CN115094104A - Method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor - Google Patents

Method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor Download PDF

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CN115094104A
CN115094104A CN202210818830.XA CN202210818830A CN115094104A CN 115094104 A CN115094104 A CN 115094104A CN 202210818830 A CN202210818830 A CN 202210818830A CN 115094104 A CN115094104 A CN 115094104A
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卢丽丽
段飞宇
王珂
文焰
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Huazhong University of Science and Technology
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Abstract

The invention relates to a method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor. The method comprises the following steps: (1) culturing yeast producing beta-galactosidase; (2) treating yeast cells with ethanol; (3) adding genipin into the yeast cells after ethanol treatment to construct a crosslinking system; reacting the crosslinking system for 0.5-4 h, and centrifuging to obtain immobilized cells; (4) and uniformly mixing galactose and alkyl alcohol, adding immobilized cells, reacting for 2-16 h, performing centrifugal solid-liquid separation, and taking supernatant to obtain the alkyl galactoside. The invention provides a green and efficient method for synthesizing alkyl galactoside, which firstly adopts immobilized yeast cells and takes monosaccharide as glycosyl donor to synthesize glucoside in one step. The reaction system only contains alkyl glycoside and residual substrate after completion, has few product types, is easy to purify, is suitable for large-scale synthesis of the alkyl glycoside, and has broad application prospect.

Description

Method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor
Technical Field
The invention relates to a method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as a glycosyl donor, belonging to the technical field of sugar engineering.
Background
The alkyl glycoside is a green novel nonionic surfactant and consists of hydrophilic glycosyl and hydrophobic fatty alkyl chain. Alkyl glycosides are traditionally used in detergents and industrial emulsifiers, exhibit good wetting and surface tension reduction, electrolyte and hard water tolerance, are stable over a wide pH range, and can act synergistically with other surfactants. With the development and research of functions, the alkyl glycoside has wide application in the fields of food processing, cosmetics, pharmaceutical industry, scientific research and the like. The alkyl glycoside is used as a food additive, is safe and nontoxic, can uniformly mix food components, has foaming and thickening effects, improves the taste of the food, and prolongs the shelf life of the food. In addition, the alkyl glycosides are mild and non-irritating, are suitable for use as cosmetic formulations, and can be added to pharmaceutical skin products as stabilizers. As a highly effective stabilizer, the alkyl glycoside may also stabilize lipid nanoparticles for drug delivery. Alkyl glycosides have been gradually applied to clinical studies in recent years, for example: as a transmucosal delivery enhancer for intranasal administration; can prevent aggregation of peptide and protein drugs, reduce immunogenicity and retain activity; enhancing the permeability of insulin through epithelial mucosa; effectively improves the bioavailability of the nasal and ocular administration calcitonin; the alkyl glycoside with a specific structure has kidney targeting. Alkyl glycosides are also used as pharmaceutical intermediates in the synthesis of certain drugs, such as alpha-amylase inhibitors based on the alkyl glycoside structure, which inhibit postprandial hyperglycemia and excessive insulin secretion in type II diabetics. In the field of basic research, alkyl glycosides, which are superior surfactants, have been widely used in the preparation and research of membrane proteins.
At present, the synthesis method of alkyl glycoside is mainly a chemical method, including a Koneigs-Knorr method, a Fischer glycosylation method and the like, multiple steps of protection, deprotection, activation and the like are needed, the energy consumption is high, and the problem of environmental pollution exists. In order to simplify the reaction steps and reduce environmental pollution, the development of a green and environment-friendly synthesis method is urgently needed. The enzymatic synthesis of alkyl glycoside can realize one-step glycoside synthesis, and is green and pollution-free. The alkyl glucoside catalytically synthesized by the enzyme method at present is mainly alkyl glucoside, and beta-glucosidase is usually adopted, and amylase and pullulanase are jointly used or aldolase is utilized to carry out catalytic synthesis. Enzymatic synthesis of the alkyl galactoside is rarely reported, the beta-galactosidase is mainly utilized to carry out catalytic synthesis by a transglycosylation reaction by taking disaccharide substrate lactose as a glycosyl donor, and a large number of byproducts are generated, so that the separation and purification of an alkyl glycoside product are not facilitated. At present, no research report exists for synthesizing alkyl galactoside by using immobilized cells as enzyme sources and monosaccharide as glycosyl donors through a one-step enzyme method.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for synthesizing alkyl galactoside by using monosaccharide as glycosyl donor in immobilized cells.
The technical scheme of the invention is as follows:
a method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor comprises the following steps:
(1) culturing yeast producing beta-galactosidase, and collecting yeast cells;
(2) suspending the yeast cells by using an ethanol solution, stirring for 1-10 min at 20-30 ℃, and centrifuging to obtain ethanol-treated yeast cells;
(3) adding genipin into the yeast cells after ethanol treatment to construct a crosslinking system; reacting the crosslinking system for 0.5-4 h at 20-30 ℃ and 100-150 rpm, and centrifuging to obtain immobilized cells;
(4) uniformly mixing galactose and alkyl alcohol, adding immobilized cells, reacting for 2-16 h at 30-50 ℃, performing centrifugal solid-liquid separation, taking supernatant, removing organic solvent, performing silica gel column chromatography separation and freeze drying to obtain the alkyl galactoside.
Preferably, in step (1), the yeast producing beta-galactosidase is Kluyveromyces lactis with the preservation number of CGMCC 2.1494.
According to the invention, in the step (2), the concentration of the ethanol solution is preferably 40-50%.
According to the invention, in the step (3), the concentration of the genipin in the crosslinking system is 0.05%, and the concentration of the ethanol-treated yeast cells is 0.05-0.2 mg/muL.
Preferably, in step (4), the concentration of galactose is 0.4-0.8M.
According to the invention, in the step (4), the concentration of the alkyl alcohol is preferably 10-40%.
Preferably, in step (4), the alkyl alcohol is methanol, ethanol, n-propanol, isopropanol, n-butanol, n-pentanol or n-hexanol.
Preferably, in the step (4), the addition amount of the immobilized cells is 80-200% of the mass of galactose.
Preferably, in step (4), the silica gel column chromatography conditions are as follows: mixing methanol: ethyl acetate 3:7 is a mobile phase, the sample loading amount is 1-2 mL, and the elution flow rate is 0.5-0.8 mL/min.
The alkyl galactoside in the invention is methyl-beta-D-galactoside, ethyl-beta-D-galactoside, n-propyl-beta-D-galactoside, isopropyl-beta-D-galactoside, n-butyl-beta-D-galactoside, n-pentyl-beta-D-galactoside or n-hexyl-beta-D-galactoside, and corresponds to the alkyl alcohol substrate.
The yeast for producing beta-galactosidase in the invention is yeast which takes galactose as a substrate to carry out synthetic reaction, and other yeast which is not described is carried out according to the prior art.
Advantageous effects
1. The invention provides a green and efficient method for synthesizing alkyl galactoside, which firstly adopts immobilized yeast cells and takes monosaccharide as glycosyl donor to synthesize glucoside in one step. In the synthesis process, the reaction system only contains alkyl glycoside and residual substrate after the reaction is finished, and the product has few varieties and is easy to purify. Compared with the process for synthesizing the alkyl glycoside by a chemical method, the method has the advantages of simple steps, no group protection and deprotection process in chemical synthesis, environmental friendliness, mild reaction conditions, easiness in operation, suitability for large-scale synthesis of the alkyl glycoside and wide application prospect.
2. The method for synthesizing the alkyl glucoside adopts the immobilized yeast cells, does not need to purify enzyme, adopts genipin for preparing the immobilized cells by crosslinking, does not need to additionally use a carrier, reduces the cost of the immobilized enzyme, can be circularly used for synthesizing the alkyl glucoside, greatly reduces the cost and has wide application prospect.
3. According to the invention, genipin and yeast cells are directly crosslinked for the first time, so that the carrier-free immobilized yeast cells are prepared, no carrier material is required to be added additionally, and the method has the advantages of low cost, easiness in operation and high stability. And the genipin is used as a cross-linking agent of natural source, and compared with the existing cross-linking agent, the toxicity is almost negligible, so that the carrier-free immobilization process is non-toxic and harmless, and is green and environment-friendly.
Drawings
FIG. 1 shows a hydrogen spectrum and a carbon spectrum of methyl-beta-D-galactoside.
FIG. 2 is a graph of the batch synthesis of methyl galactoside from carrier-free immobilized yeast cells.
FIG. 3 shows a hydrogen spectrum and a carbon spectrum of ethyl-beta-D-galactoside.
FIG. 4 shows the hydrogen and carbon spectra of n-propyl-beta-D-galactoside.
FIG. 5 shows the hydrogen and carbon spectra of isopropyl-beta-D-galactoside.
FIG. 6 shows hydrogen and carbon spectra of n-butyl-beta-D-galactoside.
FIG. 7 shows hydrogen and carbon spectra of n-pentyl-beta-D-galactoside.
FIG. 8 shows the hydrogen and carbon spectra of n-hexyl-beta-D-galactoside.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
The Kluyveromyces lactis with the preservation number of CGMCC 2.1494 is sold in China general microbiological culture Collection center.
EXAMPLE 1 preparation of immobilized Yeast cells
1. The method comprises the steps of inoculating Kluyveromyces lactis CGMCC 2.1494 to a slant culture medium containing 1% of glucose, 1% of peptone, 0.5% of yeast extract and 1.5% of agar, growing for 24 hours at 30 ℃, transferring to a liquid seed culture medium, culturing for 24 hours at 200rpm and 30 ℃, transferring to a fermentation culture medium containing 1% of glucose, 1% of peptone, 0.5% of yeast extract and 1% of lactose in a volume ratio of 3%, culturing for 32 hours at 30 ℃, centrifuging, collecting Kluyveromyces lactis cells, suspending in 50% of ethanol, uniformly mixing for 1min, and washing for 2 times by using a buffer solution to obtain the ethanol-treated Kluyveromyces lactis cells.
2. Preparing a genipin solution by using a 50mM potassium phosphate buffer solution with pH of 7.5, and uniformly mixing the genipin solution with the ethanol-treated kluyveromyces lactis cells to construct a crosslinking system. Wherein the concentration of genipin in the crosslinking system is 0.05%, and the concentration of yeast cells is 0.1 mg/mL. Then reacting for 4h at 25 ℃ and 125rpm, and centrifuging for 2min at 11000rpm to obtain the immobilized yeast cells.
EXAMPLE 2 catalytic Synthesis of methyl galactoside by immobilized Yeast cells
1. Synthesis of methyl galactoside
A method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor comprises the following steps:
A1M D-galactose solution was prepared using 50mM potassium phosphate buffer, pH 7.5. Then taking 3.5mL of methanol and 6mL of 1M D-galactose, adding the immobilized yeast cells, wherein the adding amount is 140% of the mass of the D-galactose, adding a buffer solution to enable the reaction system to be 10mL, reacting at 37 ℃ for 8h, centrifuging at 11000rpm for 2min, and remaining the supernatant to obtain the alkyl galactoside.
2. Purification and structural identification of methyl galactoside
Taking the supernatant reaction liquid in the step 1, removing the organic solvent by using a rotary evaporator, then carrying out freeze drying, dissolving the obtained powder by using 1mL of mobile phase (methanol: ethyl acetate: 3:7), adding the wet-method sample into a silica gel column which contains 200-plus-300-mesh silica gel and has the specification of 25mm multiplied by 340mm, carrying out the elution flow rate of 0.5mL/min, collecting the elution sample, carrying out thin-layer chromatography detection, combining the pure elution samples of the product, then carrying out rotary evaporation to remove the organic solvent, and carrying out freeze drying to obtain the pure glycoside product.
Dissolving 15mg of the obtained glycoside product in 450 μ L of deuterated water, and performing nuclear magnetic analysis including hydrogen spectrum ( 1 H NMR), carbon spectrum( 13 C NMR), hydrogen-hydrogen correlation spectrum (COSY), hydrocarbon direct correlation spectrum (HSQC) and hydrocarbon remote correlation spectrum (HMBC), the chemical shift of the glycoside product is determined, and the glycoside product is confirmed to be methyl-beta-D-galactoside.
As shown in fig. 1, the chemical shifts of methyl- β -D-galactoside are: 1 H NMR(400MHz,D 2 O):δ4.28(d,1H,H-1,J 1,2 ＝8.0Hz),3.89(dd,1H,H-4),3.75(m,2H,H-6,H-5),3.67(dd,1H,H-6),3.61(dd,1H,H-3),3.54(s,3H,H-7),3.47(dd,1H,H-2)； 13 C NMR(100MHz,D 2 O):δ103.79(C-1),75.11(C-5),72.74(C-3),70.71(C-2),68.64(C-4),60.95(C-6),57.13(C-7)。
the methyl-beta-D-galactoside has a molecular formula of C 7 H 14 O 6 The chemical structural formula is shown as formula (1):
Figure BDA0003741905460000041
the instrument used for the nuclear magnetic analysis was a brueck dalton 400MHz nuclear magnetic resonance spectrometer (usa).
The immobilized cells obtained by centrifugation in step 1 of the present invention were subjected to a new batch synthesis reaction according to the above conditions. As shown in FIG. 2, after 10 batches of reactions of immobilized enzyme, compared with the first batch, methyl-beta-D-galactoside still can retain over 70% of relative yield, and the relative enzyme activity of immobilized cell still remains over 60%.
EXAMPLE 3 catalytic Synthesis of Ethyl galactoside by immobilized Yeast cells
A method for synthesizing an alkylgalactoside using immobilized cells with monosaccharides as glycosyl donors, the steps and conditions are the same as example 2, except that the glycosyl acceptor is ethanol.
15mg of the alkyl galactoside product obtained in this example was dissolved in 450. mu.L of deuterated water and subjected to nuclear magnetic resonance analysis, including a hydrogen spectrum ( 1 H NMR), carbon spectrum ( 13 C NMR), hydrogen-hydrogen correlation spectrum (COSY), hydrocarbon direct correlation spectrum (HSQC) and hydrocarbon remote correlation spectrum (HMBC), determining the chemical shift of the glycoside product and confirming that the glycoside product isethyl-beta-D-galactoside.
As shown in fig. 3, the chemical shifts of ethyl- β -D-galactoside are: 1 H NMR(400MHz,D 2 O):δ4.4(d,1H,H-1,J 1,2 ＝8.0Hz),3.98(dq,1H,H-7),3.92(d,1H,H-4),3.76(m,2H,H-6),3.70(m,2H,H-7,H-5),3.64(dd,1H,H-3),3.49(dd,1H,H-2),1.23(t,3H,H-8)； 13 C NMR(100MHz,D 2 O):δ102.46(C-1),75.11(C-5),72.86(C-3),70.76(C-2),68.67(C-4),66.12(C-7),60.95(C-6),14.30(C-8)。
the above ethyl-beta-D-galactoside has a molecular formula of C 8 H 16 O 6 The chemical structural formula is shown as the following formula (2):
Figure BDA0003741905460000042
EXAMPLE 4 catalytic Synthesis of n-propylgalactoside by immobilized Yeast cells
A method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor, the steps and conditions are the same as example 2, except that glycosyl acceptor is n-propanol.
15mg of the alkyl galactoside product obtained in this example was dissolved in 450. mu.L of deuterated water and subjected to nuclear magnetic resonance analysis, including a hydrogen spectrum ( 1 H NMR), carbon spectrum ( 13 C NMR), hydrogen correlation spectrum (COSY), hydrocarbon direct correlation spectrum (HSQC) and hydrocarbon remote correlation spectrum (HMBC), and the glycoside product is confirmed to be n-propyl-beta-D-galactoside.
As shown in FIG. 4, the chemical shifts of n-propyl- β -D-galactoside are: 1 H NMR(400MHz,D 2 O):δ4.39(d,1H,H-1,J 1,2 ＝8.0Hz),3.92(d,1H,H-4),3.86(m,1H,H-7),3.75(m,2H,H-6),3.68(m,1H,H-5),3.63(m,2H,H-7,H-3),3.50(dd,1H,H-2),1.63(m,2H,H-8),0.91(t,3H,H-9)； 13 C NMR(100MHz,D 2 O):δ102.71(C-1),75.08(C-5),72.85(C-3),72.20(C-7),70.80(C-2),68.67(C-4),60.95(C-6),22.19(C-8),9.64(C-9)。
the n-propyl-beta-D-galactoside has a molecular formula of C 9 H 18 O 6 The chemical structural formula is shown as formula (3):
Figure BDA0003741905460000051
EXAMPLE 5 catalytic Synthesis of isopropyl galactoside by immobilized Yeast cells
A method for synthesizing alkyl galactoside using immobilized cells with monosaccharide as glycosyl donor, the steps and conditions are the same as example 2, except that glycosyl acceptor is isopropanol.
15mg of the alkylgalactoside product obtained in this example was dissolved in 450. mu.L of deuterated water and subjected to nuclear magnetic analysis including hydrogen spectrum (C:) 1 H NMR), carbon spectrum ( 13 C NMR), hydrogen-hydrogen correlation spectrum (COSY), hydrocarbon direct correlation spectrum (HSQC) and hydrocarbon remote correlation spectrum (HMBC), and the glucoside product is isopropyl-beta-D-galactoside.
As shown in fig. 5, the chemical shifts of isopropyl- β -D-galactoside are: 1 H NMR(400MHz,D 2 O):δ4.47(d,1H,H-1,J 1,2 ＝8.0Hz),4.11(m,1H,H-7),3.92(d,1H,H-4),3.76(m,2H,H-6),3.69(m,1H,H-5),3.64(m,1H,H-3),3.46(dd,1H,H-2),1.22(m,6H,H-8,H-9)； 13 C NMR(100MHz,D 2 O):δ100.98(C-1),75.06(C-5),73.00(C-7),72.90(C-3),70.83(C-2),68.64(C-4),60.90(C-6),22.39(C-8),20.99(C-9)。
the isopropyl-beta-D-galactoside has a molecular formula of C 9 H 18 O 6 The chemical structural formula is shown as formula (4):
Figure BDA0003741905460000061
EXAMPLE 6 catalytic Synthesis of n-butylgalactoside by immobilized Yeast cells
A method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor comprises the same steps and conditions as example 2, except that glycosyl acceptor is n-butanol, and nuclear magnetic analysis is performed by Bruk Dalton 600MHz nuclear magnetic resonance spectrometer (USA).
15mg of this example was preparedDissolving the obtained alkyl galactoside product in 450 μ L deuterated water, and performing nuclear magnetic analysis including hydrogen spectrum (b), (c) 1 H NMR), carbon spectrum ( 13 C NMR), hydrogen correlation spectrum (COSY), hydrocarbon direct correlation spectrum (HSQC) and hydrocarbon remote correlation spectrum (HMBC), and the glucoside product is confirmed to be n-butyl-beta-D-galactoside.
As shown in FIG. 6, the chemical shifts of n-butyl- β -D-galactoside are: 1 H NMR(600MHz,D 2 O):δ4.40(d,1H,H-1,J 1,2 ＝6.0Hz),3.93(m,2H,H7,H-4),3.77(qd,2H,H-6),3.69(td,2H,H-7,H-5),3.65(dd,1H,H-3),3.5(dd,1H,H-2),1.61(dt,2H,H-8),1.38(dq,2H,H-9),0.91(t,3H,H-10)； 13 C NMR(150MHz,D 2 O):δ102.73(C-1),75.07(C-5),72.82(C-3),70.77(C-2),70.35(C-7),68.65(C-4),60.91(C-6),30.88(C-8),18.46(C-9),13.08(C-10)。
the n-butyl propyl-beta-D-galactoside has molecular formula of C 9 H 18 O 6 The chemical structural formula is shown as formula (5):
Figure BDA0003741905460000062
EXAMPLE 7 catalytic Synthesis of n-pentylgalactoside by immobilized Yeast cells
A method for synthesizing an alkylgalactoside using immobilized cells with monosaccharides as glycosyl donor, the steps and conditions are the same as example 2, except that glycosyl acceptor is n-pentanol.
15mg of the alkylgalactoside product obtained in this example was dissolved in 450. mu.L of deuterated water and subjected to nuclear magnetic analysis including hydrogen spectrum (C:) 1 H NMR), carbon spectrum ( 13 C NMR), hydrogen-hydrogen correlation spectrum (COSY), hydrocarbon direct correlation spectrum (HSQC) and hydrocarbon remote correlation spectrum (HMBC), and the glucoside product is confirmed to be n-amyl-beta-D-galactoside.
As shown in FIG. 7, the chemical shifts of n-pentyl-. beta. -D-galactoside are: 1 H NMR(400MHz,D 2 O):δ4.39(d,1H,H-1,J 1,2 ＝8.0Hz),3.92(m,2H,H7,H-4),3.76(m,2H,H-6),3.65(m,3H,H-7,H-5,H-3),3.49(dd,1H,H-2),1.63(p,2H,H-8),1.33(m,4H,H-9,H-10),0.89(m,3H,H-11)； 13 C NMR(100MHz,D 2 O):δ102.73(C-1),75.07(C-5),72.83(C-3),70.77(C-2),70.64(C-7),68.64(C-4),60.90(C-6),28.44(C-8),27.31(C-9),21.77(C-10),13.26(C-11)。
the n-pentyl-beta-D-galactoside has a molecular formula of C 10 H 20 O 6 The chemical structural formula is shown as formula (6):
Figure BDA0003741905460000071
EXAMPLE 8 catalytic Synthesis of n-hexyl galactoside by immobilized Yeast cells
A method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor comprises the same steps and conditions as example 2, except that glycosyl acceptor is n-hexanol.
15mg of the alkyl galactoside product obtained in this example was dissolved in 450. mu.L of deuterated water and subjected to nuclear magnetic resonance analysis, including a hydrogen spectrum ( 1 H NMR), carbon spectrum (C: 13 c NMR), hydrogen-hydrogen correlation spectrum (COSY), hydrocarbon direct correlation spectrum (HSQC), hydrocarbon remote correlation spectrum (HMBC), confirming that the glycoside product is n-hexyl- β -D-galactoside.
As shown in fig. 8, the chemical shifts of n-hexyl- β -D-galactoside are: 1 H NMR(400MHz,D 2 O):δ4.43(d,1H,H-1,J 1,2 ＝8.0Hz),3.95(m,2H,H7,H-4),3.81(m,2H,H-6),3.71(m,2H,H-7,H-5),3.67(d,2H,H-3),3.52(dd,1H,H-2),1.65(p,2H,H-8),1.33(m,4H,H-9,H-10,H-11),0.91(m,3H,H-12)； 13 CNMR(100MHz,D 2 O):δ102.75(C-1),75.08(C-5),72.84(C-3),70.77(C-2),70.81(C-7),70.70(C-4),60.95(C-6),30.85(C-10),28.74(C-8),24.76(C-9),21.95(C-11),13.36(C-12)。
the n-hexyl-beta-D-galactoside has a molecular formula of C 11 H 22 O 6 The chemical structural formula is shown as formula (7):
Figure BDA0003741905460000072
in conclusion, the invention firstly adopts the immobilized yeast cells to synthesize methyl-beta-D-galactoside, ethyl-beta-D-galactoside, n-propyl-beta-D-galactoside, isopropyl-beta-D-galactoside, n-butyl-beta-D-galactoside, n-pentyl-beta-D-galactoside or n-hexyl-beta-D-galactoside in one step by taking monosaccharide as glycosyl donor, has simple steps, does not have the processes of protecting and deprotecting groups in chemical synthesis, is environment-friendly, has mild reaction conditions, is easy to operate, is suitable for large-scale synthesis of alkyl glycoside, and has wide application prospect.

Claims (9)

1. A method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor is characterized by comprising the following steps:
(1) culturing yeast producing beta-galactosidase, and collecting yeast cells;
(2) suspending the yeast cells by using an ethanol solution, stirring for 1-10 min at 20-30 ℃, and centrifuging to obtain ethanol-treated yeast cells;
(3) adding genipin into the yeast cells after ethanol treatment to construct a crosslinking system; and (3) reacting the crosslinking system for 0.5-4 h at the temperature of 20-30 ℃ and the rpm of 100-150, and centrifuging to obtain the immobilized cell.
(4) Uniformly mixing galactose and alkyl alcohol, adding immobilized cells, reacting for 2-16 h at 30-50 ℃, performing centrifugal solid-liquid separation, taking supernatant, removing organic solvent, performing silica gel column chromatography separation and freeze drying to obtain the alkyl galactoside.
2. The synthetic method according to claim 1, wherein in step (1), the beta-galactosidase producing yeast is Kluyveromyces lactis with the preservation number of CGMCC 2.1494.
3. The synthesis method according to claim 1, wherein in the step (2), the concentration of the ethanol solution is 40-50%.
4. The synthesis method according to claim 1, wherein in the step (3), the concentration of genipin in the crosslinking system is 0.05%, and the concentration of the ethanol-treated yeast cells is 0.05-0.2 mg/μ L.
5. The method of claim 1, wherein in step (4), the concentration of galactose is 0.4-0.8M.
6. The method of claim 1, wherein in step (4), the concentration of the alkyl alcohol is 10-40%.
7. The method of claim 1, wherein in step (4), the alkyl alcohol is methanol, ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, or n-hexanol.
8. The method of claim 1, wherein in the step (4), the amount of the immobilized cells added is 80-200% by mass of galactose.
9. The synthesis method of claim 1, wherein in the step (4), the silica gel column chromatography conditions are as follows: the method comprises the following steps of mixing methanol: ethyl acetate 3:7 is a mobile phase, the sample loading amount is 1-2 mL, and the elution flow rate is 0.5-0.8 mL/min.
CN202210818830.XA 2022-07-12 2022-07-12 Method for synthesizing alkyl galactoside by using immobilized cells and monosaccharide as glycosyl donor Pending CN115094104A (en)

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