Gly Ala
35 40 45
His Gly Val Thr Phe His Asp Asp Asp Leu Ile Pro Phe Gly Ser Ser
50 55 60
Asp Ser Glu Arg Glu Glu His Val Lys Arg Phe Arg Gln Ala Leu Asp
65 70 75 80
Asp Thr Gly Met Lys Val Pro Met Ala Thr Thr Asn Leu Phe Thr His
85 90 95
Pro Val Phe Lys Asp Gly Gly Phe Thr Ala Asn Asp Arg Asp Val Arg
100 105 110
Arg Tyr Ala Leu Arg Lys Thr Ile Arg Asn Ile Asp Leu Ala Val Glu
115 120 125
Leu Gly Ala Glu Thr Tyr Val Ala Trp Gly Gly Arg Glu Gly Ala Glu
130 135 140
Ser Gly Gly Ala Lys Asp Val Arg Asp Ala Leu Asp Arg Met Lys Glu
145 150 155 160
Ala Phe Asp Leu Leu Gly Glu Tyr Val Thr Ser Gln Gly Tyr Asp Ile
165 170 175
Arg Phe Ala Ile Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Leu
180 185 190
Leu Pro Thr Val Gly His Ala Leu Ala Phe Ile Glu Arg Leu Glu Arg
195 200 205
Pro Glu Leu Tyr Gly Val Asn Pro Glu Val Gly His Glu Gln Met Ala
210 215 220
Gly Leu Asn Phe Pro His Gly Ile Ala Gln Ala Leu Trp Ala Gly Lys
225 230 235 240
Leu Phe His Ile Asp Leu Asn Gly Gln Asn Gly Ile Lys Tyr Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ala Gly Asp Leu Arg Ala Ala Phe Trp Leu Val
260 265 270
Asp Leu Leu Glu Ser Ala Gly Tyr Ser Gly Pro Arg His Phe Asp Phe
275 280 285
Lys Pro Pro Arg Thr Glu Asp Phe Asp Gly Val Trp Ala Ser Ala Ala
290 295 300
Gly Cys Met Arg Asn Tyr Leu Ile Leu Lys Glu Arg Ala Ala Ala Phe
305 310 315 320
Arg Ala Asp Pro Glu Val Gln Glu Ala Leu Arg Ala Ser Arg Leu Asp
325 330 335
Glu Leu Ala Arg Pro Thr Ala Ala Asp Gly Leu Gln Ala Leu Leu Asp
340 345 350
Asp Arg Ser Ala Phe Glu Glu Phe Asp Val Asp Ala Ala Ala Ala Arg
355 360 365
Gly Met Ala Phe Glu Arg Leu Asp Gln Leu Ala Met Asp His Leu Leu
370 375 380
Gly Ala Arg Gly
385
<210> 2
<211> 1164
<212> DNA
<213> Artificial sequence
<400> 2
atgaactacc agccgacccc ggaagatcgc tttacttttg gcctgtggac tgtaggttgg 60
cagggtcgcg acccgttcgg cgatgctact cgtcgtgccc tggatccggt tgaatctgtg 120
caacgcctgg cggaactggg cgcacatggt gtaactttcc acgacgatga tctgatcccg 180
tttggctcca gcgactccga gcgcgaagaa cacgtgaaac gctttcgtca ggcgctggac 240
gatactggca tgaaagtccc gatggcgacg accaacctgt tcacgcaccc tgtgttcaag 300
gatggtggct tcacggctaa cgatcgtgac gttcgtcgct acgccctgcg taaaaccatt 360
cgcaacattg acctggcggt tgaactgggc gctgagacct atgttgcttg gggtggtcgt 420
gaaggtgcag aatccggtgg tgcaaaagat gtgcgtgatg ccctggatcg catgaaagaa 480
gcgttcgacc tgctgggtga atatgtcacc tctcagggtt acgatatccg ttttgctatt 540
gaaccgaaac cgaacgaacc acgtggtgac attctgctgc caaccgtagg tcacgctctg 600
gcgttcatcg agcgtctgga acgcccggaa ctgtacggtg tgaacccgga ggtcggccat 660
gagcagatgg caggtctgaa cttccctcac ggcatcgctc aggcactgtg ggctggtaaa 720
ctgttccaca ttgatctgaa cggtcagaac ggtatcaaat acgaccagga tctgcgtttc 780
ggcgctggtg atctgcgtgc agctttctgg ctggtggatc tgctggaaag cgctggttac 840
agcggtccgc gtcacttcga cttcaaaccg ccgcgtactg aagacttcga tggtgtatgg 900
gcgagcgctg cgggttgtat gcgcaattat ctgatcctga aggaacgtgc tgctgctttt 960
cgcgcggacc cggaagtaca ggaagcactg cgtgcgtctc gtctggatga gctggcgcgc 1020
cctactgctg ctgatggtct gcaggctctg ctggatgacc gctccgcttt tgaagaattc 1080
gacgtcgacg ctgccgcagc tcgtggtatg gctttcgaac gtctggatca gctggcaatg 1140
gaccatctgc tgggcgcacg tggc 1164
<210> 3
<211> 388
<212> PRT
<213> Artificial sequence
<400> 3
Met Asn Tyr Gln Pro Thr Pro Glu Asp Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
Thr Val Gly Trp Gln Gly Arg Asp Pro Phe Gly Asp Ala Thr Arg Arg
20 25 30
Ala Leu Asp Pro Val Glu Ser Val Gln Arg Leu Ala Glu Leu Gly Ala
35 40 45
His Gly Val Thr Phe His Asp Asp Asp Leu Ile Pro Phe Gly Ser Ser
50 55 60
Asp Ser Glu Arg Glu Glu His Val Lys Arg Phe Arg Gln Ala Leu Asp
65 70 75 80
Asp Thr Gly Met Lys Val Pro Lys Ala Thr Thr Asn Leu Phe Thr His
85 90 95
Pro Val Phe Lys Asp Gly Gly Phe Thr Ala Asn Asp Arg Asp Val Arg
100 105 110
Arg Tyr Ala Leu Arg Lys Thr Ile Arg Asn Ile Asp Leu Ala Val Glu
115 120 125
Leu Gly Pro Glu Thr Tyr Val Gly Trp Gly Gly Arg Glu Gly Ala Glu
130 135 140
Ser Gly Gly Ala Lys Asp Val Arg Asp Ala Leu Asp Arg Met Lys Glu
145 150 155 160
Ala Phe Asp Leu Leu Gly Glu Tyr Val Thr Ser Gln Gly Tyr Asp Ile
165 170 175
Arg Phe Ala Ile Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Leu
180 185 190
Leu Pro Thr Val Gly His Ala Leu Ala Phe Ile Glu Arg Leu Glu Arg
195 200 205
Pro Glu Leu Tyr Gly Val Asn Pro Glu Val Gly His Glu Gln Met Ala
210 215 220
Gly Leu Asn Phe Pro His Gly Ile Ala Gln Ala Leu Trp Ala Gly Lys
225 230 235 240
Leu Phe His Ile Asp Leu Asn Cys Gln Asn Gly Ile Lys Tyr Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ala Gly Asp Leu Arg Ala Ala Phe Trp Leu Val
260 265 270
Asp Leu Leu Glu Ser Ala Gly Tyr Ser Gly Pro Arg His Phe Asp Phe
275 280 285
Lys Pro Pro Arg Thr Glu Asp Phe Asp Gly Val Trp Ala Ser Ala Ala
290 295 300
Gly Cys Met Arg Asn Tyr Leu Ile Leu Lys Glu Arg Ala Ala Ala Phe
305 310 315 320
Arg Ala Asp Pro Glu Val Gln Glu Ala Leu Arg Ala Ser Arg Leu Asp
325 330 335
Glu Leu Ala Arg Pro Thr Ala Ala Asp Gly Leu Gln Ala Leu Leu Asp
340 345 350
Asp Arg Ser Ala Phe Glu Glu Phe Asp Val Asp Ala Ala Ala Ala Arg
355 360 365
Gly Met Ala Phe Glu Arg Leu Asp Gln Leu Ala Met Asp His Leu Leu
370 375 380
Gly Ala Arg Gly
385
<210> 4
<211> 1164
<212> DNA
<213> Artificial sequence
<400> 4
atgaactacc agccgacccc ggaagatcgc tttacttttg gcctgtggac tgtaggttgg 60
cagggtcgcg acccgttcgg cgatgctact cgtcgtgccc tggatccggt tgaatctgtg 120
caacgcctgg cggaactggg cgcacatggt gtaactttcc acgacgatga tctgatcccg 180
tttggctcca gcgactccga gcgcgaagaa cacgtgaaac gctttcgtca ggcgctggac 240
gatactggca tgaaagtccc gaaggcgacg accaacctgt tcacgcaccc tgtgttcaag 300
gatggtggct tcacggctaa cgatcgtgac gttcgtcgct acgccctgcg taaaaccatt 360
cgcaacattg acctggcggt tgaactgggc cctgagacct atgttggttg gggtggtcgt 420
gaaggtgcag aatccggtgg tgcaaaagat gtgcgtgatg ccctggatcg catgaaagaa 480
gcgttcgacc tgctgggtga atatgtcacc tctcagggtt acgatatccg ttttgctatt 540
gaaccgaaac cgaacgaacc acgtggtgac attctgctgc caaccgtagg tcacgctctg 600
gcgttcatcg agcgtctgga acgcccggaa ctgtacggtg tgaacccgga ggtcggccat 660
gagcagatgg caggtctgaa cttccctcac ggcatcgctc aggcactgtg ggctggtaaa 720
ctgttccaca ttgatctgaa ctgtcagaac ggtatcaaat acgaccagga tctgcgtttc 780
ggcgctggtg atctgcgtgc agctttctgg ctggtggatc tgctggaaag cgctggttac 840
agcggtccgc gtcacttcga cttcaaaccg ccgcgtactg aagacttcga tggtgtatgg 900
gcgagcgctg cgggttgtat gcgcaattat ctgatcctga aggaacgtgc tgctgctttt 960
cgcgcggacc cggaagtaca ggaagcactg cgtgcgtctc gtctggatga gctggcgcgc 1020
cctactgctg ctgatggtct gcaggctctg ctggatgacc gctccgcttt tgaagaattc 1080
gacgtcgacg ctgccgcagc tcgtggtatg gctttcgaac gtctggatca gctggcaatg 1140
gaccatctgc tgggcgcacg tggc 1164
<210> 5
<211> 44
<212> DNA
<213> Artificial sequence
<400> 5
taagaaggag atataccatg gatgaactac cagccgaccc cgga 44
<210> 6
<211> 41
<212> DNA
<213> Artificial sequence
<400> 6
gtggtggtgg tggtgctcga gttagccacg tgcgcccagc a 41
<210> 7
<211> 1179
<212> DNA
<213> Artificial sequence
<400> 7
ggaattcaac tatcaaccaa ccccagaaga cagatttaca tttggactgt ggaccgtggg 60
atggcagggt agagatcctt tcggtgatgc cacaagaaga gcacttgacc cagttgaatc 120
tgttcaaaga ttagccgaat tgggagccca cggagttact ttccatgacg atgacctaat 180
tccttttggc agtagtgact ctgagaggga ggaacatgtc aagagattta gacaagctct 240
tgacgatact ggtatgaaag tccctaaggc aaccacgaat ttgtttactc atcctgtttt 300
caaagatggt ggatttaccg caaatgatag agatgtgagg aggtatgctc tgagaaaaac 360
tatcagaaac atcgatctgg cagtcgaatt gggtccagaa acctacgttg gctggggtgg 420
aagagaggga gcagagtcag gtggtgccaa agatgtcaga gatgctctgg atcgaatgaa 480
ggaagccttt gacctgctag gagagtacgt cacctctcag ggttatgaca tcagattcgc 540
tatagaacca aaacctaatg aaccaagagg tgacatttta ttacccacag ttggtcacgc 600
tttagccttt attgaacgtt tggaaagacc tgaattgtat ggagtgaatc ctgaagttgg 660
tcacgaacag atggcaggac tgaactttcc acatggaatc gctcaggccc tgtgggccgg 720
taaattattt catattgacc tgaattgtca aaacggtatc aaatacgatc aggatttaag 780
attcggtgca ggtgacttga gagctgcttt ttggcttgtt gacttattgg aatccgcagg 840
ttactcagga cctagacact ttgacttcaa acctcccaga actgaagatt ttgatggagt 900
ttgggcttct gctgccggct gcatgagaaa ctacttgata ttgaaggaga gagcagcagc 960
ctttagggca gacccagagg tgcaagaggc tttgagagca tccaggttag atgagttggc 1020
tagaccaaca gcagcagacg gattacaggc acttttggat gacagatctg cattcgagga 1080
gtttgatgtg gatgctgctg ccgctcgtgg aatggctttc gagagactag atcagctagc 1140
tatggatcat ctgctgggcg ctagaggcta atctagagc 1179

Claims (7)

1. A glucose isomerase GIM, characterized in that the amino acid sequence is as shown in the sequence table SEQ ID NO: 3, respectively.
2. An on-line production method of F42 and F55 high fructose corn syrup, which is characterized in that the glucose isomerase of claim 1 is adopted for isomerization, and the specific steps are as follows:
(1) liquefaction: adjusting the concentration Be' 17-25 of starch slurry, adding high-temperature resistant amylase, and liquefying under the condition of pH5.0-7.0 to DE12-18, wherein the iodine test is brown;
(2) saccharification: adding saccharifying enzyme into the liquefied solution at pH of 3.8-4.5 and temperature of 58-62 deg.C for saccharifying until DX (fructose + glucose) is greater than or equal to 95%;
(3) purifying by deslagging, decoloring and filtering;
(4) f00 ion exchange: passing the sugar solution with the temperature of 52-56 ℃ from bottom to top through a cation exchange column, discharging the sugar solution from the cation exchange column, and introducing the sugar solution into an anion exchange column, so that the pH of the discharged sugar solution is 4.0-6.5, and the electric conductivity is less than or equal to 20 mu s/cm;
(5) f00 evaporation: concentrating under vacuum by an evaporator to obtain a syrup with DS of 50%;
(6) isomerization: after flash evaporation and deoxidation, adjusting the pH value of the sugar solution to 7.6-7.9, the sulfur dioxide content to 80-120ppm and the magnesium ion content to 30-80ppm, then entering an isomerase fixing column, and obtaining sugar solution F42 containing 42-44% of fructose through glucose isomerase isomerization at the temperature of 55-60 ℃;
(7) f42 ion exchange/mixed bed: passing the obtained sugar solution through a cation exchange column and an anion exchange column in sequence, and then through special resin for a mixed bed to ensure that the pH of the discharged material is more than or equal to 4.5 and the conductivity is less than or equal to 20 mus/cm;
(8) f42 evaporation: evaporating and concentrating a part of F42 fructose which passes through an F42 ion exchange/mixed bed, controlling the discharge concentration to be 71.2-71.4%, and controlling the pH to be 3.3-4.5 to obtain F42 finished product syrup;
(9) and (3) chromatography: separating the other part of F42 fructose subjected to F42 ion exchange/mixed bed into syrup AD with the fructose content of more than 85 percent and raffinate BD with the fructose content of less than or equal to 10 percent by chromatography;
(10) f55 blending and decoloring: mixing the syrup AD and a part of F42 fructose which passes through an F42 ion exchange/mixed bed into syrup with the fructose content of 55-57% in proportion, and then adsorbing by using activated carbon at the temperature of 62-65 ℃ to obtain F55 fructose;
(11) f55 deodorization: passing F55 fructose through a deodorizing column from top to bottom to remove part of protein and colored and odorous substances in the sugar;
(12) f55 mixed bed: the sugar solution is processed by special resin for mixed bed, the feeding temperature is 32-37 ℃, the discharging pH is more than or equal to 4.5, anions and cations in the sugar are removed, the ash content is reduced, the sugar solution is purified, and the storage life of the syrup is prolonged;
(13) f55 evaporation: and (3) concentrating the sugar liquid from the mixed bed of F55 to ensure that the pH value of the discharged material is 3.3-4.5, and the concentration is 77.2% -77.4%, namely F55 finished product syrup.
3. An F42, F55 co-line production method as claimed in claim 2, wherein liquefaction is carried out by means of two injection:
primary spraying: preheating an ejector and a laminar flow tank to 100 ℃, then carrying out ejection liquefaction, controlling the temperature of the ejector to be 102-110 ℃, carrying out vacuum primary flash evaporation cooling, and reducing the temperature of a system to 95-98 ℃ to enter the laminar flow tank for maintaining for 60-90 min;
secondary spraying: heating the feed liquid to 120-145 ℃ by a secondary ejector, maintaining the temperature for 3-5min by a pipeline, then carrying out vacuum secondary flash evaporation cooling, reducing the temperature of the system to 95-98 ℃, then carrying out vacuum tertiary flash evaporation cooling, reducing the temperature of the system to 78-72 ℃, reducing the temperature to 58-62 ℃ by a plate heat exchanger, entering a pH tank, adjusting the pH value, and then entering a saccharification tank.
4. The on-line production method of F42 and F55 high fructose corn syrup as claimed in claim 2, wherein the deslagging, decoloring and filtering processes in step (3) are as follows:
removing slag by using a plate frame: at the temperature of 62-65 ℃, the saccharified liquid is filtered by a plate and frame filter to remove protein in the saccharified liquid, so as to obtain clear sugar liquid;
plate frame decoloring: adding active carbon to decolorize the sugar solution at 70-75 deg.C with the addition of active carbon of 0.8-1.1 kg/tds;
filtering Ama for secondary decolorization: adding active carbon into the filtrate transfer tank, wherein the adding amount of the active carbon is 0.8-1.1kg/tds, and carrying out secondary decolorization on the sugar solution at the decolorization temperature of 65-70 ℃;
ceramic membrane filtration: filtering the decolorized solution with ceramic membrane to remove part of macromolecular protein and dextrin in the sugar solution, and retaining various microorganisms.
5. The on-line production method of F42 and F55 high fructose corn syrup as claimed in claim 2, wherein the evaporation method is a five-effect evaporator vacuum concentration method, which comprises the following steps: five effects are as follows: vacuum degree of 50-150mbar, and temperature of 35-45 deg.C; four effects are as follows: the vacuum degree is 100-; three effects are as follows: the vacuum degree is 150-; two effects are as follows: the vacuum degree is 200-; the method has the following effects: the vacuum degree is 250-.
6. The high fructose corn syrup product of F42, F55 prepared by the process of any one of claims 2 to 5.
7. The use of the glucose isomerase GIM according to claim 1.
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CN102876758A (en) * 2012-09-28 2013-01-16 浙江华康药业股份有限公司 Method for preparing fructose syrup
CN107058428A (en) * 2016-12-22 2017-08-18 河南飞天农业开发股份有限公司 Production technology that a kind of fructose, maltose are collinear
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