CN116891845A - High-activity glucose isomerase and application thereof in catalyzing high-concentration glucose - Google Patents
High-activity glucose isomerase and application thereof in catalyzing high-concentration glucose Download PDFInfo
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- CN116891845A CN116891845A CN202310839399.1A CN202310839399A CN116891845A CN 116891845 A CN116891845 A CN 116891845A CN 202310839399 A CN202310839399 A CN 202310839399A CN 116891845 A CN116891845 A CN 116891845A
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- 108700040099 Xylose isomerases Proteins 0.000 title claims abstract description 46
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 45
- 239000008103 glucose Substances 0.000 title claims abstract description 30
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 claims abstract description 34
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 claims abstract description 21
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- 238000000034 method Methods 0.000 claims abstract description 12
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- 239000007788 liquid Substances 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 238000000855 fermentation Methods 0.000 claims description 22
- 230000004151 fermentation Effects 0.000 claims description 22
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 6
- 235000011152 sodium sulphate Nutrition 0.000 claims description 6
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
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- 230000009465 prokaryotic expression Effects 0.000 claims description 2
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- 238000006555 catalytic reaction Methods 0.000 abstract description 2
- 239000012452 mother liquor Substances 0.000 abstract description 2
- 229930091371 Fructose Natural products 0.000 description 13
- 239000005715 Fructose Substances 0.000 description 13
- 235000021433 fructose syrup Nutrition 0.000 description 7
- 238000006317 isomerization reaction Methods 0.000 description 6
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- ZAQJHHRNXZUBTE-WUJLRWPWSA-N D-xylulose Chemical compound OC[C@@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-WUJLRWPWSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
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- 241000186339 Thermoanaerobacter Species 0.000 description 1
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- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
- C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
- C12Y503/01005—Xylose isomerase (5.3.1.5)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention relates to the technical field of enzyme catalysis, in particular to high-activity glucose isomerase and application thereof in catalyzing high-concentration glucose, wherein the amino acid sequence of the glucose isomerase is shown as SEQ ID NO:1. When in use, the D-glucose mother liquor is taken, PB and pure water are added, and crude enzyme liquid of glucose isomerase is added for reaction at 55-85 ℃. The high-activity glucose isomerase can catalyze D-glucose to generate D-fructose at high concentration; moreover, compared with the wild glucose isomerase, the method can be applied to high-temperature conversion of D-glucose to generate D-fructose, and has higher activity.
Description
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to high-activity glucose isomerase and application of the high-activity glucose isomerase in catalyzing high-concentration glucose.
Background
Glucose isomerase (glucose isomerase, EC 5.3)1.5), also known as D-xylose isomerase, catalyzes the isomerization of D-xylose to xylulose in the first step of xylose metabolism in many microorganisms. The conversion of xylose to xylulose to saprophytic bacteria growing on decayed plant material provides a nutritional requirement and also aids in the bioconversion of hemicellulose to ethanol. Which are formed in the presence of divalent metal ions (e.g., mg 2+ 、Co 2+ Or Mn of 2+ ) In the presence, D-glucose or D-xylose is catalyzed to be reversibly isomerized into D-fructose or D-xylulose. Glucose isomerase is involved in sugar metabolism and is widely used in the industrial production of high fructose syrup (HFCS) and bioethanol. Such isomerization reactions are important in industrial processes, such as the production of fructose syrups in the food industry. Under the push of the growing market demand for fructose and glucose syrup, glucose isomerase has been increasingly used for producing fructose and glucose syrup from corn starch hydrolysates since 1970 s, and has been juxtaposed with amylase and protease as three of the most consumed industrial enzymes. Because fructose syrups containing 55% or more fructose have a higher sweetness than sucrose, there is a greater demand for such syrups in the food and beverage industries, and as the fructose syrup market grows, so does the demand for more efficient glucose isomerase.
In the existing high fructose syrup production process, thermolabile glucose isomerase from a mesophilic microorganism is used in an immobilized enzyme reactor to produce 40% to 42% high fructose syrup at an operating temperature of 50 ℃ and 58 ℃ with a residence time of less than 1 hour. If this temperature is exceeded, undesirable browning products may be formed due to non-enzymatic reactions (e.g., maillard reactions) between the reducing sugars and the proteins. However, this temperature limitation is detrimental to high glucose conversion. In fact, the isomerisation of glucose will reach a reaction equilibrium, which is shifted towards fructose at higher temperatures. In this process, an additional expensive chromatographic step is required to obtain the desired 55% fructose syrup concentration. The fructose-glucose syrup-55 is a more desirable variety of fructose-glucose syrup for food and soft drink applications. In the process of producing the fructose-glucose syrup, the final conversion rate of D-glucose to D-fructose is highly dependent on the temperature, and the higher the temperature is, the more favorable the yield of D-fructose is. To avoid a complex concentration step downstream, higher temperatures are required to obtain fructose syrup-55 directly by promoting the equilibrium of the isomerization reaction toward D-fructose formation. Theoretically, due to the equilibrium of the isomerization reaction, isomerization at high temperatures (about 95 ℃) and lower pH (about 4.5-5.5) is required to achieve the desired fructose levels in the syrup. If this can be achieved, no additional concentration step is required.
Briefly, compared to the enzymes currently in use, industrially valuable glucose isomerase must have a lower pH, a higher temperature, and a higher pH for Ca 2+ Resistance to inhibition and higher affinity to glucose. Accordingly, there is currently a great deal of research effort directed to the identification of thermostable and acid-stable glucose isomerase that has a higher affinity for glucose. In recent years, substantial attempts have been made to increase the activity or thermostability of glucose isomerase to increase the achievable D-fructose content. Gene sequences encoding these enzymes have been reported from E.coli, B.subtilis, clostridium, streptomyces, actinomyces misilis and Thermomyces. In addition, researchers have also attempted to achieve this goal by the form of protein mutants, such as glucose isomerase mutant W139F from Thermoanaerobacter saccharolyticus strain B6A, which has an increased half-life at 80℃and pH 6.5, and a 10-fold improvement in catalytic efficiency on D-glucose over native glucose isomerase. It has also been reported that the 3 glucose isomerase mutants H99Q, V184T and D102N of Bacillus calcolyticus TK4 have higher thermostability and pH stability.
U.S. Pat. No. 3,182 and WO03/062387 describe the isolation of xylose isomerase from highly thermophilic Thermotoga. This xylose isomerase was shown to be active at a temperature of 97 ℃. However, after 2 hours at 90 ℃, their activity was only reduced to 40% and their optimal pH was still very high (above 7).
Thus, the optimal glucose isomerase for use in the food industry should meet the following conditions: it can still function efficiently under acidic conditions (i.e., a pH of about 6 or less) to avoid browning reactions; it can tolerate higher substrate and product inhibition (e.g., greater than 200g/L glucose) in order to extractHigh production efficiency per unit volume; it should remain stable at high reaction temperatures (i.e., 80 ℃ or higher) to promote high fructose conversion; the catalyst has higher catalytic activity, can reach a reaction balance point in a shorter time, and reduces energy consumption and manpower consumption; it can be free of harmful metal ions, such as Co 2+ The method comprises the steps of carrying out a first treatment on the surface of the It can be prepared by recombination in common microorganism such as Escherichia coli or Bacillus in high efficiency and large quantity, so as to facilitate production and reduce cost.
Therefore, a new glucose isomerase or mutant is developed, the high-temperature tolerance and the catalytic efficiency under the high-temperature reaction condition are improved, and the novel glucose isomerase or mutant has great practical value.
Disclosure of Invention
The invention aims to provide a high-activity glucose isomerase and an application of the high-activity glucose isomerase in catalyzing high-concentration glucose.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a high activity glucose isomerase, the amino acid sequence of which is shown in SEQ ID NO:1.
The high-activity glucose isomerase can be used for catalyzing high-concentration glucose, and the using method comprises the following steps: and (3) taking D-glucose mother liquor, adding PB and pure water, and adding crude enzyme solution of glucose isomerase at 55-85 ℃.
The preparation method of the crude enzyme liquid comprises the following steps:
(1) By a primer splicing method, SEQ ID NO:1 and cloning the corresponding coding polynucleotide sequence of the protein shown in the formula 1 into a prokaryotic expression vector to realize high expression in escherichia coli;
(2) By shake flask fermentation or fed-batch fermentation
(1) Shaking flask fermentation
E.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight;
taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1:100 in the inoculation ratio of example, 100mL of the autoclaved medium B was inoculated, cultured at 30℃until the cell OD 5-6 was reached, and the flask was immediately placed in a 25℃shaker at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours;
after the culture, the culture solution was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells; then washing the bacterial precipitate twice with distilled water, collecting bacterial precipitate, and preserving at-70 ℃; simultaneously taking a small amount of thalli for SDS-PAGE detection;
(2) fed-batch fermentation
Fed-batch fermentation was performed in a computer controlled bioreactor, a 200ml seed shake flask was prepared from a single colony of E.coli harboring the expression vector, and the bioreactor was accessed when the culture of the seed shake flask was OD 2.0; the temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% v/v orthophosphoric acid and 30% v/v aqueous ammonia; during the fermentation, when the dissolved oxygen is greatly raised, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when the OD600 is 50.0, the temperature is controlled to be 25 ℃, 0.1mM IPTG is used for inducing expression for 16 hours, and the collected thalli are centrifugally preserved at the temperature of minus 25 ℃, and when in use, 2 kg of pure water is added for each kg of wet thalli.
Wherein, the culture medium A is: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L.
Wherein, the culture medium B is: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L.
Wherein, the culture medium used for fed-batch fermentation is: 24g/L of yeast extract, 12g/L of peptone, 0.4% of glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, and the pH value is 7.0.
Compared with the prior art, the invention has the beneficial effects that:
the high-activity glucose isomerase can catalyze high-concentration D-glucose to generate D-fructose. Moreover, compared with the wild glucose isomerase, the method can be applied to high-temperature conversion of D-glucose to generate D-fructose, has stronger thermal stability, and can obtain better social benefit and economic value.
Drawings
FIG. 1 shows the results of high performance liquid chromatography of 20g/L fructose. 8.4 minutes was the product fructose peak.
FIG. 2 shows the results of high performance liquid chromatography of 20g/L glucose. The 10 minutes is the glucose peak.
FIG. 3 shows the results of the reaction of comparative example 1 for 4 hours.
FIG. 4 shows the results of the 19-hour reaction in comparative example 1.
FIG. 5 shows the results of the reaction of example 6 for 4 hours.
FIG. 6 shows the results of the 19-hour reaction in example 6.
FIG. 7 shows the results of the 19-hour reaction in example 7.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The instruments and reagents used in this example are commercially available products unless otherwise specified.
The liquid phase detection conditions referred to in the following examples are as follows:
mobile phase: acetonitrile: water = 70:30
A detector: differential detector
Flow rate: 1mL/min
Column temperature: 40 DEG C
Differential detector cell temperature: 40 DEG C
An amino column 250mm x 4.6 μm was used.
Example 1 obtaining the wild-type glucose isomerase Gene sequence
The secondary structure and codon preference of the gene are adjusted by a total gene synthesis method so as to realize high expression in escherichia coli. The Primer Premier (http:// Primer3.Ut. Ee /) and OPTIMIZER (http:// genome. Uro. Es/OPTIMIZER /) were used for design, and the difference in annealing temperature (Tm) was controlled within 3 ℃, the Primer length was controlled within 60base, the Primer sequences were as shown in Table 2, and the obtained primers were dissolved in double distilled water and then added to the following reaction system so that the final concentration of each Primer was 30nM and the final concentration of the head-to-tail primers was 0.6. Mu.M.
TABLE 1
| 2mM dNTP mix(2mM eachdNTP) | 5μl |
| 10×Pfubuffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH 2 O | So that the total volume of the reaction system was 50. Mu.l |
The prepared PCR reaction system is placed in a Bo-Japanese patent application (XP) cycler gene amplification instrument for amplification according to the following procedures: 98℃30s,55℃45s,72℃120s,35x. The DNA fragment obtained by PCR was cut and purified, and cloned into NdeI/XhoI site of pET30a by homologous recombination. The monoclonal was picked for sequencing. The DNA sequence which is sequenced successfully is SEQ ID NO:4, designated CSGIwt, having the corresponding amino acid sequence of SEQ ID NO:3.
TABLE 2
EXAMPLE 2 acquisition of the Gene sequence of glucose isomerase mutant
The high activity glucose isomerase of the present invention is derived from the sequence of SEQ ID NO:3, a wild-type glucose isomerase. Glucose isomerase mutants and polynucleotides encoding such mutants may be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by subjecting the enzyme-encoding enzyme to in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, directed evolution methods, and the like.
The secondary structure and codon preference of the gene are adjusted by a total gene synthesis method so as to realize high expression in escherichia coli. The Primer Premier (http:// Primer3.Ut. Ee /) and OPTIMIZER (http:// genome. Uro. Es/OPTIMIZER /) were used for design, and the difference in annealing temperature (Tm) was controlled within 3 ℃, the Primer length was controlled within 60base, the Primer sequences were as shown in Table 4, and the obtained primers were dissolved in double distilled water and then added to the following reaction system so that the final concentration of each Primer was 30nM and the final concentration of the head-to-tail primers was 0.6. Mu.M.
TABLE 3 Table 3
| 2mM dNTP mix(2mM eachdNTP) | 5μl |
| 10×Pfubuffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH 2 O | So that the total volume of the reaction system was 50. Mu.l |
The prepared PCR reaction system is placed in a Bo-Japanese patent application (XP) cycler gene amplification instrument for amplification according to the following procedures: 98℃30s,55℃45s,72℃120s,35x. The DNA fragment obtained by PCR was cut and purified, and cloned into NdeI/XhoI site of pET30a by homologous recombination. The monoclonal was picked for sequencing. The DNA sequence which is sequenced successfully is SEQ ID NO:2, designated CSGI1, the corresponding amino acid sequence of which is SEQ ID NO:1. compared with the sequence of CSGIwt, the sequence has four mutations of A44S, H248S, K380R and K390R.
TABLE 4 Table 4
Example 3 shake flask expression test
E.coli single colonies containing the expression vector were picked and inoculated into 10ml of autoclaved medium: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L. Culturing at 30℃and 250rpm overnight.
Taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1: an inoculation ratio of 100 was inoculated into 100ml of autoclaved medium: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L. The cells were cultured at 30℃until the cell OD 5-6 was reached, and the flask was immediately placed in a 25℃shaker at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and the incubation was continued at 25℃and 250rpm for 16 hours.
After the completion of the culture, the culture was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells. Then the bacterial cell precipitate is washed twice with distilled water, and the bacterial cells are collected and stored at-70 ℃. And simultaneously taking a small amount of thalli for SDS-PAGE detection.
Example 4 fed-batch fermentation
Fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai state of China) with a capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L dihydrogenphosphate and 12.54g/L dipotassium hydrogen phosphate, pH 7.0.
E.coli single colonies containing the expression vector were prepared into 200ml seed shake flasks and were accessed into the bioreactor when the culture of the seed shake flasks was OD 2.0. The temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration was automatically controlled at 30% by stirring rate (rpm) and aeration supply cascade, and the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation process, when the dissolved oxygen is greatly raised, the feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When OD600 was about 50.0 (wet weight: about 100 g/L), the temperature was controlled at 25℃and expression was induced with 0.1mM IPTG for 16 hours, and the cells were collected by centrifugation and stored at-25℃and used by adding 2 kg of pure water per kg of wet cells.
Example 5 Heat treatment and residual enzyme Activity detection
Each enzyme was placed in a 10ml tube (2 ml system added with 0.5ml crude enzyme solution) at 85 ℃Incubate for 0, 0.5, 1, 2 hours. Then, 500. Mu.l of the heat-treated enzyme extract and 500. Mu.l of a substrate buffer (100 mM PB buffer pH6.8, 20mM MgSO 4 ，1mM MnCl 2 360g/L D-glucose, pH 6.8) was mixed and incubated at 55℃for 4 hours to evaluate residual enzyme activity. Finally, the substrate residue and the formation of the product were detected by using a high-performance liquid chromatography, and a 0-hour CSGIwt sample was used as a standard 100%, which had a fructose concentration of 97.51g/L, a glucose concentration of 91.13g/L and a conversion of 54.17% at 4 hours. The reaction results after the heat treatment for 2 hours and for 4 hours are shown below.
Therefore, the mutant can retain more enzyme activity after high-temperature treatment, and has larger promotion than wild type protein.
Comparative example 1 high concentration reaction control example
2ml of the system, 50mM PB buffer pH6.8, 10mM MgSO, was prepared in a 10ml reaction tube 4 ，0.5mM MnCl 2 600g/L D-glucose, 50. Mu.l/ml CSGIwt crude enzyme solution. The mixture was reacted in a water bath at 55℃for 4 hours and sampled for 19 hours, respectively. As shown in FIGS. 3-4, the results of the high performance liquid phase assay after spotting and dilution 5-fold showed that the fructose concentration was 40.75g/L for 4 hours and that CSGIwt activity was severely inhibited at high substrate concentrations.
EXAMPLE 6 high concentration reaction example
2ml of the system, 50mM PB buffer pH6.8, 10mM MgSO, was prepared in a 10ml reaction tube 4 ，0.5mM MnCl 2 600g/L D-glucose, 50. Mu.l/ml CSGI4 crude enzyme solution. The mixture was reacted in a water bath at 55℃for 4 hours and sampled for 19 hours, respectively. As shown in FIGS. 5-6, the results of the high performance liquid phase detection after spotting and diluting 5 times show that the fructose concentration is 280.73g/L for 4 hours and the CSGI4 still has higher activity at high concentration of the substrate.
Example 7 high temperature high concentration reaction example
2ml of the system, 50mM PB buffer pH6.8, 10mM MgSO, was prepared in a 10ml reaction tube 4 ，0.5mM MnCl 2 600g/L D-glucose, 50. Mu.l/ml CSGI4 crude enzymeAnd (3) liquid. The reaction was carried out in a water bath at 85℃for 19 hours and sampled. As shown in FIG. 7, the results of the high performance liquid phase detection after spotting and diluting 5 times show that the fructose concentration is 305.47g/L in 19 hours, and the CSGI4 still has higher activity at high temperature and high concentration of the substrate.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A highly active glucose isomerase characterized in that: the amino acid sequence is shown in SEQ ID NO:1.
2. Use of the highly active glucose isomerase according to claim 1 for catalyzing the production of D-fructose from D-glucose.
3. The use of a highly active glucose isomerase for catalyzing high concentration glucose according to claim 2, wherein: adding PB and pure water into the D-glucose mother solution, and adding the crude enzyme solution of the glucose isomerase in the claim 1 for reaction at 55-85 ℃.
4. The use of high activity glucose isomerase for catalyzing high concentration glucose according to claim 3, wherein the preparation method of the crude enzyme solution comprises the following steps:
(1) By a primer splicing method, SEQ ID NO:1 and cloning the corresponding coding polynucleotide sequence of the protein shown in the formula 1 into a prokaryotic expression vector to realize high expression in escherichia coli;
(2) By shake flask fermentation or fed-batch fermentation
(1) Shaking flask fermentation
E.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight;
taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1:100 in the inoculation ratio of example, 100mL of the autoclaved medium B was inoculated, cultured at 30℃until the cell OD 5-6 was reached, and the flask was immediately placed in a 25℃shaker at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours;
after the culture, the culture solution was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells; then washing the bacterial precipitate twice with distilled water, collecting bacterial precipitate, and preserving at-70 ℃; simultaneously taking a small amount of thalli for SDS-PAGE detection;
(2) fed-batch fermentation
Fed-batch fermentation was performed in a computer controlled bioreactor, a 200ml seed shake flask was prepared from a single colony of E.coli harboring the expression vector, and the bioreactor was accessed when the culture of the seed shake flask was OD 2.0; the temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% v/v orthophosphoric acid and 30% v/v aqueous ammonia; during the fermentation, when the dissolved oxygen is greatly raised, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when the OD600 was 50.0, the temperature was controlled at 25℃and expression was induced with 0.1mM IPTG for 16 hours, and the cells were harvested by centrifugation and stored at-25 ℃.
5. The use of the high activity glucose isomerase for catalyzing high concentration glucose according to claim 4, wherein: in the preparation method of the crude enzyme liquid, the culture medium A is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L.
6. The use of the high activity glucose isomerase for catalyzing high concentration glucose according to claim 4, wherein: in the preparation method of the crude enzyme liquid, the culture medium B is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L.
7. The use of the high activity glucose isomerase for catalyzing high concentration glucose according to claim 4, wherein: in the preparation method of the crude enzyme liquid, the culture medium used for fed-batch fermentation is as follows: 24g/L of yeast extract, 12g/L of peptone, 0.4% of glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, and the pH value is 7.0.
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