CN104404013B - Abalone viscera [beta]-glucosidase and method for synthesizing rhodioloside thereby - Google Patents
Abalone viscera [beta]-glucosidase and method for synthesizing rhodioloside thereby Download PDFInfo
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- CN104404013B CN104404013B CN201410538493.4A CN201410538493A CN104404013B CN 104404013 B CN104404013 B CN 104404013B CN 201410538493 A CN201410538493 A CN 201410538493A CN 104404013 B CN104404013 B CN 104404013B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C12Y—ENZYMES
- 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)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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Abstract
The invention belongs to the technical field of biology and particularly discloses abalone viscera [beta]-glucosidase and a method for synthesizing rhodioloside thereby. The abalone viscera [beta]-glucosidase has an amino acid sequence represented as the SEQ ID NO.1. A preparation method comprises following steps: (S1) preparing following raw materials: 20-50 mg of glucose, 500-800 mg of p-hydroxyphenylethyl alcohol and 20-50 mg of the abalone viscera [beta]-glucosidase; (S2) adding 15-50 ml of an organic solvent and 1-3 ml of a buffer solution with mixing uniformly; (S3) performing a sealing process and carrying out a vibration reaction at 50-80 DEG C for 36-72 h to obtain the rhodioloside. In the invention, a catalytic activity of the prepared abalone viscera [beta]-glucosidase in synthesis of the rhodioloside is far strong that that of [beta]-glucosidase extracted from almonds and apple seeds. The invention provides a high-efficient catalyst for industrialized large-scale production of the rhodioloside.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of Carnis Haliotidis internal organs beta-glucosidase and its for synthesizing red
The method of Herba hylotelephii erythrosticti glycosides.
Background technology
An important component part of the beta-glucosidase as cellulase, has in medical treatment, food, biomass conversion
Important using value.Under certain condition, the transglycosylation that beta-glucosidase can also be solved against the current.Therefore it is domestic
Outer utilization beta-glucosidase synthesizing alkyl polyglycoside in organic media becomes new focus, has successfully synthesized various alkyl
Glycoside compounds, oligosaccharide, glycoside compounds of antibiotic etc., these products are in pharmacy, chemical industry, cosmetics, detergent, food
The fields such as additive are all widely used.Therefore, find the beta-glucosidase with higher inverse hydrolysing activity or to existing
Beta-glucosidase is transformed to improve its sight for having attracted numerous researcheres against hydrolysing activity.
Beta-glucosidase finds first that in 1837 the enzyme is widely distributed in nature with Semen Armeniacae Amarum, is planting
It is especially prevalent in the seed of thing and microorganism, it has also been found that the presence of the enzyme in animal and funguses body.The plant of beta-glucosidase
There are Radix Ginseng, Semen sojae atricolor etc. in thing source；Microbe-derived report is more, and such as prokaryotic micro-organisms source has flavobacterium meningosepticum
(Flavobacteriummeningosepticum), Yue Shi Flavobacteriums (Flavobacterium johnsonae) etc., eucaryon life
What thing was originated has saccharomyces sake (Candidapeltata), Phanerochaete chrysosporium（Phanerochaete
chrysosporium）Deng；The animal origin of beta-glucosidase has Apiss, Hepar Sus domestica, pig small intestine etc..β-the Fructus Vitis viniferae of separate sources
Its catalysis activity of glycosidase is also different.The research to Carnis Haliotidis internal organs beta-glucosidase is there is no to report at present.
Radix Rhodiolae is typically grown on Eurasian two state plateau band, is Crassulaceae perennial herb or semishrub plant, has
The good reputation of " plateau Radix Ginseng ".The whole world has 96 kinds of Rhodida plant, main to be distributed and East Asia, the Central Asia, Siberia and north
Beauteously area.China has 73 kinds, is distributed mainly on southwest, northwest, North, Northeast China area.The principle active component of Radix Rhodiolae is red
Herba hylotelephii erythrosticti glycosides, chemical name para hydroxybenzene ethyl-β-D-Glucose glycosides, be current most study Radix Rhodiolae in chief active into
Point, it is the primary evaluation index for evaluating Radix Rhodiolae quality.Rhodioside has significant biologic activity, and its effect is better than conventional
Radix Ginseng and the Chinese medicine such as Radix Et Caulis Acanthopanacis Senticosi, polyradical, anti-hypoxia are crossed in removing machine body, defying age, are resisted myocardial ischemia, are improved body
The aspects such as immunity have remarkable result, therefore can play the dual function of radioprotective and resisting fatigue simultaneously.
The source of rhodioside mainly has three kinds of approach at present, and first is plant origin；Second is chemosynthesis；3rd is
Enzymatic clarification.2004, Tong etc. was first as substrate with p-hydroxyphenylethanol and glucose using Apple Seeds powder beta-glucosidase
It is secondary to be synthesized, after the completion of reaction rhodioside concentration up to 11.8g/L, higher than chemical synthesiss and plant origin method.Cause
This, enzyme process becomes a kind of effective ways of synthesis rhodioside.Currently used for the beta-glucosidase for synthesizing rhodioside not
It is common, therefore, exploitation is had the beta-glucosidase for synthesizing rhodioside of higher efficiency and is groped most using the enzyme
Good reaction condition and there is greatly market using value.
The content of the invention
First technical problem to be solved by this invention is, in order to overcome above-mentioned deficiency of the prior art, there is provided one
Plant novel Carnis Haliotidis internal organs beta-glucosidase.
Second technical problem to be solved by this invention is that Carnis Haliotidis internal organs beta-glucosidase is used to synthesize rhodioside
Method.
The technical problem to be solved, is achieved by the following technical programs：
A kind of Carnis Haliotidis internal organs beta-glucosidase, with the aminoacid sequence shown in SEQ ID NO.1.
The aminoacid sequence of SEQ ID NO.1 is as follows：
Trp Gly Val Glu Tyr Ala Ser Tyr Leu Asn Glu Pro Gln Trp Asn Ser
Glu Trp Arg Glu Pro Asp His Phe Asp Thr Phe Ser Arg Asp Glu Lys
Thr Gly Tyr Asn Gly Asp Ala Cys Arg His Tyr Gly Gly His Tyr His
Arg Tyr Lys Thr Asp Val Ile His Arg Glu Glu Asp Gly Val Ala Leu
Ile Arg Phe Ser Lys Gly Trp Pro Arg Ile Val Ala Leu Glu Gly Lys
His His Leu Ala Gly Met Val Ile Pro Lys Arg Leu Val Trp Gly Lys
Leu Lys Gly Asn Ile Cys Ala Ser Ala Thr Ile Ile Gly Tyr Asp Leu
Val His Asp Ala Tyr Pro Gly Lys Gly Asp Ser Val Asn Arg Asn Lys
Arg Tyr Trp Tyr Val Glu Val Pro Lys Gly Leu Phe Glu Glu Met Leu
Tyr Asp Val Ile Pro Trp Ile Cys Ser Ala Glu Pro Trp Phe Leu Val
Ile Lys Ile Ser Tyr Gly Ile Gly Glu Ala Pro Asn His Asp Glu Tyr
Arg Glu Ala Leu Ile Val Gln Ile His Ile Leu Val Pro His Gly Glu
Ala Ser Lys Ile Phe Arg Glu Met Asn Phe Asp Lys Gly Ser Lys Ile
Ser Pro Thr Leu Asn Lys Ile Thr Ala Tyr Gly Val Ser Glu Leu Tyr
Asp Asp Lys Leu Ala Glu Tyr Tyr Ala Asn Ala Phe Ala Asn Glu Ala
Phe Leu Asp Pro Ile Phe Lys Arg Ile Lys Pro Glu Asp Met Met Glu
Glu His Lys Lys Ile Ile Asp Val Glu Val Phe Ile Lys Glu Asn Asp
Leu Lys Ser Thr Ile Val Asp Glu Asp Phe Leu Gly Val Asn Tyr Thr
Ser Glu Ser Ile Val Lys Asp Asn Asp Tyr Ser Met Leu Lys Ala Glu
Asp Glu Met Gly Pro Gly Lys Arg Thr Glu Met Gly Lys Glu Phe Trp
Pro Glu Ser Asp Leu Ile Leu Leu Lys Asn Glu Glu Phe Asp Tyr Thr
Lys Pro Gly Met Tyr Ile Thr Ser Asn Glu Asn Phe Phe Lys Asp Glu
Asp Gly Val Ala Ser Arg Val His Asn Asp Glu Tyr Trp Met Pro Lys
Glu Asn His Leu Lys Phe Lys Ile Lys Phe Glu Pro Phe Arg Gly Ile
Leu Met Gly Tyr Phe Val Trp Ser Leu Met Asp Lys Ile Phe
Carnis Haliotidis internal organs beta-glucosidase is used for the method for catalyzing and synthesizing rhodioside, comprises the steps of：
S1. take 20 ~ 50mg glucoses and 500 ~ 800mg p-hydroxyphenylethanols and 20 ~ 50mg Carnis Haliotidis internal organs β-
Glucosidase；
S2. 15 ~ 50ml organic solvents and 1 ~ 3ml buffer solution mix homogeneously are added；
S3. seal, at 50 ~ 80 DEG C, 36 ~ 72h of concussion reaction obtains final product rhodioside.
As a kind of preferred version, the glucose 40 ~ 50mg of consumption described in S1., the consumption of p-hydroxyphenylethanol is
700~800mg；The consumption of Carnis Haliotidis internal organs beta-glucosidase is 40 ~ 50mg.
As a kind of most preferably scheme, the glucose consumption 40mg described in S1., the consumption of p-hydroxyphenylethanol is
700mg；The consumption of Carnis Haliotidis internal organs beta-glucosidase is 40mg.
Used as a kind of preferred version, consumption of organic solvent described in S2. is 15 ~ 20ml；The buffer solution consumption be 1 ~
2ml。
Used as a kind of most preferably scheme, consumption of organic solvent described in S2. is 20ml；The buffer solution consumption is 2ml.
Used as a kind of preferred version, the organic solvent described in S2. is Isosorbide-5-Nitrae-dioxane；Buffer solution be citric acid-
Sodium dihydrogen phosphate buffer.
Used as a kind of preferred version, the pH value of the buffer solution of citrate-phosphate sodium dihydrogen described in S2. is 5.0 ~ 6.0.
Used as a kind of most preferably scheme, the pH value of the buffer solution of citrate-phosphate sodium dihydrogen described in S2. is 6.0.
Used as a kind of preferred version, the reaction temperature described in S3. is 70 DEG C, and the response time is 48h.
Carnis Haliotidis internal organs beta-glucosidase of the present invention is prepared via a method which to obtain：
S1. Carnis Haliotidis internal organs 50g is taken, adds appropriate amount of quartz sand and 500ml to be 4 DEG C of citrate-phosphates through the temperature of pre-cooling
Disodium hydrogen buffer（PH value be 6, ionic strength is 20mmol/L）, homogenate is fully ground on 0 DEG C of ice bath；
S2. the homogenate for S1. being prepared carries out high speed centrifugation, and centrifugal speed is 12000r/min, takes supernatant, mistake
10 kDa films retain protein, and protein is concentrated by ultrafiltration；
S3. Sephadex S-200 molecular sieve gels post on the protein after concentration is carried out into column chromatography；Gel filtration chromatography
Eluent is citrate-phosphate disodium hydrogen buffer（PH value be 6, ionic strength is 20mmol/L）, elution speed is
0.5ml/min, collects 1 with test tube and manages per 10min.
S4. the sample that S3. is collected is carried out using SDS-PAGE SDS-PAGE pure
Degree detection, the sample that the 20th ~ 25 pipe is collected after testing is same sample, is merged, lyophilizing obtains Carnis Haliotidis internal organs β-glucose
Glycosides enzyme lyophilized powder.Protein structure identification is carried out using technologies such as MALDI-TOF, is defined as in the Carnis Haliotidiss described in claim 1
Dirty beta-glucosidase.
Beneficial effect：（1）The present invention is found that the Carnis Haliotidiss with catalysis activity of novelty from Carnis Haliotidis internal organs by research
Internal organs beta-glucosidase；（2）The novel Carnis Haliotidis internal organs beta-glucosidase that the present invention is prepared is carrying out rhodioside conjunction
The more known beta-glucosidase extracted from Semen Armeniacae Amarum of catalysis activity in is eager to excel；（3）Carnis Haliotidiss of the present invention
The extracting method of internal organs beta-glucosidase is novel, and step is simple, and the Carnis Haliotidis internal organs beta-glucosidase purity for obtaining is higher, fits
Close industrialized production；（4）The present invention is from the garbage Carnis Haliotidis internal organs of Carnis Haliotidiss processing to extract beta-glucosidase, solves Bao
Fish internal organs pollute the problem of environment, turn waste into wealth.
Specific embodiment
The present invention is explained further below in conjunction with specific embodiment, but embodiment does not do any type of limit to the present invention
It is fixed.
Embodiment 1
S1. Carnis Haliotidis internal organs 50g is taken, adds appropriate amount of quartz sand and 500ml to be 4 DEG C of citrate-phosphates through the temperature of pre-cooling
Disodium hydrogen buffer（PH value be 6, ionic strength is 20mmol/L）, homogenate is fully ground on 0 DEG C of ice bath；
S2. the homogenate for S1. being prepared carries out high speed centrifugation, and centrifugal speed is 12000r/min, takes supernatant, mistake
10 kDa films retain protein, and protein is concentrated by ultrafiltration；
S3. Sephadex S-200 molecular sieve gels post on the protein after concentration is carried out into column chromatography；Gel filtration chromatography
Eluent is citrate-phosphate disodium hydrogen buffer（PH value be 6, ionic strength is 20mmol/L）, elution speed is
0.5ml/min, collects 1 with test tube and manages per 10min.
S4. the sample that S3. is collected is carried out using SDS-PAGE SDS-PAGE pure
Degree detection, the sample that the 20th ~ 25 pipe is collected after testing is same sample, is merged, lyophilizing obtains Carnis Haliotidis internal organs β-glucose
Glycosides enzyme lyophilized powder.Protein structure identification is carried out using technologies such as MALDI-TOF, is defined as in the Carnis Haliotidiss described in claim 1
Dirty beta-glucosidase.
Embodiment 2
In 100ml tool plug test tubes, 40mg glucoses are added（C6H12O6）, 700mg p-hydroxyphenylethanols, 2ml citric acids-
Sodium dihydrogen phosphate buffer（pH 6.0）, 20ml Isosorbide-5-Nitraes-dioxane and 40mg Carnis Haliotidis internal organs beta-glucosidase lyophilized powders
（Embodiment 1 is prepared）, seal after mix homogeneously, be put into shaking table（160rpm, 70 DEG C）Reaction.After reaction 48h, detection reaction
Reach balance.Use chromatograph of liquid（Inertsil ODS-4column, GL Sciences Inc.）It is dense that analysis determines product
Degree, mobile phase is methanol/water（70/30, v/v, 0.8ml/min）, 30 DEG C, 275nm ultraviolet detection.As a result measure rhodioside
Concentration be 68.6g/L.
Embodiment 3
In 100ml tool plug test tubes, 20mg glucoses are added（C6H12O6）, 500mg p-hydroxyphenylethanols, 1ml citric acids-
Sodium dihydrogen phosphate buffer（pH 5.0）, 15ml Isosorbide-5-Nitraes-dioxane and 20mg Carnis Haliotidis internal organs beta-glucosidase lyophilized powders
（Embodiment 1 is prepared）, seal after mix homogeneously, be put into shaking table（160rpm, 50 DEG C）Reaction.After reaction 72h, detection reaction
Reach balance.Use chromatograph of liquid（Inertsil ODS-4column, GL Sciences Inc.）It is dense that analysis determines product
Degree, mobile phase is methanol/water（70/30, v/v, 0.8ml/min）, 30 DEG C, 275nm ultraviolet detection.As a result measure rhodioside
Concentration be 60.4g/L.
Embodiment 4
In 100ml tool plug test tubes, 50mg glucoses are added（C6H12O6）, 800mg p-hydroxyphenylethanols, 3ml citric acids-
Sodium dihydrogen phosphate buffer（pH 6.0）, 50ml Isosorbide-5-Nitraes-dioxane and 50mg Carnis Haliotidis internal organs beta-glucosidase lyophilized powders
（Embodiment 1 is prepared）, seal after mix homogeneously, be put into shaking table（160rpm, 70 DEG C）Reaction.After reaction 36h, detection reaction
Reach balance.Use chromatograph of liquid（Inertsil ODS-4column, GL Sciences Inc.）It is dense that analysis determines product
Degree, mobile phase is methanol/water（70/30, v/v, 0.8ml/min）, 30 DEG C, 275nm ultraviolet detection.As a result measure rhodioside
Concentration be 63.6g/L.
Comparative example 1
In 100ml tool plug test tubes, 40mg glucoses are added（C6H12O6）, 700mg p-hydroxyphenylethanols, 2ml citric acids-
Sodium dihydrogen phosphate buffer（pH 6.0）, 20ml Isosorbide-5-Nitraes-dioxane and 40mg beta-glucosidases（Extract in Semen Armeniacae Amarum, purchase
From abundant bio tech ltd of upper Hisense, products C AS：9001-22-3 ）, seal after mix homogeneously, be put into shaking table
（160rpm, 70 DEG C）Reaction.After reaction 48h, detection reaction reaches balance.Use chromatograph of liquid（Inertsil ODS-
4column, GL Sciences Inc.）Analysis determines production concentration, and mobile phase is methanol/water（70/30, v/v, 0.8ml/
min）, 30 DEG C, 275nm ultraviolet detection.As a result the concentration for measuring rhodioside is 8.1g/L.
Conclusion：The Carnis Haliotidis internal organs beta-glucosidase that the present invention is prepared reaction system when rhodioside is catalyzed and synthesized
The concentration of middle Radix Rhodiolae is all higher than 60g/L, significantly larger than in prior art utilize Apple Seeds powder beta-glucosidase enzyme catalysiss when
8.1g/L during Semen Armeniacae Amarum beta-glucosidase enzyme catalysiss is utilized in 11.8g/L and comparative example.In the Carnis Haliotidiss that the present invention is prepared
There is dirty beta-glucosidase the very high rhodioside that catalyzes and synthesizes to act on.Have when rhodioside concentration is 1.3g/L in theory
There is commercial production to be worth, and the rhodioside concentration of Jing biological catalysis synthesis of the present invention is up to 50g/L, is industrialized great production
Rhodioside provides a kind of extremely efficient catalyst.
SEQUENCE LISTING
<110>Putian City converge Longhai City produce company limited
<120>A kind of Carnis Haliotidis internal organs beta-glucosidase and its method for synthesizing rhodioside
<170> PatentIn version 3.3
<210> 1
<211> 398
<212> PRT
<213>Carnis Haliotidiss（Haliotis rubra）
<400> 1
Trp Gly Val Glu Tyr Ala Ser Tyr Leu Asn Glu Pro Gln Trp Asn Ser
1 5 10 15
Glu Trp Arg Glu Pro Asp His Phe Asp Thr Phe Ser Arg Asp Glu Lys
20 25 30
Thr Gly Tyr Asn Gly Asp Ala Cys Arg His Tyr Gly Gly His Tyr His
35 40 45
Arg Tyr Lys Thr Asp Val Ile His Arg Glu Glu Asp Gly Val Ala Leu
50 55 60
Ile Arg Phe Ser Lys Gly Trp Pro Arg Ile Val Ala Leu Glu Gly Lys
65 70 75 80
His His Leu Ala Gly Met Val Ile Pro Lys Arg Leu Val Trp Gly Lys
85 90 95
Leu Lys Gly Asn Ile Cys Ala Ser Ala Thr Ile Ile Gly Tyr Asp Leu
100 105 110
Val His Asp Ala Tyr Pro Gly Lys Gly Asp Ser Val Asn Arg Asn Lys
115 120 125
Arg Tyr Trp Tyr Val Glu Val Pro Lys Gly Leu Phe Glu Glu Met Leu
130 135 140
Tyr Asp Val Ile Pro Trp Ile Cys Ser Ala Glu Pro Trp Phe Leu Val
145 150 155 160
Ile Lys Ile Ser Tyr Gly Ile Gly Glu Ala Pro Asn His Asp Glu Tyr
165 170 175
Arg Glu Ala Leu Ile Val Gln Ile His Ile Leu Val Pro His Gly Glu
180 185 190
Ala Ser Lys Ile Phe Arg Glu Met Asn Phe Asp Lys Gly Ser Lys Ile
195 200 205
Ser Pro Thr Leu Asn Lys Ile Thr Ala Tyr Gly Val Ser Glu Leu Tyr
210 215 220
Asp Asp Lys Leu Ala Glu Tyr Tyr Ala Asn Ala Phe Ala Asn Glu Ala
225 230 235 240
Phe Leu Asp Pro Ile Phe Lys Arg Ile Lys Pro Glu Asp Met Met Glu
245 250 255
Glu His Lys Lys Ile Ile Asp Val Glu Val Phe Ile Lys Glu Asn Asp
260 265 270
Leu Lys Ser Thr Ile Val Asp Glu Asp Phe Leu Gly Val Asn Tyr Thr
275 280 285
Ser Glu Ser Ile Val Lys Asp Asn Asp Tyr Ser Met Leu Lys Ala Glu
290 295 300
Asp Glu Met Gly Pro Gly Lys Arg Thr Glu Met Gly Lys Glu Phe Trp
305 310 315 320
Pro Glu Ser Asp Leu Ile Leu Leu Lys Asn Glu Glu Phe Asp Tyr Thr
325 330 335
Lys Pro Gly Met Tyr Ile Thr Ser Asn Glu Asn Phe Phe Lys Asp Glu
340 345 350
Asp Gly Val Ala Ser Arg Val His Asn Asp Glu Tyr Trp Met Pro Lys
355 360 365
Glu Asn His Leu Lys Phe Lys Ile Lys Phe Glu Pro Phe Arg Gly Ile
370 375 380
Leu Met Gly Tyr Phe Val Trp Ser Leu Met Asp Lys Ile Phe
385 390 395
Claims (9)
1. a kind of Carnis Haliotidis internal organs beta-glucosidase, it is characterised in that the aminoacid sequence of Carnis Haliotidis internal organs beta-glucosidase is
Shown in SEQ ID NO.1.
2. the Carnis Haliotidis internal organs beta-glucosidase described in claim 1 is used for the method for catalyzing and synthesizing rhodioside, and its feature exists
In comprising the steps of：
S1. 20 ~ 50mg glucoses and 500 ~ 800mg p-hydroxyphenylethanols and 20 ~ 50mg Carnis Haliotidis internal organs beta-glucosidases are taken；
S2. 15 ~ 50ml organic solvents and 1 ~ 3ml buffer solution mix homogeneously are added, wherein, organic solvent is Isosorbide-5-Nitrae-two
Six ring of oxygen, buffer solution are citrate-phosphate sodium dihydrogen buffer solution；
S3. seal, at 50 ~ 80 DEG C, 36 ~ 72h of concussion reaction obtains final product rhodioside.
3. method according to claim 2, it is characterised in that the glucose 40 ~ 50mg of consumption described in S1., to hydroxyl
The consumption of phenethanol is 700 ~ 800mg；The consumption of Carnis Haliotidis internal organs beta-glucosidase is 40 ~ 50mg.
4. method according to claim 3, it is characterised in that the glucose consumption 40mg described in S1., para hydroxybenzene
The consumption of ethanol is 700mg；The consumption of Carnis Haliotidis internal organs beta-glucosidase is 40mg.
5. method according to claim 2, it is characterised in that consumption of organic solvent described in S2. is 15 ~ 20ml；Institute
Buffer solution consumption is stated for 1 ~ 2ml.
6. method according to claim 5, it is characterised in that consumption of organic solvent described in S2. is 20ml；The buffering
Solution usage is 2ml.
7. method according to claim 2, it is characterised in that the buffer solution of citrate-phosphate sodium dihydrogen described in S2.
PH values are 5.0 ~ 6.0.
8. method according to claim 7, it is characterised in that the buffer solution of citrate-phosphate sodium dihydrogen described in S2.
PH values are 6.0.
9. the method according to claim 2, it is characterised in that the reaction temperature described in S3. is 70 DEG C, response time
For 48h.
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| CN112159803A (en) * | 2020-10-14 | 2021-01-01 | 潍坊医学院 | Method for extracting beta-glucosidase from lentinus edodes for synthesizing salidroside |
| CN114317480B (en) * | 2022-01-10 | 2022-09-13 | 湖北碳元本草生物科技有限公司 | Glycosyl transferase for catalytically synthesizing salidroside, and coding gene and application thereof |
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