CN105177085B - A kind of single-minded application for turning glycosyl alpha-galactosidase of regioselectivity - Google Patents
A kind of single-minded application for turning glycosyl alpha-galactosidase of regioselectivity Download PDFInfo
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Abstract
The present invention relates to a kind of single-minded applications for turning glycosyl alpha-galactosidase of regioselectivity, and steps are as follows：It will turn glycosyl alpha-galactosidase to mix with p-nitrophenyl-α-D- galactosides and lactose solution, and be configured to reaction system, and after reaction, terminate reaction, Globotriose is made in purifying.The expressing gene nucleotide sequence for turning glycosyl alpha-galactosidase is as shown in SEQ ID NO.1.The present invention using regioselectivity it is single-minded turn glycosyl alpha-galactosidase, using p-nitrophenyl-α-D- galactosides as glycosyl donor, using lactose as receptor, the single product oligosaccharides of one-step synthesis oligosaccharides Globotriose avoid the generation of isomerized products.This method substrate is cheap, and reaction step is simple, and product purification is easy, and the scale for being really suited for Globotriose, which is combined to and detaches, prepares sterling, has potential application prospect.
Description
Technical field
The present invention relates to a kind of single-minded application for turning glycosyl alpha-galactosidase of regioselectivity, more particularly to a kind of profits
With the application for turning glycosyl alpha-galactosidase specificity synthesis of oligose Globotriose, belong to glycosidase application field.
Technical background
Alpha-galactosidase (α-galactosidase, EC.3.2.1.22) is a kind of important glycoside hydrolase, catalysis
The hydrolysis of alpha-galactoside key, is distributed widely in ancient bacterium, bacterium, fungi, in plant and animal, according to amino acid sequence homology
Belong to glucosides enzyme family GH4, GH27, GH36, GH57, GH97 and GH110 (http://www.cazy.org/).Wherein, carefully
The alpha-galactosidase in bacterium source is distributed mainly on GH4 and GH36, and eukaryot-ic origin principally falls into GH27.The fermentoid can
Simple alpha-galactoside is hydrolyzed, and the macromolecular of the key containing alpha-galactoside can be acted on, such as polysaccharide, glycoprotein and glycolipid.This
Outside, certain alpha-galactosidases not only have hydrolysis function, also have under suitable reaction condition turn alpha-galactoside in vitro
Activity, synthesize alpha-galactooligosaccharide and galactoside compound, be applied to food and medicine industry.
α-Isosorbide-5-Nitrae galactooligosacchari(es have important medical value, if Globotriose is galactosyl with α-Isosorbide-5-Nitrae-key connection
The trisaccharide formed on to lactose, it is a kind of natural sugar chain component of important glycosyl sphingolipid Gb3 of human cell surface, is that production will is congratulated
Toxin Escherichia coli such as O157:H7 etc. invades the ligand combined when human body cell.It has proven at present artificial synthesized
Globotriose trisaccharides have affinity to shiga toxin, can be in connection before toxin enters cell, block toxin with it is thin
The combination of the natural sugar chain receptor of cellular surface prevents it from invading cell, and can excrete toxin.It is this with artificial synthesized
The therapeutic modality that receptor sugar chain neutralizes a toxin is superior to traditional antibiotherapy, it will not cause drug-resistant microorganism largely to increase
It grows, and receptor sugar chain drug can be treated just for specified microorganisms, non-sex pheromone is not suppressed, and is still existed
Beneficial functions are exercised in host, the drug resistance for eliminating or reducing normal microbial flora is horizontal.But such current sugar chain is difficult
It is the major obstacle for restricting Carbohydrate drugs development all the time largely to synthesize and obtain.Since existing synthetic method is deposited
In limitation so that the commodity price of Globotriose is very expensive always.
The glycosidic bond for the alpha-galactoside enzymatic synthesis reported at present is mainly Gal α 1-6 keys, is Gal α 1-3 and Gal α on a small quantity
1-2 keys.Patent ZL 2,011 1 0002979.2 discloses a kind of new method of alpha-galactoside enzymatic synthesis Globotriose, but
It is involved belong to GH36 alpha-galactosidase (i.e. by gene order described in patent ZL200510044898.3 or
GenBank accession number is enzyme made from the gene order of AF406640) regioselectivity is stringent, although with p-nitrophenyl-
α-D- galactosides are glycosyl donor, using lactose as in the synthetic reaction of receptor, can one-step synthesis Globotriose, but react
Product is the mixture of a variety of isomers, and other than α-Isosorbide-5-Nitrae target product, there is also α -1,3 and α -1,6 products.These isomeries
Product physical chemical property is similar, it is difficult to which a large amount of separation obtain Globotriose sterlings.
Invention content
The α-Isosorbide-5-Nitrae regioselectivity that in view of the deficiencies of the prior art, the present invention provides a kind of from GH27 is single-minded to be turned
The application of glycosyl alpha-galactosidase.
Summary of the invention
The invention is characterized in that using the single-minded alpha-galactosidase of regioselectivity with p-nitrophenyl-α-D- galas
Glucosides is glycosyl donor, and using lactose as receptor, a step Transglycosylation synthesizes the single oligosaccharides of Globotriose.
Detailed description of the invention
A kind of regioselectivity it is single-minded turn glycosyl alpha-galactosidase answering in synthesizing the single oligosaccharides of Globotriose
With the expressing gene nucleotide sequence of the alpha-galactosidase is as shown in SEQ ID NO.1.
Above application, steps are as follows：
It will turn glycosyl alpha-galactosidase to mix with p-nitrophenyl-α-D- galactosides and lactose solution, and be configured to react
System reacts 10~30min under the conditions of 35~38 DEG C, terminates reaction, and Globotriose is made in purifying.
According to currently preferred, the preparation method for turning glycosyl alpha-galactosidase is as follows：
(1) gene order shown in nucleotide sequence such as SEQ ID NO.1 is inserted into plasmid pBAD/HisA, conversion is big
Recombination bacillus coli is made in enterobacteria LMG194；
(2) recombination bacillus coli made from step (1) is cultivated through expanding, after Fiber differentiation, collects cell, carefully
Born of the same parents are broken, purify, and are made and turn glycosyl alpha-galactosidase.
After testing, turn the amino acid sequence of glycosyl alpha-galactosidase as shown in SEQ ID NO.2.This turns glucosylated-alpha-gala
Glycosidase list molecular weight subunit is 59.2kDa, and specificity hydrolyzes alpha-galactoside key, is with p-nitrophenyl-α-D- galactosides
When substrate, optimal pH is 4.5~5.0, is stablized in the ranges of pH4.0~11.0, and optimal reactive temperature is 37 DEG C~40 DEG C, low
Stablize when 40 DEG C；Ag+、Fe3+And Hg2+Significantly inhibit enzyme activity, Cu2+And Fe2+Part inhibits enzyme activity, NH4 +、K+、Na+、Mg2+、
Zn2+、Co2+、Ni2+、Mn2+、Ca2+And EDTA2+It does not make significant difference to enzyme activity.
According to the present invention it is further preferred that in the step (1), nucleotide sequence base as shown in SEQ ID NO.1
Because sequence is using the genomic DNA of bacteroides fragilis (Bacteroides fragilis) NCTC9343 as template, obtained through PCR amplification
, PCR primer nucleotide sequence is as follows：
Primers F：5'-GGCCGCTAGCCAAAACACAAATACTCCCAT-3’
Nhe I
Primer R：5'-GGCCCTCGAGTTAACGACCTTTGATGATTT-3’
Xho I
PCR system is following (50 μ L of total volume)：
10 × PCR buffer solutions, 5 μ L, 2.5mmol/L dNTP, 4 μ L, each 1 μ L of 20 μm of ol/L primers, 50 μ g/mL templates, 2 μ L,
5U/ μ L TaKaRa LA Taq 1 μ L, 36 μ L of ultra-pure water.
PCR amplification program is as follows：
95 DEG C of pre-degenerations 5 minutes；I.e. 95 DEG C of 30 cycles of reaction are denaturalized 30 seconds, and 48 DEG C are annealed 30 seconds, and 72 DEG C extend 2 points
Clock；72 DEG C extend 10 minutes.
According to the present invention it is further preferred that in the step (2), expanding condition of culture is：37 DEG C, 150~180r/
Min is cultivated to OD600It is 0.4~0.6.
According to the present invention it is further preferred that in the step (2), expansion culture medium is RM+ glucose culture solutions, is prepared
Method is as follows：
Sour 18~22g/L of hydrolyzed casein, glucose 0.18~0.22g/L, 0.09~0.10g/L MgCl2, volume basis
10 × M9 salting liquids than 10%；
10 × M9 salting liquids are prepared as follows：
Na2HPO460g/L, KH2PO430g/L、NaCl 5g/L、NH410g/L, pH7.4,121 DEG C of Cl sterilizes 20 minutes, cold
But the vitamin B1 of filtration sterilization is added afterwards to final concentration 0.3g/L.
More preferably, described to expand the ampicillin that culture medium further includes a concentration of 40~60 μ g/mL.
According to the present invention it is further preferred that in the step (2), Fiber differentiation is after expanding culture, to culture solution
Middle addition L-arabinose, make L-arabinose in culture solution mass concentration be 0.1%~0.3%, 25~28 DEG C,
100r/min continues to be incubated overnight.
According to currently preferred, in the reaction system, the reaction densities of p-nitrophenyl-α-D- galactosides is 15~
20mM, lactose reaction density are 400~500mM, and pH is 4.0~5.0.
According to currently preferred, the reaction system is prepared by the sodium acetate buffer of a concentration of 100mM, pH 4.5.
According to currently preferred, the purifying is purified using column chromatography.
Advantageous effect
Present invention firstly discovers that turning glycosyl from bacteroides fragilis (Bacteroides fragilis) NCTC9343
Alpha-galactosidase has the characteristics that the single-minded synthesis Globotriose of regioselectivity, and the enzyme is using lactose as receptor substrate
Reaction system in specificity synthesize Gal α 1-4 glycosidic bonds, single α 1-4 product oligosaccharides are obtained, there is no other isomerized products
Interference, avoid isomers purifying complex steps, the scale for being really suited for Globotriose be combined to and detach prepare it is pure
Product have potential application prospect.
Description of the drawings
Fig. 1 is the electrophoretogram for turning glycosyl alpha-galactosidase prepared by the present invention；
In figure：M, molecular weight standard；1, the granular cell enzyme solution of recombinant bacterium is compareed；2, containing the weight for turning glycosyl alpha-galactosidase
The granular cell enzyme solution of group bacterium；3, the recombinase that nickel affinity chromatography purifying obtains.
Fig. 2 is the ion chromatography spectrogram for turning glycosyl alpha-galactosidase using lactose as receptor synthetic product；
In figure, curve 1 is lactose standard, and curve 2 is gala saccharide, and curve 3 is Globotriose standard items,
Curve 4 is to inactivate reacting for recombinase and lactose and p-nitrophenyl-α-D- galactosides, and curve 5 is recombinase and lactose and right
The reaction of nitrobenzene-α-D- galactosides；
Wherein, the correspondence of retention time and saccharic composition is：5.0 minutes be galactolipin, be within 8.5 minutes lactose, 12.5
Minute is Globotriose.
Specific implementation mode
Technical scheme of the present invention is described further with reference to embodiment, but institute's protection domain of the present invention is not limited to
This.
Biological material source
Bacteroides fragilis (Bacteroides fragilis) NCTC9343 derives from Britain's Culture Collection
(National Collection of Type Cultures)；
Plasmid pBAD/HisA is purchased from Invitrogen companies.
Embodiment
A kind of regioselectivity it is single-minded turn glycosyl alpha-galactosidase answering in synthesizing the single oligosaccharides of Globotriose
With steps are as follows：
1. the preparation of alpha-galactosidase
The alpha-galactosidase gene sequence predicted according to bacteroides fragilis (Bacteroides fragilis) NCTC9343
Arrange (GenBank Accession No.CR626927) design primer F and primer R：
Primers F：5'-GGCCGCTAGCCAAAACACAAATACTCCCAT-3’
Nhe I
Primer R：5'-GGCCCTCGAGTTAACGACCTTTGATGATTT-3’
Xho I
Using the genome of bacteroides fragilis (Bacteroides fragilis) NCTC9343 as template, PCR amplification α-half
Lactose glycoside enzyme gene, amplification system are (50 μ L of total volume)：
10 × PCR buffer solutions, 5 μ L, 2.5mmol/L dNTP, 4 μ L, each 1 μ L of 20 μm of ol/L primers, 50 μ g/mL templates, 2 μ L,
5U/ μ L TaKaRa LA Taq 1 μ L, 36 μ L of ultra-pure water.
Amplification condition is：
95 DEG C of pre-degenerations 5 minutes；I.e. 95 DEG C of 30 cycles of reaction are denaturalized 30 seconds, and 48 DEG C are annealed 30 seconds, and 72 DEG C extend 2 points
Clock；30 after circulation terminates 72 DEG C extend 10 minutes.
Restriction enzyme Nhe I and Xho I was added in 37 DEG C of double digestions 3 hours in the PCR product of purifying, 16 DEG C overnight
It is connected on the plasmid pBAD/HisA carriers of same double digestion processing, construction recombination plasmid.Using CaCl2Conversion method converts place
Main bacterium Escherichia coli LMG194, transformed bacteria solution are coated with ampicillin/LB plates, and 37 DEG C are incubated overnight, picking Growth positive bacterium bacterium
It falls, extraction plasmid verification measures nucleotide sequence and sees SEQ ID No.1.
The recombination bacillus coli LMG194 containing recombinant plasmid of acquisition is inoculated in RM+ glucose culture solutions, 37 DEG C,
180r/min is cultivated to OD600About 0.6, be added final concentration 0.2% L-arabinose, 25 DEG C, 100r/min induce overnight, from
The heart is collected cell and is placed in ice-water bath with pH7.0,50mmol/L buffer solution of potassium phosphate again suspension thalline, and ultrasonic wave is broken
Chopping fine cell wall 20 minutes, 12000r/min are centrifuged 30 minutes, and supernatant is purified by nickel affinity chromatography and obtains the pure recombinase of electrophoresis,
SDS-PAGE shows that recombinase list molecular weight subunit is about 59.2kDa (as shown in Figure 1).
The RM+ glucose culture solutions ingredient is：Sour hydrolyzed casein 20g/L, glucose 0.2g/L, 0.1g/L MgCl2,
10% (v/v) 10 × M9 salting liquids.The ampicillin of filtration sterilization is added when cultivating Escherichia coli recombinant strain in culture medium, eventually
A concentration of 50 μ g/mL.
Above-mentioned 10 × M9 salting liquids ingredient is：Na2HPO460g/L, KH2PO430g/L、NaCl 5g/L、NH4Cl 10g/L,
PH7.4,121 DEG C sterilize 20 minutes, and the vitamin B1 of filtration sterilization is added after cooling to final concentration 0.3g/L.
2. the property research of alpha-galactosidase
Hydrolysing activity of the system of determination enzyme to different glycoside substrates is measured by standard enzyme activity, recombination alpha-galactosidase is single-minded
Property hydrolysis alpha-galactoside key, to the hydrolysing activity highest of p-nitrophenyl-α-D- galactosides, hydrolysis ortho-nitrophenyl-α-D-
Galactoside activity is the 37.6% of p-nitrophenyl-α-D- galactosides.The enzyme does not hydrolyze other glucosides such as ortho-nitrophenyl-β-D-
It is galactoside, p-nitrophenyl-β-D- galactosides, p-nitrophenyl-α-D-MANNOSE glycosides, p-nitrophenyl-β-D-MANNOSE glycosides, right
Nitrobenzene-alpha-D-glucose glycosides, p-nitrophenyl-β-D-Glucose glycosides, p-nitrophenyl-alpha-L-fucosidase, p-nitrophenyl-β-D-
Fucoside, p-nitrophenyl-N- acetyl-α-D-galactosamine, p-nitrophenyl-N- acetyl-β-D-galactosamine, p-nitrophenyl-
N- acetyl-β-D-Glucose amine.
With the buffer enzyme reaction system of different pH, opposite enzyme activity is measured, the results showed that turn glycosyl alpha-galactoside
For enzyme when using p-nitrophenyl-α-D- galactosides as substrate, optimal pH is 4.5~5.0, by enzyme 4 DEG C in different pH buffer solutions
Heat preservation 12 hours measures opposite enzyme activity, turns glycosyl alpha-galactosidase and stablizes in the ranges of pH4.0~11.0.In temperature appropriate
It spends in range (25 DEG C~70 DEG C), every 5 DEG C measure the opposite enzyme activity of enzyme at such a temperature for a gradient, the results showed that most suitable anti-
It is 37 DEG C~40 DEG C to answer temperature, is stablized when less than 40 DEG C.
Using p-nitrophenyl-α-D- galactosides as substrate, it is separately added into enzyme activity determination reaction system final concentration of
The various ions of 2mmol/L, Ag+、Fe3+And Hg2+Significantly inhibit enzyme activity, Cu2+And Fe2+Part inhibits enzyme activity, NH4 +、K+、Na+、
Mg2+、Zn2+、Co2+、Ni2+、Mn2+、Ca2+And EDTA2+It does not make significant difference to enzyme activity.
Above-mentioned enzyme activity determination method is that 1 μ L enzyme solutions totally 80 μ L are added in 79 μ L of 2mM substrate solutions, and 37 DEG C are reacted 10 minutes, are added
Enter 120 μ L, the termination reaction of 200mM, pH10.5 borate buffer, measures the OD of reaction solution405, calculated and hydrolyzed according to standard curve
The amount of the p-nitrophenol gone out is that 1 unit enzyme activity calculates enzyme activity with 1 μm of ol p-nitrophenol of hydrolysis per minute.
Above-mentioned difference pH buffer solutions are the extensive buffer solutions of 30mM, i.e. every liter of mixed liquor includes citric acid 6.008g, di(2-ethylhexyl)phosphate
Hydrogen potassium 3.893g, boric acid 1.769g, barbital 5.266g, required pH value is adjusted to NaOH.
3. alpha-galactoside Enzyme catalyzed synthesis oligosaccharides Globotriose
Alpha-galactosidase will be recombinated using p-nitrophenyl-α-D- galactosides as glycosyl donor, lactose is glycosyl acceptor
Carry out Transglycosylation.It is dense to have studied Transglycosylation condition such as glycosyl donor concentration of substrate (5~30mM), glycosyl acceptor substrate
Degree (100~500mM), pH (4.0~8.0), reaction temperature (30~70 DEG C) and time (10~50 minutes) are to turning glycosyl product
The influence of yield, the results showed that enzyme 37 DEG C of reaction 30min products in p-nitrophenyl-α-D- galactosides 20mM, lactose 500mM
Yield reaches highest.Reaction product is boiled into 10min and terminates reaction, 10min is centrifuged in 12000, supernatant is taken to carry out thin layer
Analysis analysis, it is single oligosaccharides that as a result display, which turns glycosyl product, further carries out ion chromatography to product, as a result shows reaction
The product oligosaccharides of generation are consistent with Globotriose standard items retention times, are 12.5 minutes, it was demonstrated that are single
Globotriose products (the results are shown in Figure 2) further use the purifying of Bio-gel P2 column chromatographies that can obtain
Globotriose sterlings.
Above-mentioned thin-layer chromatography condition is as follows：
Thin layer chromatography board point sample, in developing agent (n-butanol:Absolute ethyl alcohol:Water=5:3:2) expansion in, mist spray color developing agent
(3, the 5- dihydroxytoluenes of 20% sulfuric acid solution+0.5%) toasts 5 minutes in 120 DEG C, after sugared spot development, target product
Mobility is consistent with Globotriose standard items mobilities.
Above-mentioned ion chromatography condition is as follows：
Wear peace (Dionex) ICS2500 type ion chromatographs；Wear peace ED50 electrochemical detectors；Wear peace CarboPacTM
PA-100 ion chromatographic columns (4 × 250mm)；Mobile phase is 60mM NNaOH；Flow velocity is 1mL/min, 30 DEG C of column temperature；Analysis software
For Chromeleon 6.80SR8Build 2623 editions.Supernatant is by 1 after reaction solution boils centrifugation:49 (v/v) ratios are diluted with water,
After 0.22 μm of membrane filtration, sample introduction is analyzed.
Claims (11)
1. a kind of regioselectivity it is single-minded turn glycosyl alpha-galactosidase answering in synthesizing the single oligosaccharides of Globotriose
With the expressing gene nucleotide sequence of the alpha-galactosidase is as shown in SEQ ID NO.1.
2. application as described in claim 1, which is characterized in that steps are as follows：
It will turn glycosyl alpha-galactosidase to mix with p-nitrophenyl-α-D- galactosides and lactose solution, and be configured to reaction system,
Under the conditions of 35~38 DEG C, 10~30min is reacted, terminates reaction, Globotriose is made in purifying.
3. application as claimed in claim 2, which is characterized in that the preparation method for turning glycosyl alpha-galactosidase is as follows：
（1）Gene order shown in nucleotide sequence such as SEQ ID NO.1 is inserted into plasmid pBAD/HisA, large intestine bar is converted
Recombination bacillus coli is made in bacterium LMG194；
（2）By step（1）Recombination bacillus coli obtained is cultivated through expanding, and after Fiber differentiation, collects cell, cell is broken
Broken, purifying, is made and turns glycosyl alpha-galactosidase.
4. application as claimed in claim 3, which is characterized in that the step（1）In, nucleotide sequence such as SEQ ID NO.1
Shown in gene order with bacteroides fragilis（Bacteroides fragilis）The genomic DNA of NCTC9343 is template, warp
PCR amplification obtains, and PCR primer nucleotide sequence is as follows：
Primers F：5'-GGCCGCTAGCCAAAACACAAATACTCCCAT-3’
Nhe I
Primer R：5'-GGCCCTCGAGTTAACGACCTTTGATGATTT-3’
Xho I
PCR system is as follows, 50 μ L of total volume：
10 × PCR buffer solutions, 5 μ L, 2.5mmol/L dNTP, 4 μ L, each 1 μ L of 20 μm of ol/L primers, 50 μ g/mL templates, 2 μ L, 5U/ μ
L TaKaRa LA Taq 1 μ L, 36 μ L of ultra-pure water；
PCR amplification program is as follows：
95 DEG C of pre-degenerations 5 minutes；I.e. 95 DEG C of 30 cycles of reaction are denaturalized 30 seconds, and 48 DEG C are annealed 30 seconds, and 72 DEG C extend 2 minutes；72
DEG C extend 10 minutes.
5. application as claimed in claim 3, which is characterized in that the step（2）In, expanding condition of culture is：37℃,150
~180r/min is cultivated to OD600It is 0.4~0.6.
6. application as claimed in claim 3, which is characterized in that the step（2）In, expand culture medium and is trained for RM+ glucose
Nutrient solution, preparation method are as follows：
Sour 18~22g/L of hydrolyzed casein, glucose 0.18~0.22g/L, 0.09~0.10 g/L MgCl2, percent by volume 10%
10 × M9 salting liquids；
10 × M9 salting liquids are prepared as follows：
Na2HPO460g/L, KH2PO4 30g/L、NaCl 5g/L、NH410g/L, pH7.4,121 DEG C of Cl sterilizes 20 minutes, cooling
The vitamin B1 of filtration sterilization is added afterwards to final concentration 0.3g/L.
7. application as claimed in claim 6, which is characterized in that the expansion culture medium further includes a concentration of 40~60 μ g/mL
Ampicillin.
8. application as claimed in claim 3, which is characterized in that the step（2）In, Fiber differentiation be expand cultivate after,
L-arabinose is added into culture solution, it is 0.1%~0.3% to make the mass concentration of L-arabinose in culture solution, 25~28
DEG C, 100r/min continues to be incubated overnight.
9. application as claimed in claim 2, which is characterized in that in the reaction system, p-nitrophenyl-α-D- galactosides are anti-
It is 400~500mM to answer a concentration of 15~20mM, lactose reaction density, and pH is 4.0~5.0.
10. application as claimed in claim 2, which is characterized in that the reaction system by a concentration of 100mM acetate buffer
Liquid is prepared, pH 4.5.
11. application as claimed in claim 2, which is characterized in that the purifying is purified using column chromatography.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778928A (en) * | 2005-10-11 | 2006-05-31 | 山东大学 | Transglycosyl alpha-galactoglucosidezyme gene |
| CN102154411A (en) * | 2011-01-07 | 2011-08-17 | 山东大学 | Method for preparing Globotriose oligosaccharide |
| CN103789287A (en) * | 2014-01-20 | 2014-05-14 | 云南师范大学 | Alpha-galactosidase AgaAJB07 with transglycosylation activity and gene, recombinant carrier and recombinant strain thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778928A (en) * | 2005-10-11 | 2006-05-31 | 山东大学 | Transglycosyl alpha-galactoglucosidezyme gene |
| CN102154411A (en) * | 2011-01-07 | 2011-08-17 | 山东大学 | Method for preparing Globotriose oligosaccharide |
| CN103789287A (en) * | 2014-01-20 | 2014-05-14 | 云南师范大学 | Alpha-galactosidase AgaAJB07 with transglycosylation activity and gene, recombinant carrier and recombinant strain thereof |
Non-Patent Citations (1)
| Title |
|---|
| Bacteroides fragilis NCTC 9343, complete genome;Cerdeno-Tarraga,A.M.et al.;《GenBank: CR626927.1》;20150206;全文 * |
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