CN112094835B - Application of beta-glucosidase mutant - Google Patents
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Abstract
The invention discloses an application of a beta-glucosidase mutant, belonging to the technical field of enzyme engineering. The beta-glucosidase mutants G224A, G224N and G224V with improved transglycosylation/hydrolysis ratio are obtained through site-directed mutagenesis construction, and the yields of the prepared galactooligosaccharides are 46.7%, 43.8% and 38.4% respectively under the optimal conditions, and are 1.84 times, 1.73 times and 1.51 times of the wild type respectively; the yield of the prepared hexyl glucoside is 51.8 percent, 56.9 percent and 38.8 percent respectively, which are 2.79 times, 3.07 times and 2.09 times of the wild type respectively, and the method has better industrial application potential.
Description
Technical Field
The invention relates to an application of a beta-glucosidase mutant, belonging to the technical field of enzyme engineering.
Background
GH1 β -glucosidase, essentially a hydrolase, has as its main activity the hydrolysis of β -glycosidic bonds in oligosaccharides or glycoside derivatives. However, GH1 beta-glucosidase has been proved to have unique transglycosylation activity, shows a certain heterogeneity in glycosyl substrate recognition, and can catalyze the hydrolysis or transglycosidation synthesis of glycosidic bonds of various glycosyl units, such as sophorose by transglycosidation with glucose as a substrate and galactooligosaccharide by galactosyl transfer with lactose as a substrate. Glycosyltransferases require expensive nucleotide sugars as substrates to produce oligosaccharides compared to the synthetic activity of glycosyltransferases, whereas GH1 β -glucosidase can be synthesized using low cost starting materials, such as natural mono-or disaccharides. Therefore, GH1 beta-glucosidase has great advantages in industrial synthetic production.
Galactooligosaccharides (GOS) are conjugated oligosaccharides having 1 or more galactose molecules attached to the galactosyl side of lactose, and have high heat resistance and acid resistance, thus maintaining high stability in the digestive tract of the human body and promoting the growth of bifidobacteria. The GOS is fresh and cool in taste, low in sweetness and only half of cane sugar in heat, is an excellent low-calorific-value food ingredient, can improve the taste of dairy products, is more favorable for intestinal flora, and is widely applied to food industries such as dairy products, candies, health-care foods, cakes, infant milk powder and the like. The alkyl glycoside is an alkyl glycoside derivative synthesized by glucose and fatty alcohol, has no toxicity, small irritation to skin, safety, and remarkable thickening, tackifying and detergency. The cleaning detergent prepared from the alkyl glycoside has good solubility, mildness and degreasing capability, is widely used for preparing a tableware detergent, a bath lotion, a shampoo product, a hard surface cleaning agent, a facial cleanser, a washing powder and the like, and has obvious effect. GH1 beta-glucosidase has been widely used as an excellent biocatalyst for synthesizing high value-added oligosaccharides such as galactooligosaccharides and alkylglycosides and glycoside derivatives.
However, the reaction conditions commonly used in the enzymatic preparation of galactooligosaccharides and alkylglycosides are generally low in yield when the reaction is carried out at low water activity and high substrate concentration. The research on the synthesis mechanism of GH1 beta-glucosidase is not thorough and comprehensive at present, and the mainstream view is that the synthesis mechanism mainly depends on the transglycosylation activity of GH1 beta-glucosidase, and the enzymatic catalysis process is mainly divided into two steps, namely glycosylation and deglycosylation, wherein the deglycosylation process is the key for determining whether the transglycosylation reaction or the hydrolysis reaction occurs, and when the glycosyl acceptor is a water molecule, the hydrolysis reaction occurs; when the glycosyl acceptor is a sugar, a transglycosidation reaction occurs. And how to improve the yield of galacto-oligosaccharides and alkyl glycoside under the condition of high substrate concentration has great significance for industrial production.
Disclosure of Invention
The invention firstly provides a beta-glucosidase mutant, which is obtained by mutating the 224 th site of beta-glucosidase with an amino acid sequence shown as SEQ ID NO. 1.
In one embodiment of the present invention, the β -glucosidase mutant is: the amino acid sequence is shown as SEQ ID NO.1, the 224 th site of beta-glucosidase is obtained by mutating glycine to alanine, and the name is G224A;
or the 224 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is obtained by mutating glycine to asparagine, and is named as G224N;
or the 224 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is obtained by mutating glycine into valine, and the name is G224V.
In one embodiment of the invention, the amino acid sequence of the beta-glucosidase mutant G224A is shown as SEQ ID NO. 9; the amino acid sequence of the beta-glucosidase mutant G224N is shown in SEQ ID NO. 10; the amino acid sequence of the beta-glucosidase mutant G224V is shown in SEQ ID NO. 11.
In one embodiment of the invention, the nucleotide sequence encoding the beta-glucosidase is shown as SEQ ID NO. 2.
The invention also provides a gene for coding the beta-glucosidase mutant.
The invention also provides an expression vector carrying the gene.
The invention also provides a microbial cell carrying the gene or the expression vector.
In one embodiment of the invention, the microbial cells include, but are not limited to, bacterial, fungal cells.
The invention also provides a method for preparing galacto-oligosaccharide, which comprises the steps of adding the beta-glucosidase mutant into a reaction system containing lactose for reaction to obtain a reaction solution; and separating the galactooligosaccharide from the reaction solution.
In one embodiment of the present invention, the enzyme dosage of the β -glucosidase mutant is: 8-16U/mL.
In one embodiment of the invention, the concentration of the substrate lactose in the reaction system is 350-450 g/L.
In one embodiment of the invention, the pH of the reaction is between 5.8 and 6.2.
In one embodiment of the invention, the pH of the reaction is 6.0.
In one embodiment of the invention, the temperature of the reaction is from 75 to 85 ℃.
In one embodiment of the invention, the temperature of the reaction is 80 ℃.
In one embodiment of the invention, the reaction time is 2 to 8 hours.
In one embodiment of the invention, the reaction time is 6 h.
The invention also provides a method for preparing alkyl glycoside, which comprises the following steps: adding the beta-glucosidase mutant into a reaction system containing a glycosyl donor and a glycosyl acceptor for reaction to obtain a reaction solution; separating the reaction solution to obtain alkyl glycoside; the glycosyl donor is methyl glucoside and/or p-nitrophenylglucoside, and the glycosyl acceptor is fatty alcohol.
In one embodiment of the present invention, the beta-glucosidase mutant is added in an amount of 3.5-4.5. mu.M.
In one embodiment of the present invention, the added amount of the β -glucosidase mutant is 3.9 μ M.
In one embodiment of the invention, the concentration of the glycosyl donor is 30-40 mM.
In one embodiment of the invention, the concentration of the glycosyl donor is 34 mM.
In one embodiment of the invention, the concentration of glycosyl acceptor is 8% to 87% (v/v).
In one embodiment of the invention, the pH of the reaction is between 5.8 and 6.2.
In one embodiment of the invention, the pH of the reaction is 6.0.
In one embodiment of the invention, the temperature of the reaction is 85-95 ℃.
In one embodiment of the invention, the temperature of the reaction is 90 ℃.
In one embodiment of the invention, the reaction time is 1 to 5 hours.
In one embodiment of the invention, the reaction time is 4 h.
The invention also provides the application of the mutant or the gene or the expression vector or the microbial cell in preparing galactooligosaccharide or alkyl glucoside.
Advantageous effects
Compared with the wild type, the yield of galacto-oligosaccharides and alkyl glycoside prepared by the beta-glucosidase mutant is obviously improved. Mutants G224A, G224N, G224V produced galactooligosaccharide yields of 46.7%, 43.8% and 38.4%, respectively, which were 1.84-fold, 1.73-fold and 1.51-fold higher than the wild type. The mutants G224A, G224N and G224V produced hexyl glycoside with yields of 51.8%, 56.9% and 38.8% respectively, which were 2.79 times, 3.07 times and 2.09 times that of wild type.
Drawings
FIG. 1: SDS-PAGE electrophoresis of purified proteins of wild-type and mutant beta-glucosidase.
Wherein M is a protein molecular weight standard; 1, wild type WT; 2, mutant G224A; 3, mutant G224N; 4, mutant G224V.
FIG. 2: analysis of products from wild-type and mutant beta-glucosidase preparations of galactooligosaccharides.
FIG. 3: schematic diagram of preparation of galacto-oligosaccharide by beta-glucosidase.
Detailed Description
The 4-nitrophenyl β -D-glucopyranoside, nitrophenyl glucoside, hexanol, lactose, referred to in the following examples, were obtained from Shanghai Vitta Chemicals, Inc., and E.coli JM109 competent cells were obtained from Shanghai Producer.
The media involved in the following examples are as follows:
LB liquid medium: 5g/L of yeast powder, 10g/L of tryptone and 10g/L of NaCl.
LB solid Medium: on the basis of LB liquid medium, agar is added: 10 g/L.
TB liquid fermentation medium: 24g/L yeast powder, 5g/L glycerin, 12g/L tryptone and K 2 HPO 4 ·3H 2 O 16.43g/L，KH 2 PO 4 2.31g/L。
The detection methods referred to in the following examples are as follows:
and (3) measuring the activity of the beta-glucosidase enzyme:
the reaction system is 1mL, 960 μ L of pH 5.0 acetic acid buffer solution, 20 μ L of crude enzyme solution diluted moderately (preferably with the absorbance value of reaction solution at 405nm at 0.2-1.2) is added, 20 μ L of 100mmol/L pNPG is added, reaction is carried out in a constant temperature water bath at 60 deg.C for 10min, and 200 μ L of 1mol/L Na is added immediately after 10min 2 CO 3 The reaction was stopped with ice for 5min and the absorbance measured at 405 nm. The enzyme solution inactivated by heating was treated as a blank in the same manner.
Definition of enzyme activity unit: the enzyme activity of 1 mu mol of p-nitrophenol generated by hydrolyzing pNPG per minute per milliliter of enzyme solution is one enzyme activity unit.
The relative enzyme activity calculation method comprises the following steps: enzyme activity ═ a 405 +0.002) dilution factor of reaction system/(0.0074 reaction time plus enzyme amount)
The detection method of the content of galacto-oligosaccharide comprises the following steps:
the galacto-oligosaccharide is the sum of transfer disaccharide, transfer trisaccharide and transfer tetrasaccharide.
The product components were detected using HPLC as follows:
detection of Transfer Disaccharide (TD): agilent 1200 HPLC chromatograph, Agilent autosampler, chromatographic column Thermo Aps-2 HYPERSIL (4.6 mm. times.250 mm), differential detector Agilent 2410; the mobile phase volume fraction was 78% (v/v) acetonitrile/water solution, and the flow rate and column temperature were set to 0.8mL/min and 40 ℃ respectively.
Detection of tetrasaccharide, trisaccharide and lactose and monosaccharides in galactooligosaccharides:
agilent 1200 HPLC chromatograph, Agilent autosampler, chromatography column Hi-PlexNa (300mm × 7.7mm), differential detector Agilent 2410; the mobile phase was pure water, and the flow rate and column temperature were 0.3mL min-1 and 80 ℃ respectively.
Calculation of product conversion: yield (%) - (mass of galactooligosaccharide in product/mass of all sugars in product × 100%.
The detection method of the content of the alkyl glycoside comprises the following steps:
the detection method comprises the following steps: the detection was carried out by using a differential detector of a Waters1525EF HPLC chromatography, with the specification of an Athena C18-WP column (4.6X 250mm, 5 μm), the column temperature set at 40 ℃, the flow rate set at 0.5 mL/min-1, and the mobile phase set at 70% methanol/water solution. Results the amount of product was calculated using the external standard method.
Example 1: expression of wild-type beta-glucosidase Gene
Wild type enzyme recombinant Bacillus subtilis strain B.subtilis/pBSM mu L3-Tsbgl1A is constructed in the early stage of the laboratory (described in Chinese patent application with publication number CN 111411117A).
The expression of wild-type beta-glucosidase gene is carried out according to the patent, and the specific implementation steps are as follows: inoculating B.subtilis/pBSM mu L3-Tsbgl1A into an LB liquid culture medium (containing 100mg/L ampicillin) to grow for 8 hours from a glycerol tube preserved in the early stage of a laboratory to obtain a seed solution, inoculating the seed solution into a TB liquid fermentation culture medium (containing 10mg/L tetracycline) according to the inoculation amount of 5% (v/v), culturing for 2 hours at 37 ℃, and further transferring into a shaking table at 33 ℃ to continue to culture and ferment for 24 hours to obtain a fermentation liquid; centrifuging the fermentation liquid at 4 deg.C and 12000rpm for 10min, removing supernatant, collecting thallus, adding 50mL 50mM pH6.0 citric acid-disodium hydrogen phosphate buffer solution into thallus, fully suspending thallus, breaking cell wall with high pressure homogenizer, centrifuging at 10000rpm for 20min, collecting cell wall-broken supernatant as crude enzyme solution, OD 600 The enzyme activity of the crude enzyme solution of 5 was 10.41U/mL.
Example 2: construction and expression of beta-glucosidase single mutant
(1) Preparation of mutants
Respectively designing and synthesizing primers of G224A, G224N and G224V mutation according to the gene sequence of the beta-glucosidase with the nucleotide sequence shown as SEQ ID NO.2, carrying out site-directed mutagenesis on the beta-glucosidase TsBgl1A, and respectively sequencing to confirm whether the coding gene of the beta-glucosidase mutant is correct; and (3) introducing the vector carrying the mutant gene into bacillus subtilis to express to obtain the single mutation beta-glucosidase.
PCR amplification of site-directed mutant coding gene: using rapid PCR technology, an expression vector pBSM μ L3-Tsbgl1A (described in Chinese patent application publication No. CN 111411117A), which carries a gene encoding β -glucosidase, was used as a template.
The site-directed mutagenesis primers for introducing the G224A mutation were:
a forward primer: 5' -ATTGTTTTTAACAACGCATATTTTGAACCGGCA-3’(SEQ ID NO.3)
Reverse primer: 5' -TGCCGGTTCAAAATATGCGTTGTTAAAAACAAT-3’(SEQ ID NO.4)
The site-directed mutagenesis primers for introducing the G224N mutation were:
a forward primer: 5' -ATTGTTTTTAACAACAATTATTTTGAACCGGCA-3’(SEQ ID NO.5)
Reverse primer: 5' -TGCCGGTTCAAAATAATTGTTGTTAAAAACAAT-3’(SEQ ID NO.6)
The site-directed mutagenesis primers for introducing the G224V mutation were:
a forward primer: 5' -ATTGTTTTTAACAACGTTTATTTTGAACCGGCA-3’(SEQ ID NO.7)
Reverse primer: 5' -TGCCGGTTCAAAATAAACGTTGTTAAAAACAAT-3’(SEQ ID NO.8)
The PCR reaction systems are as follows: 5 XPS buffer 10. mu.L, dNTPs Mix (2.5 mmol. multidot.L) -1 ) 4. mu.L, 1. mu.L of forward primer (10. mu. mol. L-1), and 1. mu.L of reverse primer (10. mu. mol. L) -1 ) mu.L, template DNA 1. mu.L, Primerstar HS (5U/. mu.L) 0.5. mu.L, and distilled water was added to 50. mu.L.
The PCR amplification program was set up as: first, pre-denaturation at 94 ℃ for 5 min; then 30 cycles were entered: denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 5s, and extension at 72 deg.C for 7min for 50 s; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃. The PCR product was detected by electrophoresis on a 1% agarose gel.
Adding Dpn I into a PCR product which is verified to be correct, carrying out water bath for 2h at 37 ℃, degrading a template, then transforming escherichia coli JM109 competent cells, coating the transformation product on an LB solid culture medium containing 100mg/L ampicillin, culturing for 10-12 h at 37 ℃, selecting a positive clone, inoculating the positive clone into an LB liquid culture medium, and culturing for 8-10h at 37 ℃. Inoculating the recombinant bacteria with correct sequencing from a glycerol pipe to an LB liquid culture medium, culturing overnight, extracting plasmids, transforming and expressing host B.subtilis WS11 competent cells by the plasmids, and obtaining the recombinant strains capable of expressing mutants G224A, G224N and G224V.
(2) Expression of the mutant
Picking single colonies of the recombinant strains capable of expressing the mutants G224A, G224N and G224V obtained in the step (1), respectively inoculating the single colonies to an LB liquid culture medium (containing 100mg/L ampicillin) for growing for 8 hours to obtain seed liquid; inoculating the seed solution into a TB liquid fermentation medium (containing 10mg/L tetracycline) according to the inoculation amount of 5% (v/v), culturing at 37 ℃ for 2h, and then transferring into a shaking table at 33 ℃ for continuous culture and fermentation for 24h to obtain a fermentation solution; centrifuging the fermentation liquid at 4 deg.C and 12000rpm for 10min, removing supernatant, collecting thallus, adding 50mL 50mM pH6.0 citric acid-disodium hydrogen phosphate buffer solution into thallus, fully suspending thallus, breaking cell wall with high pressure homogenizer, centrifuging at 10000rpm for 20min, collecting cell wall-broken supernatant as crude enzyme solution, and detecting OD 600 The enzyme activity of beta-glucosidase in the crude enzyme solution is 5 hours.
The enzyme activities of beta-glucosidase in crude enzyme solution obtained by fermenting the recombinant strains capable of expressing the mutants G224A, G224N and G224V are respectively 9.9U/mL, 9.8U/mL and 9.6U/mL, and therefore the recombinant strains capable of expressing the mutants G224A, G224N and G224V are successfully expressed.
Example 3: purification of beta-glucosidase
The method comprises the following specific steps:
(1) taking 500mL of the crude enzyme solution obtained in the example 2, adding 175g of solid ammonium sulfate into the crude enzyme solution, and salting out for 12 h;
(2) centrifuging the salted-out crude enzyme solution obtained in the step (1) at 4 ℃ and 10000rpm for 20min, dissolving the precipitate with a proper amount of buffer solution A containing 20mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole and pH7.4, dialyzing in the buffer solution A for 10h, and filtering through a 0.22 mu M membrane to prepare a sample;
(3) after the Ni affinity column is balanced by the buffer solution A, the sample obtained in the step (2) is absorbed into the Ni affinity column, and is completely absorbed, then is respectively eluted by 100mL of the buffer solution A, 100mL of the buffer solution A containing 60mM of imidazole and 100mL of the buffer solution A containing 480mM of imidazole in sequence, the flow rate is 1mL/min, the target protein beta-glucosidase is eluted by the buffer solution A containing 480mM of imidazole, and the part of eluent is collected;
(4) dialyzing the eluent (480 mM imidazole-containing buffer A) obtained in the step (3) in 50mM sodium phosphate buffer solution with pH6.0 for 10h to obtain the purified beta-glucosidase enzyme product.
The purified beta-glucosidase enzyme product reaches electrophoresis purity, and the apparent molecular weight is 45000 daltons. The purified electrophoretogram is shown in FIG. 1.
The same procedure was carried out to obtain a purified β -glucosidase preparation of the β -glucosidase wild-type enzyme of example 1.
Example 4: transglycoside/hydrolysis ratio (R) of wild-type and mutant enzymes s /R h )
Determination of the transglycosylation/hydrolysis ratio (R) of the enzyme with pNPG (4-nitrophenyl beta-D-glucopyranoside) as substrate s /R h ) In the reaction product component, pNP is total reaction product, glucose is hydrolysis reaction product, pNPG 2 The transglycoside/hydrolysis ratio (R) of the transglycoside reaction product s /R h ) Comprises the following steps: pNPG 2 Ratio of the amount of glucose produced to the amount of glucose produced.
Transglycosidic hydrolysis reaction System (100. mu.L): the solvent was water, and 90. mu.L of pNPG substrate and 10. mu.L of the purified β -glucosidase preparation obtained in example 3 were added to the solvent, respectively, at a final pNPG substrate concentration of 50mM, and at a final enzyme solution concentration: 5 μ M. Transglycosidic hydrolysis reaction conditions: reacting for 10min under the conditions of pH6.0 and 80 ℃.
pNP molar production determination: 160. mu.L of the reaction solution + 40. mu.L of 1M NaCO 3 And taking 180 mu L of enzyme label plate to measure the light absorption value. Glucose massagerMeasurement of amount of formed product: and reacting for 10min by using 300 mu L of GOD +18 mu L of reaction liquid, and measuring the light absorption value by using 200 mu L of enzyme label plate. The molar yield of the transglycosidation reaction product is the difference between the total reaction product and the hydrolysis reaction product.
As a result, as shown in Table 1, it was found that the transglycoside/hydrolysis ratio (R) of the mutants G224A and G224V was not considered in the secondary hydrolysis s /R h ) All had significant increase compared with wild type.
TABLE 1 transglycosylation/hydrolysis ratio of mutant enzymes
Example 5: application of beta-glucosidase mutant in preparation of galactooligosaccharide
The method comprises the following specific steps:
using lactose as a substrate, respectively adding lactose with the final substrate concentration of 400G/L and the purified beta-glucosidase product of the beta-glucosidase wild enzyme or the beta-glucosidase mutant obtained in example 3 into a citrate-phosphate buffer solution system, wherein the enzyme adding amount of the purified beta-glucosidase product of the beta-glucosidase wild enzyme or the beta-glucosidase mutant is 16U/mL, reacting for 6h under the reaction conditions of pH6.0 and 80 ℃ to prepare galactooligosaccharide, and detecting the yield of the galactooligosaccharide produced by the beta-glucosidase wild enzyme and the mutants G224A, G224N and G224V according to the reaction principle shown in FIG. 3.
As shown in FIG. 2 and Table 2, the mutants G224A, G224N and G224V produced galactooligosaccharides with yields of 46.7%, 43.8% and 38.4% respectively, which were 1.84-fold, 1.73-fold and 1.51-fold higher than the wild type.
TABLE 2 production and yield of galactooligosaccharides from mutant enzymes
Example 6: application of beta-glucosidase mutant G224A in preparation of galactooligosaccharide
The specific embodiment was as in example 5, except that the enzyme addition amounts of the β -glucosidase preparation of purified β -glucosidase mutant G224A were adjusted to 4U/mL, 8U/mL, and 12U/mL, and the results were: the yields of galactooligosaccharides produced were 18.4%, 34.9% and 42.3%, respectively.
Example 7: application of beta-glucosidase mutant in preparation of alkyl glycoside
The method comprises the following specific steps:
and (2) taking methyl glucoside or p-nitrophenyl glucoside as a glycosyl donor, taking fatty alcohol as a glycosyl acceptor, and adding the beta-glucosidase mutant with the improved transglycosylation/hydrolysis ratio to react to prepare the alkyl glucoside.
Respectively adding p-nitrophenyl glucoside pNPG and hexanol into a reaction system, wherein the concentration of donor substrate p-nitrophenyl glucoside pNPG is 34mM, the concentration of acceptor substrate hexanol is 87% (v/v), the reaction pH is 6, placing the reaction system in a ThermoMixer thermostatic mixer at 90 ℃ to preheat for 10min at the rotation speed of 800rpm, adding 150 mu L of the purified beta-glucosidase product of the beta-glucosidase wild enzyme or the beta-glucosidase mutant obtained in the example 3 to start the reaction, wherein the adding amount of the purified beta-glucosidase product of the beta-glucosidase wild enzyme or the beta-glucosidase mutant is 3.9 mu M, taking 50 mu L of samples at different time points, diluting the samples by using 150 mu L of acetonitrile, and analyzing the final product result by high performance liquid chromatography.
The results of the experiments are shown in table 3, the mutants G224A, G224N, G224V produced hexyl glycoside with yields of 51.8%, 56.9% and 38.8% respectively, which are 2.79 times, 3.07 times and 2.09 times higher than the wild type.
TABLE 3 production and yield of galactooligosaccharides from mutant enzymes
Example 8: application of beta-glucosidase mutant G224V in preparation of alkyl glycoside
The specific embodiment was as in example 6, except that the acceptor substrate hexanol concentration (v/v) in the reaction catalyzed by the purified β -glucosidase enzyme preparation of β -glucosidase mutant G224V was adjusted to 8%, 15%, 30%, 50%, and the results were: the yields of the produced alkyl glycosides were 4.8%, 19.3%, 39.6%, 49.3%, respectively.
Comparative example 1:
the difference between the modified β -glucosidase as in example 5, which is a β -glucosidase wild-type enzyme derived from Talaromyces piceae (described in chinese patent application publication No. CN 111411117A), and a β -glucosidase wild-type enzyme derived from Talaromyces cellulolyticus (GenBank GAM40530.1), is: the yields of galactooligosaccharides produced were 11.6% and 15.3%, respectively.
Comparative example 2:
the difference between the modified β -glucosidase as in example 6, which is a β -glucosidase wild-type enzyme derived from Talaromyces piceae (described in chinese patent application publication No. CN 111411117A), and a β -glucosidase wild-type enzyme derived from Talaromyces cellulolyticus (GenBank GAM40530.1), is: the yields of alkyl glycoside produced were 12.2% and 16.6%, respectively.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university in south of the Yangtze river
Application of beta-glucosidase mutant
<130> BAA200931A
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 446
<212> PRT
<213> Artificial sequence
<400> 1
Met Ser Met Lys Lys Phe Pro Glu Gly Phe Leu Trp Gly Val Ala Thr
1 5 10 15
Ala Ser Tyr Gln Ile Glu Gly Ser Pro Leu Ala Asp Gly Ala Gly Met
20 25 30
Ser Ile Trp His Thr Phe Ser His Thr Pro Gly Asn Val Lys Asn Gly
35 40 45
Asp Thr Gly Asp Ile Ala Cys Asp His Tyr Asn Arg Trp Lys Glu Asp
50 55 60
Ile Glu Ile Met Lys Glu Leu Gly Val Lys Ala Tyr Arg Phe Ser Ile
65 70 75 80
Ser Trp Pro Arg Ile Leu Pro Glu Gly Thr Gly Arg Val Asn Gln Lys
85 90 95
Gly Ile Asp Phe Tyr Ser Arg Ile Ile Asp Thr Leu Leu Glu Gln Gly
100 105 110
Ile Thr Pro Phe Val Thr Ile Tyr His Trp Asp Leu Pro Phe Glu Leu
115 120 125
Gln Leu Lys Gly Gly Trp Ala Asn Arg Glu Val Ala Asp Trp Phe Ala
130 135 140
Glu Tyr Ser Arg Val Leu Phe Glu Asn Phe Gly Asp Arg Val Lys His
145 150 155 160
Trp Ile Thr Leu Asn Glu Pro Trp Val Val Ala Ile Val Gly His Leu
165 170 175
Tyr Gly Val His Ala Pro Gly Met Lys Asp Ile Tyr Val Ala Phe His
180 185 190
Val Val His Asn Leu Leu Arg Ala His Ala Lys Ser Val Lys Ile Phe
195 200 205
Arg Glu Ile Val Lys Asp Gly Lys Ile Gly Ile Val Phe Asn Asn Gly
210 215 220
Tyr Phe Glu Pro Ala Ser Glu Lys Glu Glu Asp Val Arg Thr Ala Glu
225 230 235 240
Phe Ala His Gln Phe Thr Asn Tyr Pro Leu Phe Leu Asn Pro Ile Tyr
245 250 255
Lys Gly Asp Tyr Pro Glu Leu Val Arg Glu Phe Ala Arg Glu Phe Leu
260 265 270
Pro Lys Asp Tyr Lys Lys Asp Met Glu Glu Ile Gln Glu Arg Ile Asn
275 280 285
Phe Val Gly Ile Asn Tyr Tyr Ser Gly His Met Val Lys Tyr Asp Pro
290 295 300
Lys Ser Pro Gly Gly Val Ser Phe Val Glu Arg Asp Leu Pro Lys Thr
305 310 315 320
Glu Met Gly Trp Glu Val Val Pro Glu Gly Leu Tyr Tyr Ile Leu Lys
325 330 335
Gly Val Lys Asp Glu Tyr Asn Pro Glu Glu Ile Tyr Val Thr Glu Asn
340 345 350
Gly Ala Ala Tyr Asn Asp Val Val Ser Glu Asp Gly Lys Val His Asp
355 360 365
Gln Asn Arg Ile Asp Tyr Leu Lys Ala His Ile Gly Gln Ala Trp Lys
370 375 380
Ala Leu Gln Asp Gly Val Pro Leu Arg Gly Tyr Phe Val Trp Ser Leu
385 390 395 400
Leu Asp Asn Phe Glu Trp Ala Glu Gly Tyr Ser Lys Arg Phe Gly Ile
405 410 415
Val Tyr Val Asp Tyr Gln Thr Gln Lys Arg Ile Ile Lys Asp Ser Gly
420 425 430
His Trp Tyr Ala Asn Val Ile Lys Asn Asn Gly Leu Glu Asp
435 440 445
<210> 2
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atgagcatga aaaagtttcc ggaaggtttt ctgtggggtg ttgcaaccgc gagctatcag 60
attgaaggta gcccgctggc cgatggtgcg ggtatgagca tttggcatac ctttagccat 120
acgccgggca atgttaaaaa tggtgatact ggtgatatcg catgcgatca ttataatcgt 180
tggaaagaag atatcgaaat catgaaagaa ctgggtgtta aagcatatcg ttttagcatc 240
agctggccgc gtatcctgcc ggaaggtaca ggtcgcgtta atcagaaagg tatcgatttt 300
tatagccgta ttatcgatac cctgctggaa cagggtatta ccccgtttgt tacaatttat 360
cattgggatc tgccgtttga actgcagctg aaaggtggct gggcaaatcg cgaagttgca 420
gattggtttg cggaatatag ccgtgtgctg tttgaaaact ttggtgatcg tgtgaaacat 480
tggattaccc tgaatgaacc ttgggttgtg gcgattgttg gtcatctgta tggtgttcat 540
gcgccgggta tgaaagatat ttatgttgcc tttcatgtgg ttcataatct gctgcgtgct 600
catgcgaaat cagtgaaaat ttttcgtgaa attgtgaaag atggcaaaat tggtattgtt 660
tttaacaacg gttattttga accggcaagc gaaaaagaag aagatgttcg tactgcagaa 720
tttgcacatc agtttaccaa ttatccgctg tttctgaatc cgatctataa aggtgattat 780
ccggaactgg ttcgtgaatt tgcacgtgaa tttctgccga aagattataa aaaggatatg 840
gaagaaatcc aggaacgcat taattttgtt ggtatcaatt attatagcgg ccacatggtt 900
aaatatgatc ctaaaagccc gggtggtgtt agctttgtgg aacgtgatct gccgaaaacc 960
gaaatgggtt gggaagttgt tccggaaggt ctgtattata tcctgaaagg tgtgaaagat 1020
gaatataatc cggaagaaat ttatgtgacc gaaaatggtg cagcatataa tgatgtggtt 1080
agcgaagatg gtaaagtgca tgatcagaat cgtattgatt atctgaaagc acatatcggt 1140
caggcgtgga aagcactgca ggatggtgtg ccgctgcgtg gttattttgt ttggagtctg 1200
ctggataatt ttgaatgggc agaaggctat agcaaacgct ttggtattgt ttatgttgat 1260
tatcagacgc agaaacgtat tattaaagat tctggtcatt ggtatgcgaa tgttattaaa 1320
aataacggcc tggaagatta a 1341
<210> 3
<211> 33
<212> DNA
<213> Artificial sequence
<400> 3
attgttttta acaacgcata ttttgaaccg gca 33
<210> 4
<211> 33
<212> DNA
<213> Artificial sequence
<400> 4
tgccggttca aaatatgcgt tgttaaaaac aat 33
<210> 5
<211> 33
<212> DNA
<213> Artificial sequence
<400> 5
attgttttta acaacaatta ttttgaaccg gca 33
<210> 6
<211> 33
<212> DNA
<213> Artificial sequence
<400> 6
tgccggttca aaataattgt tgttaaaaac aat 33
<210> 7
<211> 33
<212> DNA
<213> Artificial sequence
<400> 7
attgttttta acaacgttta ttttgaaccg gca 33
<210> 8
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<212> DNA
<213> Artificial sequence
<400> 8
tgccggttca aaataaacgt tgttaaaaac aat 33
<210> 9
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<212> PRT
<213> Artificial sequence
<400> 9
Met Ser Met Lys Lys Phe Pro Glu Gly Phe Leu Trp Gly Val Ala Thr
1 5 10 15
Ala Ser Tyr Gln Ile Glu Gly Ser Pro Leu Ala Asp Gly Ala Gly Met
20 25 30
Ser Ile Trp His Thr Phe Ser His Thr Pro Gly Asn Val Lys Asn Gly
35 40 45
Asp Thr Gly Asp Ile Ala Cys Asp His Tyr Asn Arg Trp Lys Glu Asp
50 55 60
Ile Glu Ile Met Lys Glu Leu Gly Val Lys Ala Tyr Arg Phe Ser Ile
65 70 75 80
Ser Trp Pro Arg Ile Leu Pro Glu Gly Thr Gly Arg Val Asn Gln Lys
85 90 95
Gly Ile Asp Phe Tyr Ser Arg Ile Ile Asp Thr Leu Leu Glu Gln Gly
100 105 110
Ile Thr Pro Phe Val Thr Ile Tyr His Trp Asp Leu Pro Phe Glu Leu
115 120 125
Gln Leu Lys Gly Gly Trp Ala Asn Arg Glu Val Ala Asp Trp Phe Ala
130 135 140
Glu Tyr Ser Arg Val Leu Phe Glu Asn Phe Gly Asp Arg Val Lys His
145 150 155 160
Trp Ile Thr Leu Asn Glu Pro Trp Val Val Ala Ile Val Gly His Leu
165 170 175
Tyr Gly Val His Ala Pro Gly Met Lys Asp Ile Tyr Val Ala Phe His
180 185 190
Val Val His Asn Leu Leu Arg Ala His Ala Lys Ser Val Lys Ile Phe
195 200 205
Arg Glu Ile Val Lys Asp Gly Lys Ile Gly Ile Val Phe Asn Asn Ala
210 215 220
Tyr Phe Glu Pro Ala Ser Glu Lys Glu Glu Asp Val Arg Thr Ala Glu
225 230 235 240
Phe Ala His Gln Phe Thr Asn Tyr Pro Leu Phe Leu Asn Pro Ile Tyr
245 250 255
Lys Gly Asp Tyr Pro Glu Leu Val Arg Glu Phe Ala Arg Glu Phe Leu
260 265 270
Pro Lys Asp Tyr Lys Lys Asp Met Glu Glu Ile Gln Glu Arg Ile Asn
275 280 285
Phe Val Gly Ile Asn Tyr Tyr Ser Gly His Met Val Lys Tyr Asp Pro
290 295 300
Lys Ser Pro Gly Gly Val Ser Phe Val Glu Arg Asp Leu Pro Lys Thr
305 310 315 320
Glu Met Gly Trp Glu Val Val Pro Glu Gly Leu Tyr Tyr Ile Leu Lys
325 330 335
Gly Val Lys Asp Glu Tyr Asn Pro Glu Glu Ile Tyr Val Thr Glu Asn
340 345 350
Gly Ala Ala Tyr Asn Asp Val Val Ser Glu Asp Gly Lys Val His Asp
355 360 365
Gln Asn Arg Ile Asp Tyr Leu Lys Ala His Ile Gly Gln Ala Trp Lys
370 375 380
Ala Leu Gln Asp Gly Val Pro Leu Arg Gly Tyr Phe Val Trp Ser Leu
385 390 395 400
Leu Asp Asn Phe Glu Trp Ala Glu Gly Tyr Ser Lys Arg Phe Gly Ile
405 410 415
Val Tyr Val Asp Tyr Gln Thr Gln Lys Arg Ile Ile Lys Asp Ser Gly
420 425 430
His Trp Tyr Ala Asn Val Ile Lys Asn Asn Gly Leu Glu Asp
435 440 445
<210> 10
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<212> PRT
<213> Artificial sequence
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Met Ser Met Lys Lys Phe Pro Glu Gly Phe Leu Trp Gly Val Ala Thr
1 5 10 15
Ala Ser Tyr Gln Ile Glu Gly Ser Pro Leu Ala Asp Gly Ala Gly Met
20 25 30
Ser Ile Trp His Thr Phe Ser His Thr Pro Gly Asn Val Lys Asn Gly
35 40 45
Asp Thr Gly Asp Ile Ala Cys Asp His Tyr Asn Arg Trp Lys Glu Asp
50 55 60
Ile Glu Ile Met Lys Glu Leu Gly Val Lys Ala Tyr Arg Phe Ser Ile
65 70 75 80
Ser Trp Pro Arg Ile Leu Pro Glu Gly Thr Gly Arg Val Asn Gln Lys
85 90 95
Gly Ile Asp Phe Tyr Ser Arg Ile Ile Asp Thr Leu Leu Glu Gln Gly
100 105 110
Ile Thr Pro Phe Val Thr Ile Tyr His Trp Asp Leu Pro Phe Glu Leu
115 120 125
Gln Leu Lys Gly Gly Trp Ala Asn Arg Glu Val Ala Asp Trp Phe Ala
130 135 140
Glu Tyr Ser Arg Val Leu Phe Glu Asn Phe Gly Asp Arg Val Lys His
145 150 155 160
Trp Ile Thr Leu Asn Glu Pro Trp Val Val Ala Ile Val Gly His Leu
165 170 175
Tyr Gly Val His Ala Pro Gly Met Lys Asp Ile Tyr Val Ala Phe His
180 185 190
Val Val His Asn Leu Leu Arg Ala His Ala Lys Ser Val Lys Ile Phe
195 200 205
Arg Glu Ile Val Lys Asp Gly Lys Ile Gly Ile Val Phe Asn Asn Asn
210 215 220
Tyr Phe Glu Pro Ala Ser Glu Lys Glu Glu Asp Val Arg Thr Ala Glu
225 230 235 240
Phe Ala His Gln Phe Thr Asn Tyr Pro Leu Phe Leu Asn Pro Ile Tyr
245 250 255
Lys Gly Asp Tyr Pro Glu Leu Val Arg Glu Phe Ala Arg Glu Phe Leu
260 265 270
Pro Lys Asp Tyr Lys Lys Asp Met Glu Glu Ile Gln Glu Arg Ile Asn
275 280 285
Phe Val Gly Ile Asn Tyr Tyr Ser Gly His Met Val Lys Tyr Asp Pro
290 295 300
Lys Ser Pro Gly Gly Val Ser Phe Val Glu Arg Asp Leu Pro Lys Thr
305 310 315 320
Glu Met Gly Trp Glu Val Val Pro Glu Gly Leu Tyr Tyr Ile Leu Lys
325 330 335
Gly Val Lys Asp Glu Tyr Asn Pro Glu Glu Ile Tyr Val Thr Glu Asn
340 345 350
Gly Ala Ala Tyr Asn Asp Val Val Ser Glu Asp Gly Lys Val His Asp
355 360 365
Gln Asn Arg Ile Asp Tyr Leu Lys Ala His Ile Gly Gln Ala Trp Lys
370 375 380
Ala Leu Gln Asp Gly Val Pro Leu Arg Gly Tyr Phe Val Trp Ser Leu
385 390 395 400
Leu Asp Asn Phe Glu Trp Ala Glu Gly Tyr Ser Lys Arg Phe Gly Ile
405 410 415
Val Tyr Val Asp Tyr Gln Thr Gln Lys Arg Ile Ile Lys Asp Ser Gly
420 425 430
His Trp Tyr Ala Asn Val Ile Lys Asn Asn Gly Leu Glu Asp
435 440 445
<210> 11
<211> 446
<212> PRT
<213> Artificial sequence
<400> 11
Met Ser Met Lys Lys Phe Pro Glu Gly Phe Leu Trp Gly Val Ala Thr
1 5 10 15
Ala Ser Tyr Gln Ile Glu Gly Ser Pro Leu Ala Asp Gly Ala Gly Met
20 25 30
Ser Ile Trp His Thr Phe Ser His Thr Pro Gly Asn Val Lys Asn Gly
35 40 45
Asp Thr Gly Asp Ile Ala Cys Asp His Tyr Asn Arg Trp Lys Glu Asp
50 55 60
Ile Glu Ile Met Lys Glu Leu Gly Val Lys Ala Tyr Arg Phe Ser Ile
65 70 75 80
Ser Trp Pro Arg Ile Leu Pro Glu Gly Thr Gly Arg Val Asn Gln Lys
85 90 95
Gly Ile Asp Phe Tyr Ser Arg Ile Ile Asp Thr Leu Leu Glu Gln Gly
100 105 110
Ile Thr Pro Phe Val Thr Ile Tyr His Trp Asp Leu Pro Phe Glu Leu
115 120 125
Gln Leu Lys Gly Gly Trp Ala Asn Arg Glu Val Ala Asp Trp Phe Ala
130 135 140
Glu Tyr Ser Arg Val Leu Phe Glu Asn Phe Gly Asp Arg Val Lys His
145 150 155 160
Trp Ile Thr Leu Asn Glu Pro Trp Val Val Ala Ile Val Gly His Leu
165 170 175
Tyr Gly Val His Ala Pro Gly Met Lys Asp Ile Tyr Val Ala Phe His
180 185 190
Val Val His Asn Leu Leu Arg Ala His Ala Lys Ser Val Lys Ile Phe
195 200 205
Arg Glu Ile Val Lys Asp Gly Lys Ile Gly Ile Val Phe Asn Asn Val
210 215 220
Tyr Phe Glu Pro Ala Ser Glu Lys Glu Glu Asp Val Arg Thr Ala Glu
225 230 235 240
Phe Ala His Gln Phe Thr Asn Tyr Pro Leu Phe Leu Asn Pro Ile Tyr
245 250 255
Lys Gly Asp Tyr Pro Glu Leu Val Arg Glu Phe Ala Arg Glu Phe Leu
260 265 270
Pro Lys Asp Tyr Lys Lys Asp Met Glu Glu Ile Gln Glu Arg Ile Asn
275 280 285
Phe Val Gly Ile Asn Tyr Tyr Ser Gly His Met Val Lys Tyr Asp Pro
290 295 300
Lys Ser Pro Gly Gly Val Ser Phe Val Glu Arg Asp Leu Pro Lys Thr
305 310 315 320
Glu Met Gly Trp Glu Val Val Pro Glu Gly Leu Tyr Tyr Ile Leu Lys
325 330 335
Gly Val Lys Asp Glu Tyr Asn Pro Glu Glu Ile Tyr Val Thr Glu Asn
340 345 350
Gly Ala Ala Tyr Asn Asp Val Val Ser Glu Asp Gly Lys Val His Asp
355 360 365
Gln Asn Arg Ile Asp Tyr Leu Lys Ala His Ile Gly Gln Ala Trp Lys
370 375 380
Ala Leu Gln Asp Gly Val Pro Leu Arg Gly Tyr Phe Val Trp Ser Leu
385 390 395 400
Leu Asp Asn Phe Glu Trp Ala Glu Gly Tyr Ser Lys Arg Phe Gly Ile
405 410 415
Val Tyr Val Asp Tyr Gln Thr Gln Lys Arg Ile Ile Lys Asp Ser Gly
420 425 430
His Trp Tyr Ala Asn Val Ile Lys Asn Asn Gly Leu Glu Asp
435 440 445
Claims (10)
1. A β -glucosidase mutant, wherein the β -glucosidase mutant is: mutating the 224 th position of beta-glucosidase with amino acid sequence shown as SEQ ID NO.1 from glycine to alanine; or the 224 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is obtained by mutating glycine to valine.
2. A gene encoding the β -glucosidase mutant of claim 1.
3. An expression vector carrying the gene of claim 2.
4. A microbial cell carrying the gene of claim 2 or the expression vector of claim 3.
5. A method for producing galactooligosaccharides, which comprises adding the β -glucosidase mutant of claim 1 to a reaction system containing lactose to perform a reaction, thereby obtaining a reaction solution; and (3) separating the galacto-oligosaccharide from the reaction solution, wherein the enzyme adding amount of the beta-glucosidase mutant in the reaction system is 8-16U/mL.
6. The process for preparing galactooligosaccharide according to claim 5, wherein the concentration of lactose in the reaction system is 350-450 g/L.
7. The method for preparing galactooligosaccharide of claim 5, wherein the reaction is at a pH of 5.8-6.2 and a temperature of 75-85 ℃.
8. A method of preparing an alkyl glycoside, the method comprising: adding the beta-glucosidase mutant of claim 1 into a reaction system containing a glycosyl donor and a glycosyl acceptor for reaction to obtain a reaction solution; separating the reaction solution to obtain alkyl glycoside; the glycosyl donor is methyl glucoside and/or p-nitrophenyl glucoside, and the glycosyl acceptor is fatty alcohol.
9. The method for producing an alkylglycoside according to claim 8, wherein the β -glucosidase mutant is added in an amount of 3.5 to 4.5 μ M in the reaction system in which the glycosyl donor is present in an amount of 30 to 40 mM; the concentration of glycosyl acceptor is 8-87% according to the volume ratio; the pH of the reaction is 5.8-6.2, and the temperature is 85-95 ℃.
10. Use of the mutant of claim 1 for the preparation of galactooligosaccharides or alkyl glycosides.
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| CN104818261A (en) * | 2015-04-24 | 2015-08-05 | 南京师范大学 | Heat-resisting beta-glucosidase and application of heat-resisting beta-glucosidase mutants to arctigenin preparation |
| CN104818262A (en) * | 2015-04-24 | 2015-08-05 | 南京师范大学 | Polydatin bioconversion method using heat-resisting beta-glucosidase and mutants thereof |
| CN109355275A (en) * | 2018-11-21 | 2019-02-19 | 中山大学 | High thermal stability β-glucosidase mutants and its application |
| CN111411117A (en) * | 2020-04-27 | 2020-07-14 | 江南大学 | Application of heat-resistant β -glucosidase in preparation of gentiooligosaccharide |
| CN112063605A (en) * | 2020-09-24 | 2020-12-11 | 江南大学 | Method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application of method |
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| CN104818261A (en) * | 2015-04-24 | 2015-08-05 | 南京师范大学 | Heat-resisting beta-glucosidase and application of heat-resisting beta-glucosidase mutants to arctigenin preparation |
| CN104818262A (en) * | 2015-04-24 | 2015-08-05 | 南京师范大学 | Polydatin bioconversion method using heat-resisting beta-glucosidase and mutants thereof |
| CN109355275A (en) * | 2018-11-21 | 2019-02-19 | 中山大学 | High thermal stability β-glucosidase mutants and its application |
| CN111411117A (en) * | 2020-04-27 | 2020-07-14 | 江南大学 | Application of heat-resistant β -glucosidase in preparation of gentiooligosaccharide |
| CN112063605A (en) * | 2020-09-24 | 2020-12-11 | 江南大学 | Method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application of method |
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