CN102533816B - Beta-glucuroide gene from extreme alkaliphiles bacillus and synthesis, expression and purification thereof - Google Patents
Beta-glucuroide gene from extreme alkaliphiles bacillus and synthesis, expression and purification thereof Download PDFInfo
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Abstract
The invention relates to a beta-glucuroide gene from extreme alkaliphiles bacillus and synthesis, expression and purification thereof. The beta-glucuroide gene is obtained through chemical synthesis; the nucleotide sequence of the beta-glucuroide gene is shown as in SEQ ID No 1; the overall length of the beta-glucuroide gene is 1374bp; and the coded nucleotide sequence of the beta-glucuroide gene is shown as in SEQ ID No 2. The gene is constructed into a prokaryotic expression vector and is converted into a prokaryote, and can be used for the industrial production and application of beta-glucuroide.
Description
Technical field
The present invention relates to a kind of beta-glucosidase gene from Extreme basophilic bacteria and synthetic, expression thereof and purifying, be specifically related to a kind of beta-glucosidase gene from Extreme basophilic bacteria Bacillus halodurans C-125 and synthetic, expression thereof and purifying.
Background technology
Beta-glucosidase (EC3.2.1.21) is a kind of lytic enzyme (J Struct Biol 129:69-79) that can be hydrolyzed glycosidic link between carbohydrate.Zymetology categorizing system is divided into two classes according to its aminoacid sequence by beta-glucosidase: glycoside hydrolysis enzyme family 1 (GHFl) and glycoside hydrolysis enzyme family 3 (GHF3).Being of wide application of beta-glucosidase, the application that we study it mainly concentrates on its degraded cellulose, the function of the various compounds (Mol Plant Microbe Interact 13:1041-1052) that xylan (Appl Environ Microbiol 68:1485-1490) and plant produce.In addition, also have interaction and its biologic applications between its involved in plant and pathogenic bacteria.As everyone knows, beta-glucosidase has brought huge contribution to industrial production aspect.The gene of a large amount of beta-glucosidases is separated from animal (Enz Microbial Technol43:35-42), plant (Bioresou Technol 99:6081-6087) and microorganism (J Microbiol 47:542-548), has carried out systematic research.Recently, coming from the focus that beta-glucosidase in extreme microorganism body becomes research, is mainly because these enzymes may have potential special biological function.
In ncbi database, collected the full genomic information of a lot of microorganisms, wherein the sequence of proteins encoded is all annotated.But the biochemical function of most albumen and physiological function are all unknown.Therefore the function of research annotation albumen can be improved these zymetologys, and applies in industrial application and go.
Extreme basophilic bacteria Bacillus halodurans C-125 is separated by Japanese scientist for 1977 first, and in 2000, has completed genomic order-checking (Nucleic Acids Res 28:4317-4331).Two ten years in the past, much from enzyme in this alkalescence microorganism, be separated also commercialization, wherein contain tilactase (Agri and Bio Chem 43:85-88), zytase (J Bacteriol 161:784-785), xylosidase (Appl Microbiol Biotechnol73:582-590) and acetylase (J Bacteriol 190:1375-1382) etc.Yet the beta-glucosidase from this bacterium was also never studied, also have no the report to this enzyme physiological and biochemical research.
Summary of the invention
One of technical problem to be solved by this invention, is to provide a kind of beta-glucosidase gene from Extreme basophilic bacteria, and described Extreme basophilic bacteria is Bacillus halodurans C-125.
Two of technical problem to be solved by this invention, is to provide a kind of chemical synthesis process of described beta-glucosidase gene.
Three of technical problem to be solved by this invention, is to provide expression and the application thereof of a kind of described beta-glucosidase gene in prokaryotic organism.
Technical scheme of the present invention realizes in the following manner.
Beta-glucosidase gene of the present invention, comes from Extreme basophilic bacteria Bacillus halodurans C-125, and its nucleotide sequence is as shown in SEQ ID No 1.
Described beta-glucosidase gene is prepared from by chemical synthesis process.The method adopt a large amount of overlapping primers by two-step pcr carry out full gene synthetic (PTDS) (Nucleic Acids Research, 2004,32:e98).First by overlapping extension, obtain the fragment of synthetic gene, then by outermost primer, the gene amplification of total length is obtained to goal gene.Obvious advantage of the method is exactly not need primer to carry out phosphatizing treatment.And by high-throughout DNA synthesizer device, can obtain comparatively expediently a large amount of primers.
The synthetic method of described beta-glucosidase gene, comprises the following steps:
1, design of primers
Design length is probably 34 of the primer sequences of oligonucleotide of the whole beta-glucosidase gene of covering of about 60mer, and every two adjacent Oligonucleolide primers sequences have the repetition of 20 bases.
Utilize PCR to carry out beta-glucoside enzymatic amplification, in 100 μ l reaction systems, the P2-P33 addition of totally 32 primers is 2ng, and outside primer P1 and P34 addition are 30ng, and amplification condition is: 94 ℃ of preheating 1min; 94 ℃, 30s, 50 ℃, 30s, 72 ℃, 10min, the Taq archaeal dna polymerase of use is KOD FX taq enzyme (Toyobo company, Japan), totally 25 circulations.
After PCR finishes, 1% agarose gel reclaims fragment, gets 10 μ l be directly connected with T/A cloning vector (Dalian treasured biotech firm).4 ℃ of connections are spent the night, in Efficient Conversion DH5 α competence.The positive colony of SEQ ID No 1 sequence that acquisition length is 1374bp, is beta-glucosidase gene of the present invention.
Above-mentioned SEQ ID No 1 sequence is directly connected with prokaryotic expression carrier pYM4087, and 4 ℃ connect 2 hours.Then by these carrier transformed competence colibacillus intestinal bacteria TOP10 (Invitrogen).Bacterium liquid is coated after the solid LB substratum (15g/L agar, 5g/L yeast extract, 5g/L NaCl, 10g/L Tryptones, phosphoric acid buffer pH7.5) that contains 50 μ g/mL penbritins is cultivated and obtained light blue bacterium colony.
Beneficial effect of the present invention
The present invention, by clone, efficiently synthesizes beta-glucosidase, and it is the very wide biology enzyme of a class application.By to the prokaryotic expression of this gene and purifying, can be applied in the suitability for industrialized production of beta-glucosidase.
Accompanying drawing explanation
Fig. 1 is the chemosynthesis policy map of beta-glucosidase gene of the present invention.
Fig. 2 expresses cell schematics with pYM4087 plasmid construction DNA.
Fig. 3 is the optimum temperuture of enzyme and the stability of temperature, and wherein A is optimum temperuture, the stability that B is temperature.
Fig. 4 is the stability of optimal pH and the pH of enzyme.
Fig. 5 is the impact that metal ion is lived on enzyme.
Fig. 6 is the impact that glucose, wood sugar, sucrose and semi-lactosi are lived on enzyme.
Embodiment
Below in conjunction with accompanying drawing, describe technical scheme of the present invention in detail.Embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement the technical scheme of invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.
If the reagent unexplained reference that the present invention is used, all purchased from Sigma-aldrich (Sigma-Aldrich) company.
The present invention relates to molecular biology experiment, as not dated especially, all with reference to from < < molecular cloning > > mono-book, (J. Pehanorm Brooker, E.F. be Ritchie, T. Manny A Disi work not, 1994, Science Press.)
The chemosynthesis of embodiment 1 beta-glucosidase gene
Referring to Fig. 1, with method for synthesizing gene (Nucleic Acids Research, 2004,32, e98) clone synthetic beta-glucosidase gene of the present invention.The primer of design is as follows respectively:
P1：Tm＝54，60mer
GGA，TCC，ATG，TCT，ATC，ATT，CAG，TTT，CCA，AAG，GAG，ATG，AAG，TGG，GGT，GTT，GCT，ACT，GCT，TCT
P2：Tm＝54，60mer
CAC，CTC，TAC，CAC，CAG，CAT，TGA，TAG，CAC，CTT，CGA，TCT，GGT，AAG，AAG，CAG，TAG，CAA，CAC，CCC
P3：Tm＝54，60mer
CAA，TGC，TGG，TGG，TAG，AGG，TGC，TTC，CAT，CTG，GGA，TGT，CTT，TGC，CAA，GAC，TCC，AGG，TAA，GGT
P4：Tm＝54，60mer
ATA，GGA，GTC，ACA，GGC，AAC，ATC，ACC，ATT，GTC，ACC，GTT，CTT，GAC，CTT，ACC，TGG，AGT，CTT，GGC
P5：Tm＝54，60mer
ATG，TTG，CCT，GTG，ACT，CCT，ATC，ACA，GAT，ACG，AAG，AAG，ACA，TTG，AGA，TCA，TGA，AGG，ATC，TTG
P6：Tm＝54，60mer
ATT，CTT，GGC，CAA，GCA，ACA，GAG，AAT，CTG，TAC，ATG，TCA，ACA，CCA，AGA，TCC，TTC，ATG，ATC，TCA
P7：Tm＝54，60mer
TCT，GTT，GCT，TGG，CCA，AGA，ATC，TTT，CCA，AAC，GGT，ACT，GGT，GAA，GTC，TCC，AGA，GAA，GGT，CTT
P8：Tm＝54，60mer
CAT，TCT，CGG，TGA，GTC，TGT，CAA，CGA，GTC，TGT，GAT，AGT，AAT，CAA，GAC，CTT，CTC，TGG，AGA，CTT
P9：Tm＝54，60mer
TGA，CAG，ACT，CAC，CGA，GAA，TGG，TAT，TCA，ACC，AAT，GTG，TAC，TCT，CTA，CCA，TTG，GGA，TCT，GCC
P10：Tm＝54，60mer
GTC，TCT，GTT，GTC，CCA，ACC，ACC，TTT，CTC，TTG，CAG，AGC，TTG，TGG，CAG，ATC，CCA，ATG，GTA，GAG
P11：Tm＝54，60mer
GTG，GTT，GGG，ACA，ACA，GAG，ACA，CCA，TCG，ATG，CCT，TTG，TCA，GAT，ACG，CTG，AAG，TCA，TGT，TCA
P12：Tm＝54，60mer
TTG，AAG，GTG，ATC，CAG，TGG，TTG，ATC，TTG，TCA，CCA，AAC，TCC，TTG，AAC，ATG，ACT，TCA，GCG，TAT
P13：Tm＝54，60mer
AAC，CAC，TGG，ATC，ACC，TTC，AAC，GAA，CTG，TGG，TGT，GTT，TCC，TTC，CTC，TCC，AAC，TAC，ATT，GGT
P14：Tm＝54，60mer
TGG，TAG，CAA，GCT，GGA，GAT，CGG，TAT，TGC，CTG，GAG，CAT，GGA，CAC，CAA，TGT，AGT，TGG，AGA，GGA
P15：Tm＝54，60mer
CGA，TCT，CCA，GCT，TGC，TAC，CAA，CGT，TGC，TCA，CCA，TCT，TCT，TGT，TGC，TCA，TGG，TAA，GGC，TGT
P16：Tm＝54，60mer
ACC，AAT，CTG，ACC，ATC，AAG，ACC，CAT，CTT，TCT，GTA，GGA，CTG，AAC，AGC，CTT，ACC，ATG，AGC，AAC
P17：Tm＝54，60mer
GTC，TTG，ATG，GTC，AGA，TTG，GTT，ATG，CTC，CTA，ACG，TCG，AGT，GGA，ACG，AAC，CAT，TCT，CCA，ATC
P18：Tm＝54，60mer
CAG，CCA，TTG，CCT，CTC，TTA，CAG，GCT，TCA，GCA，TCT，TCC，ATC，TGA，TTG，GAG，AAT，GGT，TCG，TTC
P19：Tm＝54，60mer
TGT，AAG，AGA，GGC，AAT，GGC，TGG，TTC，ATC，GAG，TGG，TTC，ATG，GAC，CCA，GTC，TTC，AAA，GGT，GCC
P20：Tm＝54，60mer
TGC，CTT，TCT，TCT，CGA，ACC，ATT，CGA，CCA，AGA，AGG，ATG，GGT，AGG，CAC，CTT，TGA，AGA，CTG，GGT
P21：Tm＝54，60mer
ATG，GTT，CGA，GAA，GAA，AGG，CAT，CAC，TGT，TCC，AAT，TGA，AGC，TGG，TGA，TAT，GGA，GAC，CAT，CCA
P22：Tm＝54，60mer
ACC，AGT，GTA，GTA，GTT，GAT，GCC，AAG，GAA，GTC，GAT，TGG，TTG，TTG，GAT，GGT，CTC，CAT，ATC，ACC
P23：Tm＝54，60mer
GCA，TCA，ACT，ACT，ACA，CTG，GTT，CTG，TTG，CCA，GAT，ACA，AGG，AGA，ACG，AAG，GTC，TCT，TTG，ACC
P24：Tm＝54，60mer
CCA，ATG，TCA，GTC，TTC，TCA，TAG，CCA，GCA，TCG，ACT，TTC，TCC，AGG，TCA，AAG，AGA，CCT，TCG，TTC
P25：Tm＝54，60mer
TAT，GAG，AAG，ACT，GAC，ATT，GGC，TGG，AAC，ATC，TAT，CCA，GAA，GGC，TTC，TAC，AAG，GTC，CTG，TAC
P26：Tm＝54，60mer
TGA，TGT，AGA，TTG，GAA，TCT，GAC，CAT，ACT，GTT，CAG，TGA，TGT，AGT，ACA，GGA，CCT，TGT，AGA，AGC
P27：Tm＝54，60mer
TCA，GAT，TCC，AAT，CTA，CAT，CAC，TGA，GAA，TGG，TTC，CTG，CTA，CAA，CGA，CGA，ACC，AGT，CAA，TGG
P28：Tm＝54，60mer
CTG，AGA，CAG，GTA，TCT，GAT，TCT，ACC，TTC，ATC，CTT，GAC，TTG，ACC，ATT，GAC，TGG，TTC，GTC，GTT
P29：Tm＝54，60mer
GAA，TCA，GAT，ACC，TGT，CTC，AGC，ACC，TGA，CTG，CTC，TCA，AGA，GAA，GCA，TGG，AGT，CTG，GTG，TCA
P30：Tm＝54，60mer
AAG，TTG，TCG，AGC，AGG，GAC，CAA，GCC，ATG，TAG，CCT，TTG，ATG，TTG，ACA，CCA，GAC，TCC，ATG，CTT
P31：Tm＝54，60mer
TGG，TCC，CTG，CTC，GAC，AAC，TTT，GAA，TGG，GCT，GAA，GGC，TAC，TCT，ATG，AGA，TTC，GGT，ATC，GTT
P32：Tm＝54，60mer
AGT，CCT，TCT，TGG，TTC，TCT，CAA，GGG，TTC，TGT，AGT，TGA，CGT，GAA，CGA，TAC，CGA，ATC，TCA，TAG
P33：Tm＝54，60mer
TGA，GAG，AAC，CAA，GAA，GGA，CTC，TTT，CTA，CTG，GTA，CAA，ACA，GAT，GAT，TGC，CAA，CCA，GTT，CTT
P34：Tm＝54，54mer
GAG，CTC，TTA，ATG，GTG，ATG，GTG，ATG，GTG，GAG，TTC，GAA，GAA，CTG，GTT，GGC，AAT，CAT
Utilize PCR to carry out the amplification of beta-glucosidase gene, in 100 μ l reaction systems, the P2-P33 addition of totally 32 primers is 2ng, and outside primer P1 and P34 addition are 30ng, and amplification condition is: 94 ℃ of preheating 1min; 94 ℃, 30s, 50 ℃, 30s, 72 ℃, 10min, the TaqDNA polysaccharase of use is KOD FXtaq enzyme (Toyobo company, Japan), totally 25 circulations.
After PCR finishes, 1% agarose gel reclaims, and gets 10 μ l be directly connected with T/A cloning vector (Dalian treasured biotech firm).4 ℃ of connections are spent the night, in Efficient Conversion DH5 α competence.Obtain positive colony, be beta-glucosidase gene of the present invention, its sequence is as shown in SEQ ID No 1.This SEQ ID No 1 sequence and the beta-glucosidase gene from Extreme basophilic bacteria Bacillus haloduransC-125 are compared, and homology is 79%, specific as follows:
1 GTGGATATGTATCGCTTCTCTGTTGCTTGGCCGCGAATTTTTCCGAATGGTACAGGGGAG
|| || ||||| | ||||||||||||||||| |||| ||||| || ||||| || ||
223 GTTGACATGTACAGATTCTCTGTTGCTTGGCCAAGAATCTTTCCAAACGGTACTGGTGAA
61 GTGTCACGTGAAGGATTAGATTATTACCATCGACTTGTTGATCGTTTAACCGAAAACGGG
|| || | ||||| | ||||| || || |||| ||||| | | ||||| || ||
283 GTCTCCAGAGAAGGTCTTGATTACTATCACAGACTCGTTGACAGACTCACCGAGAATGGT
121 ATTCAACCGATGTGTACTCTCTACCATTGGGATTTGCCGCAAGCATTGCAAGAAAAGGGC
|||||||| |||||||||||||||||||||||| |||| ||||| ||||||| || ||
343 ATTCAACCAATGTGTACTCTCTACCATTGGGATCTGCCACAAGCTCTGCAAGAGAAAGGT
181 GGCTGGGATAATCGCGACACGATCGACGCTTTTGTCCGTTATGCTGAGGTCATGTTCAAA
|| ||||| || | ||||| ||||| || |||||| | || ||||| |||||||||||
403 GGTTGGGACAACAGAGACACCATCGATGCCTTTGTCAGATACGCTGAAGTCATGTTCAAG
241 GAGTTCGGTGATAAAATTAACCACTGGATTACTTTTAATGAGCTGTGGTGCGTTTCTTTC
||||| ||||| || || ||||||||||| || || || || |||||||| ||||| |||
463 GAGTTTGGTGACAAGATCAACCACTGGATCACCTTCAACGAACTGTGGTGTGTTTCCTTC
301 CTTTCAAATTATATCGGTGTCCATGCACCTGGAAATACCGATCTCCAGCTAGCGACAAAT
|| || || || || ||||||||||| || || ||||||||||||||||| || || ||
523 CTCTCCAACTACATTGGTGTCCATGCTCCAGGCAATACCGATCTCCAGCTTGCTACCAAC
361 GTGGCCCACCATCTTCTAGTTGCTCATGGAAAAGCGGTTCAGTCGTATCGAAAAATGGGG
|| || ||||||||||| ||||||||||| || || |||||||| || |||| |||||
583 GTTGCTCACCATCTTCTTGTTGCTCATGGTAAGGCTGTTCAGTCCTACAGAAAGATGGGT
421 CTTGATGGACAAATCGGGTACGCGCCGAATGTAGAATGGAATGAGCCTTTCAGCAATCAA
|||||||| || || || || || || || || || ||||| || || ||| ||||||
643 CTTGATGGTCAGATTGGTTATGCTCCTAACGTCGAGTGGAACGAACCATTCTCCAATCAG
481 ATGGAGGATGCTGAAGCTTGTAAGCGGGGGAATGGTTGGTTTATCGAATGGTTTATGGAT
||||| ||||||||||| |||||| | || ||||| ||||| ||||| ||||| |||||
703 ATGGAAGATGCTGAAGCCTGTAAGAGAGGCAATGGCTGGTTCATCGAGTGGTTCATGGAC
541 CCAGTCTTCAAGGGAGCCTACCCATCTTTCTTAGTCGAATGGTTTGAGAAGAAAGGGATT
||||||||||| || ||||||||||| ||||| ||||||||||| ||||||||||| ||
763 CCAGTCTTCAAAGGTGCCTACCCATCCTTCTTGGTCGAATGGTTCGAGAAGAAAGGCATC
601 ACCGTACCGATTGAGGCTGGAGATATGGAGACAATCCAACAACCAATCGACTTTTTAGGA
|| || || ||||| ||||| ||||||||||| |||||||||||||||||||| | ||
823 ACTGTTCCAATTGAAGCTGGTGATATGGAGACCATCCAACAACCAATCGACTTCCTTGGC
661 ATTAACTATTATACGGGAAGTGTTGCCCGCTACAAGGAGAACGAAGGACTATTTGATCTA
|| ||||| || || || ||||||| | ||||||||||||||||| || ||||| ||
883 ATCAACTACTACACTGGTTCTGTTGCCAGATACAAGGAGAACGAAGGTCTCTTTGACCTG
721 GAAAAAGTAGATGCAGGTTATGAGAAAACGGATATTGGCTGGAACATTTATCCTGAAGGC
|| ||||| ||||| || |||||||| || || |||||||||||||| ||||| ||||||
943 GAGAAAGTCGATGCTGGCTATGAGAAGACTGACATTGGCTGGAACATCTATCCAGAAGGC
781 TTTTACAAAGTGTTGTACTATATAACTGAGCAATATGGACAAATCCCGATTTATATTACG
|| ||||| || ||||||| || ||||| || ||||| || || || || || || ||
1003 TTCTACAAGGTCCTGTACTACATCACTGAACAGTATGGTCAGATTCCAATCTACATCACT
841 GAAAATGGGTCTTGTTACAATGATGAACCTGTGAATGGGCAAGTGAAGGATGAGGGGCGA
|| ||||| || || ||||| || ||||| || ||||| ||||| |||||||| || ||
1063 GAGAATGGTTCCTGCTACAACGACGAACCAGTCAATGGTCAAGTCAAGGATGAAGGTAGA
901 ATCCGCTACTTGAGCCAGCATTTGACGGCACTAAAGCGTAGCATGGAGTCAGGTGTCAAC
||| | ||| || ||||| |||| || || ||| | ||||||||||| |||||||||
1123 ATCAGATACCTGTCTCAGCACCTGACTGCTCTCAAGAGAAGCATGGAGTCTGGTGTCAAC
961 ATTAAAGGATACATGGCGTGGTCATTGCTCGATAATTTTGAATGGGCCGAGGGCTACAGT
|| ||||| |||||||| ||||| ||||||| || ||||||||||| || |||||| |
1183 ATCAAAGGCTACATGGCTTGGTCCCTGCTCGACAACTTTGAATGGGCTGAAGGCTACTCT
1021 ATGAGGTTTGGCATCGTTCACGTCAATTATCGAACGTTGGAACGGACGAAGAAGGATAGT
||||| || || |||||||||||||| || |||| | || | || |||||||| |
1243 ATGAGATTCGGTATCGTTCACGTCAACTACAGAACCCTTGAGAGAACCAAGAAGGACTCT
1081 TTCTATTGGTATAAACAAATGATTGCCAATCAGTTCTTTGAACTTTAA
||||| ||||| ||||| ||||||||||| |||||||| ||||| |
1303 TTCTACTGGTACAAACAGATGATTGCCAACCAGTTCTTCGAACTCCACCATCACCATCAC
Embodiment 2 beta-glucosidase genes are at expression in escherichia coli
Referring to Fig. 2, above-mentioned SEQ ID No 1 sequence is directly connected with prokaryotic expression carrier pYM4087,4 ℃ connect 2 hours.Then by these carrier transformed competence colibacillus intestinal bacteria TOP10 (Invitrogen).Bacterium liquid is coated to the solid LB substratum (15g/L agar, 5g/L yeast extract, 5g/L NaCl, 10g/L Tryptones, phosphoric acid buffer pH7.5) that contains 50 μ g/mL penbritins, cultivated 16 hours for 37 ℃, until there is nattier blue bacterium colony to grow.A picking 3-5 bacterium colony, is inoculated in 50ml liquid LB substratum (5g/L yeast extract, 5g/L NaCl, 10g/L Tryptones, phosphoric acid buffer pH7.5), and 37 ℃ of shaking tables stop cultivation at 1.0 o'clock until the concentration of bacterium liquid reaches OD600.
The purifying of embodiment 3 beta-glucosidase genes
By the final nutrient solution centrifugal 5min under 5,000g universal gravity constant obtaining of embodiment 2, sterile water wash 1 time.The cell precipitation of gained is resuspended with 1mL phosphoroclastic cleavage damping fluid, is then transferred in Eppendorf tube.Use ultrasonoscope cracking bacterium liquid 30min, with the centrifugal 30min of 12,000g.Use the centrifugal good supernatant of filtering with microporous membrane, upper to Ni-Agarose post, be purified into target protein.By using albumen to preserve liquid after target protein dialysis, save.
The enzyme kinetics specificity analysis of embodiment 4 beta-glucosidase genes
The enzyme kinetics specificity analysis of beta-glucosidase mainly contains three substrates.They are respectively: oNPGlu, oNPGal and pNPGal.Reaction adopts the system in embodiment 3.The main K of enzyme to different substrates that measure
mwith maximum reaction velocity V
max.Namely, under the constant condition of temperature, pH and enzyme concn, concentration of substrate has a great impact the speed of enzymatic reaction.When concentration of substrate is very low, the speed of enzymatic reaction (v) increases sharply with the increase of concentration of substrate; Along with the continuation increase of concentration of substrate, the increase of speed of response starts to slow down; When concentration of substrate is increased to a certain degree, speed of response reaches a ultimate value (V
max).
The available Michaelis-Menten equation of this relation of concentration of substrate and speed of response represents:
In formula: v--speed of response; K
m--Michaelis-Menton constant; V
max--enzyme reaction top speed; [S]--concentration of substrate.
Adopt Linewaver-Burk graphing method to measure K
m, V
max, this method is the form reciprocal according to Michaelis-Menton equation, with 1/v to 1/[S] mapping, obtain straight line.The intercept of straight line on transverse axis is-1/K
m, vertical intercept is 1/V
max, obtain K
mwith V
max.
Here our concentration of three substrates used is: oNPGlu (from 0.1mM to 2mM), oNPGal (from 0.8mM to 2.8mM) and pNPGal (from 0.4mM to 2.4mM).Resulting result is as shown in table 1.
Table 1
The Analysis of The Physiological And Biochemical Properties of embodiment 5 beta-glucosidase genes
The liquid of protease of preserving in embodiment 3 is done to specific Analysis of The Physiological And Biochemical Properties.This analysis process is to carry out in following enzyme reaction system: 195 μ l Zm damping fluid (100mM potassium phosphate buffer, 10mM KCl, 1mM MgSO
4, the reaction substrate oNPGlu that contains 2mM in pH7.0).Reaction is to add the enzyme liquid of 5 μ l to start, and reaction is in the water-bath of 50 ℃, to carry out 10min, to add the 1M Na of 60 μ l
2cO
3termination reaction.Then at 420nm, go out to measure the burst size of p-NP.The protein concentration that the enzyme that here we define some amount is 1U. beta-glucosidase at the vigor of the p-NP of per minute catalysis 1 μ M is analyzed with Bradford test kit.
The physio-biochemical characteristics that we analyze mainly contain the optimum temperuture of enzyme, the stability of the temperature of enzyme, the optimal pH of enzyme, the stability of pH, the specificity of substrate, the impact that metal ion is lived on enzyme, the impact that the impact that organic solvent is lived on enzyme and different sugar are lived on enzyme.
When the temperature of the optimal reaction of studying enzyme, the temperature of reaction is since 15 ℃, every 5 ℃, until 60 ℃.Every individual system is done 3 revision tests (each reaction is to repeat for 3 times below); When the stability of the temperature of studying enzyme, mainly measure enzyme 30,40,50 and 60 ℃ of these four treatment of different temperature enzyme liquid are then measured the residual activity of enzyme under optimum temperuture; When the pH of the optimal reaction of studying enzyme, select different damping fluids, mainly contain citric acid-sodium phosphate buffer (0.1M, pH 3.0-5.5), Zm damping fluid (0.1M, pH 6.0-8.0), glycine-NaOH damping fluid (0.2M, pH 8.5-10.6); When the stability of the pH of studying enzyme, be first enzyme first to be processed in the damping fluid of different pH to 24h, then in the residual activity of the mensuration enzyme of the optimal pH of enzyme; In the specificity of substrate, the impact that metal ion is lived on enzyme, the impact that the impact that organic solvent is lived on enzyme and different sugar are lived on enzyme is that enzyme is processed by different influence factors, then in the optimum response system of enzyme, measures the residual activity of enzyme.
Through experimental verification, enzyme is at 50 ℃, and during pH 7, activity is the highest, although the thermostability of enzyme is not high, at 30 ℃, processes after 30min, and the activity of enzyme only has 80%.But enzyme has a good pH stability, after alkaline condition is processed, still has very high activity (referring to Fig. 3,4).Metal ion impact on enzyme when lower concentration is little, when concentration of metal ions is increased to 5mM, and Cd
2+, Zn
2+and Fe
3+can suppress respectively 87%, 88% and 92% activity.In addition, through this experimental study, find, the activity of this enzyme can be activated by four kinds of sugar such as glucose, wood sugar, sucrose and semi-lactosi, and when sugared concentration reaches 1M, still can improve the activity (referring to Fig. 5,6) of enzyme.
Claims (1)
1. the synthetic method from the beta-glucosidase gene of Extreme basophilic bacteria, wherein, the nucleotide sequence of beta-glucosidase gene is as shown in SEQ ID No1, the aminoacid sequence of its coding is as shown in SEQ ID No2, it is characterized in that, adopt overlapping primer to carry out the synthetic of full gene by two-step pcr, first design 34 primers, by overlapping extension, obtain the fragment of synthetic gene, then by outermost primer, the gene amplification of total length is obtained to goal gene;
The sequence of described primer is specific as follows:
P1:Tm=54,60mer
GGA,TCC,ATG,TCT,ATC,ATT,CAG,TTT,CCA,AAG,GAG,ATG,AAG,TGG,GGT,GTT,GCT,ACT,GCT,TCT
P2:Tm=54,60mer
CAC,CTC,TAC,CAC,CAG,CAT,TGA,TAG,CAC,CTT,CGA,TCT,GGT,AAG,AAG,CAG,TAG,CAA,CAC,CCC
P3:Tm=54,60mer
CAA,TGC,TGG,TGG,TAG,AGG,TGC,TTC,CAT,CTG,GGA,TGT,CTT,TGC,CAA,GAC,TCC,AGG,TAA,GGT
P4:Tm=54,60mer
ATA,GGA,GTC,ACA,GGC,AAC,ATC,ACC,ATT,GTC,ACC,GTT,CTT,GAC,CTT,ACC,TGG,AGT,CTT,GGC
P5:Tm=54,60mer
ATG,TTG,CCT,GTG,ACT,CCT,ATC,ACA,GAT,ACG,AAG,AAG,ACA,TTG,AGA,TCA,TGA,AGG,ATC,TTG
P6:Tm=54,60mer
ATT,CTT,GGC,CAA,GCA,ACA,GAG,AAT,CTG,TAC,ATG,TCA,ACA,CCA,AGA,TCC,TTC,ATG,ATC,TCA
P7:Tm=54,60mer
TCT,GTT,GCT,TGG,CCA,AGA,ATC,TTT,CCA,AAC,GGT,ACT,GGT,GAA,GTC,TCC,AGA,GAA,GGT,CTT
P8:Tm=54,60mer
CAT,TCT,CGG,TGA,GTC,TGT,CAA,CGA,GTC,TGT,GAT,AGT,AAT,CAA,GAC,CTT,CTC,TGG,AGA,CTT
P9:Tm=54,60mer
TGA,CAG,ACT,CAC,CGA,GAA,TGG,TAT,TCA,ACC,AAT,GTG,TAC,TCT,CTA,CCA,TTG,GGA,TCT,GCC
P10:Tm=54,60mer
GTC,TCT,GTT,GTC,CCA,ACC,ACC,TTT,CTC,TTG,CAG,AGC,TTG,TGG,CAG,ATC,CCA,ATG,GTA,GAG
P11:Tm=54,60mer
GTG,GTT,GGG,ACA,ACA,GAG,ACA,CCA,TCG,ATG,CCT,TTG,TCA,GAT,ACG,CTG,AAG,TCA,TGT,TCA
P12:Tm=54,60mer
TTG,AAG,GTG,ATC,CAG,TGG,TTG,ATC,TTG,TCA,CCA,AAC,TCC,TTG,AAC,ATG,ACT,TCA,GCG,TAT
P13:Tm=54,60mer
AAC,CAC,TGG,ATC,ACC,TTC,AAC,GAA,CTG,TGG,TGT,GTT,TCC,TTC,CTC,TCC,AAC,TAC,ATT,GGT
P14:Tm=54,60mer
TGG,TAG,CAA,GCT,GGA,GAT,CGG,TAT,TGC,CTG,GAG,CAT,GGA,CAC,CAA,TGT,AGT,TGG,AGA,GGA
P15:Tm=54,60mer
CGA,TCT,CCA,GCT,TGC,TAC,CAA,CGT,TGC,TCA,CCA,TCT,TCT,TGT,TGC,TCA,TGG,TAA,GGC,TGT
P16:Tm=54,60mer
ACC,AAT,CTG,ACC,ATC,AAG,ACC,CAT,CTT,TCT,GTA,GGA,CTG,AAC,AGC,CTT,ACC,ATG,AGC,AAC
P17:Tm=54,60mer
GTC,TTG,ATG,GTC,AGA,TTG,GTT,ATG,CTC,CTA,ACG,TCG,AGT,GGA,ACG,AAC,CAT,TCT,CCA,ATC
P18:Tm=54,60mer
CAG,CCA,TTG,CCT,CTC,TTA,CAG,GCT,TCA,GCA,TCT,TCC,ATC,TGA,TTG,GAG,AAT,GGT,TCG,TTC
P19:Tm=54,60mer
TGT,AAG,AGA,GGC,AAT,GGC,TGG,TTC,ATC,GAG,TGG,TTC,ATG,GAC,CCA,GTC,TTC,AAA,GGT,GCC
P20:Tm=54,60mer
TGC,CTT,TCT,TCT,CGA,ACC,ATT,CGA,CCA,AGA,AGG,ATG,GGT,AGG,CAC,CTT,TGA,AGA,CTG,GGT
P21:Tm=54,60mer
ATG,GTT,CGA,GAA,GAA,AGG,CAT,CAC,TGT,TCC,AAT,TGA,AGC,TGG,TGA,TAT,GGA,GAC,CAT,CCA
P22:Tm=54,60mer
ACC,AGT,GTA,GTA,GTT,GAT,GCC,AAG,GAA,GTC,GAT,TGG,TTG,TTG,GAT,GGT,CTC,CAT,ATC,ACC
P23:Tm=54,60mer
GCA,TCA,ACT,ACT,ACA,CTG,GTT,CTG,TTG,CCA,GAT,ACA,AGG,AGA,ACG,AAG,GTC,TCT,TTG,ACC
P24:Tm=54,60mer
CCA,ATG,TCA,GTC,TTC,TCA,TAG,CCA,GCA,TCG,ACT,TTC,TCC,AGG,TCA,AAG,AGA,CCT,TCG,TTC
P25:Tm=54,60mer
TAT,GAG,AAG,ACT,GAC,ATT,GGC,TGG,AAC,ATC,TAT,CCA,GAA,GGC,TTC,TAC,AAG,GTC,CTG,TAC
P26:Tm=54,60mer
TGA,TGT,AGA,TTG,GAA,TCT,GAC,CAT,ACT,GTT,CAG,TGA,TGT,AGT,ACA,GGA,CCT,TGT,AGA,AGC
P27:Tm=54,60mer
TCA,GAT,TCC,AAT,CTA,CAT,CAC,TGA,GAA,TGG,TTC,CTG,CTA,CAA,CGA,CGA,ACC,AGT,CAA,TGG
P28:Tm=54,60mer
CTG,AGA,CAG,GTA,TCT,GAT,TCT,ACC,TTC,ATC,CTT,GAC,TTG,ACC,ATT,GAC,TGG,TTC,GTC,GTT
P29:Tm=54,60mer
GAA,TCA,GAT,ACC,TGT,CTC,AGC,ACC,TGA,CTG,CTC,TCA,AGA,GAA,GCA,TGG,AGT,CTG,GTG,TCA
P30:Tm=54,60mer
AAG,TTG,TCG,AGC,AGG,GAC,CAA,GCC,ATG,TAG,CCT,TTG,ATG,TTG,ACA,CCA,GAC,TCC,ATG,CTT
P31:Tm=54,60mer
TGG,TCC,CTG,CTC,GAC,AAC,TTT,GAA,TGG,GCT,GAA,GGC,TAC,TCT,ATG,AGA,TTC,GGT,ATC,GTT
P32:Tm=54,60mer
AGT,CCT,TCT,TGG,TTC,TCT,CAA,GGG,TTC,TGT,AGT,TGA,CGT,GAA,CGA,TAC,CGA,ATC,TCA,TAG
P33:Tm=54,60mer
TGA,GAG,AAC,CAA,GAA,GGA,CTC,TTT,CTA,CTG,GTA,CAA,ACA,GAT,GAT,TGC,CAA,CCA,GTT,CTT
P34:Tm=54,54mer
GAG,CTC,TTA,ATG,GTG,ATG,GTG,ATG,GTG,GAG,TTC,GAA,GAA,CTG,GTT,GGC,AAT,CAT；
Wherein, every two adjacent primer sequences have the repetition of 20 bases;
Primer P2-P33 is inner side primer, and P1, P34 are outside primer.
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| Title |
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| NP_242798.1;Takami et al.;《GenBank》;20091109;1-3 * |
| Takami et al..NP_242798.1.《GenBank》.2009,1-3. |
| 合成人降钙素基因实验研究的初步报告;王秀利等;《河南医学研究》;20041231;第13卷(第4期);第294页左栏第2-6段，右栏第1-5段 * |
| 灰绿曲霉β-葡萄糖苷酶的分离及特性;邓敏等;《生物技术》;20091231;第19卷(第6期);61-63 * |
| 王秀利等.合成人降钙素基因实验研究的初步报告.《河南医学研究》.2004,第13卷(第4期),第294页左栏第2-6段，右栏第1-5段. |
| 邓敏等.灰绿曲霉β-葡萄糖苷酶的分离及特性.《生物技术》.2009,第19卷(第6期),61-63. |
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