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CN110004099A - A kind of fermentation method for producing of rhodioside - Google Patents

A kind of fermentation method for producing of rhodioside Download PDF

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Publication number
CN110004099A
CN110004099A CN201810008237.2A CN201810008237A CN110004099A CN 110004099 A CN110004099 A CN 110004099A CN 201810008237 A CN201810008237 A CN 201810008237A CN 110004099 A CN110004099 A CN 110004099A
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glucosidase
genetic engineering
rhodioside
apple
engineering bacterium
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CN110004099B (en
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孙敬方
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Sun Jing Fang
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Square Bio Tech Ltd Anhui
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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 present invention provides a kind of using apple beta-glucosidase as new catalyst, enzymatic conversion fermenting and producing and the method for preparing rhodioside.Using gene recombination technology, efficient apple beta-glucosidase, 50mM~1000mM tyrosol of addition are prepared, 10mM~500mM glucose reacts 20~50h at 37~50 DEG C.Obtain rhodioside.

Description

A kind of fermentation method for producing of rhodioside
Technical field
The present invention relates to a kind of methods for preparing rhodioside using β-glucosidase mutants fermenting and producing, belong to base Because of engineering and field of microbial fermentation.
Background technique
Rhodioside (salidroside) is the principle active component of Crassulaceae Rhodida plant.Root of kirilow rhodiola is for many years It is big to be mainly grown in 1600~4000 meters of height above sea level high and cold, dry, anoxic, strong ultraviolet radiation, day and night temperature for raw herbaceous plant Area, have extremely strong adaptive capacity to environment and vitality.Rhodioside have enhancing immune function, protection cardiovascular and cerebrovascular, Promote sexual function, promote memory and a variety of physiological and pharmacological activity of anticancer, antidepression, attention, mental and physical strength can be improved, And then central nervous system is influenced, human body debilitated state is particularly effective, cell or body can be significantly improved to external toxic The immunity of substance can also prevent heart overstrain or arrhythmia cordis, prevent the growth and diffusion of malignant tumour in liver.
The traditional production method of rhodioside is to extract to separate using plant root of kirilow rhodiola.That is: with root of kirilow rhodiola root and rhizome, powder It is broken into coarse powder, is extracted with 70% alcohol reflux, divides and takes extracting solution, ethyl alcohol is recovered under reduced pressure, gained concentrate adds equivalent amount of water stirring equal It is even, it stands, filtering, petroleum ether, chloroform, ethyl acetate, extracting n-butyl alcohol, ethyl acetate and just are successively used in filtrate decompression concentration Solvent is separately recovered in butanol fraction, obtains rhodioside crude product.
Since environmental degradation and artificial excessively excavation, the quantity of wild root of kirilow rhodiola are reduced rapidly, people also explore always logical Plant callus and cell culture technology are crossed come a possibility that obtaining sachalin rhodiola rhizome product.
Using the culture of dense callus architectural study rhodioside high yield, can from the root of R.sachalinensis, Stem, leaf, cotyledon chorista, which obtain several callus, sets about studying its speed of growth, Determination of Salidroside and culture breeding item Part determines the optimum condition for generating high yield rhodioside.Rhodioside yield can reach dry weight 57.72mg/g, be wild plant 5~10 times of object content, corresponding rhodioside yield are 555.13mg/L, are very suitable for industrialized production.
Using plant cell culture technology come when obtaining gadoloside, due in cell culture, sachalin rhodiola rhizome Secondary metabolites yield is usually lower, this makes its economic value be under suspicion.Therefore, the content of secondary metabolites how is improved extremely It closes important.Han Aiming et al. is to growth regulatory substance during sachalin rhodiola rhizome cell suspension cultures and the shadow of main nutrient composition Sound is inquired into, and is cell large-scale culture to find the preferable condition of culture for being suitble to cell growth and rhodioside accumulation It prepares.Under experiment condition, using the Optimal Medium most beneficial for sachalin rhodiola rhizome cell culture and rhodioside accumulation, carefully After born of the same parents cultivate 24d, biomass reaches 14.04g/L, and Determination of Salidroside is only 5.66mg/g in stem cell.With cometabolism way Rhodioside production can be improved in the research and application of diameter and metabolic engineering by plant cell large-scale culture and bioconversion Amount.In exponential phase of growth every being repeated 3 times addition 3mmol/L tyrosol for 24 hours, the yield of rhodioside is up to 516 μm of ol/L, as a result There is a possibility that industrialized production.
The present invention is to produce rhodioside using apple beta-glucosidase as new catalyst.Using genetic recombination Apple β-glucosidase mutants are expressed at Escherichia coli (E.coli), add junket alcohol and glucose, enzymatic conversion hair by technology Ferment produces rhodioside.
Summary of the invention
The object of the present invention is to provide a kind of methods of enzymatic conversion fermenting and producing rhodioside.
As one aspect of the present invention, the present invention provides a kind of genetic engineering bacterium of high yield apple beta-glucosidase, Apple β-glucosidase mutants are recombinantly expressed acquisition by the genetic engineering bacterium in Escherichia coli；Wherein, the apple The amino acid sequence of β-glucosidase mutants is as shown in SEQ ID NO.1 or the sequence is through replacement, missing or addition one Or several amino acids formed amino acid sequences with same function.Encode the apple β-glucosidase mutants gene Nucleotide sequence is as shown in SEQ ID NO.2 or the sequence is substituted, lacks and/or increases one or more nucleotide, and Express the nucleotide sequence of identical function protein；Or the nucleotide sequence hybridized under given conditions with SEQ ID NO.2.
Further, the genetic engineering bacterium is prepared as follows: apple β-glucosidase mutants MBGLM is packed into In carrier pUC57；With being tapped and recovered after NdeI and EcoRI double digestion, connect with pET-24d (+) carrier of NdeI and EcoRI digestion It connects, converts Escherichia coli, obtain genetic engineering bacterium.
As one aspect of the present invention, the present invention provides a kind of method of enzymatic conversion fermenting and producing rhodioside, the party Method includes the following steps:
Step A produces beta-glucosidase with above-mentioned engineering bacteria fermentation, obtains recombination beta-glucosidase fermentation Liquid；
Step B, the fermentation liquid obtained using step A synthesize rhodioside as catalyst, by substrate of junket alcohol and glucose；
Further, beta-glucosidase is produced with the engineering bacteria fermentation of high-yield beta-glucosidase described in step A, The following steps are included: genetic engineering bacterium is inoculated in the LB liquid medium containing Kan, 8~10h is cultivated at 35~40 DEG C；With 5 ~10% inoculum concentration is forwarded to containing LB liquid medium, 35~40 DEG C of 1~5h of culture；It is induced with IPTG, is cooled to 25 DEG C of constant temperature It cultivates 40~48h and induces producing enzyme, after fermentation, thalline were collected by centrifugation, ultrasonication.Centrifugation, supernatant after being induced, i.e., For fermentation liquid.
Further, step B is that junket alcohol and glucose is first added, and adjusts pH to 9.5~10.5, adds step A acquisition Fermentation liquid is reacted.
Further, the initial concentration of the tyrosol is 50mM~1000mM；
The initial concentration of the glucose is 10mM~500mM；
The concentration of the fermentation liquid is 0.2u/m1~9.0u/m1；
The reaction temperature is 37~50 DEG C, and the reaction time is 20~50h.
The present invention obtains the higher fermenting enzyme of unit enzyme activity by selecting the bacterial strain of high the substrate transformation rate to carry out heterogenous expression Liquid compensates for the low disadvantage of wild mushroom enzyme-activity unit, lays the foundation for a large amount of enzymes processed of low cost.Meanwhile the β-that the present invention obtains Glucuroide has good activity and stability under alkaline pH reaction condition.Meanwhile good thermal stability, at 50 DEG C Half-life period is up to 150h, compensates for other enzymes in the disadvantage of basic reaction conditions stability inferior difference.Enzyme reaction is carried out with the enzyme, Have many advantages, such as that reaction method is easy, it is short not need to ask when providing additional bio-energy, reaction.Under different concentration of substrate, The high the substrate transformation rate under corresponding concentration of substrate can be obtained, is laid the foundation for industrial amplification production.
Specific embodiment
The acquisition of 1 high yield apple beta-glucosidase gene engineering bacteria of embodiment
(1) according to apple beta-glucosidase gene sequence, base optimization and artificial synthesized its gene mutation body MBGLM (for its amino acid sequence as shown in SEQ ID NO.1, it is wild-type strain enzyme activity which, which is 198IU/ml, 9 times), be fitted into carrier pUC57.
(2) it will be tapped and recovered after target gene NdeI and EcoRI double digestion, the pET-24d with NdeI and EcoRI digestion The connection of (+) carrier, converts e. coli bl21 (DE3)；
Transformation mixture is coated on picking transformant after the kalamycin resistance LB plate containing 50mg/L, 37 DEG C of cultures 6 ~8h, picking single colonie obtain MBGLM-pET-24d (+)/E.coli BL21 (DE3)。
After cultivating 8~10h in LB/Kan fluid nutrient medium, culture presevation is carried out.
2 enzymatic production of embodiment
Genetic engineering bacterium MBGLM-pET-24d (+)/E.coli BL21 (DE3) is inoculated in the training of the LB liquid containing Kan Base is supported, cultivates 8~10h at 37 DEG C；It is forwarded to 5% inoculum concentration containing LB liquid medium, 37 DEG C of culture 3h；Use 0.4mM/L IPTG induction is cooled to 25 DEG C of 40~48h of constant temperature incubation induction producing enzymes, and after fermentation, thalline were collected by centrifugation for hair, and ultrasound is broken It is broken.Centrifugation, supernatant after being induced, as crude enzyme liquid.
3 Enzyme catalyzed synthesis rhodioside of embodiment
250mM tyrosol is weighed, 50mM glucose, 1.25U/ml crude enzyme liquid is miscible to be mixed in buffer/n-hexane (1/1, V/V) In zoarium system.At 50 DEG C, under conditions of 180r/min, concussion reaction is for 24 hours.After reaction, a certain amount of aqueous phase reactions are extracted Liquid is diluted with the distilled water of 2 times of volumes, and through filtering with microporous membrane, the filtrate was concentrated to dryness, obtains white solid powder.mp 156- 159 DEG C, IR (KBr) vcm-: 268,1630,1609；13CNMR (CDOD) 6:35.0 (Ar-CH2-)69.0(-CH2-O)；61.1 (6), 70.7 (4), 71.2 (3), 73.6 (2), 75.2 (5), 103.6 (Gal-1)；114.8 (2), 129.5 (3), 155.3 (Ar), MSm/z:306.8(M+), 324.8 (M+H2O) 287.4 (M-H2O+H+).With document (the compound such as Duan Jingyun, Zhang Dianzeng, Fan Yin section Root of kirilow rhodiola piece pharmacodynamic study [J] Chinese patent drug, 1999,21 (11): 588-591) data are consistent.
Product salidroside is taken, is analyzed on high performance liquid chromatograph: chromatographic condition: Sinochrom ODS-BP column (4.6mm × 200mm, 5um), UV230+ type UV detector, P230 type high pressure constant flow pump, 30 DEG C of column temperature, Detection wavelength 278nm, 20 μ l of sample volume, mobile phase are the methanol/water (30/70, V/V) of 0.8mL/min.It the results are shown in Table 1.
The amount and inversion rate of glucose of rhodioside are generated in 1 reaction product of table
Reaction time (h) Rhodioside (mg/L) Inversion rate of glucose (%)
1 3.50 11.0
10 4.55 14.3
20 5.25 16.5
30 6.07 19.1
40 7.38 23.2
50 7.16 22.5
The result shows that apple β-glucosidase mutants MBGLM can react tyrosol with glucose enzymatic reaction 40h Entirely.
Sequence table
<110>Anhui Biotechnology Co., Ltd, pros
<120>a kind of fermentation method for producing of rhodioside
<160> 2
<170> SIPOSequenceListing 1.0
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Met Ala Thr Lys Leu Gly Ser Leu Leu Leu Cys Val Leu Leu Leu Asn
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Gly Phe Ala Leu Thr Asn Thr Lys Ala Ala Asn Pro Asp Arg Pro Ile
20 25 30
Val Arg Asn Ser Leu Asp Arg Thr Lys Phe Asp Ala Leu Lys Pro Gly
35 40 45
Phe Val Phe Gly Ala Ala Ser Ala Ala Tyr Gln Val Glu Gly Ala Trp
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Asn Glu Asp Gly Arg Gly Pro Ser Ile Trp Asp Thr Phe Thr His Asn
65 70 75 80
His Pro Glu Lys Ile Thr Asp Arg Ser Asn Gly Asp Val Ala Ile Asp
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Gln Tyr His Leu Tyr Lys Lys Asp Val Ala Ile Met Lys Asp Met Lys
100 105 110
Leu Asp Ala Tyr Arg Phe Ser Ile Ser Trp Pro Arg Leu Leu Pro Asn
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Ile Phe His Trp Asp Val Pro Gln Ala Leu Glu Asp Ala Tyr Gly Gly
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Phe Leu Ser Ala Ser Ile Val Asp Asp Phe Lys Asp Tyr Ala Glu Leu
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Gly Val Ile Gly Ile Thr Leu Val Ser His Trp Tyr Glu Pro Ala Ser
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Lys Gly Ile His Asp Phe Val Leu Tyr Thr Lys Asn Lys Tyr Asp Asp
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atggcaacga agttgggctc tttgctcttg tgtgtcttgc tactcaatgg ctttgcattg 60
acaaacacca aagctgctaa cccagatcga cccattgtcc gtaactcact tgacaggacc 120
aagtttgatg ctctaaaacc agggttcgtc tttggtgcag cttcagcagc ttaccaggta 180
gaaggtgcat ggaacgaaga tggtagagga ccaagcatat gggacacctt cacccacaac 240
catccagaaa aaatcactga tcgcagcaat ggagatgtcg ccattgatca ataccacctc 300
tataagaaag atgtagcaat tatgaaggat atgaagttgg atgcttatag gttctctatc 360
tcatggccca gattgttacc aaatggcacg ctaagtgggg gtgtcaacag gaaaggaatt 420
gaatattacg acaatctcat caatgaactc cttcgcaatg gcatacaacc atttgtgaca 480
atctttcact gggatgttcc ccaagcgtta gaagatgcat atggtggttt cttaagcgct 540
agtattgtcg atgactttaa agactacgca gaactttgtt tttcactttt tggtgatcgg 600
gtgaagcact ggatcacgtt gaatgagcca tataccttca gtaaccatgc atatacaatc 660
gggatccacg caccgggacg atgctctgct tggcaagacc caacctgcct cggtggagat 720
acggctactg aaccctattt ggtaacacac caccaactcc ttgctcatgc agctgctgta 780
aaagtataca aggataaatt tcaggcatat caaaatgggg tgataggaat aacactagtg 840
tcacattggt atgagcctgc ttcagatgca aaggaagata tagatgctgc aaatcgagct 900
ttggatttta tgtttggatg gtttatggat ccaattacaa gaggtgacta cccgtacaac 960
atgcgatgcc ttgttagaga acgattgcca aaattcacgg aagaagaatc caagatgtta 1020
actgggtctt ttgattttgt tggattgaac tattattctg ctagatatgc aactgatgta 1080
cctaagaatt attctgaacc tgcaagttac ttatacgatc cacatgttac tacactgact 1140
gaacgtgatg gcattcctat tggtcctcag gctgcttcag actggttata tgtttatcca 1200
aaaggaattc acgattttgt actctacacg aagaataagt atgatgatcc aatcatttac 1260
attactgaga atggcgttga tgaggtcaat aattccacct tatcactcga cgatgccctc 1320
tatgatacca ataggactga ctactacaat cgccacctct gttaccttca agcagcaatc 1380
aagaagggta gtaatgtgaa aggatacttt gcatggtcaa ttttagacaa ctttgaatgg 1440
agtgaaggct acacagttcg atttggtatt aactatgtgg attatgacaa tggactccaa 1500
aggtacccaa aactttcgac ctattggttc aaaaatttcc tcaagaagcg caaaggaagt 1560
tcaaatattt tggccgatta tgttggagac actaagtctg tgtattaa 1608

Claims (10)

1. a kind of genetic engineering bacterium of high yield apple beta-glucosidase, which is characterized in that the genetic engineering bacterium is by apple β- Glucoside enzyme mutant recombinantly expresses acquisition in Escherichia coli；Wherein, the ammonia of the apple β-glucosidase mutants Base acid sequence is as shown in SEQ ID NO.1 or the sequence is through replacement, missing or addition is one or several amino acids formed has The amino acid sequence of same function.
2. genetic engineering bacterium as described in claim 1, which is characterized in that encode the apple β-glucosidase mutants base The nucleotide sequence of cause is as shown in SEQ ID NO.2 or the sequence is substituted, lacks and/or increases one or more nucleotide, And express the nucleotide sequence of identical function protein；Or the nucleotide sequence hybridized under given conditions with SEQ ID NO.2.
3. genetic engineering bacterium as described in claim 1, which is characterized in that the genetic engineering bacterium is prepared as follows: will Apple β-glucosidase mutants are fitted into carrier pUC57；With being tapped and recovered after NdeI and EcoRI double digestion, with NdeI and PET-24d (+) carrier of EcoRI digestion connects, and converts Escherichia coli, obtains genetic engineering bacterium.
4. a kind of method of enzymatic conversion fermenting and producing rhodioside, which is characterized in that described method includes following steps:
Step A produces beta-glucosidase with engineering bacteria fermentation described in claim 1, obtains recombination beta-glucosidase Enzyme fermentation liquid；
Step B, the fermentation liquid obtained using step A synthesize rhodioside as catalyst, by substrate of junket alcohol and glucose.
5. method as claimed in claim 4, which is characterized in that produce β-grape described in step A with engineering bacteria fermentation Glycosidase, comprising the following steps: genetic engineering bacterium is inoculated in the LB liquid medium containing Kan, culture 8 at 35~40 DEG C~ 10h；It is forwarded to 5~10% inoculum concentrations containing LB liquid medium, 35~40 DEG C of 1~5h of culture；It is induced with IPTG, is cooled to 25 DEG C 40~48h of constant temperature incubation induces producing enzyme, and after fermentation, thalline were collected by centrifugation, ultrasonication, centrifugation, after being induced on Clear liquid, as fermentation liquid.
6. method as claimed in claim 4, which is characterized in that step B is that junket alcohol and glucose is first added, and adjusts pH to 9.5 ~10.5, the fermentation liquid of step A acquisition is added, is reacted.
7. method as claimed in claim 6, which is characterized in that the initial concentration of the tyrosol is 50mM~1000mM.
8. method as claimed in claim 6, which is characterized in that the initial concentration of the glucose is 10mM~500mM.
9. method as claimed in claim 6, which is characterized in that the concentration of the fermentation liquid is 0.2u/m1~9.0u/m1.
10. method as claimed in claim 6, which is characterized in that the reaction temperature be 37~50 DEG C, the reaction time be 20~ 50h。
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