CN105779522A - Method for producing L-4-hydroxyisoleucine by microbial enzyme conversion method - Google Patents
Method for producing L-4-hydroxyisoleucine by microbial enzyme conversion method Download PDFInfo
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Abstract
The invention relates to a method for producing L-4-hydroxyisoleucine by a microbial enzyme conversion method. The method comprises the following steps: 1, culturing a mature bacterial strain; 2, grafting mature escherichia coli recombinant bacteria which are capable of inducing production of L-4-hydroxyisoleucine into a fermentation tank, and carrying out fermentation culture, wherein the temperature of the fermentation process is controlled in two stages, i.e., 4 to 36 DEG C in the earlier stage and 30 to 32 DEG C in the later stage; 3, collecting thallus after fermentation, adding phosphate buffer and washing, then adding alpha-oxoglutarate, isoleucine, ferrous sulfate, magnesium sulfate and ascorbic acid, and carrying out forced ventilation for catalysis; 4, removing bacteria from the catalytic liquid with a ceramic membrane, filtering with an ultrafiltration membrane, decoloring with activated carbon, desalting with faintly acid cation exchange resin, adsorbing effluent with 732 cation exchange resin and eluting with ammonia water, carrying out evaporation concentration and recrystallization to obtain the L-4-hydroxyisoleucine. The preparation method disclosed by the invention is simple and efficient in reaction system and simple and feasible in extraction process. The conversion rate is up to over 95.4 percent, and the purity of the product can reach over 98 percent.
Description
Technical field
The present invention relates to technical field of microbial fermentation, be specifically related to a kind of microbial enzyme conversion method production L-4-hydroxyl different
Leucic method.
Background technology
L-4-hydroxyisoleucine (4-hydroxyisoleucine) is the nonprotein amino that a kind of nature exists
Acid.Research finds, L-4-hydroxyisoleucine has promotion insulin secretion, improves insulin resistant and regulation blood fat etc. in many ways
Face acts on.L-4-hydroxyisoleucine can not only promote that the picked-up of glucose is utilized by surrounding tissue, additionally it is possible to reduces liver
Glucose output is to improve the insulin resistant of peripheral tissues.Compare the medicine having been used to now treat T2DM, L-4-hydroxyl
The insulin synthesis of isoleucine regulation and control is strictly to rely on concentration of glucose.Therefore, L-4-hydroxyisoleucine will not cause class
Like side effect such as hypoglycemia.The most also find that L-4-hydroxyisoleucine can effectively reduce triglyceride and cholesterol in blood
Amount, has acceleration lipid metabolism, blood fat reducing and the effect of liver function protecting.Accordingly, as effectively prevention and treatment diabetes and
The ideal medicament of obesity, L-4-hydroxyisoleucine is with a wide range of applications and the market demand.
Being limited by technology and cost, the yield of China's L-4-hydroxyisoleucine can not meet domestic and foreign export
Demand.The L-4-hydroxyisoleucine production method of report includes extraction method, chemical synthesis and enzyme process at present, but only extracts
Method for industrialized production and there is extraction ratio difficulty low, isolated and purified, Raw Material Demand amount is big and high in cost of production is not enough, tradition
Chemical method whether organic synthesis or chemical resolution all lose competitiveness because production cost is high.Because of enzyme catalysis in biological method
Method preparation method efficiency is of a relatively high, but still suffers from complex operation, expensive starting materials, the problem that production cost is too high, and current
Lack the relevant Technology that can be used for industrialization large-scale production.
Summary of the invention
It is an object of the invention to the deficiency for solving above-mentioned technical problem, it is provided that a kind of microbial enzyme conversion method produces L-4-
The method of hydroxyisoleucine, uses high density fermentation and enzyme to live high, and reaction system is simply efficiently and extraction simple flow is easy,
Yield is up to 75% and product purity is more than 98%, and the method efficiency is high, low cost, it is easy to industrialization produces.
The present invention solves above-mentioned technical problem, the technical scheme provided is: a kind of microbial enzyme conversion method produces L-
The method of 4-hydroxyisoleucine, comprises the following steps:
(1), the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine will can be induced to access the kind equipped with seed tank culture base
In sub-tank, at temperature 33 DEG C-37 DEG C, ph 6.5-7.5, dissolved oxygen cultivates the OD600 to bacterium solution under conditions of controlling more than 25%
Value is 12-16: obtain the Escherichia coli recombinant strain of the induced production L-4-hydroxyisoleucine of maturation；
The construction method of the described Escherichia coli recombinant strain inducing production L-4-hydroxyisoleucine is: according to Su Yun gold spore
In bacillus YL-03, gene I/D O(i.e. encodes the gene of ILE hydroxylase) it is carried out manually modified, it is thus achieved that gene order
As shown in SEQ NO.1；Clone this gene, and by this gene constructed enter plasmid pET28a, obtain recombiant plasmid, matter of recombinating
Import e. coli bl21 after Li, i.e. obtain inducing the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine.
(2), the Escherichia coli recombinant strain of ripe induced production L-4-hydroxyisoleucine is accessed equipped with fermentation tank
In the fermentation tank of culture medium, carry out deep liquid ventilating fermentation cultivation；In incubation, when cultivating 5h in fermentation medium one
Secondary property puts into the IPTG(isopropyl-β-D-thiogalactoside of final concentration of 0.1-0.3mmol/L) to induce, dissolved oxygen controls
At 30-50%, pH6.5-7.0, after end sugar exhausts, stream adds glycerine water solution that mass concentration is 10-80% as carbon source, ferments
The control of Cheng Wendu two rank, before adding IPTG induction, control temperature is at 34-36 DEG C, after pending abduction delivering, adjustment temperature to 30-
Cultivate for 32 DEG C；
(3), after fermentation ends, by butterfly centrifugal machine cryogenic collector thalline, and use phosphate buffer that thalline is washed
Wash, remove phage surface impurity, then with pure water preparation catalyst system and catalyzing: a-ketoglutaric acid 30-35g/L, isoleucine 26-
30g/L, ferrous sulfate 0.5-4g/L, magnesium sulfate 0.1-1g/L, ascorbic acid 0.01-0.2g/L, add in catalyst system and catalyzing and wash
Thalline after washing, forced ventilation is catalyzed, and maintenance pH is 6.0-6.5, and catalytic temperature is 28-32 DEG C, obtains catalytic liquid；Described thalline
Addition be: every 100ml catalyst system and catalyzing adds 0.3g thalline；
(4), that catalytic liquid first passes through ceramic membrane filter is degerming, after the ultrafilter membrane of 2-4Kd molecular weight, and through activated carbon decolorizing, adopts
After 122 weak-acid cation-exchange resin desalinations, 732 cationic exchange resin adsorption use ammonia eluting on effluent, steam
Send out and be concentrated to give L-4-hydroxyisoleucine crude product, be then recrystallized to give L-4-hydroxyisoleucine.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
The formula stating seed tank culture base is: glucose 0.5-20g/L, yeast powder 0.5-10g/L, peptone 0.5-5g/L, ammonium sulfate
0.5-10g/L, potassium dihydrogen phosphate 0.5-3g/L, magnesium sulfate 0.5-3g/L, vitamin B 10-20mg/L, biotin 0.5-
10mg/L, ferrous sulfate 0.5-50mg/L, manganese sulfate 0.5-50mg/L, defoamer 0-0.5ml/L, remaining is water.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
The formula stating fermentation tank culture medium is: glucose 0.5-10g/L, yeast powder 0.5-10g/L, peptone 0.5-5g/L, ammonium sulfate
0.5-10g/L, potassium dihydrogen phosphate 0.5-3g/L, magnesium sulfate 0.5-3g/L, VB1 0.5-20mg/L, VH 0.5-10mg/L, sulphuric acid
Ferrous 0.5-50mg/L, manganese sulfate 0.5-50mg/L, Semen Maydis pulp 0.5-10mL, sodium citrate 0.5-2g/L. defoamer 0-
0.5ml/L, remaining is water.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
State catalytic liquid in step (4) and be first heated to 60-80 DEG C before ceramic membrane filter is degerming, and keep 10-40min.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
Adding the 0.1-5% that charcoal amount is material liquid volume when stating activated carbon decolorizing in step (4), temperature retention time is 30-60min.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
Stating the ammonia mass concentration of eluting in step (4) is 1%-3%, and elution flow rate is 1-1.5BV/h.
The further improvements in methods of L-4-hydroxyisoleucine are produced: step as the present invention a kind of microbial enzyme conversion method
Suddenly evaporation and concentration described in (4), the final concentration of 500-600g/L of 4-hydroxyisoleucine crude product after evaporation and concentration.
Beneficial effect
One, applicant is for the deficiency in current fermentation process, in University Of Science and Technology Of Tianjin, China's aminoacid technical service
Under the help of the heart is instructed, by fermentating formula and process control, microbial enzyme are converted scheme and control technique, extraction scheme and
Control the improvement of technique so that extraction process is simple, and conversion ratio is up to more than 95.4%, and product purity is up to more than 98%, pole
Improve greatly production capacity and the efficiency of L-4-hydroxyisoleucine, the heavy industrialization of L-4-hydroxyisoleucine can be met
Produce, there is higher Social and economic benef@；
Two, the progress on the method and technology in the present invention includes following aspect: 1, fermentation culture: during the fermentation to fermenting
Cheng Wendu carries out two rank controls, and earlier fermentation i.e. adds and controls temperature before IPTG pre-induction at 34-36 DEG C, pending abduction delivering
After, adjust temperature to 30-32 DEG C cultivation and carry out cooling expression；Reduce temperature after induction and can reduce thalline specific growth rate, fall
Low plasmid Loss Rate, is of value to the formation of enzymatic structure, ensures that enzyme divides after preventing the expression of formation inclusion body, beneficially genes of interest
The biological activity of son.Culture medium increases Semen Maydis pulp as organic nitrogen source, for thalli growth and expression of enzymes provide aminoacid, growth because of
Son etc..It is simultaneously introduced sodium citrate suppression EMP Embden Meyerbof Parnas pathway, reduces carbon metabolism flow loss, reduce the generation of the metabolic by-products such as acetic acid,
It is beneficial to the expression of albumen.Current existing report focuses mostly in shaking flask level, and has no that to carry out 4-hydroxyl with glycerol for carbon source different
Leucic fermentation culture, after reducing, after induction, the expression that temperature can reduce thalline specific growth rate, beneficially genes of interest
Ensure the biological activity of enzyme molecule.Stream glycerol adding is cultivated, and cell yield is significantly higher than glucose, and beneficially genes of interest
Abduction delivering, enzyme total amount is high, and economic benefit is obviously improved.One time fermentation can meet the catalysis of 3 batches.2, enzymatic conversion:
More conventional test is compared, and the enzyme catalysis cycle is short, and in 4h, enzyme reaction terminates, and saves the production time, and Conventional catalytic conversion ratio typically exists
About 85%, there is more isoleucine remaining, be unfavorable for subsequent extracted purification, and cause substrate to waste, have a strong impact on economic effect
Benefit.The present invention adds enough α-ketoglutaric acid, relatively art methods and compares, and reaction rate and conversion ratio significantly improve.Cross
On the one hand the α-ketoglutaric acid of amount meets thalline own metabolism system consumption, simultaneously facilitates the carrying out of this enzymatic reaction.Drop simultaneously
Low enzymatic reaction temperature, increases Mg2+Strengthen enzyme activity, reduce iron ion content and alleviate follow-up extraction pressure.3, extract: existing
In technology studying extraction scheme and mainly start with from plant extract, the extraction to catalyst system and catalyzing relates to the fewest.Through 122
After acidulous cation resin desalination, 732 resin absorption amounts can be increased substantially, and decrease subsequent cell separating difficulty.Warp
Crossing in the ethanol of 75% after recrystallization, product liquid phase purity is obviously improved, up to more than 98%.
Detailed description of the invention
A kind of microbial enzyme conversion method produces the method for L-4-hydroxyisoleucine, comprises the following steps:
(1), the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine will can be induced to access the kind equipped with seed tank culture base
In sub-tank, at temperature 33 DEG C-37 DEG C, ph 6.5-7.5, dissolved oxygen cultivates the OD600 to bacterium solution under conditions of controlling more than 25%
Value is 12-16: obtain the Escherichia coli recombinant strain of the induced production L-4-hydroxyisoleucine of maturation；
The construction method of the described Escherichia coli recombinant strain inducing production L-4-hydroxyisoleucine is: according to Su Yun gold spore
In bacillus YL-03, gene I/D O(i.e. encodes the gene of ILE hydroxylase) it is carried out manually modified, it is thus achieved that gene order
As shown in SEQ NO.1；Clone this gene, and by this gene constructed enter plasmid pET28a, obtain recombiant plasmid, matter of recombinating
Import e. coli bl21 after Li, i.e. obtain inducing the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine.
(2), the Escherichia coli recombinant strain of ripe induced production L-4-hydroxyisoleucine is accessed equipped with fermentation tank
In the fermentation tank of culture medium, carry out deep liquid ventilating fermentation cultivation；In incubation, when cultivating 5h in fermentation medium one
Secondary property puts into the IPTG(isopropyl-β-D-thiogalactoside of final concentration of 0.1-0.3mmol/L) to induce, dissolved oxygen controls
At 30-50%, pH6.5-7.0, after end sugar exhausts, stream adds glycerine water solution that mass concentration is 10-80% as carbon source, ferments
The control of Cheng Wendu two rank, before adding IPTG induction, control temperature is at 34-36 DEG C, after pending abduction delivering, adjustment temperature to 30-
Cultivate for 32 DEG C；
(3), after fermentation ends, by butterfly centrifugal machine cryogenic collector thalline, and use phosphate buffer that thalline is washed
Wash, remove phage surface impurity, then with pure water preparation catalyst system and catalyzing: a-ketoglutaric acid 30-35g/L, isoleucine 26-
30g/L, ferrous sulfate 0.5-4g/L, magnesium sulfate 0.1-1g/L, ascorbic acid 0.01-0.2g/L, add in catalyst system and catalyzing and wash
Thalline after washing, forced ventilation is catalyzed, and maintenance pH is 6.0-6.5, and catalytic temperature is 28-32 DEG C, obtains catalytic liquid；Described thalline
Addition be: every 100ml catalyst system and catalyzing adds 0.3g thalline；
(4), that catalytic liquid first passes through ceramic membrane filter is degerming, after the ultrafilter membrane of 2-4Kd molecular weight, and through activated carbon decolorizing, adopts
After 122 weak-acid cation-exchange resin desalinations, 732 cationic exchange resin adsorption use ammonia eluting on effluent, steam
Send out and be concentrated to give L-4-hydroxyisoleucine crude product, be then recrystallized to give L-4-hydroxyisoleucine.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
The formula stating seed tank culture base is: glucose 0.5-20g/L, yeast powder 0.5-10g/L, peptone 0.5-5g/L, ammonium sulfate
0.5-10g/L, potassium dihydrogen phosphate 0.5-3g/L, magnesium sulfate 0.5-3g/L, vitamin B 10-20mg/L, biotin 0.5-
10mg/L, ferrous sulfate 0.5-50mg/L, manganese sulfate 0.5-50mg/L, defoamer 0-0.5ml/L, remaining is water.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
The formula stating fermentation tank culture medium is: glucose 0.5-10g/L, yeast powder 0.5-10g/L, peptone 0.5-5g/L, ammonium sulfate
0.5-10g/L, potassium dihydrogen phosphate 0.5-3g/L, magnesium sulfate 0.5-3g/L, VB1 0.5-20mg/L, VH 0.5-10mg/L, sulphuric acid
Ferrous 0.5-50mg/L, manganese sulfate 0.5-50mg/L, Semen Maydis pulp 0.5-10mL, sodium citrate 0.5-2g/L. defoamer 0-
0.5ml/L, remaining is water.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
State catalytic liquid in step (4) and be first heated to 60-80 DEG C before ceramic membrane filter is degerming, and keep 10-40min.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
Adding the 0.1-5% that charcoal amount is material liquid volume when stating activated carbon decolorizing in step (4), temperature retention time is 30-60min.
Further improvements in methods as the present invention a kind of microbial enzyme conversion method production L-4-hydroxyisoleucine: institute
Stating the ammonia mass concentration of eluting in step (4) is 1%-3%, and elution flow rate is 1-1.5BV/h.
The further improvements in methods of L-4-hydroxyisoleucine are produced: step as the present invention a kind of microbial enzyme conversion method
Suddenly evaporation and concentration described in (4), the final concentration of 500-600g/L of 4-hydroxyisoleucine crude product after evaporation and concentration.
In the present invention, the calculation of conversion ratio is as follows:
The ILE of 1mol can be catalyzed the L-4-hydroxyisoleucine producing 1mol in theory.
In terms of the conversion situation of L-4-hydroxyisoleucine content
Conversion ratio (%)=(L-4-hydroxyisoleucine yield)/(theoretical L-4-hydroxyisoleucine content) * 100%
The content use of L-4-hydroxyisoleucine, after DNFB column front derivation, liquid phase detects.
Below in conjunction with embodiment, the present invention is further illustrated:
Embodiment 1
A kind of microbial enzyme conversion method produces the method for L-4-hydroxyisoleucine, comprises the following steps:
(1), the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine will can be induced to access the kind equipped with seed tank culture base
In sub-tank, temperature 36 DEG C, ph7.0, the OD600 value that dissolved oxygen is cultivated under conditions of controlling more than 25% to bacterium solution is 14, to obtain final product
Escherichia coli recombinant strain to ripe induced production L-4-hydroxyisoleucine；
The present embodiment can be induced the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine obtained by purchase；Its structure side
Method is, clones its gene order gene as shown in SEQ NO.1；By this gene constructed enter plasmid pET28a, obtain recombinate matter
Grain, will import e. coli bl21 after recombiant plasmid, i.e. obtain inducing the escherichia coli weight producing L-4-hydroxyisoleucine
Group bacterium；
Seed tank culture based formulas: glucose 20g/L, yeast powder 10g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate
2g/L, magnesium sulfate 1 g/L, VB1 10mg/L, VH 0.5mg/L, ferrous sulfate 20mg/L, manganese sulfate 20mg/L and defoamer
0.4ml/L, remaining is water.
(2), ripe strain is accessed in fermentation tank, cultivate under the following conditions: ventilated by deep liquid and send out
Ferment, it is different that fermentation 5h disposable stream in fermentation medium adds the final concentration (calculating with the fermentating liquid volume) IPTG(as 0.1mmol/L
Propyl group-β-D-Thiogalactopyranoside) induce.Sweat temperature section controls, and earlier fermentation controls temperature 36
DEG C, after derivant to be added, adjusting temperature 30 DEG C cultivation, after end sugar has consumed, stream adds 50% glycerol and ferments；
Fermentation tank tank formula: glucose 5g/L, yeast powder 5g/L, peptone 5g/L, ammonium sulfate 3g/L, potassium dihydrogen phosphate 2.5g/
L, magnesium sulfate 2g/L, VB1(vitamin B1) 10mg/L, VH (biotin) 2mg/L, ferrous sulfate 50mg/L, manganese sulfate
50mg/L, Semen Maydis pulp 10mL, sodium citrate 1g/L. defoamer 0.5ml/L, remaining is water.
(3), fermented after, by butterfly centrifugal machine cryogenic collector thalline, and use phosphate buffer that thalline is entered
Row washing, removes phage surface impurity, then with pure water preparation catalyst system and catalyzing: a-ketoglutaric acid 30g/L, isoleucine 25g/
L, ferrous sulfate 3g/L, magnesium sulfate 1 g/L, ascorbic acid 0.1 g/L, add the thalline after washing, bacterium in catalyst system and catalyzing
The addition of body is: every 100ml catalyst system and catalyzing adds 0.3g thalline, and forced ventilation is catalyzed, and maintains pH 6.5, and catalytic temperature is 30
Degree.In 4h, substrate reactions is complete, and conversion ratio reaches 95.4%；More conventional test is compared, and this programme enzyme catalysis cycle is short, and 4h endoenzyme is anti-
Should terminate, conventional method catalytic conversion, typically about 85%, has more isoleucine remaining, is unfavorable for that subsequent extracted is pure
Change, and cause substrate to waste, affect economic benefit.
(4), catalytic liquid degerming through ceramic membrane, remove a part of pigment and albumen after the ultrafilter membrane of 3Kd molecular weight, and
Through 1% activated carbon insulation decolouring, after using 122 weak-acid cation-exchange resin desalinations, upper 732 cationic exchange resin adsorption
And with 1% ammonia eluting, evaporation and concentration (the final concentration of 500g/L of evaporation and concentration) obtains L-4-hydroxyisoleucine crude product, 75%
Ethanol in recrystallization, purity is 98.8%.
Embodiment 2
A kind of microbial enzyme conversion method produces the method for L-4-hydroxyisoleucine, comprises the following steps:
(1), the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine will can be induced to access the kind equipped with seed tank culture base
In sub-tank, temperature 37 DEG C, ph 6.5, the OD600 value that dissolved oxygen is cultivated under conditions of controlling more than 25% to bacterium solution is 12:
Escherichia coli recombinant strain to ripe induced production L-4-hydroxyisoleucine；
Seed tank culture based formulas: glucose 20g/L, yeast powder 10g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate
2g/L, magnesium sulfate 1 g/L, VB1 10mg/L, VH 0.5mg/L, ferrous sulfate 20mg/L, manganese sulfate 20mg/L, defoamer
0.4ml/L, remaining is water.
(2), ripe strain is accessed in fermentation tank, cultivate under the following conditions: ventilated by deep liquid and send out
Ferment, fermentation 5h disposable stream in fermentation medium adds IPTG(isopropyl-beta D-thio galactopyranoside) 0.2mmol/L.
Sweat temperature section controls, and earlier fermentation control temperature, at 36 DEG C, after derivant to be added, adjusts temperature 30 DEG C cultivation,
After end sugar has consumed, stream adds 60% glycerol and ferments；
Fermentation tank tank formula: glucose 5g/L, yeast powder 5g/L, peptone 5g/L, ammonium sulfate 3g/L, potassium dihydrogen phosphate 2.5g/
L, magnesium sulfate 2g/L, VB1 10mg/L, VH 2mg/L, ferrous sulfate 50mg/L, manganese sulfate 50mg/L, Semen Maydis pulp 10 mL, Fructus Citri Limoniae
Acid sodium 1g/L, defoamer 0.5ml/L, remaining is water.
(3), fermented after, by butterfly centrifugal machine cryogenic collector thalline, and use phosphate buffer that thalline is entered
Row washing, removes phage surface impurity, then prepares catalyst system and catalyzing with pure water: add a-ketoglutaric acid 30g/L, isoleucine
26g/L, ferrous sulfate 3g/L, magnesium sulfate 0.5g/L, ascorbic acid 0.05 g/L, forced ventilation is catalyzed, and maintains pH 6.5, urges
Changing temperature is 30 degree.In 4h, substrate reactions is complete, and it is 27.86 g/L that L-4-hydroxyisoleucine produces acid, and theoretical L-4-hydroxyl is different
It is 29.2 g/L that leucine produces acid, and conversion ratio reaches 95.4%, and more conventional test is compared, and the enzyme catalysis cycle is short, enzyme reaction knot in 4h
Bundle, method catalytic conversion, about 85%, has more isoleucine remaining, is unfavorable for subsequent extracted purification, and causes substrate
Waste, affects economic benefit.
(4), catalytic liquid degerming through ceramic membrane, remove a part of pigment and albumen after the ultrafilter membrane of 3Kd molecular weight, and
Through 1% activated carbon insulation decolouring, after using 122 weak-acid cation-exchange resin desalinations, upper 732 cationic exchange resin adsorption
And with 1% ammonia eluting, evaporation and concentration (the final concentration of 600g/L of evaporation and concentration) obtains L-4-hydroxyisoleucine crude product, 75%
Ethanol in recrystallization, purity is 98.6%.
Embodiment 3
A kind of microbial enzyme conversion method produces the method for L-4-hydroxyisoleucine, comprises the following steps:
(1), the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine will can be induced to access the kind equipped with seed tank culture base
In sub-tank, temperature 36 DEG C, ph 6.8, the OD600 value that dissolved oxygen is cultivated under conditions of controlling more than 25% to bacterium solution is 13:
Escherichia coli recombinant strain to ripe induced production L-4-hydroxyisoleucine；
Seed tank culture based formulas: glucose 10g/L, yeast powder 8g/L, peptone 3g/L, ammonium sulfate 4g/L, potassium dihydrogen phosphate
1g/L, magnesium sulfate 2g/L, VB1 15mg/L, VH 5mg/L, ferrous sulfate 30mg/L, manganese sulfate 30mg/L, defoamer 0.4ml/
L, remaining is water.
(2), ripe strain is accessed in fermentation tank, cultivate under the following conditions: ventilated by deep liquid and send out
Ferment, fermentation 5h disposable stream in fermentation medium adds IPTG(isopropyl-beta D-thio galactopyranoside) 0.2mmol/L.
Sweat temperature section controls, and earlier fermentation control temperature, at 35 DEG C, after derivant to be added, adjusts temperature 31 DEG C cultivation,
After end sugar has consumed, stream adds 80% glycerol and ferments；
Fermentation tank tank formula: glucose 5g/L, yeast powder 5g/L, peptone 5g/L, ammonium sulfate 3g/L, potassium dihydrogen phosphate 2.5g/
L, magnesium sulfate 2g/L, VB1 10mg/L, VH 2mg/L, ferrous sulfate 50mg/L, manganese sulfate 50mg/L, Semen Maydis pulp 10 mL, Fructus Citri Limoniae
Acid sodium 1g/L, defoamer 0.5ml/L, remaining is water.
(3), fermented after, by butterfly centrifugal machine cryogenic collector thalline, and use phosphate buffer that thalline is entered
Row washing, removes phage surface impurity, then with pure water preparation catalyst system and catalyzing: a-ketoglutaric acid 32g/L, isoleucine 28g/
L, ferrous sulfate 2g/L, magnesium sulfate 0.5g/L, ascorbic acid 0.05 g/L, add the thalline after washing in catalyst system and catalyzing,
The addition of thalline is: every 100ml catalyst system and catalyzing adds 0.3g thalline, and forced ventilation is catalyzed, and maintains pH 6.2, and catalytic temperature is
28℃.In 4h, substrate reactions is complete, and conversion ratio reaches 96.4%
(4), catalytic liquid is degerming through ceramic membrane, after the ultrafilter membrane a part of pigment of removing and the albumen of 3Kd molecular weight, and through 1%
Activated carbon insulation decolouring, after using 122 weak-acid cation-exchange resin desalinations, upper 732 cationic exchange resin adsorption are also used
1% ammonia eluting, evaporation and concentration (the final concentration of 500g/L of evaporation and concentration) obtains L-4-hydroxyisoleucine crude product, in the second of 75%
Recrystallization in alcohol, purity is 98.3%.
The above, be only presently preferred embodiments of the present invention, and the present invention not makees any pro forma restriction, though
So the present invention is disclosed above with preferred embodiment, but is not limited to the present invention, any technology people being familiar with this specialty
Member, in the range of without departing from technical solution of the present invention, when the technology contents of available the disclosure above makes a little change or modification
For the Equivalent embodiments of equivalent variations, as long as being without departing from technical solution of the present invention content, according to the technical spirit pair of the present invention
Any simple modification, equivalent variations and the modification that above example is made, all still falls within the range of technical solution of the present invention.
Relevant comparative's test example:
This test uses the method in method of the prior art and the present invention to carry out the different bright ammonia of fermenting and producing L-4-hydroxyl respectively
Acid, then contrasts the fermentation results of each group.Strain employed in fermentation is " can induce production L-4-hydroxyl different bright
The Escherichia coli recombinant strain of propylhomoserin ", the construction method of this bacterium is: clone its gene order gene as shown in SEQ NO.1；
By this gene constructed enter plasmid pET28a, obtain recombiant plasmid, by after recombiant plasmid import e. coli bl21, i.e. obtain luring
Lead the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine；”；By each to art methods and the inventive method point two groups,
I.e. art methods A group, art methods B group, the inventive method A group and the inventive method B group.The wherein method of each group
Operating condition is as follows:
Art methods A group:
Being accessed by strain in seed tank, cultivate under the following conditions: cultivation temperature 36 DEG C, ph7.0, dissolved oxygen controls 25%
Above, during subcultivation, OD600 value is 14, obtains the strain of maturation；
Seed tank culture based formulas: glucose 20g/L, yeast powder 10g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate
2g/L, magnesium sulfate 1 g/L, VB1 10mg/L, VH 0.5mg/L, ferrous sulfate 10mg/L, manganese sulfate 10mg/L, defoamer
0.4ml/L, surplus is water.
Ripe strain is accessed in fermentation tank, cultivates under the following conditions: by deep liquid ventilating fermentation, send out
Ferment 5h disposable stream in fermentation medium adds IPTG(isopropyl-beta D-thio galactopyranoside) 0.1mmol/L.Fermented
Journey, ferment control temperature is at 36 DEG C, after end sugar has consumed, flows liquid feeding sugar fermentation；
Fermentation medium: glucose 5g/L, yeast powder 5g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 2g/L, sulfur
Acid magnesium 2g/L, VB1 2mg/L, VH 1mg/L, ferrous sulfate 10mg/L, manganese sulfate 5mg/L, defoamer 0.3ml/L, surplus is
Water..When fermentation period about 21h is the highest, OD600 value is 35.
After having fermented, by butterfly centrifugal machine cryogenic collector thalline, and add brine, thalline is carried out pre-
Process.After 30 degree of incubations process, add a-ketoglutaric acid 28g/L, isoleucine 25g/L, ferrous sulfate 5g/L, ascorbic acid
0.02 g/L, forced ventilation is catalyzed, and maintains pH 7, and catalytic temperature is 32 degree.In 18h, substrate reactions is complete, and L-4-hydroxyl is different bright
It is 24.3g/L that propylhomoserin produces acid, and it is that 28.05 g/L conversion ratios reach 86.6% that theoretical L-4-hydroxyisoleucine produces acid；
Catalytic liquid is degerming through ceramic membrane, and through 1% activated carbon insulation decolouring, the ethanol precipitation of 90%, upper 732 cationes after evaporation
Exchanger resin adsorbs and uses 1% ammonia eluting, resin absorption amount 30g/L, and evaporation and concentration (the final concentration of 500g/L of evaporation and concentration) obtains
To L-4-hydroxyisoleucine crude product, the 4-hydroxyisoleucine content obtained is up to 40%.Product yield 40.4%.
Art methods B group:
During just strain accesses seed tank, cultivate under the following conditions: cultivation temperature 37 DEG C, ph7.0, dissolved oxygen controls
More than 25%, during subcultivation, OD600 value is 15, obtains the strain of maturation；
Seed tank culture based formulas: glucose 20g/L, yeast powder 10g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate
2g/L, magnesium sulfate 1 g/L, VB1 10mg/L, VH 0.5mg/L, ferrous sulfate 20mg/L, manganese sulfate 10mg/L, defoamer
0.4ml/L, surplus is water.
Ripe strain is accessed in fermentation tank, cultivates under the following conditions: by deep liquid ventilating fermentation, send out
Ferment 4h disposable stream in fermentation medium adds IPTG(isopropyl-beta D-thio galactopyranoside) 0.1mmol/L.Fermented
Journey, ferment control temperature is at 36 DEG C, after end sugar has consumed, flows liquid feeding sugar fermentation；
Fermentation medium: glucose 7g/L, yeast powder 5g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 2g/L, sulfur
Acid magnesium 2g/L, VB1 2mg/L, VH 1mg/L, ferrous sulfate 10mg/L, manganese sulfate 5mg/L, defoamer 0.3ml/L, surplus is
Water.Fermentation period about 21h the highest OD600 value is 40.
After having fermented, by butterfly centrifugal machine cryogenic collector thalline, and add carbonate buffer solution washing, thalline is entered
Row pretreatment.After 25 degree of incubations process, add a-ketoglutaric acid 23g/L, isoleucine 20g/L, ferrous sulfate 3g/L, anti-
Bad hematic acid 0.05 g/L, forced ventilation is catalyzed, and maintains pH 7, and catalytic temperature is 32 degree.In 16h, substrate reactions is complete, L-4-hydroxyl
It is 19.1g/L that base isoleucine produces acid, and it is 22.4 g/L that theoretical L-4-hydroxyisoleucine produces acid, and conversion ratio reaches 85.3%.
Catalytic liquid is degerming through ceramic membrane, and through 0.5% activated carbon insulation decolouring, upper 732 cationic exchange resin adsorption are also
By 2% ammonia eluting, resin absorption amount 25g/L, evaporation and concentration, obtaining L-4-hydroxyisoleucine crude product, the 4-hydroxyl obtained is different
Leucine content is up to 35%.Purifying crude product with silica gel column chromatography, purity is up to 80.1%.Product yield 32.6%.
The inventive method A group:
(1), strain is accessed in seed tank, cultivate under the following conditions: cultivation temperature 36 DEG C, ph7.0, dissolved oxygen controls
More than 25%, during subcultivation, OD600 value is 14, obtains the strain of maturation；
Seed tank culture based formulas: glucose 20g/L, yeast powder 10g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate
2g/L, magnesium sulfate 1 g/L, VB1 10mg/L, VH 0.5mg/L, ferrous sulfate 20mg/L, manganese sulfate 20mg/L, defoamer
0.4ml/L, surplus is water.
(2), ripe strain is accessed in fermentation tank, cultivate under the following conditions: ventilated by deep liquid and send out
Ferment, fermentation 5h disposable stream in fermentation medium adds IPTG(isopropyl-beta D-thio galactopyranoside) 0.1mmol/L.
Sweat temperature section controls, and earlier fermentation control temperature, at 36 DEG C, after derivant to be added, adjusts temperature 30 DEG C cultivation,
After end sugar has consumed, stream adds 50% glycerol and ferments；
Fermentation tank tank formula: glucose 5g/L, yeast powder 5g/L, peptone 5g/L, ammonium sulfate 3g/L, potassium dihydrogen phosphate 2.5g/
L, magnesium sulfate 2g/L, VB1 10mg/L, VH 2mg/L, ferrous sulfate 50mg/L, manganese sulfate 50mg/L, Semen Maydis pulp 10mL, Fructus Citri Limoniae
Acid sodium 1g/L, defoamer 0.5ml/L, surplus is water.Under 21h, tank, thalline the highest OD600 value are 65.
(3), fermented after, by butterfly centrifugal machine cryogenic collector thalline, and add phosphate buffer washing, thalline is entered
Row pretreatment.Use phosphate buffer is buffer system, adds a-ketoglutaric acid 30g/L, isoleucine 25g/L, sulphuric acid
Ferrous 3g/L, magnesium sulfate 1 g/L, ascorbic acid 0.1 g/L, forced ventilation is catalyzed, and maintains pH 6.5, and catalytic temperature is 30
Degree.In 4h, substrate reactions is complete, and conversion ratio reaches 95.4%；
(4), catalytic liquid is degerming through ceramic membrane, after the ultrafilter membrane a part of pigment of removing and the albumen of 3Kd molecular weight, and through 1%
Activated carbon insulation decolouring, after using 122 weak-acid cation-exchange resin desalinations, upper 732 cationic exchange resin adsorption are also used
1% ammonia eluting, evaporation and concentration (the final concentration of 500g/L of evaporation and concentration) obtains L-4-hydroxyisoleucine crude product, in the second of 75%
Recrystallization in alcohol, purity is 98.8%.Product yield 60.5%.
B group of the present invention:
(1), strain is accessed in seed tank, cultivate under the following conditions: cultivation temperature 36 DEG C, ph7.0, dissolved oxygen controls
More than 25%, during subcultivation, OD600 value is 15, obtains the strain of maturation；
Seed tank culture based formulas: glucose 20g/L, yeast powder 10g/L, peptone 5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate
2g/L, magnesium sulfate 1 g/L, VB1 10mg/L, VH 0.5mg/L, ferrous sulfate 20mg/L, manganese sulfate 20mg/L, defoamer
0.4ml/L, surplus is water.
(2), ripe strain is accessed in fermentation tank, cultivate under the following conditions: ventilated by deep liquid and send out
Ferment, fermentation 5h disposable stream in fermentation medium adds IPTG(isopropyl-beta D-thio galactopyranoside) 0.2mmol/L.
Sweat temperature section controls, and earlier fermentation control temperature, at 36 DEG C, after derivant to be added, adjusts temperature 30 DEG C cultivation,
After end sugar has consumed, stream adds 40% glycerol and ferments；Under 21h, tank, thalline the highest OD600 value are 70.
Fermentation tank tank formula: glucose 5g/L, yeast powder 5g/L, peptone 5g/L, ammonium sulfate 3g/L, potassium dihydrogen phosphate
2.5g/L, magnesium sulfate 2g/L, VB1 10mg/L, VH 2mg/L, ferrous sulfate 50mg/L, manganese sulfate 50mg/L, Semen Maydis pulp 10
ML, sodium citrate 1g/L, defoamer 0.5ml/L, surplus is water.
(3), fermented after, by butterfly centrifugal machine cryogenic collector thalline, and add phosphate buffer washing, to bacterium
Body carries out pretreatment.Use phosphate buffer is buffer system, adds a-ketoglutaric acid 30g/L, isoleucine
26g/L, ferrous sulfate 3g/L, magnesium sulfate 0.5g/L, ascorbic acid 0.05 g/L, forced ventilation is catalyzed, and maintains pH 6.5, urges
Changing temperature is 30 degree.In 4h, substrate reactions is complete, and conversion ratio reaches 95.6%；
(4), catalytic liquid is degerming through ceramic membrane, after the ultrafilter membrane a part of pigment of removing and the albumen of 3Kd molecular weight, and through 1%
Activated carbon insulation decolouring, after using 122 weak-acid cation-exchange resin desalinations, upper 732 cationic exchange resin adsorption are also used
1% ammonia eluting, evaporation and concentration (the final concentration of 600g/L of evaporation and concentration) obtains L-4-hydroxyisoleucine crude product, in the second of 75%
Recrystallization in alcohol, purity is 98.6%.Product yield 62.4%.
Contrast and experiment is as follows:
Comparative test result shows, the inventive method can make conversion ratio be up to more than 95.4%, and product purity is up to more than 98%,
Significantly improve production capacity and the efficiency of L-4-hydroxyisoleucine, there is preferable application prospect.
SEQUENCE LISTING
<110>Henan Julong Bio-Engineering Co., Ltd.
<120>a kind of method that microbial enzyme conversion method produces L-4-hydroxyisoleucine
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1875
<212> DNA
<213>artificial sequence
<400> 1
aagctaccta cttcttttgc aacccactcc catggtgtga cgggcggtgt gtacaaggcc 60
cgggaacgta ttcaccgtgg cattctgatc cacattacta gcgattccga cttcatggag 120
tcgagttgca gactccaatc cggactacga cgcactttat gaggtccgct tgctctcgcg 180
aggtcgcttc tctttgtatg cgccattgta gcacgtgtgt agccctgtcg taagggccat 240
gatgacttga cgtcatcccc accttcctcc agtttatcac tggcagtctc ctttgagttc 300
ccggccggac cgctggcaac aaaggataag ggttgcgctc gttgcgggac ttaacccaac 360
atttcacaac acgagctgac gacagccatg cagcacctgt ctcacggttc ccgaaggcac 420
attctcatct ctgaaaactt ccgtggatgt caagaccagg taaggttctt cgcgttgcat 480
cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcatt tgagttttaa 540
ccttgcggcc gtactcccca ggcggtcgac ttaacgcgtt agctccggaa gccacgcctc 600
aagggcacaa cctccaagtc gacatcgttt acggcgtgga ctaccagggt atctaatcct 660
gtttgctccc cacgctttcg cacctgagcg tcagtcttcg tccagggggc cgccttcgcc 720
accggtattc ctccagatct ctacgcattt caccgctaca cctggaattc tacccccctc 780
tacgagactc aagcttgcca gtatcagatg cagttcccag gttgagcccg gggatttcac 840
atctgactta acaaaccgcc tgcgtgcgct ttacgcccag taattccgat taacgcttgc 900
accctccgta ttaccgcggc tgctggcacg gagttagccg gtgcttcttc tgcgggtaac 960
gtcaatgagc aaaggtatta actttactcc cttcctcccc gctgaaagta ctttacaacc 1020
cgaaggcctt cttcatacac gcgggcatgg gctgcatcag gcttgcgccc attgtgcaat 1080
attccccact gctgcctccc gtaggagtct ggaccgtgtc tcagttccag tgtggctggt 1140
catcctctca gaatgaaaat gagtgggttt agcatagaag aaaaggtaca tgaatttgaa 1200
tctaaagggt ttcttgaaat ctcaaatgaa atctttttac aagaggaaga gaatcatagt 1260
ttattaacac aagcacagtt agattattat aatttggaag atgatgcgta cggtgaatgc 1320
cgtgctagat cttattcaag gtatataaag tatgttgatt caccagatta tattttagat 1380
aatagtaatg attacttcca atctaaagaa tataactatg atgatggcgg gaaagttaga 1440
cagttcaata gcataaatga tagcttttta tgtaatcctt taattcaaaa tatcgtgcgt 1500
ttcgatactg agtttgcatt taaaacaaat ataatagata aaagtaaaga tttaattata 1560
ggcttacatc aagtaagata taaagctact aaagaaagac catcttttag ttcacctatt 1620
tggttacata aagatgatga accagtagta tttttacacc ttatgaattt aagtaataca 1680
gctatcggcg gagataattt aatagctaat tctcctcggg aaattaatca gtttataagt 1740
ttgaaggagc ctttagaaac tttagtattt ggacaaaagg tcttccatgc cgtaacgcca 1800
cttggaacag aatgtagtac ggaggctttt cgtgatattt tattagtaac attttcttat 1860
aaagaaacca aataa 1875
Claims (7)
1. the method that a microbial enzyme conversion method produces L-4-hydroxyisoleucine, it is characterised in that: comprise the following steps:
(1), the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine will can be induced to access the kind equipped with seed tank culture base
In sub-tank, at temperature 33 DEG C-37 DEG C, ph 6.5-7.5, dissolved oxygen cultivates the OD600 to bacterium solution under conditions of controlling more than 25%
Value is 12-16: obtain the Escherichia coli recombinant strain of the induced production L-4-hydroxyisoleucine of maturation；
The described construction method inducing the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine is to clone its gene sequence
Row gene as shown in SEQ NO.1；By this gene constructed enter plasmid pET28a, obtain recombiant plasmid, will after recombiant plasmid import
E. coli bl21, i.e. obtains inducing the Escherichia coli recombinant strain producing L-4-hydroxyisoleucine；
(2), the Escherichia coli recombinant strain of ripe induced production L-4-hydroxyisoleucine is accessed equipped with fermentor cultivation
In the fermentation tank of base, carry out deep liquid ventilating fermentation cultivation；In incubation, cultivate during 5h disposable in fermentation medium
The IPTG putting into final concentration of 0.1-0.3mmol/L induces, and dissolved oxygen controls at 30-50%, pH6.5-7.0, and end sugar exhausts
After, stream adds the glycerine water solution that mass concentration is 10-80% and controls as carbon source, sweat temperature two rank, adds IPTG induction
Front control temperature is at 34-36 DEG C, after pending abduction delivering, adjusts temperature and cultivates to 30-32 DEG C；
(3), after fermentation ends, by butterfly centrifugal machine cryogenic collector thalline, and use phosphate buffer that thalline is washed
Wash, remove phage surface impurity, then with pure water preparation catalyst system and catalyzing: a-ketoglutaric acid 30-35g/L, isoleucine 26-
30g/L, ferrous sulfate 0.5-4g/L, magnesium sulfate 0.1-1g/L, ascorbic acid 0.01-0.2g/L, add in catalyst system and catalyzing and wash
Thalline after washing, forced ventilation is catalyzed, and maintenance pH is 6.0-6.5, and catalytic temperature is 28-32 DEG C, obtains catalytic liquid；Described thalline
Addition be: every 100ml catalyst system and catalyzing adds 0.3g thalline；
(4), that catalytic liquid first passes through ceramic membrane filter is degerming, after the ultrafilter membrane of 2-4Kd molecular weight, and through activated carbon decolorizing, adopts
After 122 weak-acid cation-exchange resin desalinations, 732 cationic exchange resin adsorption use ammonia eluting on effluent, steam
Send out and be concentrated to give L-4-hydroxyisoleucine crude product, be then recrystallized to give L-4-hydroxyisoleucine.
2. the method that microbial enzyme conversion method produces L-4-hydroxyisoleucine as claimed in claim 1, it is characterised in that: described
The formula of seed tank culture base is: glucose 0.5-20g/L, yeast powder 0.5-10g/L, peptone 0.5-5g/L, ammonium sulfate
0.5-10g/L, potassium dihydrogen phosphate 0.5-3g/L, magnesium sulfate 0.5-3g/L, vitamin B 10-20mg/L, biotin 0.5-
10mg/L, ferrous sulfate 0.5-50mg/L, manganese sulfate 0.5-50mg/L, defoamer 0-0.5ml/L, remaining is water.
3. the method that microbial enzyme conversion method produces L-4-hydroxyisoleucine as claimed in claim 1, it is characterised in that: described
The formula of fermentation tank culture medium is: glucose 0.5-10g/L, yeast powder 0.5-10g/L, peptone 0.5-5g/L, ammonium sulfate
0.5-10g/L, potassium dihydrogen phosphate 0.5-3g/L, magnesium sulfate 0.5-3g/L, VB1 0.5-20mg/L, VH 0.5-10mg/L, sulphuric acid
Ferrous 0.5-50mg/L, manganese sulfate 0.5-50mg/L, Semen Maydis pulp 0.5-10mL, sodium citrate 0.5-2g/L and defoamer 0-
0.5ml/L, remaining is water.
4. the method that microbial enzyme conversion method produces L-4-hydroxyisoleucine as claimed in claim 1, it is characterised in that: described
In step (4), catalytic liquid is first being heated to 60-80 DEG C before ceramic membrane filter is degerming, and keeps 10-40min.
5. the method that microbial enzyme conversion method produces L-4-hydroxyisoleucine as claimed in claim 1, it is characterised in that: described
Adding the 0.1-5% that charcoal amount is material liquid volume in step (4) during activated carbon decolorizing, temperature retention time is 30-60min.
6. the method that microbial enzyme conversion method produces L-4-hydroxyisoleucine as claimed in claim 1, it is characterised in that: described
In step (4), the ammonia mass concentration of eluting is 1-3%, and elution flow rate is 1-1.5BV/h.
7. the method that microbial enzyme conversion method produces L-4-hydroxyisoleucine as claimed in claim 1, it is characterised in that: step
(4) evaporation and concentration described in, the final concentration of 500-600g/L of 4-hydroxyisoleucine crude product after evaporation and concentration.
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| CN108299220A (en) * | 2018-03-27 | 2018-07-20 | 河南巨龙生物工程股份有限公司 | A kind of method that L-4- hydroxyisoleucines are extracted in fermentation catalytic liquid |
| CN110499343A (en) * | 2019-09-11 | 2019-11-26 | 鲁东大学 | A kind of method that enzyme process prepares 4- phenylac epsilontaldshyde |
| CN113233991A (en) * | 2021-05-27 | 2021-08-10 | 无锡晶海氨基酸股份有限公司 | Method for extracting 4-hydroxyisoleucine from whole-cell catalytic liquid |
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| CN110499343A (en) * | 2019-09-11 | 2019-11-26 | 鲁东大学 | A kind of method that enzyme process prepares 4- phenylac epsilontaldshyde |
| CN113233991A (en) * | 2021-05-27 | 2021-08-10 | 无锡晶海氨基酸股份有限公司 | Method for extracting 4-hydroxyisoleucine from whole-cell catalytic liquid |
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Inventor after: Liu Shuai Inventor after: Zhang Chenglin Inventor after: Xu Qingyang Inventor after: Li Jing Inventor after: Yue Guilong Inventor after: Zhang Mengtao Inventor before: Cao Huajie Inventor before: Liu Shuai Inventor before: Li Jing Inventor before: Yue Guilong Inventor before: Zhang Mengtao |
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Effective date of registration: 20170622 Address after: Pingdingshan City, Henan province Ruzhou city overpass west 467500 Co-patentee after: Tianjin University of Science & Technology Patentee after: Henan Julong Bio-Engineering Co.,Ltd. Address before: Pingdingshan City, Henan province Ruzhou city overpass west 467500 Patentee before: Henan Julong Bio-Engineering Co.,Ltd. |