CN106498000B - A kind of method that the fixed trehalose synthase catalysis maltose of resin produces trehalose - Google Patents
A kind of method that the fixed trehalose synthase catalysis maltose of resin produces trehalose Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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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
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Abstract
The invention discloses the methods that a kind of fixed trehalose synthase catalysis maltose of resin produces trehalose.Realization recycling is fixed with resin in trehalose synthase by the present invention.Carrier is the resin with epoxy group, and the epoxy group on trehalose synthase on amino and resin passes through Covalent bonding together.Immobilised enzymes initial conversion is 58% or so, and almost the same with resolvase, when being recycled to 18 batches, conversion ratio may also stay at 72% or so of initial conversion substantially.When being recycled to 14 batches, conversion ratio may also stay at 80% or so of initial conversion substantially.The maltose substrate that immobilized enzyme catalysis mass percent concentration is 20% generates trehalose, and the concentration of substrate is higher, can industrial applications.Immobilised enzymes is reduced compared to resolvase, by-product glucose amount.Immobilised enzymes initially can reach conversion ratio identical with resolvase, in addition reusable certain batch, saves cost, is directly immobilized using thick enzyme, and process for fixation is simple and easy to do, have industrial applications prospect.
Description
Technical field
The present invention relates to technical field of enzyme immobilization more particularly to a kind of fixed trehalose of resin with epoxy group to close
The new method of enzymatic maltose production trehalose.The process for fixation is simple and easy to do, is directly immobilized using thick enzyme, and nothing
Crosslinking agent need to be added.Immobilised enzymes is suitable for high concentration substrate and can mention significantly successive reaction 14-18 days in 50 DEG C of condition
The high stability of enzyme.In addition, by-product glucose is reduced compared to resolvase, there is good industrial applications prospect.
Background technique
Trehalose is that had with irreducibility disaccharide made of 1,1- glucosides key connection to organism by two molecule glucoses
Very important biological significance is protein and biomembrane molecule in adverse circumstances such as dehydration, high temperature, oxygen radical, low temperature
In stabilizer and protective agent.Again because trehalose has sugariness moderate, property is stablized, and is not easily decomposed, no reproducibility etc. is special
Property is therefore widely used in food processing industry, pharmaceutical sector, agricultural, biochemical product industry and cosmetic industry.
In the industrial production of trehalose, trehalose synthase compared with other trehalose synthetases with greater advantage,
Only need a kind of enzyme one-step reaction that can obtain trehalose, simply, fast, and its raw materials for production is more cheap malt
Sugar, some by-product are glucose.The self-produced trehalose synthase production trehalose in laboratory has realized industrialization at present, and enzyme is answered
It is resolvase with form.But the trehalose synthetase stability of free state is poor, and easy in inactivation cannot reuse, and after reaction
Also it is not easy to separate with product.
Immobilised enzymes has important application value in the industrial production, by fixed enzyme not only there is high catalysis to imitate
Rate and high specificity, and immobilised enzymes improves enzyme to the stability of soda acid and temperature to a certain extent, increases enzyme
Service life.For industrialization, it can also simplify technique, easily be separated with reaction product after reaction, reduce product separation
The difficulty of purifying, repeatability substantially reduce industrial production cost.
Enzyme immobilization method can be generally divided into two kinds, include it is carrier immobilized (by enzyme in conjunction with carrier or with carry
Body embeds enzyme) and carrier-free immobilization (being crosslinked between enzyme molecule).2003, Wang Jun etc. was using chitosan as carrier, and penta
Dialdehyde is the fixed trehalose synthase of crosslinking agent, and content of trehalose can be up to 40%.2005, the Youn-Jeung Cho of South Korea
Trehalose synthase is immobilized with EupergitC250L etc. reporting, immobilised enzymes specific ionization enzyme influenced by pH value it is smaller,
Thermal stability improves, more easy to store.2011, the trehalose synthase with histidine tag was passed through gold by Tsung-Ta Wu et al.
Belong to ion sequestration to immobilize, the concentration of substrate maltose is 100Mm, and 24 batches may be reused, and is retained original
The 80% of enzyme activity, immobilised enzymes have stronger tolerance to pH and temperature compared to resolvase.2013, Anna Panek etc.
People uses Fe3O4Magnetic nano-particle immobilizes trehalose synthase, and reusable 12 times, enzyme activity is maintained at 80% or so.
2014, Ying Chen et al. immobilized the cross-linked aggregates of trehalose synthase with water-in-oil emulsion, considerably increased
The stability of enzyme, including the tolerance to high temperature, pH, in addition, immobilised enzymes is in Tris, urea, guanidine hydrochloride and Zn2+,Cu2 +,Al3+,Fe2+In environment, equal specific ionization enzyme is stablized.
In conclusion trehalose synthase immobilization can be improved to the stability of enzyme, and it can reuse, if investment work
Industry production, can substantially reduce industrial production cost.
The present invention provides a kind of with resin by trehalose synthase immobilization, reacts in the substrate of high concentration and carries out weight
The method used again.The carrier used is that Xi'an indigo plant knows LX-1000EP resin, fixation of the carrier in the trehalose synthase of report
In change method, it is not investigated.
Experiments have shown that the process for fixation is simple and easy to do, enzyme can be bound directly with carrier, be not required to addition auxiliary crosslinking agent or
Person's activator, it is easier than general process for fixation.Immobilised enzymes property is stablized, can be anti-in the high concentration substrate of 20%-30%
It answers.In addition, easy microbiological contamination, reaction can avoid the problem in higher temperature because substrate and product are carbohydrate, immobilization sea
Algae sugar synthase can be used continuously -18 days 14 days in 50 DEG C of environment.Have compared to the immobilization trehalose synthase of report
Better stability and industrial advantages.Compared to resolvase, by-product glucose is reduced after immobilization.
The present invention provides a kind of, and the resin-immobilized trehalose synthase with epoxy group carries out catalysis reaction.This immobilization
Enzyme can not only be reused more multiple batches of, and immobilised enzymes is relatively stable, and immobilised enzymes and resolvase reach maximum conversion
Time consistency, immobilised enzymes are reduced compared to resolvase, by-product glucose.This easy immobilization side with excellent results
Method is never mentioned or is disclosed.
Summary of the invention
The technical problem to be solved by the present invention is to be directed to the existing enzyme in laboratory, a kind of easy-to-use fixation is provided
Change method improves the utilization rate of enzyme by reusing, and reduces production cost.
Technical scheme is as follows:
A kind of method that the fixed trehalose synthase catalysis maltose of resin produces trehalose, which is characterized in that including walking as follows
It is rapid:
(1) selection and pretreatment of resin
Carrier is resin of the LX-1000EP with epoxy group of Xi'an Lan Xiao company；Under alkaline environment, in enzyme molecule
Amino group is easier to attack carbon atom, so that covalent bond occurs for resin and enzyme；
Claim 100g carrier that 400ml is added, the phosphate buffer that 0.1M, pH are 8,200rpm stirs 15min at room temperature, surveys pH
Value maintains pH to filter the resin drained after being activated after 7.8-8.2,1h；
(2) enzyme immobilizatio
Escherichia coli with trehalose synthase target gene are fermented, thallus is obtained after centrifugation, volume, which is added, is
Thallus is resuspended the PBS of fermentating liquid volume 1/5, and clasmatosis liquid is obtained after ultrasonication, is immobilized with clasmatosis liquid；
Process for fixation are as follows: it is broken that 1ml cell is added in the resin 0.4g after weighing activation in 10ml phosphoric acid buffer liquid system
Broken liquid, under room temperature, the oscillation of 150rpm shaking table immobilizes；It is filtered after immobilization 5-6h, solid matter is immobilization
Enzyme, load capacity 70-80%；
(3) immobilised enzymes reacts
0.4g immobilised enzymes is added in the maltose substrate that the mass percent concentration of 20ml is 20%, in 50 DEG C of shaking tables
150rpm reacts for 24 hours；It is filtered after reaction, reaction solution its component content of high performance liquid chromatography detection, mobile phase is
Acetonitrile: water=87:13, flow velocity 1ml/min, column temperature are 25 DEG C；
(4) recycling of immobilised enzymes
Reaction terminates to obtain immobilised enzymes after filtering, and the mass percent of new 20ml is put into after being rinsed with deionized water
Next batch reaction is carried out in the maltose substrate that concentration is 20%, is recycled to realize.
Further, it is Thermus thermophilus that trehalose synthase used, which is the source of Yan Li et al. people research,
HB27, by transformation expression in escherichia coli enzyme；Trehalose synthase sequence used is NO.1.
Trehalose synthase is fixed on the resin with epoxy group and carries out catalysis reaction by the present invention, reusable excessively certain
Batch improves the utilization rate of trehalose synthase, substantially reduces production cost.
Detailed description of the invention
Fig. 1 is influence of the different carriers amount to conversion ratio
Fig. 2 is influence of the different enzyme amount to conversion ratio
Fig. 3 is influence of the different immobilization times to conversion ratio
Influence of Fig. 4 difference pH to conversion ratio
Influence of Fig. 5 different temperatures to conversion ratio
Fig. 6 is circulation batch
The amount of resolvase and immobilised enzymes malaga sugar under Fig. 7 differential responses time
Specific embodiment
By the following embodiment, above content of the invention is remake and is further explained in detail and Zhan Shu.
The selection of 1 resin of embodiment and enzyme immobilizatio
(1) selection and pretreatment of resin
The present invention relates to trehalose synthase be laboratory be transformed bacterial strain produce enzyme, have been carried out industrialization at present.It examines
Worry is applied to industrial production, is screened to some carriers, chooses property and stablizes, is easily isolated, industrialized production is more square
Just carrier.The carrier of final choice is resin of the LX-1000EP with epoxy group of Xi'an Lan Xiao company, and epoxy resin is one
With enzyme covalent bond can occur for resin of the kind containing epoxy group, and essential information be shown in Table 1.
Resin properties are relatively stable, are saved under the conditions of 4 DEG C.Resin needs to pre-process, and processing method is 100g load
400ml is added in body, and the phosphate buffer that 0.1M, pH are 8,200rpm stirs 15min at room temperature, surveys pH value, maintains pH in 7.8-
The resin after being activated is drained in filtering after 8.2,1h.
(2) enzyme immobilizatio
The produced trehalose synthase band histidine tag of this experiment, can purify, but in order to industrialize conveniently, directly attempt to make
It is immobilized with clasmatosis liquid.
Process for fixation are as follows: 2ml clasmatosis is added in the resin after weighing 1g activation in 10ml phosphoric acid buffer liquid system
Liquid (carrier and enzyme are all excessive at this time, and clasmatosis liquid protein content is about 20mg/ml), under room temperature, the oscillation of 150rpm shaking table
It immobilizes.It filters after immobilization 4h, is rinsed three times with deionized water.
(3) immobilised enzymes reacts
Immobilised enzymes is added in the maltose substrate that the mass percent concentration of 20ml is 20%, 50 DEG C of reactions are for 24 hours.Reaction
After filter obtain enzyme after immobilization, put into after being rinsed with deionized water in next batch reaction.Using high performance liquid chromatography
Detected, mobile phase is acetonitrile: water=87:13, flow velocity 1ml/min, column temperature are 25 DEG C.
2. fixing condition optimizes
The preliminary identification process for fixation has certain effect and can recycle certain batch, further to its immobilization
Condition and reaction condition are optimized.
(1) carrier amount optimizes
It is fixed that 1ml clasmatosis liquid-liquid-solid is added in carrier 0.2g, 0.4g, 0.6g, 0.8g, 1.0g after weighing activation respectively
Filtering is drained after 4h, is put into maltose substrate and is reacted, surveys conversion ratio after reaction for 24 hours.The immobilised enzymes of different carriers amount
Conversion ratio such as Fig. 1.
(2) enzyme amount optimizes
0.4g carrier is separately added into the clasmatosis liquid of 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 2.0ml, oscillation
It puts into maltose substrate and reacts after fixed 4h, reaction for 24 hours terminates to survey conversion ratio.The conversion ratio of the immobilised enzymes of different enzyme amount is such as
Fig. 2.
(3) immobilization is time-optimized
The clasmatosis liquid of 1.0ml is added in 0.4g carrier, 1h, 2h, 3h, 4h, 5h, 6h is vibrated respectively at room temperature, after suction filtration
It is reacted in investment maltose substrate, reaction for 24 hours terminates to survey conversion ratio.Conversion ratio such as Fig. 3 of different immobilization times.
3. reaction condition optimization
The reaction condition of immobilised enzymes is optimized, including anti-pH and reaction temperature.
(1) reaction pH optimization experiment
It is reacted under conditions of pH is 6,6.5,7,7.5,8, reaction for 24 hours terminates to survey conversion ratio.Under condition of different pH
The conversion ratio of reaction such as Fig. 4.
(2) reaction temperature optimization experiment
It is reacted respectively under conditions of 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C, reaction for 24 hours terminates to survey and turn
Rate.The conversion ratio reacted under condition of different pH such as Fig. 5.
4. the circulation batch of immobilised enzymes
Trehalose synthase is fixed under conditions of after optimization, and is reacted under optimum condition, and every batch of secondary response terminates
Immobilised enzymes filtering is drained afterwards, next batch reaction is put into after being rinsed with deionized water.
Resin after claiming 0.4g activation, adds 1ml clasmatosis liquid liquid, vibrates fixed 5-6h at room temperature, obtains immobilised enzymes
It is reacted under conditions of 50 DEG C, pH are 7 afterwards for 24 hours, sampling, HPLC detects conversion ratio.It is as shown in Figure 6 to recycle batch.
Can be seen that the thick enzyme i.e. original transformation rate of clasmatosis liquid from Fig. 1 to Fig. 6 can after 56%-60%, immobilization
It is recycled.The result shows that have certain effect with resin-immobilized, optimize fixing condition and reaction condition the results show that plus
Enter 1ml clasmatosis liquid liquid to immobilize, can achieve preferable as a result, being differed after reaction for 24 hours with the conversion ratio of resolvase
Less.Optimal carrier amount is 0.4g, and when the immobilization time is 5-6h, immobilization effect is preferable.PH be 7 when conversion ratio relatively
Height, while considering industrial applications, use water as reaction dissolvent.Enzyme activity is with respect to highest at 50 DEG C, when temperature is lower, for 24 hours cannot
Reach maximum conversion, when temperature is higher, the amount of by-product glucose can be greatly increased.50 DEG C comparatively, and by-product is less
And it can reach maximum conversion for 24 hours.When Fig. 7 can be seen that 50 DEG C, compared to resolvase, the by-product glucose of immobilised enzymes
Amount is reduced.
It is recycled, initial conversion is 58% or so, and when being recycled to 18 batches, conversion ratio may also stay at initially substantially
72% or so of conversion ratio.When being recycled to 14 batches, conversion ratio may also stay at 80% or so of initial conversion substantially.
Therefore after trehalose synthase being fixed on the resin with epoxy group, it can reach conversion ratio identical with resolvase,
If being in addition maintained at 80% or more conversion ratio, reusable 14 batches or so, production cost is greatlyd save.
Table 1 is the essential information of epoxy resin
GTGGACCCCCTCTGGTACAAGGACGCGGTGATCTACCAGCTCCACGTCCGCTCCTTCTTTGACGCCAAC
AACGACGGCTACGGGGACTTTGAGGGCCTGAGGCGGAAGCTTCCCTACTTGGAGGAGCTCGGGGTCAACATCCTCTG
GCTCATGCCCTTCTTCCAGTCCCCCTTGAGGGACGACGGATACGACATCTCCGACTACTACCAGATCCTCCCCGTCC
ACGGGACCCTGGAGGACTTCAAGCGCTTCCTGGACGAGGCCCACGGCCGGGGGATGAAGGTGATCATTGAGCTCGTC
CTGAACCACACCTCCATTGACCACCCCTGGTTCCAGGAAGCGAGGAAGCCAGGAAGCCCCATGCGGGACTGGTACGT
GTGGAGCGACACCCCGGAGAAGTACAAGGGGGTCCGGGTCATCTTCAAGGACTTTGAAACCTCCAACTGGACCTTTG
ACCCCGTGGCCAAGGCCTACTACTGGCACCGCTTCTACTGGCACCAGCCCGACCTCAACTGGGACAACCCCGAGGTG
GAGAAGGCCATCCACCAGGTCATGTTCTTCTGGGCCGACCTGGGGGTGGACGGCTTCCGCCTGGACGCCATCCCCTA
CCTCTACGAGCGGGAGGGGACCTCCTGCGAGAACCTCCCCGAGACCATTGAGGCGGTGAAGCGCCTGAGGAAGGCCC
TGGAGGAGCGCTACGGCCCCGGGAAGATCCTCCTCGCCGAGGCCAACATGTGGCCGGAGGAGACCCTCCCCTACTTC
GGGGACGGGGACGGGGTCCACATGGCCTACAACTTCCCCCTGATGCCCCGGATCTTCATGGCCCTAAGGCGGGAGGA
CCGGGGGCCCATTGAAACCATGCTCAAGGAGACGGAGGGGATCCCCGAGACCGCCCAGTGGGCCCTCTTCCTCCGCA
ACCACGACGAGCTCACCCTGGAGAAGGTCACGGAGGAGGAGCGGGAGTTCATGTACGAGGCCTACGCCCCCGACCCC
AAGTTCCGCATCAACCTGGGGATCCGCCGCCGCCTCATGCCCCTCCTCGGGGGCGACCGCAGGCGGTACGAGCTCCT
CACCGCCCTCCTCCTCACCCTGAAGGGCACGCCCATCGTCTACTACGGGGACGAGATCGGCATGGGGGACAACCCCT
TCCTCGGGGACCGGAACGGGGTCCGGACCCCCATGCAGTGGTCCCAAGACCGCAACGCCGGCTTCTCCCGCGCCCCC
TACCACGCCCTCTTCCTTCCCCCCGTGAGCGAGGGGCCCTACAGCTACCACTTCGTCAACGTGGAGGCCCAGCGGGA
AAACCCCCACTCCCTCCTGAGCTTCAACCGCCGCTTCCTCGCCCTGAGGAACCAGCACGCCAAGATCTTCGGCCGGG
GGAGCCTCACCCTTCTTCCCGTGGAGAACCGGCGCGTCCTCGCCTACCTGAGGGAGCACGAGGGGGAGCGGGTCCTG
GTGGTGGCCAACCTCTCCCGCTACACCCAGGCCTTTGACCTCCCCTTGGAGGCCTACCAAGGCCTCGTCCCCGTGGA
GCTCTTCTCGCAGCAACCCTTCCCCCCGGTGGAGGGGCGCTACCGCCTGACCCTGGGCCCCCACGGTTTCGCCCTCT
TCGCCCTGAAACCCGTGGAGGCGGTGCTCCACCTCCCCTCCCCCGACTGGGCCGAGGAGCCCGCCCCCGAGGAGGCC
GACCTGCCCCGGGTCCACATGCCCGGGGGGCCGGAGGTCCTCCTGGTGGACACCCTGGTCCACGAAAGGGGGCGGGA
GGAGCTCTTAAACGCCCTCGCCCAGACCCTGAAGGAGAAGAGCTGGCTCGCCCTCAAGCCGCAGAAGGTGGCCCTCC
TGGACGCCCTCCGCTTCCAGAAGGACCCGCCCCTTTACCTCACCCTGCTCCAGCTGGAGAACCACAGGACCCTCCAG
GTCTTCCTCCCCCTCCTCTGGTCCCCCCAGAGGAGGGAAGGCCCCGGCCTCTTCGCCCGCACCCACGGCCAGCCCGG
CTACTTCTACGAGCTCTCCTTGGACCCGGGCTTCTACCGCCTCCTCCTCGCCCGCCTCAAGGAGGGGTTTGAGGGGC
GGAGCCTCCGGGCCTACTACCGCGGCCGCCACCCGGGTCCCGTGCCCGAGGCCGTGGACCTCCTCCGGCCGGGACTC
GCGGCGGGGGAGGGGGTCTGGGTCCAGCTCGGCCTCGTCCAAGACGGGGGCCTGGACCGCACGGAGCGGGTCCTCCC
CCGCCTGGACCTCCCCTGGGTCCTCCGGCCCGAAGGGGGCCTCTTCTGGGAGCGGGGCGCCTCCAGAAGGGTCCTCG
CCCTCACGGGAAGCCTCCCCCCGGGCCGCCCCCAGGACCTCTTCGCCGCCCTGGAGGTCCGGCTCCTGGAAAGCCTT
CCCCGCCTCCGGGGGCACGCCCCCGGGACCCCAGGCCTCCTTCCCGGGGCCCTGCACGAGACCGAGGCCCTGGTCCG
CCTCCTCGGGGTGCGCCTCGCCCTCCTCCACCGGGCCCTTGGGGAGGTGGAGGGGGTGGAGGGGGGCCACCCCCTCC
TAGGCCGCGGCCTCGGGGCCTTCCTGGAGCTGGAGGGGGAGGTGTACCTCGTGGCCCTGGGCGCGGAGAAGCGGGGC
GCGGTGGAGGAGGACCTGGCCCGCCTGGCCTACGACGTGGAGCGGGCCGTGCACCTCGCCCTCGAGGCCCTGGAGGC
GGAGCTTTGGGCCTTCGCCGAGGAGGTGGCCGACTACCTCCACGCCGCCTTCCTCCAAGCCTACCGCTCCGCCCTCC
CCGAGGAGGCCCTGGAGGAGGCGGGCTGGACGCGGCACATGGCCGAGGTGGCGGCGGAGCACCTCCACCGGGAGGAG
AGGCCCGCCCGCAAGCGCATCCACGAGCGCTGGCAGGCCAAGGCCGGAAAAGCCTAG
Claims (3)
1. the method that a kind of fixed trehalose synthase catalysis maltose of resin produces trehalose, which comprises the steps of:
(1) selection and pretreatment of resin
Carrier is resin of the LX-1000EP with epoxy group of Xi'an Lan Xiao company；
Claim 100g carrier that 400ml is added, the phosphate buffer that 0.1M, pH are 8,200rpm stirs 15min at room temperature, pH value is surveyed,
PH is maintained to filter the resin drained after being activated after 7.8-8.2,1h；
(2) enzyme immobilizatio
Escherichia coli with trehalose synthase target gene are fermented, thallus is obtained after centrifugation, it is fermentation that volume, which is added,
Thallus is resuspended the PBS of liquid product 1/5, and clasmatosis liquid is obtained after ultrasonication, is immobilized with clasmatosis liquid；
Process for fixation are as follows: 1ml clasmatosis is added in the resin 0.4g after weighing activation in 10ml phosphoric acid buffer liquid system
Liquid, under room temperature, the oscillation of 150rpm shaking table immobilize；It being filtered after immobilization 5-6h, solid matter is immobilised enzymes,
Load capacity is 70-80%；
(3) immobilised enzymes reacts
0.4g immobilised enzymes is added in the maltose substrate that the mass percent concentration of 20ml is 20%, in 50 DEG C of shaking tables
150rpm reacts for 24 hours；Trehalose synthase sequence used is NO.1.
2. according to the method described in claim 1, it is characterized by:
Immobilised enzymes is filtered after reaction, and reaction solution its component content of high performance liquid chromatography detection, mobile phase is second
Nitrile: water=87:13, flow velocity 1ml/min, column temperature are 25 DEG C.
3. according to the method described in claim 1, it is characterized by:
Immobilised enzymes reaction terminates to obtain immobilised enzymes after filtering, and the quality hundred of new 20ml is put into after being rinsed with deionized water
Divide in the maltose substrate that specific concentration is 20% and carry out next batch reaction, is recycled to realize.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343117A (en) * | 2013-07-03 | 2013-10-09 | 北京科技大学 | Preparation method of immobilized cephalosporin C acylase |
| CN105838700A (en) * | 2016-03-25 | 2016-08-10 | 浙江工业大学 | Immobilized halohydrin dehalogenase and application thereof |
-
2016
- 2016-12-19 CN CN201611177093.0A patent/CN106498000B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343117A (en) * | 2013-07-03 | 2013-10-09 | 北京科技大学 | Preparation method of immobilized cephalosporin C acylase |
| CN105838700A (en) * | 2016-03-25 | 2016-08-10 | 浙江工业大学 | Immobilized halohydrin dehalogenase and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Immobilization of thermostable trehalose synthase for the production of trehalose;Youn-Jeung Cho等;《Enzyme and Microbial Technology》;Elsevier Inc.;20061231;第39卷;108-113 * |
| 海藻糖合酶的高效表达及固定化;陈慧;《中国优秀硕士学位论文全文数据库 基础科学辑》;20180415(第04期);A006-219 * |
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