CN106480005A - A kind of preparation method of the 3 epimerase immobilised enzymes of D psicose with side chain - Google Patents
A kind of preparation method of the 3 epimerase immobilised enzymes of D psicose with side chain Download PDFInfo
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- CN106480005A CN106480005A CN201610898960.3A CN201610898960A CN106480005A CN 106480005 A CN106480005 A CN 106480005A CN 201610898960 A CN201610898960 A CN 201610898960A CN 106480005 A CN106480005 A CN 106480005A
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- psicose
- epimerase
- side chain
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Abstract
The invention provides a kind of preparation method of the 3 epimerase immobilised enzymes of D psicose with side chain, it is engineering bacteria of the construction expression with 3 epimerase of D psicose of side chain, carry out fermented and cultured and obtain thalline, then smudge cells obtains the supernatant of 3 epimerase of D psicose with side chain, spent ion exchange resin adsorptive enzyme, obtains the 3 epimerase immobilised enzymes of D psicose with side chain again.Sharp immobilised enzymes in this way, without the need for isolating and purifying to enzyme, the carried charge of zymoprotein can be changed according to the size for adjusting pH value, and the charge adsorption effect between the charged oligomeric amino acid side chain using enzyme molecule connection and ion exchange resin is fixed on zymoprotein on resin, simple to operate, and the adhesion between enzyme and carrier is difficult for drop-off by force, repeated multiple times can use, can be used for continuous production on the basis of reduces cost or be stirred reaction in the reactor, have a extensive future.
Description
Technical field
The invention belongs to enzyme engineering field, and in particular to a kind of D-Psicose 3- epimerism enzyme immobilization with side chain
The preparation method of enzyme.
Background technology
Being worth for 0.007kcal/g for D-Psicose, is referred to as the sweetener of noenergy；Enteron aisle phlorose can be suppressed
Glycosides enzymatic activity, prevents blood glucose rise, used as the auxiliary therapeutical agent of type II diabetes people；Fatty synthesis enzymatic activity can be reduced, suppression
Intra abdominal fat is piled up, reduction blood fat, and can pre- preventing obesity；There is protective effect to nerve.In view of D-Psicose is good
Property, United States food and drug administration guidelines confirmed in the security to D-Psicose in 2011, and official approval its be
GRAS food, it is allowed to be applied in food and dietary supplements and pharmaceutical preparation, development prospect is extremely wide.But,
Its content in nature is few, is difficult to extract from nature, and the method accessory substance of chemical synthesis is many, it is pure to be difficult
Change；And the research of biological catalysis predominantly stays in laboratory level at present, the report of large-scale production is there is no；Grinding at this stage
Study carefully the application that D-Psicose cannot be met in food and dietary supplements and pharmaceutical preparation, although the market of psicose
Demand increasingly increases, but fancy price limits their market scale and application.Therefore, production is greatly reduced from every side
Cost, accomplishing scale production becomes of crucial importance.
Water miscible enzyme is difficult to separate from reaction system, is unfavorable for control and automated production；Using physics or
The method of chemistry makes enzyme be combined with macromolecular carrier, makes enzyme immobilization on the particle of good mechanical property, can increase the steady of enzyme
Qualitative, the repeated multiple times use of energy, can be used for continuous production on the basis of reduces cost or be stirred in the reactor anti-
Should；So, immobilized transformation of D-levulose generates the weight that the enzyme of D-Psicose is in D-Psicose large-scale industrial production
Want condition.But, traditional enzyme immobilizatio method is related to isolating and purifying for enzyme, and preparation process is complicated, high cost.
Content of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention is to provide a kind of simple, efficient and inexpensive band
The preparation method of the D-Psicose 3- epimerase immobilised enzymes of side chain.
The preparation method of the D-Psicose 3- epimerase immobilised enzymes with side chain of the present invention, step is：
(1) engineering bacteria of the construction expression with the D-Psicose 3- epimerase of side chain；
(2) engineering bacteria of step (1) is inoculated in LB culture medium by 1% inoculum concentration, at 37 DEG C, 200rpm condition is sent out
Ferment 2～4h of culture, after OD value reaches 0.6, with the IPTG of final concentration of 0.1mM, 37 DEG C, 200rpm overnight induction, Ran Houzai
In 4 DEG C, 8000rpm is collected by centrifugation thalline, and the phosphate buffer washing thalline with the 0.1M that pH value is 8.0, then 4 DEG C,
8000rpm is collected by centrifugation and obtains thalline；
(3) it is 8.0 to add pH value in the thalline for obtaining in the ratio for adding 10mL phosphate buffer in every 1g thalline
0.1M phosphate buffer, crushed after 15 ± 5min, at 4 DEG C using cell crushing instrument, 8000rpm pelleted by centrifugation, collect
The supernatant of the D-Psicose 3- epimerase with side chain；
(4) ion exchange resin is proportionally added in the supernatant of the D-Psicose 3- epimerase with side chain,
At 4 DEG C, 120rpm condition 10 ± 3h of adsorption of immobilization, obtain being adsorbed with the tree of the D-Psicose 3- epimerase with side chain
Fat；
(5) resin 3～5 times for preparing is washed with the phosphate buffer of the 0.1M that pH value is 8.0, to wash away free enzyme
Liquid, obtains the D-Psicose 3- epimerase immobilised enzymes with side chain, and 4 DEG C preserve, standby；
It is characterized in that：
Method of step (1) construction expression with the D-Psicose 3- epimerism enzyme engineering bacteria of side chain be：
1. with D-Psicose 3- epimerase nucleotide sequence (the nucleotide sequence such as SEQ ID No.1 institute of report
Show) it is template, primer is designed, enters performing PCR cloning reaction, obtain the gene sequence of the D-Psicose 3- epimerase with side chain
Row；The nucleotide sequence of wherein relevant primer is：
DPE-12-NdeI upstream：5’-GGGAATTCCATATGAAATATGGTATTTATTACGCT-3’；
DPE-12-XhoI downstream：5’-CCGCTCGAGTCAATCTTCGTCCTCATCTTCGTCCTCATCTTCGTCGTCGA
CTTCAAATACATGTT-3’；
The unnamed gene of the D-Psicose 3- epimerase with side chain for 2. obtaining is DPE-12, its nucleotide sequence
As shown in SEQ ID No.2, genes of interest product is reclaimed using DNA purifying QIAquick Gel Extraction Kit；
3. the genes of interest product and plasmid vector pET22b with restriction enzyme NdeI and XhoI respectively to reclaiming enters
Row double digestion, is separately recovered the DPE-12 after digestion and plasmid fragments using DNA purifying QIAquick Gel Extraction Kit, is then connected using T4
DPE-12 and plasmid fragments are attached by kit；
4. the connection liquid in 3. is converted E. coli DH5 α, using Amp-LB plate screening, the positive of acquisition
Clone carries out sequencing analysis, and errorless positive colony Amplification Culture is sequenced, and extracts plasmid, is named as expression plasmid pET22b-
DPE-12；
5. expression plasmid pET22b-DPE-12 is converted e. coli bl21, is obtained using Amp-LB plate screening positive
Transformant, verifies that errorless positive transformant is named as BL21-pET22b-DPE-12, that is, obtain D- A Luo of the expression with side chain
The engineering bacteria of ketose 3- epimerase；
Step (4) ion exchange resin is alkalescence anion-exchange resin；Its addition is added by 1mL enzyme supernatant
The ratio of 1g resin adds ion exchange resin in the supernatant of the D-Psicose 3- epimerase with side chain.
D-Psicose 3- epimerase with side chain of the present invention be by D-Psicose 3- epimerase
C-terminal with the addition of charged oligomeric amino acid side chain composition containing 12 polyglutamic acids and aspartic acid, described with side chain
D-Psicose 3- epimerase compared with common D-Psicose 3- epimerase, under the conditions of same pH, | pI-
PH | bigger, the electric charge of protein band is more, more firm with the absorption of ion exchange resin.Sharp immobilised enzymes in this way, need not
Enzyme is isolated and purified, the carried charge of zymoprotein can be changed according to the size for adjusting pH value, and using enzyme molecule connection
Charge adsorption effect between charged oligomeric amino acid side chain and ion exchange resin is fixed on zymoprotein on resin, behaviour
Make simply, and the adhesion between enzyme and carrier is difficult for drop-off by force.
The immobilised enzymes obtained by the inventive method, stability are increased, it is possible to repeated multiple times use, in reduces cost
On the basis of can be used for continuous production or be stirred reaction in the reactor, have a extensive future.
Description of the drawings
Fig. 1 is the D-Psicose 3- epimerase genetic modification plasmid construction figure with side chain.
Fig. 2 is the SDS-PAGE result figure of BL21-pET22b-DPE-12 transformant fermented supernatant fluid；
Wherein：Swimming lane 1 is BL21-pET22b blank；Swimming lane 2 is not induced for BL21-pET22b-DPE-12-；Swimming lane
3 is BL21-pET22b-DPE-12- holoprotein；Swimming lane 4 is the broken precipitation of BL21-pET22b-DPE-12-；Swimming lane 5 is BL21-
PET22b-DPE-12- crushes albumen supernatant.
Fig. 3 is immobilised enzymes recycling design sketch.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
Engineering bacteria of 1 construction expression of embodiment with the D-Psicose 3- epimerase of side chain
(1) D-Psicose 3- epimerase nucleotide sequence (the nucleotide sequence such as SEQ ID No.1 to have reported
Shown) it is template, primer is designed, enters performing PCR cloning reaction, obtain the gene of the D-Psicose 3- epimerase with side chain
Sequence；The nucleotide sequence of the primer for wherein designing is：
DPE-12-NdeI upstream：
GGGAATTCCATATGAAATATGGTATTTATTACGCT (dashed part is NdeI restriction enzyme site)；
DPE-12-XhoI downstream：
CCGCTCGAGTCAATCTTCGTCCTCATCTTCGTCCTCATCTTCGTCGTCGACTTCAAATACATGTT (draws
Line part is XhoI restriction enzyme site),
PCR amplification condition：94 DEG C of 2min of denaturation；98℃10sec；59 DEG C of 15sec of annealing；Extend 72 DEG C of 1min；30
Circulation；72 DEG C of extension 10min；12 DEG C of insulations；
(2) unnamed gene of the D-Psicose 3- epimerase with side chain for obtaining is DPE-12, its nucleotides sequence
Row are as shown in SEQ ID No.2.Right using DNA purifying QIAquick Gel Extraction Kit (buying in TIANGEN Biotech (Beijing) Co., Ltd.)
Genes of interest product is reclaimed, concrete operation method reference reagent box operation manual, is verified using 1% agarose gel electrophoresis
Amplification, genes of interest product DPE-12 stripe size are 911bp；
(3) genes of interest product DPE-12 and plasmid vector of the restriction enzyme NdeI and XhoI respectively to recovery are used
PET22b carries out double digestion, is separately recovered the DPE-12 after digestion and plasmid fragments, Ran Houli using DNA purifying QIAquick Gel Extraction Kit
DPE-12 and plasmid fragments are attached with T4 connection kit, concrete digestion and Connection Step, reference operation specification；
(4) public affairs limited in full formula gold biotechnology are bought the connection liquid conversion E. coli DH5 α in (3) (
Department), the positive colony of acquisition delivers to Invitrogen (Shanghai) trade Co., Ltd carries out sequencing analysis, through the errorless positive is sequenced
Clone's Amplification Culture, extracts plasmid, is named as expression plasmid pET22b-DPE-12, and building process is as shown in Figure 1；
(5) expression plasmid pET22b-DPE-12 conversion e. coli bl21, obtains positive turning using Amp-LB plate screening
Beggar, verifies that errorless positive transformant is named as BL21-pET22b-DPE-12, that is, obtain D- A Luo ketone of the expression with side chain
The engineering bacteria of sugared 3- epimerase.
Preparation method of the embodiment 2 with the D-Psicose 3- epimerase immobilised enzymes of side chain
(1) engineering bacteria of embodiment 1 is inoculated in LB culture medium by 1% inoculum concentration, at 37 DEG C, 200rpm condition is trained
After foster 2h, OD value reaches 0.6, with the IPTG of final concentration of 0.1mM, 37 DEG C, after 200rpm overnight induction, then 4 DEG C, 8000rpm
Thalline is collected by centrifugation, using the phosphate buffer washing thalline of the 0.1M that pH value is 8.0, then 4 DEG C, 8000rpm is collected by centrifugation
Obtain thalline；
(2) it is 8.0 to add pH value in the thalline for obtaining in the ratio for adding 10mL phosphate buffer in every 1g thalline
0.1M phosphate buffer, crushed after 10min, at 4 DEG C using cell crushing instrument, 8000rpm pelleted by centrifugation, collection belt side
The supernatant of the D-Psicose 3- epimerase of chain；SDS-PAGE analyzing proteins are carried out after broken end, destination protein
Size near 38kD, as shown in Figure 2；
(3) pretreatment of alkalescence anion-exchange resin：Processing method is with reference to country of the GB5476-85 People's Republic of China (PRC)
Standard ion exchange resins preprocess method；
1. resin is loaded in chromatographic column of the internal diameter for 3cm, post bed height is 30cm, is rinsed with pure water repeatedly, directly
To Wu visible mechanical admixture and going out clarification of water in sample；
2. successively 100mL1N hydrochloric acid solution, 200mL pure water, 100mL1N sodium hydroxide solution and 200mL pure water are used, from upper
And down by resin bed, reagent flow is 2.0mL/min, pure water flow is 10mL/min, in each transferring reagent, makes liquid level
It is higher by resin bed 1cm, it is ensured that bubble-free in resin bed；This operation carries out 2 times；
3. the resin through 2. processing passes through resin bed with 400mL1N sodium hydroxide solution, and flow is 10mL/min, then
With pure water, until stopping washing when being colourless with instructions phenolphthalein solution inspection efflux；
(4) add the ratio of 1g resin in 1mL enzyme supernatant in the supernatant of the D-Psicose 3- epimerase with side chain
Alkalescence anion-exchange resin is added in liquid, at 4 DEG C, 120rpm condition adsorption of immobilization 10h, obtain being adsorbed with the D- with side chain
The resin of psicose 3- epimerase；
(5) resin 3 times for preparing is washed with the phosphate buffer of the 0.1M that pH value is 8.0, to wash away free enzyme liquid,
The D-Psicose 3- epimerase immobilised enzymes with side chain is obtained, 4 DEG C preserve, standby；
(6) fructose soln with 500g/L, is added as in every 100mL fructose soln as substrate with immobilised enzymes as catalyst
The ratio catalysis fructose for entering 45g catalyst generates D-Psicose, and reaction completes once to wash fixation afterwards with phosphate buffer
Change enzyme, second catalytic reaction is carried out, is carried out successively, until the enzyme activity for detecting immobilised enzymes is reduced to the 50% of protoenzyme, such as
Shown in Fig. 3.
Preparation method of the embodiment 3 with the D-Psicose 3- epimerase immobilised enzymes of side chain
(1) engineering bacteria of embodiment 1 is inoculated in LB culture medium by 1% inoculum concentration, at 37 DEG C, 200rpm condition is trained
After foster 4h, OD value reaches 0.6, with the IPTG of final concentration of 0.1mM, 37 DEG C, after 200rpm overnight induction, then 4 DEG C, 8000rpm
Thalline is collected by centrifugation, using the phosphate buffer washing thalline of the 0.1M that pH value is 8.0, then 4 DEG C, 8000rpm is collected by centrifugation
Obtain thalline；
(2) it is 8.0 to add pH value in the thalline for obtaining in the ratio for adding 10mL phosphate buffer in every 1g thalline
0.1M phosphate buffer, crushed after 20min, at 4 DEG C using cell crushing instrument, 8000rpm pelleted by centrifugation, collection belt side
The supernatant of the D-Psicose 3- epimerase of chain；
(3) add the ratio of 1g resin in 1mL enzyme supernatant in the supernatant of the D-Psicose 3- epimerase with side chain
Alkalescence anion-exchange resin is added in liquid, at 4 DEG C, 120rpm condition adsorption of immobilization 13h, obtain being adsorbed with the D- with side chain
The resin of psicose 3- epimerase；
(4) resin 5 times for preparing is washed with the phosphate buffer of the 0.1M that pH value is 8.0, to wash away free enzyme liquid,
The D-Psicose 3- epimerase immobilised enzymes with side chain is obtained, 4 DEG C preserve, standby.
The present invention is using the charged oligomeric amino acid of the connection of the D-Psicose 3- epimerism enzyme molecule with side chain
Charge adsorption effect between side chain and ion exchange resin is fixed on zymoprotein on resin, simple to operate, and enzyme and load
Adhesion between body is difficult for drop-off by force.And the D-Psicose 3- epimerase immobilised enzymes with side chain is repeated and is made
With therefore, the technology has great superiority, industrially has using the immobilised enzymes production D-Psicose huge
Using value.
Claims (2)
1. a kind of preparation method of the D-Psicose 3- epimerase immobilised enzymes with side chain, step is：
(1) engineering bacteria of the construction expression with the D-Psicose 3- epimerase of side chain；
(2) engineering bacteria of step (1) is inoculated in LB culture medium by 1% inoculum concentration, at 37 DEG C, the fermentation of 200rpm condition is trained
2～4h is supported, after OD value reaches 0.6, with the IPTG of final concentration of 0.1mM, 37 DEG C, 200rpm overnight induction, then then at 4
DEG C, 8000rpm is collected by centrifugation thalline, and the phosphate buffer washing thalline with the 0.1M that pH value is 8.0, then 4 DEG C,
8000rpm is collected by centrifugation and obtains thalline；
(3) it is 8.0 to add pH value in the ratio for adding 10mL phosphate buffer in every 1g thalline in the thalline for obtaining
The phosphate buffer of 0.1M, is crushed after 15 ± 5min, at 4 DEG C using cell crushing instrument, 8000rpm pelleted by centrifugation, collection belt
The supernatant of the D-Psicose 3- epimerase of side chain；
(4) ion exchange resin is proportionally added in the supernatant of the D-Psicose 3- epimerase with side chain, 4
DEG C, 120rpm condition 10 ± 3h of adsorption of immobilization, obtain being adsorbed with the resin of the D-Psicose 3- epimerase with side chain；
(5) resin 3～5 times for preparing is washed with the phosphate buffer of the 0.1M that pH value is 8.0, to wash away free enzyme liquid,
The D-Psicose 3- epimerase immobilised enzymes with side chain is obtained, 4 DEG C preserve, standby；
It is characterized in that：
Method of step (1) construction expression with the D-Psicose 3- epimerism enzyme engineering bacteria of side chain be：
1. arranged as template with the D-Psicose 3- epimerase nucleotides sequence that reports, primer is designed, enter performing PCR clone anti-
Should, obtain the gene order of the D-Psicose 3- epimerase with side chain；The nucleotide sequence of wherein relevant primer is：
DPE-12-NdeI upstream：5’-GGGAATTCCATATGAAATATGGTATTTATTACGCT-3’；
DPE-12-XhoI downstream：5’-CCGCTCGAGTCAATCTTCGTCCTCATCTTCGTCCTCATCTTCGTCGTCGACTTCA
AATACATGTT-3’；
The unnamed gene of the D-Psicose 3- epimerase with side chain for 2. obtaining is DPE-12, and its nucleotide sequence is such as
Shown in SEQ ID No.2, genes of interest product is reclaimed using DNA purifying QIAquick Gel Extraction Kit；
3. respectively the genes of interest product that reclaims and plasmid vector pET22b is carried out with restriction enzyme NdeI and XhoI double
Digestion, is separately recovered the DPE-12 after digestion and plasmid fragments using DNA purifying QIAquick Gel Extraction Kit, then connects reagent using T4
DPE-12 and plasmid fragments are attached by box；
4. the connection liquid in 3. is converted E. coli DH5 α, using Amp-LB plate screening, the positive colony of acquisition
Sequencing analysis are carried out, errorless positive colony Amplification Culture is sequenced, plasmid is extracted, is named as expression plasmid pET22b-DPE-12；
5. expression plasmid pET22b-DPE-12 is converted e. coli bl21, positive transformants are obtained using Amp-LB plate screening
Son, verifies that errorless positive transformant is named as BL21-pET22b-DPE-12, that is, obtain D-Psicose of the expression with side chain
The engineering bacteria of 3- epimerase；
Step (4) ion exchange resin is alkalescence anion-exchange resin；Its addition is to add 1g tree by 1mL enzyme supernatant
The ratio of fat adds ion exchange resin in the supernatant of the D-Psicose 3- epimerase with side chain.
2. as claimed in claim 1, the preparation method of the D-Psicose 3- epimerase immobilised enzymes with side chain, its feature
It is, the D-Psicose 3- epimerase with side chain is with the addition of by the C-terminal of D-Psicose 3- epimerase
Charged oligomeric amino acid side chain composition containing 12 polyglutamic acids and aspartic acid, the D-Psicose 3- with side chain
, compared with common D-Psicose 3- epimerase, under the conditions of same pH, | pI-pH | is bigger, albumen for epimerase
The electric charge of band is more, more firm with the absorption of ion exchange resin.
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| CN110373408A (en) * | 2019-08-12 | 2019-10-25 | 山东星光首创生物科技有限公司 | One kind is with poly-dopamine-magnetic Fe3O4The method of nanoparticle immobilization D-Psicose 3- epimerase |
| CN111458396A (en) * | 2019-01-18 | 2020-07-28 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
| WO2021244005A1 (en) * | 2020-06-03 | 2021-12-09 | 中国科学院天津工业生物技术研究所 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
| CN114958815A (en) * | 2022-04-29 | 2022-08-30 | 西北工业大学 | D-psicose 3-epimerase with high substrate conversion rate and immobilization method thereof |
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| CN105637089A (en) * | 2013-09-03 | 2016-06-01 | 罗盖特兄弟公司 | Improved variant of d-psicose 3-epimerase and uses thereof |
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| CN102869783A (en) * | 2009-09-30 | 2013-01-09 | Cj第一制糖株式会社 | Immobilization of psicose-epimerase and method of producing d-psicose using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111458396A (en) * | 2019-01-18 | 2020-07-28 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
| CN111458396B (en) * | 2019-01-18 | 2022-07-08 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
| CN110373408A (en) * | 2019-08-12 | 2019-10-25 | 山东星光首创生物科技有限公司 | One kind is with poly-dopamine-magnetic Fe3O4The method of nanoparticle immobilization D-Psicose 3- epimerase |
| CN110373408B (en) * | 2019-08-12 | 2021-06-25 | 山东星光首创生物科技有限公司 | Method for immobilizing D-psicose 3-epimerase by polydopamine-magnetic Fe3O4 nanoparticles |
| WO2021244005A1 (en) * | 2020-06-03 | 2021-12-09 | 中国科学院天津工业生物技术研究所 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
| CN114958815A (en) * | 2022-04-29 | 2022-08-30 | 西北工业大学 | D-psicose 3-epimerase with high substrate conversion rate and immobilization method thereof |
| CN114958815B (en) * | 2022-04-29 | 2023-07-25 | 西北工业大学 | D-psicose 3-epimerase and immobilization method thereof |
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