CN106085989A - One Bacillus species β 1,3 1,4 glucanase and encoding gene thereof and application - Google Patents
One Bacillus species β 1,3 1,4 glucanase and encoding gene thereof and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2448—Licheninase (3.2.1.73)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01039—Glucan endo-1,3-beta-D-glucosidase (3.2.1.39)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract
The present invention relates to Bacillus species β 1,3 1,4 glucanase and encoding gene thereof and application.The protein that the present invention provides has β 1,3 Isosorbide-5-Nitrae dextranase activities, specific enzyme activity power 431.8U mg‑1, optimal reaction pH is 6.0, keeps stable at pH 3.5 9；Optimal reactive temperature is 55 DEG C, keeps high enzyme vigor, have preferable thermostability below 55 DEG C；Substratspezifitaet is relatively strong, has good β 1,3 Isosorbide-5-Nitrae glucanase zymologic property；There is in food, feedstuff industry good using value.This protein can significantly reduce viscosity during malt amylase, shorten filtration time；β Portugal oligosaccharide yield can be significantly improved, during to the bran of Fagopyrum esculentum Moench hydrolysis of exogenous enzyme inactivation, it is possible to obtain Portugal's trisaccharide that relative amount is higher and Portugal's tetrose in bran of Fagopyrum esculentum Moench beta glucan hydrolytic process；Can be also used for preparation as β 1,3 glucanase and can obtain right oligosaccharide.
Description
Technical field
The present invention relates to biological technical field, specifically Bacillus species β-1,3-1,4-glucanase and volume thereof
Code gene and application.
Background technology
1,4 beta-glucanase is that a class can be degraded the enzyme of beta glucan in corn, including β-1,3-1,4-glucanase
(lichenase, EC 3.2.1.73), β-Isosorbide-5-Nitrae-glucanase (cellulase, EC 3.2.1.4), β-1,3-glucanase
(laminarinase, EC 3.2.1.39) and β-1,3 (4)-glucanase (EC 3.2.1.6) (McCarthy, et al.,
International Journal of Biological Macromolecules,2003,33:141-148).Wherein, β-1,
3-1,4-glucanase has strict substrate specificity, and the 3-O-of its hydrolysis beta glucan replaces β-1 on glucose residue,
4-glycosidic bond, generates and mainly comprises 3-O-beta fibers diglycosyl-D-Glucose and the water of 3-O-beta fibers three glycosyls-D-Glucose
Hydrolysis products (Yang, et al., Journal of Agricultural and Food Chemistry, 2008,56:5345-
5351)。
Beta-1,3-1,4-dextranase is widely used in Beer Production and feed industry.In Beer Production, β-1,3-
Isosorbide-5-Nitrae-glucanase can single-minded decomposition barley beta-glucan, make barley endosperm cell wall loosen, promote cellular content excessive,
And then the utilization rate of raising Fructus Hordei Vulgaris, make converted mash viscosity reduce simultaneously, greatly reduce the filtration time of beerwort, make beer production
Increase, improve the turbidity of medicated beer, improve medicated beer quality (Furtado, et al., Process Biochemistry,
2011,46(5):1202-1206.).In feed industry, β-1,3-1,4-glucanase can be specifically by β-1,3-1,4-
Glucosan is hydrolyzed to low molecular sugar, makes the structure of plant cell wall be destroyed, and then discharges nutrient substance.Owing to not having
Hydrophilic and stickiness, the beta glucan after degraded will not expand in animal and bird intestines and be adhered, therefore make the digestibility of chyme
All increase (Smits, et al., Worlds Poultry Science Journal with the capacity usage ratio of feedstuff
1996,52(2):203-221).It addition, beta glucan hydrolysate can improve growth (Jaskari, the et of beneficial bacteria of intestinal tract
Al.Applied Microbiology and Biotechnology, 1998,49 (2): 175-181), it is also possible to delaying senility,
Improve nutritious compound absorption speed and anti-hypercholesterolemia effect (Bode, 2009,67 (11): S183-S191) is provided.
Due to β-1,3-1,4-glucanase has important industrial application value, and it has been carried out greatly by domestic and international researcher
Quantity research.At present, having separated and identified β-1 from multiple-microorganism, 3-1,4-glucanase, wherein great majority derive from carefully
Bacterium, including bacillus cereus (Bacillus species), as bacillus subtilis (B.subtilis) (Tang, et al.,
Bioresource Technology, 2004,93 (2): 175-181), bacillus amyloliquefaciens
(B.amyloliquefaciens) (Hofemeister, et al., Gene, 1986,49:177-187), Bacillus licheniformis
(B.licheniformis) (Chaari, et al., Process Biochemistry, 2012,47 (3): 509-516) etc.；Or
Other antibacterial, such as Clostridium thermocellum (Clostridium thermocellum) (Ribeiro, et al., British Poultry
Science, 2012,53 (2): 224-234) etc..In some funguses, also separate and identify β-1,3-1,4-glucanase, as
Paecilomyces thermaphila (Paecilomyces thermophila) (Yang, et al., Journal of Agricultural and
Food Chemistry, 2008,56 (13): 5345-5351), rhizomucor miehei (Rhizomucor miehei) (Tang, et
Al., Journal of Agricultural and Food Chemistry, 2012,60 (9): 2354-2361) etc..
In order to improve β-1, the yield of 3-1,4-glucanase and heat stability, have expressed in different hosts and carrier
Antibacterial or fungus beta-1,3-1,4-dextranase gene.Such as, at expression in escherichia coli from bacillus subtilis
(B.subtilis)(Qiao,et al.,Applied Biochemistry and Biotechnology,2009,152(2):
334-342), bacillus amyloliquefaciens (B.amyloliquefaciens) (Sun, et al., Annals of
Microbiology, 2012,62 (3): 1235-1242), Paenibacillus polymyxa (Paenibaccillus polymyxa)
β-1,3-1,4-the Portugal of (Wen, et al., Scientia Agricultura Sinica, 2010,43 (22): 4614-4623)
Xylanase gene；Pichia sp. (Pichia pastoris) have expressed from bacillus subtilis (B.subtilis)
(Qiao, et al., Biologia, 2010,65 (2): 191-196), product succinic acid thread bacillus (Fibrobacter
succinogenes)(Huang,et al.,Applied Microbiology and Biotechnology,2008,78(1):
95-103) with Paecilomyces thermaphila (Paecilomyces thermophila) (Hua, et al., Applied Microbiology
And Biotechnology, 2010,88 (2): 509-518) beta-1,3-1,4-dextranase gene.Some patents disclose
Beta-1,3-1,4-dextranase gene and expression thereof.Such as, disclose from bacillus subtilis in CN 104561060 A
(B.subtilis) beta-1,3-1,4-dextranase gene and the expression in escherichia coli thereof；In CN 101775385 A
Disclose from the beta-1,3-1,4-dextranase gene of Paecilomyces thermaphila and the table in Pichia sp. (P.pastoris) thereof
Reach.In order to make β-1,3-1,4-glucanase can preferably adapt to industrial process conditions, and some patents further disclose and change
The method of the character of kind beta-1,3-1,4-dextranase.Such as, CN 103045560 A discloses the acidity that orthomutation obtains
β-1,3-1,4-glucanase, in the case of keeping other zymologic property to be basically unchanged, its optimum pH is changed into 5.0 by 7.0；
CN 101684475 A discloses and expresses by bacillus amyloliquefaciens in Pichia sp. (P.pastoris)
(B.amyloliquefaciens) 107 amino acid residues of the aminoterminal of beta-1,3-1,4-dextranase gene and leaching fiber crops spore
Bacillus (B.macerans) β-1, the heterozygous genes of 107 amino acid residue compositions of the c-terminus of 3-1,4-glucanase gene,
Its heat stability in sour environment is made to be much higher than single wild type 1,4 beta-glucanase.
Series bacillus (Paenibacillus sp.) is the class facultative anaerobic bacteria that nature is widely distributed, at present
Report from Paenibacillus sp.S09 (Cheng, et al., 2014, Biotechnology Letters, 36:797-
803), Paenibacillus sp.X4 (Na, et al., 2015, Biotechnology Letters, 37:643-655) and
Paenibacillus sp.F-40(Yang,et al.,Journal of Microbiology and Biotechnology,
Clone in 2007,17:58-66) and have expressed some beta-1,3-1,4-dextranases.Osman et al. (Osman, et al.,
International Journal of Systematic and Evolutionary Microbiology,2006,56:
1509-1514) from jet propulsion laboratory aircraft assembly plant (Jet Propulsion Laboratory Spacecraft
Assembly Facility) isolate balun Pueraria lobota hereby series bacillus (Paenibacillus barengoltzii).Originally grind
Study carefully group from South China Sea, isolated balun Pueraria lobota hereby series bacillus CAU904, and therefrom clone and have expressed two kinds of chitins
Enzyme (Yang, et al., Food Chemisty, 2016,192:1041-1048；Fu et al.,Biotechnology for
Biofuels,2014,7)。
Although having carried out many β-1 at present, the research of 3-1,4-glucanase, but owing to the industry such as food, feedstuff is raw
That produces is actually needed, and clones and express novel β-1 with good zymologic property and substrate specificity, 3-1,4-glucanase pair
Above-mentioned industry has great importance.
Summary of the invention
For defect present in prior art, it is an object of the invention to provide Bacillus species β-1,3-1,4-
Glucanase and encoding gene thereof and application.
β-1 provided by the present invention, 3-1,4-glucanase, from balun Pueraria lobota hereby series bacillus (Paenibacillus
Barengoltzii), it is following (1) or (2) or (3) or the protein of (4):
(1) protein of the aminoacid sequence composition shown in sequence 2；
(2) sequence 2 is from the protein of the 31st to 416 shown aminoacid sequence composition of amino terminal；
(3) carboxyl terminal in described (2) adds the protein of Poly-His label；It is specially the carboxyl in described (2)
End with the addition of the protein of AAALEHHHHHH；
(4) by the aminoacid sequence of described (1) or (2) or (3) through the replacement of one or several amino acid residue and/or
What disappearance and/or interpolation were obtained has the protein derivative by (1) or (2) or (3) of identical function.
In order to make the protein in (1) or (2) or (3) or (4) be easy to purification, can be by the ammonia shown in sequence 2
The amino terminal of the protein of base acid sequence composition or carboxyl terminal connect upper label as shown in table 1.
The sequence of table 1 label
| Label | Residue number | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned protein can synthetic, it is possible to first synthesize its encoding gene, then carries out biological expression and obtain.Above-mentioned albumen
The encoding gene of matter can be by the password by lacking one or several amino acid residue in the DNA sequence shown in sequence 1
Son, and/or carry out the missense mutation of one or several base pair, and/or connect the mark shown in table 1 at its 5 ' end and/or 3 ' ends
The coded sequence signed obtains.
The present invention protects the encoding gene of above-mentioned protein.
Described encoding gene is the DNA molecular of following (a) or (b) or (c) or (d):
DNA molecular shown in (a) sequence 1；
91st to 1248 the shown DNA molecular of (b) sequence 1；
C DNA molecule hybridize and the DNA of code for said proteins that () is limited with (a) or (b) under strict conditions divide
Son；
D DNA molecular that () and (a) or (b) or (c) limit has sequence iden and the encoding said proteins of at least 75%
The DNA molecular of matter.
Above-mentioned stringent condition can be at 6 × SSC, in the solution of 0.5%SDS, hybridizes at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Present invention protection contains the recombinant vector (i.e. recombiant plasmid) of encoding gene, expression cassette described in any of the above, turns base
Because of cell line or recombinant bacterium.
Described recombinant vector concretely carrier pET-28a (+) Nco I and Not I site between insert sequence table
Sequence 1 obtains after the 91st to 1248 shown DNA fragmentation side recombiant plasmid；
Described recombinant bacterium concretely contains bacillus coli DH 5 alpha or the e. coli bl21 (DE3) of described recombinant vector.
The present invention protects encoding gene total length described in any of the above or the primer pair of its any fragment.
The present invention protects protein described in any of the above as the application in beta-1,3-1,4-dextranase.
This application includes the step being hydrolyzed β-1,3-1,4-glucosan；
And/or, described β-1,3-1,4-glucosan be in barley beta-glucan, avenabeta glucosan and lichenin extremely
Few one.
The present invention protects protein described in any of the above as the application in beta-1,3-glucanase.
This application includes the step being hydrolyzed beta-1,3-dextran；
And/or, described β-1,3-glucosan is at least one in laminarin and the right polysaccharide of Ke get.
In above-mentioned application,
The pH value of described hydrolysis can be 3 10.5, or 3.5 9.0, such as 3.0,3.5,4.0,4.5,5.0,5.5,6.0,
6.5、7.0、7.5、8.0、8.5、9.0、9.5、10.0、10.5。
And/or, the temperature of described hydrolysis can be 40 75 DEG C, or 40 55 DEG C, as 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60
℃、65℃、70℃、75℃。
In above-mentioned application, described application is the application in malt amylase；
And/or, the application in bran of Fagopyrum esculentum Moench hydrolyzes；
And/or, the application in right oligosaccharide can be obtained in preparation.
The present invention protects described encoding gene and described recombinant vector, expression cassette, transgenic cell line or recombinant bacterium in system
Application in standby beta-1,3-1,4-dextranase and/or beta-1,3-glucanase.
It is demonstrated experimentally that protein provided by the present invention (being specially recombiant protein R-PbBglu16A in an embodiment) tool
There are β-1,3-1,4-dextranase activity, there is 431.8U mg-1Specific enzyme activity power, optimal reaction pH is 6.0, and wider
PH in the range of (pH3.5 9) keep stable；Optimal reactive temperature is 55 DEG C, and keeps high enzyme vigor below 55 DEG C,
There is preferable thermostability；Substratspezifitaet is relatively strong, add during malt amylase this protein can significantly reduce viscosity,
Shorten filtration time；In bran of Fagopyrum esculentum Moench beta glucan hydrolytic process, add this protein, β-Portugal's oligosaccharide can be significantly improved and obtain
Rate, during the hydrolysis of bran of Fagopyrum esculentum Moench that exogenous enzyme is inactivated by this protein, it is possible to obtain Portugal's trisaccharide that relative amount is higher and Portugal's tetrose；
It addition, this protein is as β-1,3 glucanase can be also used for preparation can obtain right oligosaccharide.The protein that the present invention provides has
Good beta-1,3-1,4-dextranase zymologic property；There is in food, feedstuff industry good using value.
Accompanying drawing explanation
The present invention has a drawings described below:
Fig. 1 is SDS-PAGE, wherein swimming lane M: low-molecular-weight standard protein；Swimming lane 1: crude enzyme liquid；Swimming lane 2: pure
The solution of the recombiant protein R-PbBglu16A changed.
Fig. 2 is the optimum pH measurement result of recombiant protein R-PbBglu16A.
Fig. 3 is the pH Stability Determination result of recombiant protein R-PbBglu16A.
"●" in Fig. 2 and Fig. 3 represents citric acid-citrate buffer solution；" ▲ ", represents MES buffer；" ■ " represents
MOPS buffer；"○" represents Tris-HCl buffer；" " represents glycine-NaOH buffer.
Fig. 4 is the optimum temperature measurement result of recombiant protein R-PbBglu16A.
Fig. 5 is the temperature stability measurement result of recombiant protein R-PbBglu16A.
Fig. 6 is recombiant protein R-PbBglu16A hydrolysis bran of Fagopyrum esculentum Moench and the Herba bromi japonici of endogenous enzymes inactivation under different hydrolysis time
The β of wheat bran-Portugal's oligosaccharide yield determination result.Wherein, "●" represents the recombiant protein R-adding 150U/g bran of Fagopyrum esculentum Moench
During PbBglu16A, β-Portugal's oligosaccharide yield after bran of Fagopyrum esculentum Moench hydrolysis；"○" represents the recombiant protein R-adding 150U/g bran of Fagopyrum esculentum Moench
During PbBglu16A, β-Portugal's oligosaccharide yield after the bran of Fagopyrum esculentum Moench hydrolysis of endogenous enzymes inactivation；" ■ " expression is not added with recombiant protein R-
During PbBglu16A, β-Portugal's oligosaccharide yield after bran of Fagopyrum esculentum Moench hydrolysis；When " " expression is not added with recombiant protein R-PbBglu16A, endogenous
β-Portugal's oligosaccharide yield after the bran of Fagopyrum esculentum Moench hydrolysis of enzyme inactivation.
Fig. 7 is thin layer chromatography (the Thin Layer of hydrolyzate after recombiant protein R-PbBglu16A hydrolysis bran of Fagopyrum esculentum Moench
Chromatography, TLC) analysis result.Wherein, G1, G2, G3 and G4 represent glucose, cellobiose, cellotriose respectively
And cellotetrose；Gn represents oligosaccharide standard；The hydrolyzate of bran of Fagopyrum esculentum Moench when OB+ represents addition recombiant protein R-PbBglu16A；
OB-represents the hydrolyzate of bran of Fagopyrum esculentum Moench when not adding recombiant protein R-PbBglu16A；DOB+ represents addition recombiant protein R-
The hydrolyzate of the bran of Fagopyrum esculentum Moench of endogenous enzymes inactivation during PbBglu16A.
Fig. 8 is that recombiant protein R-PbBglu16A hydrolysis can obtain the TLC analysis result of hydrolyzate after right polysaccharide.Wherein,
G1, L2, L3, L4, L5 and L6 represent glucose, 3-O-beta-D-Glucopyranosyl-D-glucose, laminaritriose, Thallus Laminariae (Thallus Eckloniae) tetrose, Thallus Laminariae (Thallus Eckloniae) pentasaccharides and Thallus Laminariae (Thallus Eckloniae) six respectively
Sugar；M represents oligosaccharide standard；0,15,30,60,120,240min refers to recombiant protein R-under different hydrolysis times respectively
PbBglu16A hydrolysis can obtain the hydrolyzate of right polysaccharide.
Detailed description of the invention
Experimental technique used in following example is conventional method if no special instructions.
Material used in following example, reagent etc., be if no special instructions and commercially obtain.
In following example, with reference to (Yang, et al., Journal of Industrial such as Yang
Microbiology&Biotechnology 2014,41 (10): 1487-1495) method, measure β-1,3-1,4-glucosan
The enzyme activity of enzyme: add the barley beta-glucan of 50 μ L 1% (w/v) in small test tube, 55 DEG C of preheating 3min, it is subsequently adding 150
The enzyme liquid to be measured (i.e. the solution of testing protein) that μ L suitably dilutes, adds 200 μ L DNS reagent (every 1L after 55 DEG C of reaction 10min
Aqueous solution comprises 10g 3,5-dinitrosalicylic acid (DNS), 10g NaOH and 2g phenol), add 200 μ L after boiling 15min and satisfy
And potassium sodium tartrate solution, under 540nm wavelength, measure light absorption value after cooling, using glucose as standard.
The definition of beta-1,3-1,4-dextranase unit of activity is: hydrolysis barley beta-glucan the most per minute
Generate the enzyme amount required for 1 μm ol glucose.
Balun Pueraria lobota hereby series bacillus (Paenibacillus barengoltzii) CAU904 is at document " Fu X.et
Al., Biotechnology for Biofuels.2014,7:174 " disclosed in, the public can obtain from China Agricultural University.
Agarose Ni-IDA affinity column is purchased from GE company of the U.S., and catalog number is 17-0575-01.
Embodiment 1. gene PbBglu16A and the acquisition of albumen PbBglu16A
Design forward primer PbBglu16AncoF:
5’-TGACTCCATGG(underscore shows Nco I restriction enzyme site to GCGCTCCCAACTGGCAGTTG-3 ', after underscore
The first two base is used for the glycine reading frame on completion purpose carrier, the 3rd to last base with sequence 1 in the
91 108 identical)；
With downstream primer PbBglu16AnotR:
5’-TGACTGCGGCCGCGTTTACCTTCGTAAACATCCACTT-3 ' (underscore shows Not I restriction enzyme site, under
After line, sequence matches with the 1225th 1248 in sequence 1)；
And with the genomic DNA of balun Pueraria lobota hereby series bacillus CAU904 as template, the aminoacid of PCR amplification maturation protein
Coding gene sequence.
PCR amplification condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54 DEG C of annealing 30s, 72 DEG C extend 80s, circulation
35 times；5min is extended after last 72 DEG C.
PCR primer reclaims through 1% agarose gel electrophoresis, with Nco I and Not I double digestion.After this double digestion
Product with cross through identical double digestion prokaryotic expression carrier pET-28a (+) vector backbone segment carry out with T4 DNA ligase
Connect, convert to host e. coli DH5 α.Choosing colony PCR (primer used by PCR and amplification condition and this section of aforementioned PCR
Identical) be verified as the positive transformant order-checking.
Result: the recombiant plasmid in positive transformant be carrier pET-28a (+) Nco I and Not I site between insert
91st to 1248 shown DNA fragmentation in sequence 1, described positive transformant i.e. recombinant bacterium A is containing described heavy
The bacillus coli DH 5 alpha of group plasmid.
It is gene PbBglu16A by the 1st to 1251 shown unnamed gene in sequence 1, this gene is compiled
The albumen named albumen PbBglu16A of code, its aminoacid sequence is as shown in sequence 2.
The expression of embodiment 2. recombiant protein R-PbBglu16A and purification
Extract the described recombiant plasmid in the recombinant bacterium A that embodiment 1 obtains, translation table up to host e. coli BL21
(DE3).Positive transformant is seeded to 1L LB fluid medium (containing 50 μ g mL-1Kanamycin).At 37 DEG C, 200rpm condition
Lower cultivation is to OD600Between 0.6-0.8, addition IPTG (isopropyl-β-D-thiogalactoside) to final concentration of 1mM, 30 DEG C
Overnight induction.Centrifugal collect thalline after, by thalline according to the ratio of 1:10 (v/v), by buffer A, (20mM pH 8.0 phosphoric acid delays
Rush liquid, 0.5M NaCl, 20mM imidazoles) resuspension, then ultrasonication in ice-water bath (200W, ultrasonic 3s, intermittently 4s, 120
Secondary), more centrifugal supernatant of collecting is crude enzyme liquid.
Based on carrier pET28-a (+) in have coding His-Tag label protein sequence, use agarose Ni-IDA affine
(its aminoacid sequence is the 31st to 416 shown aminoacid in sequence 2 to column purification recombiant protein R-PbBglu16A
The carboxyl terminal of sequence with the addition of AAALEHHHHHH).Concrete purification step is as follows:
Crude enzyme liquid is splined on Ni-IDA post be purified.Purge process is for (flow velocity is 1mL min-1): first use buffer A
(20mM pH 8.0 phosphate buffer, 0.5M NaCl, 20mM imidazoles) is eluted to OD280Less than 0.05, then use buffer B
(20mM pH 8.0 phosphate buffer, 0.5M NaCl, 50mM imidazoles) is eluted to OD280Less than 0.1, finally use buffer C
(50mM pH 8.0 phosphate buffer, 0.5M NaCl, 200mM imidazoles) eluting.Collect buffer C elution fraction, obtain purification
The solution of recombiant protein R-PbBglu16A.
Purity of protein (figure is detected through SDS-PAGE (Laemmli, U.K.Nature, 1970,227 (5259): 680-685)
1).Total protein concentration, β-1 in the solution of the recombiant protein R-PbBglu16A of mensuration purification and the most purified corresponding crude enzyme liquid,
The enzyme activity of 3-1,4-glucanase and specific enzyme activity power；With total enzyme activity of crude enzyme liquid for 100%, calculate the purification response rate；
With the purification of crude enzyme liquid for 1, calculating purification, result is as shown in table 2.
Wherein, the mensuration of protein content uses lowry method (Lowry, O.H.et al., Journal of Biological
Chemistry, 1951,193 (1): 265-275), and use bovine serum albumin as standard protein.
Table 2 recombiant protein R-PbBglu16A purification table
Embodiment 3. recombiant protein R-PbBglu16A is as the character of beta-1,3-1,4-dextranase
One, optimum pH measures
Recombiant protein R-PbBglu16A and substrate barley beta-glucan are dissolved in following 5 kinds respectively there is different pH value
Buffer system: citric acid/sodium citrate buffer (pH 3.0-6.0)；MES (2-(N-morpholino) ethyl sulfonic acid) buffer (pH
5.5-7.0)；MOPS (2-(N-morpholino) propane sulfonic acid) buffer (pH 6.0-8.5)；Tris-HCl buffer (pH 7.0-
9.0)；Glycine/NaOH buffer (pH 8.5-10.5).Then, mensuration β-1 at 55 DEG C, 3-1,4-glucanase enzyme activity,
Using enzyme activity peak as 100%, calculating the enzyme activity measured under each pH respectively, result is as shown in Figure 2.
Fig. 2 shows: recombiant protein R-PbBglu16A is 6.0 (Fructus Citri Limoniaes as the optimum pH of beta-1,3-1,4-dextranase
Acid/sodium citrate buffer solution).
Two, pH Stability Determination
Recombiant protein R-PbBglu16A is diluted respectively by step one 5 kinds of buffer systems with different pH value, will dilution
Good recombiant protein R-PbBglu16A solution is handled as follows: be incubated 30min in 50 DEG C of thermostat water baths, then at ice
Water-bath cools down 30min；
Measuring β-1 under the conditions of 55 DEG C and pH 6.0,3-1,4-glucanase enzyme activity, to dilute through identical buffer system
But the beta-1,3-1,4-dextranase enzyme activity without the recombiant protein R-PbBglu16A solution diluted of above-mentioned process is right
According to；Calculate the residual enzyme activity after different buffer systems process respectively.The percentage ratio of comparison enzyme activity is accounted for residual enzyme activity
Calculate enzyme activity.
Result is as shown in Figure 3.Fig. 3 shows: recombiant protein R-PbBglu16A has relatively as beta-1,3-1,4-dextranase
Wide pH stability range, when pH is 3.5 9.0, remaining enzyme activity is all more than 80%.
Three, optimal reactive temperature measures
Recombiant protein R-PbBglu16A is suitably diluted, so in 50mM citric acid/sodium citrate buffer (pH6.0)
Rear β-1 that measures at a temperature of 40-75 DEG C respectively, the enzyme activity of 3-1,4-glucanase, using the peak of enzyme activity as
100%, the enzyme activity measured at a temperature of calculating each respectively, result is as shown in Figure 4.
Fig. 4 shows: recombiant protein R-PbBglu16A is 55 DEG C as the optimum temperature of beta-1,3-1,4-dextranase.
Four, temperature stability measures
Recombiant protein R-PbBglu16A is suitably diluted in the citric acid/sodium citrate buffer (pH 6.0) of 50mM
After, carry out following heat treatment: at 40-75 DEG C, be incubated 30min respectively, in ice-water bath, immediately cool down 30min；
Finally measuring β-1 under the conditions of 55 DEG C and pH 6.0, the enzyme activity of 3-1,4-glucanase, with the most thermally treated
Recombiant protein R-PbBglu16A solution after above-mentioned dilution, as comparison, calculates under different temperatures after heat treatment respectively through upper
State the residual enzyme activity in the recombiant protein R-PbBglu16A solution after dilution.The hundred of comparison enzyme activity are accounted for residual enzyme activity
Proportion by subtraction calculates enzyme activity.Result is as shown in Figure 5.
Fig. 5 shows: recombiant protein R-PbBglu16A is as β-1, and 3-1,4-glucanase is relatively stable below 55 DEG C,
Enzyme activity can keep more than 80%.
Five, substrate specificity
Paranitrophenol-the glucosides (pNP-glucosides) using multiple polysaccharide and synthetic measures recombiant protein as substrate
The substrate specificity of R-PbBglu16A.
Polysaccharide includes: barley beta-glucan, lichenin, avenabeta glucosan, laminarin, birch xylan, Pu Lu
Blue sugar, soluble starch, locust bean gum and sodium carboxymethyl cellulose；
PNP-glucosides includes: pNP-β-xylopyranoside, pNP-beta galactose pyranoside, pNP-α-galactose pyranose
Glycosides, pNP-β-pyranglucoside and pNP-α-pyranglucoside.
Concrete assay method is as follows:
For polysaccharide as substrate, using above-mentioned different polysaccharide as test substrate 50mM pH 6.0 citric acids/Fructus Citri Limoniae
Acid sodium buffer is the concentration of 1% (w/v), and according to above-mentioned β-1, the assay method of 3-1,4-glucanase enzyme activity is surveyed
Fixed；
For the pNP-glucosides of synthetic as substrate, by the pNP-glucosides 50mM of above-mentioned different synthetic
PH 6.0 citric acid/sodium citrate buffer becomes 5mM concentration, reacts 10min, measure the extinction at 410nm at 55 DEG C
Value, now enzyme activity unit (U) is defined as hydrolysis substrate the most per minute and discharges required for 1 μm ol pNP
Enzyme amount.
With barley beta-glucan for the enzyme activity of mensuration during substrate for comparison (100%), under the conditions of calculating other substrate
Enzyme activity, result is as shown in table 3, when the above-mentioned substrate listed the most in table 3 represents with it for substrate, recombiant protein
The enzyme activity of R-PbBglu16A is less than 5%.
Result in table 3 shows: recombiant protein R-PbBglu16A is to being made up of β-1,3 glycosidic bond and β-1,4 glycosidic bond
Barley beta-glucan, avenabeta glucosan and lichenin show greater activity, enzyme activity is respectively 100%,
92.3% and 49.8%.Recombiant protein R-PbBglu16A also can hydrolyze by β-1, and the laminarin that 3 glycosidic bonds connect, it is relative
Enzyme activity is 20.5%.But recombiant protein R-PbBglu16A can not hydrolyzed xylan, sodium carboxymethyl cellulose, pulullan,
Other polysaccharide such as soluble starch and locust bean gum, can not hydrolyze the pNP-glucosides of synthetic simultaneously.
Table 3 recombiant protein R-PbBglu16A is as the substrate specificity of beta-1,3-1,4-dextranase
| Substrate kind | Specific enzyme activity (U mg<sup>-1</sup>) | Enzyme activity (%) |
| Barley beta-glucan | 425±26 | 100 |
| Avenabeta glucosan | 392±11 | 92.3 |
| Lichenin | 212±18 | 49.8 |
| Laminarin | 87±5 | 20.5 |
Embodiment 4. recombiant protein R-PbBglu16A application in malt amylase
One, the preparation of beerwort
Beerwort preparation uses agreement saccharifying, specific as follows:
Weigh 50.0g Fructus Hordei Germinatus fine powder (particle diameter < 0.2mm) in the saccharifying cup of known weight, add the water of 200mL 45 DEG C,
(experimental group adds the water-soluble of a certain amount of recombiant protein R-PbBglu16A according to table 4 to be incubated 30min in 45 DEG C of water-baths
Liquid), it is stirred continuously.Then make mash be warming up to 70 DEG C in 25min with the speed of 1 DEG C/min, then add in saccharifying cup
The water that 100mL is 70 DEG C, and at 70 DEG C, it is incubated 1h.After saccharifying terminates, mash is rapidly cooled in 10-15min room temperature,
Mash is mended heavy to 450.0g with distilled water, then filters with Medium speed filter paper, the 100ml filtrate initially collected is refunded and heavily filters,
Till filtering layer surface does not has liquid, collected filtrate is agreement beerwort.
Two, the mensuration of wort filtration time
After agreement saccharifying in step one terminates, the mash of benefit weight to 450.0g is poured over rapidly and is covered with Medium speed filter paper
Funnel in (filter paper totally 18 folding, Funnel diameter 15cm), the 100mL filtrate initially collected is refunded and heavily filters, record collect 200mL
Time needed for filtrate is the filtration time of beerwort.
Three, beerwort proportion and the mensuration of extract content
Beerwort extract content is measured according to EBC (association is brewageed in Europe) standard method, specific as follows:
The water being cooled to 15 DEG C after boiling fills dry, the density bottle (m) of constant weight, is placed in 20 DEG C of water-baths to permanent
Temperature, weighs the weight (m of the density bottle filling water1).In identical density bottle, add in " embodiment 4 step one " and prepare
Beerwort, weigh the weight (m of density bottle filling beerwort in the same way2).Beerwort 20 DEG C proportion according under
Formula (1) calculates:
Wherein,Represent the proportion of beerwort 20 DEG C.
According to obtaining beerwort extract content G than reviewing relative density and extract content synopsis.
Four, the mensuration of Wort viscosity
Use HAAKE falling ball viscometer to measure, record falling sphere from falling sphere pipe upper end graduation mark to dropping down onto lower end graduation mark
The required time.The viscosity of beerwort calculates according to following formula (2):
η=K (ρ1-ρ2)t (2)
Wherein, K represents the falling sphere coefficient of falling ball viscometer, 0.01006mPa s cm3/g·s；ρ1Represent fall-ball type
The falling sphere density of viscometer, 2.221g/cm3；ρ2Represent the density (g/cm of beerwort3), it can pass throughIt is multiplied by the close of water
Degree obtains；T represents falling sphere time (s).
According to the following formula (3), the viscosity conversion calculating acquisition above becoming beerwort percentage concentration is viscosity when 8.6:
Wherein, E represents that beerwort percentage concentration is the viscosity (mPa s) when 8.6；η represents that falling ball viscometer determines
The Wort viscosity (mPa s) come；G represents the extract content (g) of beerwort.
Five, result
Recombiant protein R-PbBglu16A is as shown in table 4 on the impact of malt amylase.Result shows along with recombiant protein R-
The increase of PbBglu16A consumption, filtration time and the viscosity of wheat juice all decrease, when this recombiant protein R-PbBglu16A's
When addition is 80U/g Fructus Hordei Germinatus, Wort viscosity decreases 3.38%, and the rate of filtration improves 14.04%.
The table 4 recombiant protein R-PbBglu16A impact on malt amylase
Embodiment 5. recombiant protein R-PbBglu16A application in hydrolysis bran of Fagopyrum esculentum Moench beta glucan
Bran of Fagopyrum esculentum Moench used by the present embodiment is conventional removing oil bran of Fagopyrum esculentum Moench, and particle diameter is between 10-200 mesh.
One, the method for enzymolysis bran of Fagopyrum esculentum Moench beta glucan
The present embodiment compares recombiant protein R-PbBglu16A hydrolysis bran of Fagopyrum esculentum Moench and the bran of Fagopyrum esculentum Moench of endogenous enzymes inactivation
The effect of middle beta glucan.Wherein, the bran of Fagopyrum esculentum Moench of endogenous enzymes inactivation is by bran of Fagopyrum esculentum Moench is added 80% with 1:10 (w/v)
Ethanol, and it is heated to reflux what 2h prepared at 75 DEG C.
Hydrolytic process is specific as follows: the bran of Fagopyrum esculentum Moench that 1.5g bran of Fagopyrum esculentum Moench or endogenous enzymes inactivate is added 30ml 50mM lemon
Lemon acid/sodium citrate rushes in liquid (pH 6.0), is then not added with or adds recombiant protein R-with the ratio of 150U/g bran of Fagopyrum esculentum Moench
PbBglu16A.Mixture is placed in 50 DEG C of water-bath vibration 12h be hydrolyzed.
Two, hydrolyzate detection
In step one hydrolysis time be 0,0.5,1,2,4,8 and during 12h separately sampled.Sample is kept in boiling water bath
5min makes enzyme inactivate, and is then centrifuged for supernatant and passes through DNS method mensuration content of reducing sugar.β-Portugal's oligosaccharide yield is defined as 1g swallow
The reducing sugar that Testa Tritici skin is obtained, and calculate according to below equation:
By TLC com-parison and analysis hydrolyzate.It is as follows that TLC analyzes operation: takes 1 μ L difference hydrolyzed solution point sample respectively in TLC
Chromatoplate, is placed in TLC chromatoplate in developing agent (n-butyl alcohol: acetic acid: water=2:1:1).With developer (sulfur after exhibition layer terminates
Acid: methanol=5:95) 130 DEG C of baking colour developings.
Three, result
As shown in Figure 6, for bran of Fagopyrum esculentum Moench, add recombiant protein R-PbBglu16A150U/g bran of Fagopyrum esculentum Moench hydrolysis Herba bromi japonici
Wheat bran 8h can obtain the highest β-Portugal's oligosaccharide yield (9.5%), when being not added with recombiant protein R-PbBglu16A, β-Portugal's oligosaccharide yield
It is 5.3% (due to the effect of endogenous enzymes).And for the bran of Fagopyrum esculentum Moench of endogenous enzymes inactivation, add the restructuring of 150U/g bran of Fagopyrum esculentum Moench
Albumen R-PbBglu16A hydrolysis bran of Fagopyrum esculentum Moench 8h can obtain the highest β-Portugal's oligosaccharide yield (7.0%), and is not added with recombiant protein R-
β during PbBglu16A-Portugal's oligosaccharide yield is less than 1%.
As it is shown in fig. 7, for bran of Fagopyrum esculentum Moench hydrolyzate, hydrolyzate when adding recombiant protein R-PbBglu16A includes
A series of oligosaccharide such as disaccharide, trisaccharide and tetrose, and hydrolyzate when being not added with recombiant protein R-PbBglu16A be mainly disaccharide and
Trisaccharide, and comprise a small amount of tetrose；And for the bran of Fagopyrum esculentum Moench of endogenous enzymes inactivation, add the hydrolysis of recombiant protein R-PbBglu16A
Product is based on trisaccharide and tetrose.
Result above shows: for the hydrolytic process of beta-glucosan from oat bran, by adding recombiant protein R-
PbBglu16A can be effectively improved β-Portugal's oligosaccharide yield, and the hydrolysis of the beta-glucosan from oat bran for endogenous enzymes inactivation
For process, can obtain of a relatively high β-Portugal's oligosaccharide yield by adding recombiant protein R-PbBglu16A, β-Portugal is few simultaneously
The composition of sugar has preferable industrial application value based on functional trisaccharide and tetrose, the production thus for β-Portugal's oligosaccharide.
Embodiment 6. recombiant protein R-PbBglu16A can obtain the application in right oligosaccharide in preparation
One, enzymolysis can obtain right polysaccharide method
Recombiant protein R-PbBglu16A has broad substrate specificity, and not only to β-1,3-1,4-glucosan is (as greatly
Wheat beta glucan, lichenin) there is the highest hydrolysis vigor, also to β-1,3-glucosan (such as laminarin) has hydrolysis vigor.This
Embodiment is that the beta-1,3-glucanase activity hydrolysis utilizing recombiant protein R-PbBglu16A can obtain right polysaccharide (English name
Curdlan, Chinese translation have can obtain right polysaccharide, can blue polysaccharide, curdlan, can blue glue, curdlan, heat setting glue many
Sugar) prepare oligosaccharide.
Concrete operations are, 5g can obtain right polysaccharide and add in 100ml 20mM acetate buffer (pH 6.0), 100rpm
Under the conditions of, 50 DEG C of preheating 30min so that mixture becomes colloidal suspensions, is subsequently added the water-soluble of recombiant protein R-PbBglu16A
Liquid, make the initial concentration of recombiant protein R-PbBglu16A be 0.5U/mL (in terms of laminarin enzyme activity, the work of laminarinase
Power assay method and definition and above-mentioned β-1, the enzyme activity determination method of 3-1,4-glucanase and define identical, but substrate is changed
For laminarin), after mixing, mixture is placed in 50 DEG C of water-bath 4h and is hydrolyzed.
Two, hydrolyzate detection
In step one hydrolysis time be 0,15,30,60,120,240min samples, respectively by sample in boiling from hydrolyzed solution
Water-bath keeps 5min make enzyme inactivate, be then centrifuged for supernatant, by TLC com-parison and analysis hydrolyzate.TLC analyzes operation:
Take 1 μ L difference hydrolyzed solution respectively point sample in TLC chromatoplate, TLC chromatoplate is placed in developing agent (n-butyl alcohol: acetic acid: water=2:1:
1) in.Exhibition layer develops the color with developer (sulphuric acid: methanol=5:95) 130 DEG C baking after terminating.
Three, result
As shown in Figure 8, the right polysaccharide suspension that obtains of 5% (w/v) is adding after recombiant protein R-PbBglu16A, about 4h
Can be by complete hydrolysis.TLC analyzes display recombiant protein R-PbBglu16A hydrolysis can obtain the oligosaccharide hydrolysis product that right polysaccharide obtains
Along with hydrolysis gradually accumulates, final hydrolyzate is mainly containing glucose, 3-O-beta-D-Glucopyranosyl-D-glucose, laminaritriose, Thallus Laminariae (Thallus Eckloniae) tetrose
With a small amount of more high polymerization degree oligosaccharide.
The content not being described in detail in this specification belongs to prior art known to professional and technical personnel in the field.
Claims (10)
1. a protein, is following (1) or (2) or (3) or the protein of (4):
(1) protein of the aminoacid sequence composition shown in sequence 2；
(2) sequence 2 is from the protein of the 31st to 416 shown aminoacid sequence composition of amino terminal；
(3) carboxyl terminal in described (2) adds the protein of Poly-His label；
(4) aminoacid sequence of described (1) or (2) or (3) is passed through replacement and/or the disappearance of one or several amino acid residue
And/or add the protein derivative by (1) or (2) or (3) with identical function obtained.
2. the encoding gene of protein described in claim 1.
3. encoding gene as claimed in claim 2, it is characterised in that: described encoding gene be following (a) or (b) or (c) or
The DNA molecular of (d):
DNA molecular shown in (a) sequence 1；
91st to 1248 the shown DNA molecular of (b) sequence 1；
Protein described in c DNA molecule hybridize that () is limited with (a) or (b) under strict conditions and coding claim 1
DNA molecular；
D DNA molecular that () and (a) or (b) or (c) limit has sequence iden and coding claim 1 institute of at least 75%
State protein DNA molecule.
4. contain the recombinant vector of encoding gene described in Claims 2 or 3, expression cassette, transgenic cell line or recombinant bacterium.
5. protein described in claim 1 is as the application in beta-1,3-1,4-dextranase.
Apply the most as claimed in claim 5, it is characterised in that: described application includes that, to β-1,3-1,4-glucosan is hydrolyzed
Step；
And/or, described β-1,3-1,4-glucosan is at least in barley beta-glucan, avenabeta glucosan and lichenin
Kind.
7. protein described in claim 1 is as the application in beta-1,3-glucanase.
Apply the most as claimed in claim 7, it is characterised in that: described application includes β-1, the step that 3-glucosan is hydrolyzed
Suddenly；
And/or, described β-1,3-glucosan is at least one in laminarin and the right polysaccharide of Ke get.
9. the application as described in arbitrary in claim 58, it is characterised in that: the pH value of described hydrolysis is 3 10.5；
And/or, the temperature of described hydrolysis is 40 75 DEG C；
And/or, described application is the application in malt amylase；
And/or, described application is the application in bran of Fagopyrum esculentum Moench hydrolyzes；
And/or, described application is can to obtain the application in right oligosaccharide in preparation.
10. recombinant vector described in encoding gene described in claim 2 and 3 and claim 4, expression cassette, transgenic cell line or
Recombinant bacterium application in preparing beta-1,3-1,4-dextranase and/or beta-1,3-glucanase.
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| CN107893061A (en) * | 2017-11-15 | 2018-04-10 | 中国农业大学 | A kind of dextranases of β 1,3 in rhizomucor miehei source and its application |
| CN110628750A (en) * | 2019-09-25 | 2019-12-31 | 广西大学 | Beta-1, 3-1, 4-glucan glucohydrolase and application thereof |
| CN113785039A (en) * | 2019-04-03 | 2021-12-10 | 诺维信公司 | Polypeptides having beta-glucanase activity, polynucleotides encoding same and use thereof in cleaning and detergent compositions |
| CN114317636A (en) * | 2021-12-29 | 2022-04-12 | 苏州科宁多元醇有限公司 | Preparation method of oligosaccharide |
| CN114561418A (en) * | 2020-11-27 | 2022-05-31 | 潍坊康地恩生物科技有限公司 | Aspergillus niger mutant strain for high yield of beta-1, 3-glucanase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107893061A (en) * | 2017-11-15 | 2018-04-10 | 中国农业大学 | A kind of dextranases of β 1,3 in rhizomucor miehei source and its application |
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| CN110628750A (en) * | 2019-09-25 | 2019-12-31 | 广西大学 | Beta-1, 3-1, 4-glucan glucohydrolase and application thereof |
| CN110628750B (en) * | 2019-09-25 | 2022-03-11 | 广西大学 | Beta-1, 3-1, 4-glucan glucohydrolase and application thereof |
| CN114561418A (en) * | 2020-11-27 | 2022-05-31 | 潍坊康地恩生物科技有限公司 | Aspergillus niger mutant strain for high yield of beta-1, 3-glucanase |
| CN114561418B (en) * | 2020-11-27 | 2023-09-29 | 潍坊康地恩生物科技有限公司 | Aspergillus niger mutant strain for high yield of beta-1, 3-glucanase |
| CN114317636A (en) * | 2021-12-29 | 2022-04-12 | 苏州科宁多元醇有限公司 | Preparation method of oligosaccharide |
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