CN105603016B - A method of S-Leucine is prepared using leucine dehydrogenase coupling glucose dehydrogenase - Google Patents
A method of S-Leucine is prepared using leucine dehydrogenase coupling glucose dehydrogenase Download PDFInfo
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Abstract
The invention discloses a kind of methods for preparing S-Leucine using leucine dehydrogenase coupling glucose dehydrogenase, belong to enzyme engineering and chiral medicinal intermediate preparation technical field.The method of the present invention includes the addition substrate trimethyl pyruvic acid into reaction system, glucose, the pH value of regulation system is in 8.0-9.0, it (includes leucine dehydrogenase that the crude enzyme liquid that the coexpression bacterial strain by culture medium cultures of substances such as additional niacin is crushed, which is added, glucose dehydrogenase and coenzyme) start to react later, by the temperature of control reaction process at 25-30 DEG C, pH value produces S-Leucine in 8.0-9.0.The features such as present invention is coupled glucose dehydrogenase system by using leucine dehydrogenase, and the coenzyme contained using thallus itself carries out S-Leucine preparation, has single batch reaction substrate concentration high, and reaction efficiency is high, does not need coenzyme addition.
Description
Technical field
The present invention relates to a kind of methods for preparing S-Leucine using leucine dehydrogenase coupling glucose dehydrogenase, belong to
In enzyme engineering and chiral medicinal intermediate preparation technical field.
Background technique
Optical voidness S-Leucine, it is stronger hydrophobic that not only there is space steric effect also to have for contained tert-butyl group
Property, to be widely used as the template of asymmetry catalysis synthesis.Its amino acid as a kind of non-natural boundary simultaneously, is also usually answered
In pharmaceutically active peptides.Studies have reported that S-Leucine is used for anti-tumor drug reagent, the suppression of anti-AIDS protease
The drugs such as preparation.Just because of the extensive use of S-Leucine, a large amount of synthetic method is reported by exploitation.Wherein mainly include
Chemical resolution method, chemical dissymmetric synthesis, enzyme Split Method and enzyme reductive amination synthetic method (Bommarius et al.,
1995).Due to the enhancing of people's environmental consciousness in recent years, there are severe reaction conditions, reaction process is complicated, the low change of reaction efficiency
Synthetic method is learned gradually to be replaced by biological synthesis process.It is split in synthetic method in enzyme, some enzymes mainly used include mould
Plain acylase, lipase, protease etc. (Abderhalden, Faust , &Haase, 1934；Agosta et al.,2006；
Turner,Winterman,McCague,Parratt,&Taylor,1995).Since that there is operating procedures is numerous for enzymatic resolution method
Trivial and theoretical yield only has for 50% the reason of, never the report of correlation technique industrial application.
Enzyme restores amino synthetic method, has the characteristics that easy to operate and theoretical yield 100%.It mainly uses at present
Enzyme includes two kinds, is side-chain amino group transferase and leucine dehydrogenase respectively.Hong etc., which is reported, utilizes side-chain amino group transferase
Synthesize S-Leucine, conversion ratio be greater than 90%, but be catalyzed concentration of substrate it is too low be not achieved industrial requirements (Hong, Cha,
Yun,&Kim,2010).Another is using leucine dehydrogenase synthesis S-Leucine in the nineties by Degussa (Germany)
Company's exploitation, and it is applied to industrial production.This method uses enzyme mebrane reactor, and leucine dehydrogenase and hydrogenlyase are fixed
On enzyme membrane, wherein hydrogenlyase can be catalyzed NAD+NADH is generated, the regeneration of coenzyme is realized, reduces the dosage of coenzyme
(Liese,Seelbach,&Wandrey,2006).Leucine dehydrogenase catalysis trimethyl pyruvic acid synthesis S-Leucine is recognized
To be the synthesis path for most having industrial prospect.
The patent report about leucine dehydrogenase synthesis path included Chinese patent 201110202325.4 in recent years,
201210508084.0 201410695848.0,201310044292.4 etc..These synthesis paths all have the following problems: 1.
Reaction needs additional coenzyme, and coenzyme is expensive, less economical, if it is possible to if being added without coenzyme in synthesis engineering,
The cost of industrial processes will be greatly reduced；2. formate dehydrogenase enzymatic activity is low, need to add during asymmetric transformation
Add a large amount of hydrogenlyase, leucine dehydrogenase can be given full play to and catalyze and synthesize effect, increases the cost of enzyme investment；
3. reaction efficiency is lower, just because of the lower hydrogenlyase of activity used in reaction process, coenzyme transformation efficiency is influenced,
Increase the reaction time required for complete conversion of substrate, so that reaction efficiency is lower.
Summary of the invention
In order to overcome the above problem, the present invention is broken using coexpression leucine dehydrogenase and glucose dehydrogenase bacterial strain
Crude enzyme liquid carries out catalysis trimethyl Pyruvate production S-Leucine as catalyst.The glucose dehydrogenase that the present invention uses
Opposite hydrogenlyase has higher enzyme activity, to improve coenzyme utilization efficiency.It can be neglected using the reaction formation of crude enzyme liquid
Slightly inhibition of the substrate for somatic cells, improves concentration of substrate.By the outer plus niacin in thallus incubation, improve intracellular auxiliary
Enzyme concentration, and realize zero coenzyme addition in reaction.Finally, it realizes using leucine dehydrogenase coupling glucose dehydrogenase come high
Effect prepares S-Leucine.
It is to contain glucose, substrate trimethyl the object of the present invention is to provide a kind of method for preparing S-Leucine
The clasmatosis crude enzyme liquid of addition coexpression leucine dehydrogenase and the recombinant bacterium of glucose dehydrogenase in pyruvic acid reaction system,
It is stirred to react.
In one embodiment of the invention, substrate trimethyl concentrations of pyruvate 0.5-1.2M, grape in the method
Sugared concentration is 1.0-1.3 times of concentration of substrate.
In one embodiment of the invention, at 20-35 DEG C, pH is controlled in 7.5- for the temperature control of the reaction system
9.0.Preferable temperature is 30 DEG C, pH 8.5.
In one embodiment of the invention, the pH is controlled by ammonium hydroxide.
In one embodiment of the invention, the time of the reaction is 1-2.5 hours.Preferred reaction time is
1.5h。
In one embodiment of the invention, every L contains the thin of 20-60g (weight in wet base) recombinant bacterium in the reaction system
Born of the same parents are crushed liquid, preferably 30g/L.
In one embodiment of the invention, the revolving speed of the stirring is 100-300r/min, preferably 200r/min.
In one embodiment of the invention, the substrate trimethyl concentrations of pyruvate 1M, concentration of glucose is 1.2M,
Crude enzyme liquid is that 30g/L (weight in wet base) cell obtains, and reaction temperature is 30 DEG C, and pH is 8.5, time 1.5h.
In one embodiment of the invention, the leucine dehydrogenase and glucose dehydrogenase are respectively derived from
Bacillus cereus and Bacillus sp.
In one embodiment of the invention, the preparation of the crude enzyme liquid: (1) building co-expresses amino acid sequence such as
The recombinant bacterium of leucine dehydrogenase and the amino acid sequence such as glucose dehydrogenase of SEQ ID NO.2 of SEQ ID NO.1；(2)
Recombinant bacterium is cultivated, inducible strain expresses leucine dehydrogenase and glucose dehydrogenase, obtains fermentation liquid；It (3) will fermentation
Liquid centrifuging and taking cell, washing, then ultrasonication is to get crude enzyme liquid.
In one embodiment of the invention, the step (1) is specifically: in glucose dehydrogenase GDH and leucine
SD-AS sequence is added between dehydrogenase LDH and obtains gene GDH-SD-AS-LDH, is then attached on pET28a plasmid, then convert
Into e. coli bl21 host, recombinant bacterium is obtained.
In one embodiment of the invention, the SD-AS sequence is GGAGATATACC (shown in SED ID NO.3).
In one embodiment of the invention, in the step (2) recombinant bacterium cultivated used in culture medium
Added with the precursor substance in coenzyme synthesis path to improve endobacillary coenzyme concentration.When being added with precursor substance, it is used for
When conversion production S-Leucine, cell additive amount can be reduced to 20-30g/L (weight in wet base)；When not adding precursor substance, cell
Additive amount is improved to 40g/L or more, and reaction process does not need addition coenzyme, high conversion rate.
In one embodiment of the invention, the precursor substance is niacin, niacinamide, L-Trp or L- asparagus fern ammonia
In acid any one or it is a variety of；Preferably, the precursor substance is niacin.
In one embodiment of the invention, the culture medium of the step (2) is LB culture medium (peptone 10g/L, ferment
Female extract 5g/L, NaCl 10g/L, pH 7.0, remaining is water, uses preceding 50 μ g/mL of addition kanamycin sulfate)；The training
Supporting is to cultivate in 37 DEG C, 200r/min to thallus OD600Reach 0.6-2.0, be then added the IPTG of final concentration of 0.1mM in
18 DEG C of culture 2-24h；
In one embodiment of the invention, the culture medium of the step (2) is self-induction culture medium (DEXTROSE ANHYDROUS
0.5g/L, glycerol 5g/L, KH2PO46.8g/L, MgSO40.48g/L, Na2HPO47.1g/L, Na2SO40.71g/L, NH4Cl
2.67g/L, albumen powder 10g/L, yeast powder 5g/L；Inducer alpha-lactose 10g/L, pH 7.60, is configured with deionized water, is used
Preceding 50 μ g/mL of addition kanamycin sulfate), the culture is that incubation time is 24-48h at 30-37 DEG C, 200r/min.
In one embodiment of the invention, the method is specifically: (1) in glucose dehydrogenase GDH and leucine
Sequence SD-AS sequence as shown in SEQ ID NO.3 is added between dehydrogenase LDH and obtains gene GDH-SD-AS-LDH, then connects
It is connected on pET28a plasmid, then is transformed into e. coli bl21 host, obtain recombinant bacterium；(2) recombinant bacterium is being contained into 1g/L
Cultivated in the culture medium of niacin, and inducible strain expression leucine dehydrogenase and glucose dehydrogenase, taken after culture somatic cells,
Washing；(3) in the reaction system, substrate trimethyl concentrations of pyruvate 1M is added, concentration of glucose is 1.2M, 30g/L cell
Crude enzyme liquid, reaction temperature are 30 DEG C, control pH 8.5 with ammonium hydroxide, react 1.5h.
Beneficial effect
1, the recombinant bacterium that the present invention utilizes includes leucine dehydrogenase, glucose dehydrogenase and coenzyme, in catalysis trimethyl
During pyruvic acid synthesizes S-Leucine, the effective coenzyme using contained by thallus itself realizes coenzyme circular regeneration,
It during being catalyzed reaction does not need that coenzyme additionally is added, realizes zero addition of coenzyme, save the cost of coenzyme.
2, in single batch reaction, concentration of substrate reaches 130g/L and carries out catalysis reaction (for the single batch of report the present invention
Highest concentration of substrate), conversion ratio reaches 99% or more, and the reaction time shortens within 1.5h, and space-time yield reaches 2096gL-1d-1。
3, the present invention carries out catalysis reaction using the crude enzyme liquid of single recombinant bacterium, has catalyst preparation process simple, urges
The characteristics of agent usage amount is few, high catalytic efficiency is suitble to industrialized production.
Detailed description of the invention
Fig. 1 is reaction process schematic diagram of the present invention.
Specific embodiment
The testing conditions of substrate are as follows: SB-Aq Column (4.6*150mm, 5um), flow velocity 1ml/min, UV detector
210nm, mobile phase are 20mM potassium phosphate/acetonitrile solution (99.5:0.5v/v, pH2.0), sample volume 5ul.
The testing conditions of product are as follows: C18Column (4.6*250mm, 5um), using OPA derivatization, flow velocity 1ml/min,
UV detector 338nm, mobile phase are 50mM NaAc/ acetonitrile solution (80:20v/v, pH7.2), sample volume 10ul.
Embodiment 1: the E. coli recombinant stain of building coexpression leucine dehydrogenase and glucose dehydrogenase
Using the gene order design primer in ncbi database, transfer respectively from Bacillus cereus and
Two kinds of enzyme genes of leucine dehydrogenase and glucose dehydrogenase of Bacillus sp, utilize restriction enzyme XhoI and NheI
Double digestion is carried out to target gene and expression plasmid pET28a, target gene obtains recombinant plasmid after connecting with carrier
PET28a-LDH and pET28a-GDH.Again from the expression plasmid of two kinds of genes, existed using overlap extension pcr, two
SD-AS sequence (GGAGATATACC) is added between gene.Design includes the primer of SD-AS sequence, and base is obtained after two-wheeled PCR
Because of GDH-SD-AS-LDH, which is connected on expression vector pET28a, is transferred in escherichia coli host BL21, is weighed
Group bacterium E.coli BL21/pET28a-G-SD-AS-L, to realize two kinds of enzymes expressing in series on the same plasmid.
Embodiment 2:LB culture medium inducing expressing gene engineering strain
Fermented and cultured is carried out to recombinant bacterium using LB culture medium.LB medium component is peptone 10g/L, yeast extract
5g/L, NaCl 10g/L, pH7.0, are prepared with deionized water, use preceding 50 μ g/mL of addition kanamycin sulfate.Condition of culture is
Initial temperature is 37 DEG C, shaking speed 200r/min, as thallus OD600Reach 0.6-2.0, is added final concentration of 0.1mM's
IPTG, in 18 DEG C of culture 17h.
Embodiment 3: the LB culture medium inducing expressing gene engineering strain of the substances such as additional niacin
Niacin (or the coenzyme synthesis path such as niacinamide, L-Trp, L-Aspartic acid of 1g/L is added in LB culture medium
In precursor substance).LB medium component is peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH7.0, to go
Ionized water is prepared, and preceding 50 μ g/mL of addition kanamycin sulfate is used.Condition of culture is that initial temperature is 37 DEG C, shaking speed
200r/min, as thallus OD600Reach 0.6-2.0, the IPTG of final concentration of 0.1mM is added, in 18 DEG C of culture 17h.
Embodiment 4: self-induction culture medium inducing expressing gene engineering strain
Fermented and cultured is carried out to recombinant bacterium using self-induction culture.The ingredient of self-induction culture medium is DEXTROSE ANHYDROUS
0.5g/L, glycerol 5g/L, KH2PO46.8g/L, MgSO40.48g/L, Na2HPO47.1g/L, Na2SO40.71g/L, NH4Cl
2.67g/L, albumen powder 10g/L, yeast powder 5g/L, inducer alpha-lactose 10g/L, pH 7.60 are configured with deionized water, are used
Preceding 50 μ g/mL of addition kanamycin sulfate.Condition of culture is 37 DEG C of temperature, revolving speed 200r/min, incubation time 48h.
Embodiment 5: the self-induction culture medium inducing expressing gene engineering strain of the substances such as additional niacin
In self-induction culture medium be added 1g/L niacin (or the coenzyme such as niacinamide, L-Trp, L-Aspartic acid synthesis
Precursor substance in path is applied to biocatalysis and synthesizes S-Leucine, and effect is similar).The ingredient of self-induction culture medium is
DEXTROSE ANHYDROUS 0.5g/L, glycerol 5g/L, KH2PO46.8g/L, MgSO40.48g/L, Na2HPO47.1g/L, Na2SO40.71g/
L, NH4Cl 2.67g/L, albumen powder 10g/L, yeast powder 5g/L, inducer alpha-lactose 10g/L, pH 7.60 are matched with deionized water
It sets, uses preceding 50 μ g/mL of addition kanamycin sulfate.Condition of culture is 30 DEG C of temperature, revolving speed 200r/min, and incubation time is
24h。
Embodiment 6: the preparation of biocatalyst
Fermentation liquid is centrifuged, collection thallus, after brine, recombinant bacterium is resuspended in 0.1M by 1:1-1:10
It in the Tris-HCl buffer of pH7.2, is crushed using sonicator, obtains bacterial strain and be crushed crude enzyme liquid, as biology
Catalyst.It simultaneously include leucine dehydrogenase, glucose dehydrogenase and coenzyme NAD (H) in the catalyst.
Embodiment 7: biocatalysis synthesis
Configuration 100mL reaction system: a certain amount of water and ammonium hydroxide, the substrate front three of final concentration 0.5M are added into reactor
Benzylacetone acid reaches 8.0 or so using the pH that ammonium hydroxide adjusts mixed liquor, 1.2 times of relative substrate concentration of glucose, 4g is added
Thallus (being broken into crude enzyme liquid) prepared by (weight in wet base) embodiment 2, while opening mechanical stirring and starting to react.In reaction process,
The temperature of reaction system is controlled at 35 DEG C, pH value prepares S-Leucine 7.5 or so.Sample in reaction process is sharp respectively
With ethyl alcohol and trichloroacetic acid precipitation albumen, the liquid phase of progress substrate and product detection respectively after certain multiple is then diluted.Instead
1h between seasonable, the substrate transformation rate are greater than 99%.Spatial-temporal Transformation rate is 1039gL-1d-1。
Embodiment 8: biocatalysis synthesis
Configuration 100mL reaction system: a certain amount of water and ammonium hydroxide, the substrate front three of final concentration 0.5M are added into reactor
Benzylacetone acid reaches 8.0-9.0 using the pH that ammonium hydroxide adjusts mixed liquor, 1 times of relative substrate concentration of glucose is added, and 6g is (wet
Thallus (being broken into crude enzyme liquid) prepared by embodiment 4 again), while opening mechanical stirring and starting to react.In reaction process, control
For the temperature of reaction system at 25 DEG C, pH value prepares S-Leucine 9.0.Sample in reaction process be utilized respectively ethyl alcohol and
Then trichloroacetic acid precipitation albumen dilutes the liquid phase of progress substrate and product detection respectively after certain multiple.Reaction time 1h,
The substrate transformation rate is greater than 99%.Spatial-temporal Transformation rate is 1043gL-1d-1。
Embodiment 9: biocatalysis synthesis
Configuration 100mL reaction system: a certain amount of water and ammonium hydroxide, the substrate front three of final concentration 0.5M are added into reactor
Benzylacetone acid reaches 8.0 using the pH that ammonium hydroxide adjusts mixed liquor, 1.3 times of relative substrate concentration of glucose, 2g (weight in wet base) is added
Thallus (being broken into crude enzyme liquid) prepared by embodiment 3, while opening mechanical stirring and starting to react.In reaction process, control is anti-
Answer the temperature of system at 30 DEG C, pH value prepares S-Leucine 8.0.Sample in reaction process is utilized respectively ethyl alcohol and three
Then monoxone protein precipitation dilutes the liquid phase of progress substrate and product detection respectively after certain multiple.Reaction time 2.5h,
The substrate transformation rate is greater than 99%.
Embodiment 10: biocatalysis synthesis
Configuration 100mL reaction system: a certain amount of water and ammonium hydroxide, the substrate trimethyl of final concentration 1M are added into reactor
Pyruvic acid reaches 8.5 using the pH that ammonium hydroxide adjusts mixed liquor, and 1.2 times of relative substrate concentration of glucose is added, and 2g (weight in wet base) is real
The thallus (being broken into crude enzyme liquid) of the preparation of example 2 is applied, while opening mechanical stirring and starting to react.In reaction process, control reaction
For the temperature of system at 25-30 DEG C, pH value prepares S-Leucine 8.0.Sample in reaction process is utilized respectively ethyl alcohol and three
Then monoxone protein precipitation dilutes the liquid phase of progress substrate and product detection respectively after certain multiple.Reaction time 1.5h,
The substrate transformation rate is greater than 99%.Intensity is produced up to 84.25gL-1h-1, Spatial-temporal Transformation rate is 2076gL-1d-1。
Embodiment 11: biocatalysis synthesis
Configuration 100mL reaction system: a certain amount of water and ammonium hydroxide, the substrate front three of final concentration 1.2M are added into reactor
Benzylacetone acid, is reached between 8.0-9.0 using the pH that ammonium hydroxide adjusts mixed liquor, and 1.2 times of relative substrate concentration of glucose is added,
Thallus (being broken into crude enzyme liquid) prepared by 2g (weight in wet base) embodiment 5, while opening mechanical stirring and starting to react.Reaction process
In, the temperature of reaction system is controlled at 28 DEG C, and pH value prepares S-Leucine between 8.0-9.0.Sample in reaction process
It is utilized respectively ethyl alcohol and trichloroacetic acid precipitation albumen, then dilutes the liquid phase of progress substrate and product inspection respectively after certain multiple
It surveys.Reaction time 1h.Intensity is produced up to 82.6gL-1h-1, Spatial-temporal Transformation rate 1982gL-1d-1。
Embodiment 11: biocatalysis synthesis
Configuration 100mL reaction system: a certain amount of water and ammonium hydroxide, the substrate trimethyl of final concentration 1M are added into reactor
Pyruvic acid reaches 8.5 or so using the pH that ammonium hydroxide adjusts mixed liquor, 1.2 times of relative substrate concentration of glucose is added, and 3g is (wet
Thallus (being broken into crude enzyme liquid) prepared by embodiment 3 again), while opening mechanical stirring and starting to react.In reaction process, control
For the temperature of reaction system at 30 DEG C, pH value prepares S-Leucine 8.5 or so.Sample in reaction process is utilized respectively second
Then pure and mild trichloroacetic acid precipitation albumen dilutes the liquid phase of progress substrate and product detection respectively after certain multiple.When reaction
Between 1.5h, the substrate transformation rate be greater than 99%, produce intensity up to 87.3gL-1h-1, Spatial-temporal Transformation rate is 2096gL-1d-1。
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (8)
1. a kind of method for preparing S-Leucine, which is characterized in that the method is to contain glucose, substrate trimethyl third
The crude enzyme liquid of addition coexpression leucine dehydrogenase and the recombinant bacterium of glucose dehydrogenase, is stirred to react in ketone acid reaction system；
The preparation of the crude enzyme liquid: (1) building coexpression the amino acid sequence such as leucine dehydrogenase of SEQ ID NO.1 and amino acid sequence
The recombinant bacterium of the column such as glucose dehydrogenase of SEQ ID NO.2；(2) recombinant bacterium is cultivated, and inducible strain expresses bright ammonia
Acidohydrogenase and glucose dehydrogenase；(3) by fermentation liquid centrifuging and taking cell, washing, then ultrasonication is to get crude enzyme liquid；Institute
Stating step (1) is: sequence being added between glucose dehydrogenase GDH and leucine dehydrogenase LDH as shown in SEQ ID NO.3
SD-AS sequence obtains gene GDH-SD-AS-LDH, is then attached on pET28a plasmid, then is transformed into e. coli bl21 place
In master, recombinant bacterium is obtained.
2. the method according to claim 1, wherein substrate trimethyl concentrations of pyruvate 0.5- in the method
1.2M, concentration of glucose are 1.0-1.3 times of concentration of substrate.
3. pH is controlled the method according to claim 1, wherein the temperature control of the reaction system is at 20-35 DEG C
System is in 7.5-9.0.
4. the method according to claim 1, wherein the time of the reaction is 1-2.5h.
5. the method according to claim 1, wherein used in recombinant bacterium is cultivated in the step (2)
Added with the precursor substance in coenzyme synthesis path in culture medium.
6. according to the method described in claim 5, it is characterized in that, the precursor substance be niacin, niacinamide, L-Trp or
In L-Aspartic acid any one or it is a variety of.
7. according to the method described in claim 5, which is characterized in that the precursor substance is niacin.
8. the method according to claim 1, wherein the method is specifically: (1) in glucose dehydrogenase GDH
Sequence SD-AS sequence as shown in SEQ ID NO.3 is added between leucine dehydrogenase LDH and obtains gene GDH-SD-AS-
LDH is then attached on pET28a plasmid, then is transformed into e. coli bl21 host, and recombinant bacterium is obtained；(2) by recombinant bacterium
It is cultivated in the culture medium containing 1g/L niacin, and inducible strain expression leucine dehydrogenase and glucose dehydrogenase, after culture
Take somatic cells, washing；(3) in the reaction system, substrate trimethyl concentrations of pyruvate 1M is added, concentration of glucose is 1.2M,
The crude enzyme liquid of 30g/L cell, reaction temperature are 30 DEG C, control pH 8.5 with ammonium hydroxide, react 1.5h.
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Non-Patent Citations (3)
| Title |
|---|
| Accession No：AID61663，leucine dehydrogenase [Bacillus cereus];Yan,X.;《GenBank Database》;20140630;ORIGIN部分 |
| Accession No：WP_013351020，MULTISPECIES：glucose-1-dehydrogenase [Bacillus subtilis group];none;《GenBank Database》;20150122;ORIGIN部分 |
| 亮氨酸脱氢酶的基因发掘、催化性能及其应用研究;李静;《中国优秀硕士学位论文全文数据库 基础科学辑》;20140915(第9期);正文第36页第1段、图4.1，第38页第4.2.6节，第40页第4.2.6.2节，第41页第1段、第4.2.8.1节 |
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