CN102220301A - Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof - Google Patents
Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof Download PDFInfo
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Abstract
The invention relates to an Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof. The invention provides an alpha-galactosidase AgaAJB13 derived from Sphingobium sp., a coding gene agaAJB13 for encoding the alpha-galactosidase AgaAJB13, a recombinant vector containing the gene agaAJB13 and a recombination strain containing the gene agaAJB13, wherein, the amino acid sequence of the alpha-galactosidase AgaAJB13 is shown in SEQ ID No.1. The alpha-galactosidase AgaAJB13 has the following properties that: the optimal PH is 5.0; the optimal temperature is 60 DEG C, the enzyme activity of more than 10% and the enzyme activity of more than 20% can be achieved respectively at the temperature of 10 DEG C and 20 DEG C; the activity of about 40% can be maintained after processing for 1h at the temperature of 37 DEG C by using a buffer with 0.1M and pH 11.0; and the alpha-galactosidase AgaAJB13has good thermal stability, good proteinase resistance and good hydrolysis to various natural supports, thus being capable of being used as a feed or food additive in fields of feed and food.
Description
Technical field
The present invention relates to gene engineering technology field, specifically a kind of alkali resistance low temperature alpha-galactosidase A gaAJB13 and gene thereof.
Background technology
Alpha-galactosidase be melibiose (1,6-α-d-galactoside galactohydrolase; α-galactosidase; Melibiase; EC 3.2.1.22), the terminal irreducibility D-semi-lactosi of α-connection in the removable different substrates comprises saccharan substrates such as oligose substrates such as melibiose, raffinose, stachyose and verbascose and polygalactomannan.These polysaccharide extensively are present in food and the feedstuff raw material, particularly in the seeds of leguminous crops, as (Karr-Lilienthal et al. Livest Prod Sci, 2005,97:1 – 12.) such as dregs of beans, cotton dregs and the dish dregs of rice.
Alpha-galactosidase can be applicable in feed, food, papermaking and the medical industry.In the feed industry, the alpha galactosides zymin can promote the digestion of nutritive substance and elimination or reduce the side effect of the antinutritional factor of feed in forming oligose such as () raffinoses to nutrient digestion, thereby improved the production performance of animal, reduced feed cost; In grocery trade, the alpha galactosides zymin can reduce the content in soymilk such as raffinose, increases human absorption to beans nutrition; Alpha-galactosidase also can improve (Cao et al. Appl Microbiol Biotechnol, 2009,83:875 – 884.) such as the bleaching effect of paper and treatment Fabry diseases.
Alpha-galactosidase extensively is present in bacterium, actinomycetes, fungi and the plant.According to amino acid sequence similarity, alpha-galactosidase is divided into glycoside hydrolase the 4th, 27,36 and 57 families, and most alpha-galactosidases belong to glycoside hydrolase the 27th and 36 families.The alpha-galactosidase majority in eukaryote source belongs to 27 families, and prokaryotic organism source alpha-galactosidase substantially all belongs to 36 families (Finn et al. Nucleic Acids Res, 2008,36:D281 – D288.).But, temperature or high temperature enzyme and acid resistance or the enzyme of anti-neutral pH the during the present alpha-galactosidase of reporting mostly is, (20 ℃ of the 0 –) alpha-galactosidase that has alkali resistance and low temperature active was not simultaneously also reported.
Summary of the invention
The purpose of this invention is to provide a kind of alkali resistance low temperature alpha-galactosidase.
A further object of the present invention provides the gene of the above-mentioned alpha-galactosidase of coding.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another object of the present invention provides the recombinant bacterial strain that comprises said gene.
Alpha-galactosidase A gaAJB13 of the present invention can derive from Sphingol single-cell (
SphingobiumSp.), as
Sphingobium estrogenivoransATCC BAA-1367.The aminoacid sequence of AgaAJB13 is shown in SEQ ID NO. 1.
This enzyme contains 738 amino acid altogether, wherein 23 signal peptide sequences " MVMRRWGAALAAATMLAAAPAHA " (SEQ ID NO. 2) that amino acid is its prediction of N end.
Therefore, the theoretical molecular of sophisticated alpha-galactosidase A gaAJB13 is 80.0kDa, and its aminoacid sequence is shown in SEQ ID NO. 3.
The optimum pH of alpha-galactosidase A gaAJB13 of the present invention is 5.0, keeps the enzymic activity more than 80% in the scope of pH4.5 – 6.0; Damping fluid through pH4.0 – 11.0 is handled 1h, and enzyme is lived residue more than 35%; Optimum temperuture is 60 ℃, lives at 10 ℃ and the 20 ℃ enzymes that have respectively more than 10% and 20%, has the low-temperature catalyzed characteristic of cold-adapted enzyme; Transformation period under 37 ℃ and 60 ℃〉60min, have good thermostability; Hydrolyzable dregs of beans, cotton dregs and raffinose; Behind trypsinase and Proteinase K processing 1h, AgaAJB13 still can keep 102.1% and 114.0% enzyme work respectively.
The invention provides the gene of the above-mentioned alpha-galactosidase of coding
AgaAJB13, this gene order is shown in SEQ ID NO. 4.
The structural dna sequence analytical results shows, alpha-galactosidase gene
AgaAJB13The nucleotide sequence of coded signal peptide is shown in SEQ ID NO. 5.
Alpha-galactosidase gene
AgaAJB13The nucleotide sequence of encoding mature peptide is shown in SEQ ID NO. 6.
The method separating clone of the present invention by PCR the encoding gene of alpha-galactosidase A gaAJB13
AgaAJB13, its total length 2217bp, initiation codon are ATG, termination codon is TAG.Through BLAST comparison, this alpha-galactosidase gene
AgaAJB13Among amino acid sequence coded and the GenBank
AcidobacteriumSp. the potential alpha-galactosidase (EFI56085) in MP5ACTX8 source has the highest consistence, is 59.2%; Active with conclusive evidence
Lichtheimia corymbiferaThe consistence of IFO 8084 source alpha-galactosidases (AAF68953) is 40.1%.Illustrate that alpha-galactosidase A gaAJB13 is a kind of new alpha-galactosidase.
The present invention also provides and has comprised above-mentioned alpha-galactosidase gene
AgaAJB13Recombinant vectors, be preferably pET-
AgaAJB13Alpha-galactosidase gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, its nucleotide sequence is connected with expression regulation sequence.As the most preferred embodiment of the present invention, alpha-galactosidase gene of the present invention is inserted into plasmid pET-28a(+) on
EcoRI and
HindBetween the III restriction enzyme site, obtain expression of recombinant e. coli plasmid pET-
AgaAJB13
The present invention also provides and has comprised above-mentioned alpha-galactosidase gene
AgaAJB13Recombinant bacterial strain, preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably recombinant bacterial strain
BL21 (DE3)/
AgaAJB13
The method for preparing alpha-galactosidase A gaAJB13 of the present invention is carried out according to the following steps:
1), gets recombinant bacterial strain with above-mentioned recombinant vectors transformed host cell;
2) cultivate recombinant bacterial strain, induce the reorganization alpha-galactosidase to express;
3) reclaim the also expressed alpha-galactosidase A gaAJB13 of purifying.
Wherein, preferred described host cell is a Bacillus coli cells, preferably with expression of recombinant e. coli plasmid transformation escherichia coli cell BL21(DE3), obtain recombinant bacterial strain
BL21 (DE3)/
AgaAJB13
The invention provides a new alpha-galactosidase gene, the alpha-galactosidase optimal pH 5.0 of its coding; Live at the enzyme that has respectively under 10 ℃ and 20 ℃ more than 10% and 20%; Handle 1h for 37 ℃ through 0.1M pH11.0 damping fluid, still can keep about 40% activity, the operative temperature scope is wider, good thermostability and protease resistant; The ability of the various natural substrates of hydrolysis can be widely used in industries such as feed and food preferably.
Description of drawings
Fig. 1: the SDS-PAGE at the reorganization alpha-galactosidase of expression in escherichia coli analyzes, wherein, and M: low molecular weight protein Marker; 1: the reorganization alpha-galactosidase of purifying.
Fig. 2: the optimal pH of reorganization alpha-galactosidase.
Fig. 3: the pH stability of reorganization alpha-galactosidase.
Fig. 4: the optimum temperuture of reorganization alpha-galactosidase.
Fig. 5: the thermostability of reorganization alpha-galactosidase.
Embodiment
Test materials and reagent
1, bacterial strain and carrier: Sphingol single-cell (
SphingobiumSp.) with document report bacterial classification character, as
Sphingobium estrogenivoransATCC BAA-1367; Intestinal bacteria
Escherichia coliBL21(DE3) and expression vector pET-28a(+) can purchase company in Novagen.
2, enzyme and other biochemical reagents: restriction enzyme, archaeal dna polymerase, ligase enzyme and dNTP are available from TaKaRa company;
pNPG(
p-nitrophenyl-α-d-galactopyranoside) available from Sigma company; Other all is domestic reagent (all can buy from common biochemical reagents company and obtain).
3, substratum:
The LB substratum: Peptone 10g, Yeast extract 5g, NaCl 10g, adding distil water are to 1000ml, and pH is (being about 7) naturally.Solid medium adds 2.0%(w/v on this basis) agar.
Illustrate: make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
Embodiment 1: alpha-galactosidase gene
AgaAJB13The clone
Extract the Sphingol single-cell genomic dna: with the bacterium liquid centrifuging and taking thalline of liquid culture 2d, add the 1mL N,O-Diacetylmuramidase, handle 60min for 37 ℃, add lysate again, 70 ℃ of water-bath cracking 60min, every the 10min mixing once, at 4 ℃ of centrifugal 5min of following 10000rpm.Get supernatant extrct foreigh protein removing in phenol/chloroform, get supernatant again and add the equal-volume Virahol, after room temperature leaves standstill 5min, 4 ℃ of centrifugal 10min of following 10000rpm.Abandon supernatant, precipitation is with 70% washing with alcohol twice, and vacuum-drying adds an amount of TE and dissolves, place-20 ℃ standby.
Degenerated primer GH36F and GH36R(table 1 have been synthesized in design according to the conserved sequence of glycoside hydrolase the 36th family ([F/L/V]-[L/V]-[L/M/V]-D-D-G-W-F and E-P-E-M-[V/I]-[N/S]-[P/E])).
With the total DNA of Sphingol single-cell is that template is carried out pcr amplification.The PCR reaction parameter is: 94 ℃ of sex change 5min; 94 ℃ of sex change 30sec then, 43 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 30 back 72 ℃ of insulation 10min of circulation.Obtain an about 172bp fragment, this fragment is reclaimed the back link to each other, send Beijing Liuhe Huada Genomics Technology Co., Ltd's Guangzhou Branch order-checking then with pMD 18-T carrier.
According to the nucleotide sequence that order-checking obtains, design each two of upstream and downstream TAIL-PCR Auele Specific Primers respectively: design direction is for needing the zone of ignorance direction of amplification, and the Position Design of sp2 is in the inboard of sp1.Distance between per two primers does not have strict regulation, and primer length is generally 20 – 30nt, and annealing temperature is 70 ℃ of 65 –.And with them difference called after usp1 and usp2(upstream Auele Specific Primer) and dsp1 and dsp2(downstream Auele Specific Primer; Table 1).
Obtain the flanking sequence of known sequence by TAIL-PCR, amplified production send Beijing Liuhe Huada Genomics Technology Co., Ltd's Guangzhou Branch order-checking.Sequencing result splices mutually with the known sequence fragment, obtains alpha-galactosidase gene
AgaAJB13, this gene order is shown in SEQ ID NO. 4.
Embodiment 2: the preparation of reorganization alpha-galactosidase
With expression vector pET-28a(+) carry out double digestion (
EcoRI and
HindIII), will encode the simultaneously gene of alpha-galactosidase
AgaAJB13Carry out double digestion (
EcoRI and
HindIII), the alpha-galactosidase that above-mentioned enzyme is cut
AgaAJB13With expression vector pET-28a(+) be connected, obtain to contain alpha-galactosidase gene
AgaAJB13Recombinant plasmid pET-
AgaAJB13And transformed into escherichia coli BL21(DE3), obtain recombinant escherichia coli strain
BL21 (DE3)/
AgaAJB13
Get and contain recombinant plasmid pET-
AgaAJB13 E. coliBL21(DE3) bacterial strain and only contain pET-28a(+) empty plasmid
E. coliBL21(DE3) bacterial strain, the inoculum size with 0.1% are inoculated in LB(and contain 50 μ g/mL Kan) in the nutrient solution, 37 ℃ of quick oscillation 16h.Then this activatory bacterium liquid being inoculated into fresh LB(with 1% inoculum size and containing 50 μ g/mL Kan) in the nutrient solution, quick oscillation is cultivated about 2 – 3h(OD
600Reach 0.6 – 1.0) after, the IPTG that adds final concentration 0.7mM induces, and continues the about 20h of shaking culture or 26 ℃ of about 8h of shaking culture in 20 ℃.The centrifugal 5min of 12000rpm collects thalline.Behind an amount of pH7.0 Tris-Hcl damping fluid suspension thalline, ultrasonic disruption thalline under the low temperature water-bath.,, draw supernatant and use Nickel-NTA Agarose purifying target protein behind the centrifugal 10min of 000rpm through 13 with spissated enzyme liquid just in the upper eye lid.SDS-PAGE result (Fig. 1) shows that the reorganization alpha-galactosidase has obtained expression in intestinal bacteria, be single band behind Nickel-NTA Agarose purifying.
Embodiment 3: the activation analysis of reorganization alpha-galactosidase
Activity determination method adopts
pThe NPG method.Will
pNPG is dissolved in the 0.1M damping fluid, and making its final concentration is 2mM.Reaction system contains an amount of enzyme liquid of 50 μ L, the 2mM substrate of 450 μ L.Substrate behind the preheating 5min, adds enzyme liquid and reacts 10min again under temperature of reaction, adds 1.5mL 1M Na then
2CO
3Termination reaction is cooled to measure under the 405nm wavelength after the room temperature and discharges
pNP.1 enzyme unit alive (U) is defined as per minute and decomposes
pNPG produces 1 μ mol
pThe enzyme amount that NP is required.Activity determination method to substrate raffinose, dregs of beans and cotton dregs adopts 3, and 5-dinitrosalicylic acid (DNS) method: substrate is dissolved in the 0.1M damping fluid, and making its final concentration is 0.5%(w/v); Reaction system contains an amount of enzyme liquid of 100 μ L, 900 μ L substrates; Substrate reacts 120min after adding enzyme liquid under temperature of reaction behind the preheating 5min again, adds 1.5mL DNS termination reaction then, and boiling water boils 5min, measures the OD value after being cooled to room temperature under the 540nm wavelength.1 enzyme unit alive (U) is defined as the required enzyme amount of per minute bottom exploded deposits yields 1 μ mol semi-lactosi under given condition.
Embodiment 4: the property testing of reorganization alpha-galactosidase A gaAJB13
1, the measuring method of the optimal pH of reorganization alpha-galactosidase A gaAJB13 and pH stability is as follows:
The optimal pH of enzyme is measured: the alpha-galactosidase A gaAJB13 of embodiment 2 purifying is carried out enzymatic reaction under the damping fluid of 37 ℃ and pH3.0 – 8.0.The pH of enzyme stability is measured: the enzyme liquid of purifying is placed the 0.1M damping fluid of pH2.0 – 12.0, handle down more than the 1h at 37 ℃, carry out enzymatic reaction then under pH5.0 and 37 ℃, with untreated enzyme liquid in contrast.Damping fluid is: 0.1M McIlvaine buffer(pH2.0 – 8.0) and 0.1M glycine-NaOH(pH9.0 – 12.0).With
pNPG is a substrate, reaction 10min, the zymologic property of the AgaAJB13 of mensuration purifying.The result shows: the optimal pH of AgaAJB13 is 5.0, keeps the enzymic activity (Fig. 2) more than 80% in the scope of pH4.5 – 6.0; Damping fluid through pH4.0 – 11.0 is handled 1h, and enzyme is lived residue more than ~ 40% or 40% (Fig. 3).
2, optimum temperuture and the thermal stability determination method of reorganization alpha-galactosidase A gaAJB13 are as follows:
The optimum temperuture of enzyme is measured: in the damping fluid of pH5.0, carry out enzymatic reaction under 70 ℃ of 0 –.The thermal stability determination of enzyme: after placing the temperature (37 ℃, 60 ℃ or 70 ℃) of setting to handle 0 – 60min the enzyme liquid of same enzyme amount, under pH5.0 and 37 ℃, carry out enzymatic reaction, in contrast with untreated enzyme liquid.With
pNPG is a substrate, reaction 10min, the zymologic property of the AgaAJB13 of mensuration purifying.The result shows: the optimum temperuture of AgaAJB13 is 60 ℃, at 10 ℃ and the 20 ℃ enzymes (Fig. 4) alive that have respectively more than 10% and 20%; The transformation period of 60 ℃ of following AgaAJB13〉60min, very fast inactivation (Fig. 5) under 70 ℃.
3, the kinetic parameter measuring method of reorganization alpha-galactosidase A gaAJB13 is as follows:
The kinetic parameter first order reaction timing of enzyme: under pH5.0 and 60 ℃, with 0.5mM
pNPG is a substrate, and termination reaction and measure enzymic activity in 1 – 30min of enzymatic reaction calculates the ratio in enzymic activity and reaction times successively, if this ratio keeps stable within a certain period of time, then this time is the first order reaction time.With 0.05 – 0.5mM
pNPG is a substrate,, measures according to the Lineweaver-Burk method under the time in pH5.0,60 ℃ and first order reaction
Km,
VmaxWith
KcatAfter measured, under 60 ℃ of pH5.0 conditions, AgaAJB13 is right
pNPG's
K m,
V MaxWith
k CatBe respectively 2.75 mM
1, 1250.00 μ mol min
1Mg
1With 1792.08 s
1
4, different metal ion and chemical reagent to the AgaAJB13 enzyme live to influence measuring method as follows:
In enzymatic reaction system, add metal ion and the chemical reagent of 10mM, study its influence enzymic activity.Under 37 ℃, pH5.0 condition, measure enzymic activity.Result's (table 2) shows, the Ag of 10mM
+And Hg
2+Can suppress AgaAJB13 fully; SDS is to the inhibition of AgaAJB13 strong (residual enzyme lives ~ 7%); Fe
2+To the inhibition of AgaAJB13 weak (residual enzyme lives ~ 70%); Ca
2+And Pb
2+Can make the enzyme work of AgaAJB13 improve about 0.2 times and about 0.3 times respectively; All the other metal ions and chemical reagent are very little to the influence of AgaAJB13.
Table 2. metal ion and chemical reagent are to the influence of reorganization alpha-galactosidase A gaAJB13 vigor
5, the protease inhibitor ability of alpha-galactosidase A gaAJB13
The protease resistant of enzyme: with being equivalent to the trypsin pH7.5 of recombinase 10 times (w/w)) and Proteinase K (pH7.5) at 37 ℃ to recombinase processing 1h, under pH5.0 and 37 ℃, carry out enzymatic reaction then, with in the corresponding pH damping fluid of proteolytic enzyme but the enzyme liquid that does not add proteolytic enzyme in contrast.Behind trypsinase and Proteinase K processing 1h, AgaAJB13 still can keep 102.1% and 114.0% enzyme work respectively.
6, alpha-galactosidase A gaAJB13 is to the degraded of natural substrate
Under 37 ℃ of pH5.0 conditions, AgaAJB13 is to 1.0%(w/v) dregs of beans and the ratio work of cotton dregs be respectively 6.99 ± 0.21 and 2.80 ± 0.21 U mg
1, the ratio work of the raffinose to 0.5% is 1.67 ± 0.01 U mg
1
Sequence table
＜110〉Yunnan Normal University
＜120〉a kind of alkali resistance low temperature alpha-galactosidase A gaAJB13 and gene
<160> 6
<170> PatentIn?version?3.3
<210> 1
<211> 738
<212> PRT
＜213〉Sphingol single-cell (Sphingobium sp.)
<400> 1
Met?Val?Met?Arg?Arg?Trp?Gly?Ala?Ala?Leu?Ala?Ala?Ala?Thr?Met?Leu
1 5 10 15
Ala?Ala?Ala?Pro?Ala?His?Ala?Ser?Ala?Gly?Tyr?Asp?Ala?Lys?Thr?Arg
20 25 30
Met?Phe?Arg?Leu?Asp?Gly?Gly?Gly?Thr?Thr?Tyr?Ala?Phe?Gly?Val?Thr
35 40 45
Asp?Asp?Gly?Tyr?Leu?Gln?Ala?Ala?Tyr?Trp?Gly?Gly?Arg?Leu?Gly?Ala
50 55 60
Asp?Asp?Pro?Ile?Arg?Leu?Thr?Lys?Ala?Gln?Gly?Leu?Ser?Gly?Phe?Asp
65 70 75 80
Leu?Val?Asn?Ser?Ile?Leu?Pro?Gln?Glu?Phe?Pro?Gly?Gln?Gly?Ala?Gly
85 90 95
Leu?Tyr?Thr?Glu?Pro?Ala?Leu?Lys?Val?Ala?Trp?Pro?Asp?Gly?Asn?Arg
100 105 110
Asp?Leu?Val?Leu?Lys?Tyr?Val?Ser?His?Lys?Met?Ser?Arg?Asp?His?Val
115 120 125
Glu?Ile?Val?Leu?Lys?Asp?Ile?Glu?Arg?Pro?Leu?Phe?Val?Thr?Leu?Asp
130 135 140
Tyr?Ser?Ile?Asp?Pro?Asp?Thr?Gly?Val?Val?Gly?Arg?Ser?Ala?Arg?Ile
145 150 155 160
Glu?Asn?Arg?Ser?Asp?Thr?Asp?Val?Arg?Ile?Asp?Gln?Ala?Glu?Ala?Gly
165 170 175
Ala?Leu?Thr?Leu?Pro?Val?Ala?His?Asp?Tyr?Arg?Leu?His?Tyr?Leu?Thr
180 185 190
Gly?Arg?Trp?Ala?Ala?Glu?Trp?Thr?Leu?Gln?Asp?Arg?Pro?Leu?Thr?Pro
195 200 205
Gly?Ala?Thr?Val?Leu?Glu?Ser?Arg?Arg?Gly?Ser?Thr?Gly?Ser?Glu?Asn
210 215 220
Asn?Pro?Trp?Phe?Ala?Ile?Thr?Arg?Asp?His?Asp?Ala?Gly?Glu?Glu?Tyr
225 230 235 240
Gly?Pro?Val?Trp?Phe?Gly?Ala?Leu?Ala?Trp?Ser?Gly?Ser?Trp?Arg?Ile
245 250 255
Thr?Val?Asp?Gln?Asp?Pro?Ala?Gly?Glu?Val?Arg?Val?Val?Gly?Gly?Phe
260 265 270
Asn?Pro?Phe?Asp?Phe?Ala?Tyr?Arg?Leu?Lys?Pro?Gly?Glu?Ser?Leu?Asp
275 280 285
Thr?Pro?Thr?Phe?Tyr?Ala?Gly?Tyr?Ser?Asp?His?Gly?Met?Gly?Gly?Ala
290 295 300
Ser?Arg?Leu?Leu?His?Arg?Phe?Glu?Arg?Asp?Thr?Ile?Leu?Pro?His?Asp
305 310 315 320
Ala?Asp?Gly?Lys?Leu?Pro?Leu?Arg?Pro?Val?Leu?Tyr?Asn?Ser?Trp?Glu
325 330 335
Ala?Thr?Gly?Phe?Asp?Val?Asp?Glu?Ala?Gly?Gln?Ile?Ala?Leu?Ala?Glu
340 345 350
Lys?Ala?Ala?Lys?Ile?Gly?Val?Glu?Arg?Phe?Val?Met?Asp?Asp?Gly?Trp
355 360 365
Phe?Gly?Ala?Arg?Asn?Asp?Asp?His?Ala?Gly?Leu?Gly?Asp?Trp?Thr?Val
370 375 380
Asn?Arg?Thr?Lys?Phe?Pro?Asn?Gly?Leu?Lys?Pro?Leu?Ile?Asp?Lys?Val
385 390 395 400
His?Gly?Leu?Gly?Met?Gln?Phe?Gly?Leu?Trp?Val?Glu?Pro?Glu?Met?Thr
405 410 415
Asn?Pro?Asp?Ser?Asp?Leu?Tyr?Arg?Ala?His?Pro?Asp?Trp?Val?Met?Asn
420 425 430
Tyr?Thr?Gly?Arg?Pro?Arg?Thr?Glu?Gly?Arg?Asn?Gln?Leu?Val?Leu?Asn
435 440 445
Leu?Ala?Arg?Thr?Asp?Val?Arg?Asp?Tyr?Ile?Phe?Lys?Val?Leu?Asp?Asp
450 455 460
Leu?Leu?Asp?Glu?Asn?Asp?Ile?Gln?Phe?Leu?Lys?Trp?Asp?Tyr?Asn?Arg
465 470 475 480
Asn?Trp?Ser?Glu?Pro?Gly?Trp?Pro?Glu?Ala?Asp?Val?Ala?Asp?Gln?Gln
485 490 495
Gln?Ile?Tyr?Val?Lys?Tyr?Val?Arg?Asn?Leu?Tyr?Trp?Ile?Ile?Asp?Lys
500 505 510
Leu?Arg?Ala?Arg?His?Pro?Lys?Leu?Glu?Ile?Glu?Ser?Cys?Ser?Gly?Gly
515 520 525
Gly?Gly?Arg?Val?Asp?Leu?Gly?Ile?Met?Ser?Arg?Thr?Asp?Glu?Val?Trp
530 535 540
Pro?Ser?Asp?Asn?Thr?Asp?Pro?Phe?Asp?Arg?Leu?Thr?Ile?Gln?Asn?Gly
545 550 555 560
Phe?Thr?Tyr?Ala?Tyr?Pro?Pro?Ala?Ala?Met?Met?Ala?Trp?Val?Thr?Ala
565 570 575
Ser?Pro?Asn?Trp?Val?Asn?Asn?Arg?Ala?Thr?Ser?Leu?Asp?Tyr?Arg?Phe
580 585 590
Leu?Ser?Ala?Met?Gln?Gly?Gly?Leu?Gly?Ile?Gly?Ala?Asp?Leu?Asn?Lys
595 600 605
Trp?Ser?Asp?Ala?Glu?Phe?Ala?Glu?Ala?Ser?Arg?Met?Val?Ala?Ala?Tyr
610 615 620
Lys?Arg?Val?Arg?Ala?Thr?Val?Gln?Gln?Gly?Asp?Leu?Tyr?Arg?Leu?Ile
625 630 635 640
Ile?Pro?Asn?Gly?Ile?Asp?Arg?Asp?Asp?Arg?Val?Ala?Asn?Leu?Ser?Val
645 650 655
Ser?Pro?Asp?Lys?Gln?Gln?Ala?Val?Leu?Phe?Ala?Phe?Leu?His?Ser?Ser
660 665 670
Gln?Glu?Leu?Asp?Arg?Leu?Ser?Ala?Ile?Arg?Leu?Arg?Gly?Leu?Ala?Pro
675 680 685
Lys?Lys?Asn?Tyr?Arg?Val?Ala?Arg?Ile?Asp?Gly?Arg?Pro?Leu?Ala?Asp
690 695 700
Asp?Thr?Pro?Ala?Lys?Ala?Ser?Gly?Ala?Tyr?Trp?Met?Ala?Arg?Gly?Ile
705 710 715 720
Asp?Val?Pro?Leu?Ile?Gly?Asp?Phe?Asp?Ala?Ala?Gly?Tyr?Ile?Phe?Gln
725 730 735
Ala?Ile
<210> 2
<211> 23
<212> PRT
＜213〉Sphingol single-cell (Sphingobium sp.)
<400> 2
Met?Val?Met?Arg?Arg?Trp?Gly?Ala?Ala?Leu?Ala?Ala?Ala?Thr?Met?Leu
1 5 10 15
Ala?Ala?Ala?Pro?Ala?His?Ala
20
<210> 3
<211> 715
<212> PRT
＜213〉Sphingol single-cell (Sphingobium sp.)
<400> 3
Ser?Ala?Gly?Tyr?Asp?Ala?Lys?Thr?Arg?Met?Phe?Arg?Leu?Asp?Gly?Gly
1 5 10 15
Gly?Thr?Thr?Tyr?Ala?Phe?Gly?Val?Thr?Asp?Asp?Gly?Tyr?Leu?Gln?Ala
20 25 30
Ala?Tyr?Trp?Gly?Gly?Arg?Leu?Gly?Ala?Asp?Asp?Pro?Ile?Arg?Leu?Thr
35 40 45
Lys?Ala?Gln?Gly?Leu?Ser?Gly?Phe?Asp?Leu?Val?Asn?Ser?Ile?Leu?Pro
50 55 60
Gln?Glu?Phe?Pro?Gly?Gln?Gly?Ala?Gly?Leu?Tyr?Thr?Glu?Pro?Ala?Leu
65 70 75 80
Lys?Val?Ala?Trp?Pro?Asp?Gly?Asn?Arg?Asp?Leu?Val?Leu?Lys?Tyr?Val
85 90 95
Ser?His?Lys?Met?Ser?Arg?Asp?His?Val?Glu?Ile?Val?Leu?Lys?Asp?Ile
100 105 110
Glu?Arg?Pro?Leu?Phe?Val?Thr?Leu?Asp?Tyr?Ser?Ile?Asp?Pro?Asp?Thr
115 120 125
Gly?Val?Val?Gly?Arg?Ser?Ala?Arg?Ile?Glu?Asn?Arg?Ser?Asp?Thr?Asp
130 135 140
Val?Arg?Ile?Asp?Gln?Ala?Glu?Ala?Gly?Ala?Leu?Thr?Leu?Pro?Val?Ala
145 150 155 160
His?Asp?Tyr?Arg?Leu?His?Tyr?Leu?Thr?Gly?Arg?Trp?Ala?Ala?Glu?Trp
165 170 175
Thr?Leu?Gln?Asp?Arg?Pro?Leu?Thr?Pro?Gly?Ala?Thr?Val?Leu?Glu?Ser
180 185 190
Arg?Arg?Gly?Ser?Thr?Gly?Ser?Glu?Asn?Asn?Pro?Trp?Phe?Ala?Ile?Thr
195 200 205
Arg?Asp?His?Asp?Ala?Gly?Glu?Glu?Tyr?Gly?Pro?Val?Trp?Phe?Gly?Ala
210 215 220
Leu?Ala?Trp?Ser?Gly?Ser?Trp?Arg?Ile?Thr?Val?Asp?Gln?Asp?Pro?Ala
225 230 235 240
Gly?Glu?Val?Arg?Val?Val?Gly?Gly?Phe?Asn?Pro?Phe?Asp?Phe?Ala?Tyr
245 250 255
Arg?Leu?Lys?Pro?Gly?Glu?Ser?Leu?Asp?Thr?Pro?Thr?Phe?Tyr?Ala?Gly
260 265 270
Tyr?Ser?Asp?His?Gly?Met?Gly?Gly?Ala?Ser?Arg?Leu?Leu?His?Arg?Phe
275 280 285
Glu?Arg?Asp?Thr?Ile?Leu?Pro?His?Asp?Ala?Asp?Gly?Lys?Leu?Pro?Leu
290 295 300
Arg?Pro?Val?Leu?Tyr?Asn?Ser?Trp?Glu?Ala?Thr?Gly?Phe?Asp?Val?Asp
305 310 315 320
Glu?Ala?Gly?Gln?Ile?Ala?Leu?Ala?Glu?Lys?Ala?Ala?Lys?Ile?Gly?Val
325 330 335
Glu?Arg?Phe?Val?Met?Asp?Asp?Gly?Trp?Phe?Gly?Ala?Arg?Asn?Asp?Asp
340 345 350
His?Ala?Gly?Leu?Gly?Asp?Trp?Thr?Val?Asn?Arg?Thr?Lys?Phe?Pro?Asn
355 360 365
Gly?Leu?Lys?Pro?Leu?Ile?Asp?Lys?Val?His?Gly?Leu?Gly?Met?Gln?Phe
370 375 380
Gly?Leu?Trp?Val?Glu?Pro?Glu?Met?Thr?Asn?Pro?Asp?Ser?Asp?Leu?Tyr
385 390 395 400
Arg?Ala?His?Pro?Asp?Trp?Val?Met?Asn?Tyr?Thr?Gly?Arg?Pro?Arg?Thr
405 410 415
Glu?Gly?Arg?Asn?Gln?Leu?Val?Leu?Asn?Leu?Ala?Arg?Thr?Asp?Val?Arg
420 425 430
Asp?Tyr?Ile?Phe?Lys?Val?Leu?Asp?Asp?Leu?Leu?Asp?Glu?Asn?Asp?Ile
435 440 445
Gln?Phe?Leu?Lys?Trp?Asp?Tyr?Asn?Arg?Asn?Trp?Ser?Glu?Pro?Gly?Trp
450 455 460
Pro?Glu?Ala?Asp?Val?Ala?Asp?Gln?Gln?Gln?Ile?Tyr?Val?Lys?Tyr?Val
465 470 475 480
Arg?Asn?Leu?Tyr?Trp?Ile?Ile?Asp?Lys?Leu?Arg?Ala?Arg?His?Pro?Lys
485 490 495
Leu?Glu?Ile?Glu?Ser?Cys?Ser?Gly?Gly?Gly?Gly?Arg?Val?Asp?Leu?Gly
500 505 510
Ile?Met?Ser?Arg?Thr?Asp?Glu?Val?Trp?Pro?Ser?Asp?Asn?Thr?Asp?Pro
515 520 525
Phe?Asp?Arg?Leu?Thr?Ile?Gln?Asn?Gly?Phe?Thr?Tyr?Ala?Tyr?Pro?Pro
530 535 540
Ala?Ala?Met?Met?Ala?Trp?Val?Thr?Ala?Ser?Pro?Asn?Trp?Val?Asn?Asn
545 550 555 560
Arg?Ala?Thr?Ser?Leu?Asp?Tyr?Arg?Phe?Leu?Ser?Ala?Met?Gln?Gly?Gly
565 570 575
Leu?Gly?Ile?Gly?Ala?Asp?Leu?Asn?Lys?Trp?Ser?Asp?Ala?Glu?Phe?Ala
580 585 590
Glu?Ala?Ser?Arg?Met?Val?Ala?Ala?Tyr?Lys?Arg?Val?Arg?Ala?Thr?Val
595 600 605
Gln?Gln?Gly?Asp?Leu?Tyr?Arg?Leu?Ile?Ile?Pro?Asn?Gly?Ile?Asp?Arg
610 615 620
Asp?Asp?Arg?Val?Ala?Asn?Leu?Ser?Val?Ser?Pro?Asp?Lys?Gln?Gln?Ala
625 630 635 640
Val?Leu?Phe?Ala?Phe?Leu?His?Ser?Ser?Gln?Glu?Leu?Asp?Arg?Leu?Ser
645 650 655
Ala?Ile?Arg?Leu?Arg?Gly?Leu?Ala?Pro?Lys?Lys?Asn?Tyr?Arg?Val?Ala
660 665 670
Arg?Ile?Asp?Gly?Arg?Pro?Leu?Ala?Asp?Asp?Thr?Pro?Ala?Lys?Ala?Ser
675 680 685
Gly?Ala?Tyr?Trp?Met?Ala?Arg?Gly?Ile?Asp?Val?Pro?Leu?Ile?Gly?Asp
690 695 700
Phe?Asp?Ala?Ala?Gly?Tyr?Ile?Phe?Gln?Ala?Ile
705 710 715
<210> 4
<211> 2217
<212> DNA
＜213〉Sphingol single-cell (Sphingobium sp.)
<400> 4
atggtgatga?ggcgatgggg?ggcagccctt?gcggccgcga?cgatgctggc?ggcggcgccg 60
gcgcatgcgt?cggcgggcta?cgacgcgaag?acccgcatgt?tccggctcga?cggcggcggc 120
accacctacg?cgttcggggt?gaccgacgac?ggctatctcc?aggccgccta?ttggggcggg 180
cgactcggcg?ccgacgaccc?gatccggctg?accaaggcgc?aagggctgag?cggcttcgat 240
ctggtcaact?cgatcctgcc?gcaggaattt?cccgggcaag?gcgccggcct?ctataccgag 300
ccggcgctca?aggtcgcctg?gcccgacggc?aaccgcgatc?tcgtgctcaa?atacgtctcg 360
cacaagatgt?ccagggacca?tgttgagatc?gtgctcaagg?atatcgagcg?accgttgttc 420
gtcacgctcg?actacagcat?cgatcccgat?accggcgtgg?tcggccgctc?ggcgcgtatc 480
gaaaaccgca?gcgataccga?cgtgcggatc?gatcaggccg?aggcgggcgc?gctcaccctg 540
cccgtcgcgc?acgattaccg?gctgcactat?ctcaccggcc?gctgggccgc?cgagtggacg 600
ctgcaggatc?gcccgctgac?cccgggcgcg?accgtcctcg?aaagccgccg?cggctcgacc 660
ggctcggaaa?acaacccctg?gttcgcgatc?acccgcgatc?acgatgccgg?cgaggagtac 720
gggcccgtct?ggttcggcgc?gctggcgtgg?agcggatcgt?ggcggatcac?ggtcgaccag 780
gatccggccg?gcgaggtccg?cgtcgtcggc?gggttcaacc?cgttcgactt?cgcctatcgc 840
ctcaagcccg?gcgaatcgct?cgacacgccg?accttctacg?ccggctattc?ggatcacggc 900
atgggcggcg?cctcgcggct?gctccaccgc?ttcgagcgcg?acacgatcct?gccccacgat 960
gccgacggca?agctgccgct?gcgccccgtc?ctctacaaca?gctgggaagc?gaccgggttc 1020
gatgtcgacg?aggccggcca?gatcgcgctt?gccgaaaagg?cggcgaagat?tggcgtcgag 1080
cgcttcgtga?tggacgacgg?ctggttcggc?gcgcgcaacg?acgatcatgc?cgggctcggc 1140
gactggaccg?tcaaccgcac?caaattcccc?aacggcctca?aaccgctgat?cgacaaggtc 1200
cacggcctcg?gcatgcagtt?cgggctgtgg?gtcgagcccg?agatgaccaa?tcccgacagc 1260
gatctctatc?gcgcgcatcc?cgattgggtg?atgaactata?ccggccgccc?gcgcaccgag 1320
gggcgtaacc?agctcgtcct?caatctcgcg?cgaaccgacg?tgcgcgatta?catcttcaag 1380
gtgctcgacg?acctgctcga?cgagaacgac?atccagttcc?tcaaatggga?ttacaaccgc 1440
aactggagcg?agcccggctg?gcccgaggcc?gatgtcgccg?accagcagca?gatctacgtc 1500
aaatacgtcc?gcaacctcta?ttggatcatc?gacaagctgc?gcgccaggca?tcccaagctc 1560
gagatcgaat?cgtgctcggg?cggcggcggc?cgcgtcgatc?tcggcattat?gagccgcacc 1620
gacgaggtgt?ggccgtcgga?caataccgat?ccgttcgatc?ggctgacgat?ccagaacggc 1680
tttacttacg?cctatccgcc?ggccgcgatg?atggcgtggg?tgacggcgtc?gcccaattgg 1740
gtcaataatc?gcgctacctc?gctcgattat?cgcttcctgt?cggcgatgca?aggcgggctc 1800
ggtattggcg?ccgacctcaa?taaatggagc?gatgcagaat?ttgcggaggc?gagtcgcatg 1860
gtggcggcct?ataagcgtgt?ccgagcgacg?gtgcagcaag?gcgacctgta?tcggttgatt 1920
atcccgaacg?gaatcgatcg?tgacgaccgc?gtcgccaatc?tctcggtatc?tccagacaag 1980
cagcaggcgg?tgctgttcgc?gtttctgcac?agcagccagg?agctcgatcg?gctttctgct 2040
atccgactgc?gcgggctcgc?tcctaagaag?aactaccgcg?tcgcccggat?cgatggccgc 2100
ccgctggccg?acgacacccc?agctaaggcg?agcggcgctt?attggatggc?gcgtggcatc 2160
gacgttccat?taatcggcga?cttcgacgcc?gctggctata?tctttcaggc?catctag 2217
<210> 5
<211> 69
<212> DNA
＜213〉Sphingol single-cell (Sphingobium sp.)
<400> 5
atggtgatga?ggcgatgggg?ggcagccctt?gcggccgcga?cgatgctggc?ggcggcgccg 60
gcgcatgcg 69
<210> 6
<211> 2145
<212> DNA
＜213〉Sphingol single-cell (Sphingobium sp.)
<400> 6
tcggcgggct?acgacgcgaa?gacccgcatg?ttccggctcg?acggcggcgg?caccacctac 60
gcgttcgggg?tgaccgacga?cggctatctc?caggccgcct?attggggcgg?gcgactcggc 120
gccgacgacc?cgatccggct?gaccaaggcg?caagggctga?gcggcttcga?tctggtcaac 180
tcgatcctgc?cgcaggaatt?tcccgggcaa?ggcgccggcc?tctataccga?gccggcgctc 240
aaggtcgcct?ggcccgacgg?caaccgcgat?ctcgtgctca?aatacgtctc?gcacaagatg 300
tccagggacc?atgttgagat?cgtgctcaag?gatatcgagc?gaccgttgtt?cgtcacgctc 360
gactacagca?tcgatcccga?taccggcgtg?gtcggccgct?cggcgcgtat?cgaaaaccgc 420
agcgataccg?acgtgcggat?cgatcaggcc?gaggcgggcg?cgctcaccct?gcccgtcgcg 480
cacgattacc?ggctgcacta?tctcaccggc?cgctgggccg?ccgagtggac?gctgcaggat 540
cgcccgctga?ccccgggcgc?gaccgtcctc?gaaagccgcc?gcggctcgac?cggctcggaa 600
aacaacccct?ggttcgcgat?cacccgcgat?cacgatgccg?gcgaggagta?cgggcccgtc 660
tggttcggcg?cgctggcgtg?gagcggatcg?tggcggatca?cggtcgacca?ggatccggcc 720
ggcgaggtcc?gcgtcgtcgg?cgggttcaac?ccgttcgact?tcgcctatcg?cctcaagccc 780
ggcgaatcgc?tcgacacgcc?gaccttctac?gccggctatt?cggatcacgg?catgggcggc 840
gcctcgcggc?tgctccaccg?cttcgagcgc?gacacgatcc?tgccccacga?tgccgacggc 900
aagctgccgc?tgcgccccgt?cctctacaac?agctgggaag?cgaccgggtt?cgatgtcgac 960
gaggccggcc?agatcgcgct?tgccgaaaag?gcggcgaaga?ttggcgtcga?gcgcttcgtg 1020
atggacgacg?gctggttcgg?cgcgcgcaac?gacgatcatg?ccgggctcgg?cgactggacc 1080
gtcaaccgca?ccaaattccc?caacggcctc?aaaccgctga?tcgacaaggt?ccacggcctc 1140
ggcatgcagt?tcgggctgtg?ggtcgagccc?gagatgacca?atcccgacag?cgatctctat 1200
cgcgcgcatc?ccgattgggt?gatgaactat?accggccgcc?cgcgcaccga?ggggcgtaac 1260
cagctcgtcc?tcaatctcgc?gcgaaccgac?gtgcgcgatt?acatcttcaa?ggtgctcgac 1320
gacctgctcg?acgagaacga?catccagttc?ctcaaatggg?attacaaccg?caactggagc 1380
gagcccggct?ggcccgaggc?cgatgtcgcc?gaccagcagc?agatctacgt?caaatacgtc 1440
cgcaacctct?attggatcat?cgacaagctg?cgcgccaggc?atcccaagct?cgagatcgaa 1500
tcgtgctcgg?gcggcggcgg?ccgcgtcgat?ctcggcatta?tgagccgcac?cgacgaggtg 1560
tggccgtcgg?acaataccga?tccgttcgat?cggctgacga?tccagaacgg?ctttacttac 1620
gcctatccgc?cggccgcgat?gatggcgtgg?gtgacggcgt?cgcccaattg?ggtcaataat 1680
cgcgctacct?cgctcgatta?tcgcttcctg?tcggcgatgc?aaggcgggct?cggtattggc 1740
gccgacctca?ataaatggag?cgatgcagaa?tttgcggagg?cgagtcgcat?ggtggcggcc 1800
tataagcgtg?tccgagcgac?ggtgcagcaa?ggcgacctgt?atcggttgat?tatcccgaac 1860
ggaatcgatc?gtgacgaccg?cgtcgccaat?ctctcggtat?ctccagacaa?gcagcaggcg 1920
gtgctgttcg?cgtttctgca?cagcagccag?gagctcgatc?ggctttctgc?tatccgactg 1980
cgcgggctcg?ctcctaagaa?gaactaccgc?gtcgcccgga?tcgatggccg?cccgctggcc 2040
gacgacaccc?cagctaaggc?gagcggcgct?tattggatgg?cgcgtggcat?cgacgttcca 2100
ttaatcggcg?acttcgacgc?cgctggctat?atctttcagg?ccatc 2145
Claims (8)
1. an alkali resistance low temperature alpha-galactosidase A gaAJB13 is characterized in that its aminoacid sequence is shown in SEQ ID NO. 1.
2. alkali resistance low temperature alpha-galactosidase A gaAJB13 as claimed in claim 1, the signal peptide sequence that it is characterized in that described alpha-galactosidase A gaAJB13 is shown in SEQ ID NO. 2.
3. alkali resistance low temperature alpha-galactosidase A gaAJB13 as claimed in claim 1, the mature peptide sequence that it is characterized in that described alpha-galactosidase A gaAJB13 is shown in SEQ ID NO. 3.
4. the alpha-galactosidase gene of the described alkali resistance low temperature of the claim 1 of encoding an alpha-galactosidase A gaAJB13
AgaAJB13, it is characterized in that its nucleotide sequence is shown in SEQ ID NO. 4.
5. alpha-galactosidase gene as claimed in claim 4
AgaAJB13, it is characterized in that described alpha-galactosidase gene
AgaAJB13The nucleotide sequence of coded signal peptide is shown in SEQ ID NO. 5.
6. alpha-galactosidase gene as claimed in claim 4
AgaAJB13, it is characterized in that described alpha-galactosidase gene
AgaAJB13The nucleotide sequence of encoding mature peptide is shown in SEQ ID NO. 6.
7. one kind comprises the described alpha-galactosidase gene of claim 4
AgaAJB13Recombinant vectors.
8. one kind comprises the described alpha-galactosidase gene of claim 4
AgaAJB13Recombinant bacterial strain.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321599A (en) * | 2011-10-25 | 2012-01-18 | 云南师范大学 | Low-temperature alpha-galactosidase AgaAGN14 and gene thereof |
| CN110317820A (en) * | 2018-11-06 | 2019-10-11 | 东莞泛亚太生物科技有限公司 | A kind of alpha-galactosidase LrgalA gene |
| CN110678478A (en) * | 2017-04-11 | 2020-01-10 | 科·汉森有限公司 | Lactase with improved activity at low temperatures |
| CN111440782A (en) * | 2020-04-22 | 2020-07-24 | 青岛大学 | Novel β -galactosidase GalA and application thereof |
| CN113106082A (en) * | 2021-05-27 | 2021-07-13 | 云南师范大学 | Alanine racemase from animal manure metagenome as well as preparation and application thereof |
| CN113481185A (en) * | 2021-08-05 | 2021-10-08 | 云南师范大学 | Salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof |
| CN113637660A (en) * | 2021-08-05 | 2021-11-12 | 云南师范大学 | Beta-galactosidase GalNC3-89 and preparation method and application thereof |
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| CN1932004A (en) * | 2005-09-16 | 2007-03-21 | 新疆农业科学院微生物应用研究所 | Low temperature beta-galactosidase strain, low temperature bata-galactosidase and its production process |
| WO2010092057A1 (en) * | 2009-02-10 | 2010-08-19 | Kobenhavns Universitet | Cold-active beta-galactosidase, a method of producing same and use of such enzyme |
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| CN1932004A (en) * | 2005-09-16 | 2007-03-21 | 新疆农业科学院微生物应用研究所 | Low temperature beta-galactosidase strain, low temperature bata-galactosidase and its production process |
| WO2010092057A1 (en) * | 2009-02-10 | 2010-08-19 | Kobenhavns Universitet | Cold-active beta-galactosidase, a method of producing same and use of such enzyme |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321599A (en) * | 2011-10-25 | 2012-01-18 | 云南师范大学 | Low-temperature alpha-galactosidase AgaAGN14 and gene thereof |
| CN102321599B (en) * | 2011-10-25 | 2013-01-09 | 云南师范大学 | Low-temperature alpha-galactosidase AgaAGN14 and gene thereof |
| CN110678478A (en) * | 2017-04-11 | 2020-01-10 | 科·汉森有限公司 | Lactase with improved activity at low temperatures |
| CN110317820A (en) * | 2018-11-06 | 2019-10-11 | 东莞泛亚太生物科技有限公司 | A kind of alpha-galactosidase LrgalA gene |
| CN111440782A (en) * | 2020-04-22 | 2020-07-24 | 青岛大学 | Novel β -galactosidase GalA and application thereof |
| CN113106082A (en) * | 2021-05-27 | 2021-07-13 | 云南师范大学 | Alanine racemase from animal manure metagenome as well as preparation and application thereof |
| CN113481185A (en) * | 2021-08-05 | 2021-10-08 | 云南师范大学 | Salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof |
| CN113637660A (en) * | 2021-08-05 | 2021-11-12 | 云南师范大学 | Beta-galactosidase GalNC3-89 and preparation method and application thereof |
| CN113637660B (en) * | 2021-08-05 | 2023-09-08 | 云南师范大学 | Beta-galactosidase GalNC3-89, and preparation method and application thereof |
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