WO2022062501A1 - Method for preparing gentiooligosaccharide by catalyzing cellulose by using compound enzyme, and use thereof - Google Patents

Method for preparing gentiooligosaccharide by catalyzing cellulose by using compound enzyme, and use thereof Download PDF

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WO2022062501A1
WO2022062501A1 PCT/CN2021/101171 CN2021101171W WO2022062501A1 WO 2022062501 A1 WO2022062501 A1 WO 2022062501A1 CN 2021101171 W CN2021101171 W CN 2021101171W WO 2022062501 A1 WO2022062501 A1 WO 2022062501A1
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glucosidase
preparing
gentiosaccharide
cellulose
reaction
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吴敬
夏伟
许俊勇
徐星豪
黄燕
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江南大学
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    • 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)
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12P19/12—Disaccharides
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    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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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)
    • 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
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the invention relates to a method for preparing oligosaccharide gentisose from cellulose catalyzed by a compound enzyme and its application, and belongs to the technical field of enzyme engineering.
  • Gentiose oligosaccharides are oligosaccharides formed by the connection of glucose by ⁇ -1,6 glycosidic bonds.
  • the main component is gentiobiose, and contains a small amount of gentiotriose and gentiotetraose. It is a new type of functional Sexual oligosaccharides.
  • gentiosaccharides Compared with functional oligosaccharides such as isomaltooligosaccharides, fructooligosaccharides, and galactooligosaccharides, gentiosaccharides have low viscosity, good water holding capacity, It is very stable to pH and heat, and can be used in some other foods that are intolerant of oligosaccharides; and has a unique refreshing bitter taste, which can increase the richness of food taste. Gentianose oligosaccharide has been widely used in the food that is very popular among the public, and it is a high value-added sugar product with great development prospects.
  • Rates are roughly distributed between 5-15%.
  • the use of ⁇ -glucosidase transglycosidase reaction to produce gentiosaccharides with cellobiose as substrate has gradually appeared.
  • this transglycosidation reaction The system has obvious advantages in catalytic efficiency and conversion rate, and the conversion rate is about 19.6%, but the substrate cellobiose is expensive and not suitable for large-scale production; how to develop a cheap substrate that can effectively utilize and efficiently
  • the method for preparing oligogentisose has become an urgent problem to be solved.
  • the present invention establishes a reaction system for preparing oligosaccharide gentisose by converting cellulose to catalyzed by a compound enzyme.
  • a reaction system for preparing oligosaccharide gentisose by converting cellulose to catalyzed by a compound enzyme Under the condition that a certain concentration of glucose inhibits the hydrolysis activity of ⁇ -glucosidase, the ⁇ -glucosidase with high transglycosidic activity is used to replace the traditional ⁇ -glucosidase.
  • the high hydrolytic activity ⁇ -glucosidase in the cellulose degrading enzyme system can convert cellobiose into oligosaccharide gentisose and eliminate the product inhibition of cellobiose; the hydrolysis of cellulose by upstream cellulase can fill the fiber Disaccharide consumption.
  • high-efficiency preparation of gentiosaccharide can be achieved.
  • the present invention first provides a ⁇ -glucosidase mutant, which is obtained by mutating the 34th and/or 200th position of the ⁇ -glucosidase whose amino acid sequence is shown in SEQ ID NO.1 .
  • the ⁇ -glucosidase mutant is obtained by mutating the 34th position of the ⁇ -glucosidase whose amino acid sequence is shown in SEQ ID NO.1 from glutamine to glycine , named Q34G;
  • the 34th position of the ⁇ -glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is mutated from glutamine to glycine, and the 34th position of the ⁇ -glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is changed simultaneously.
  • the 200-position was obtained by mutating tyrosine to phenylalanine and named Q34G/Y200F.
  • the amino acid sequence of the ⁇ -glucosidase mutant Q34G is shown in SEQ ID NO. 9, and the amino acid sequence of the ⁇ -glucosidase mutant Q34M is shown in SEQ ID NO. 10, the amino acid sequence of the ⁇ -glucosidase mutant Y200F is shown in SEQ ID NO.11, and the amino acid sequence of the ⁇ -glucosidase mutant Q34G/Y200F is shown in SEQ ID NO.12.
  • nucleotide sequence encoding the ⁇ -glucosidase is shown in SEQ ID NO.2.
  • the present invention also provides a gene encoding the above-mentioned ⁇ -glucosidase mutant.
  • the present invention also provides an expression vector carrying the above gene.
  • the present invention also provides a microbial cell carrying the above-mentioned gene or the above-mentioned expression vector.
  • the microbial cells are bacteria or fungi.
  • the present invention also provides a method for preparing oligosaccharide gentisose by compounding multiple enzymes.
  • the method comprises adding cellulase and the above-mentioned ⁇ -glucosidase mutant to a reaction system containing cellulose and glucose for reaction. , the reaction solution is obtained; the oligosaccharide gentisose are separated from the reaction solution.
  • the cellulase is one of endocellulase, ⁇ -glucanase, type I cellobiohydrolase, and type II cellobiohydrolase or variety.
  • the concentration of the cellulase added to the reaction system is 10-50 U/g of cellulose filter paper enzymatic activity.
  • the concentration of the cellulase added to the reaction system is 25-50 U/g of cellulose filter paper enzymatic activity.
  • the concentration of the ⁇ -glucosidase mutant added to the reaction system is 100-400 U/g glucose.
  • the concentration of the ⁇ -glucosidase mutant added to the reaction system is 300-400 U/g glucose.
  • the concentration of the substrate cellulose is 200 g/L.
  • the concentration of the substrate glucose is 100g/L-400g/L.
  • the temperature of the reaction is 60°C.
  • the pH of the reaction is 5.0.
  • the reaction time is 24-96 h.
  • the reaction time is 96h.
  • the processing method of the substrate cellulose is as follows: the cellulose raw material is subjected to phosphoric acid expansion pretreatment to obtain phosphoric acid swollen cellulose.
  • the method is as follows: adding cellulose swollen with phosphoric acid to a pH 5.0 buffer to a concentration of 200 g/L, and adding glucose to a concentration of 400 g/L to obtain a reaction system , add 50U/g cellulose filter paper enzymatically active cellulase and the above-mentioned 400U/g glucose ⁇ -glucosidase mutant into the reaction system, and react at 60°C for 96h.
  • the buffer is a citric acid-phosphate buffer.
  • the present invention also provides the application of the above mutant or the above gene or the above expression vector or the above microbial cell or the above preparation method in the preparation of gentiosaccharide oligosaccharides.
  • the compound enzyme of the present invention can effectively convert cellulose to generate gentiosaccharide oligosaccharides; when 20% of phosphoric acid swollen cellulose (PASC) and 40% of glucose are used as substrates, the enzyme-catalyzed conversion to prepare gentiosaccharide oligosaccharides,
  • PASC phosphoric acid swollen cellulose
  • the overall conversion rates of compound cellulase and ⁇ -glucosidase mutants Q34G, Q34M, Y200F or Q34G/Y200F were 17.6%, 16.0%, 16.7% and 19.1%, respectively, compared with wild-type ⁇ -glucosidase, respectively. Improvements of 60.6%, 46.1%, 52.7% and 74.7%.
  • the compound enzyme with the highest conversion rate uses cellulose and glucose as raw materials to prepare oligosaccharides with a yield of up to 114.7g/L, and cellobiose conversion The rate can reach 57.3%.
  • the present invention realizes the preparation of gentiosaccharide oligosaccharides with wider and cheaper cellulose substrates, which greatly reduces the production cost of gentisaccharide oligosaccharides in industry. Therefore, the technical solution of the present invention has a high Industrial application value.
  • Figure 1 Schematic diagram of the synthesis of oligosaccharides from cellulose catalyzed by complex enzymes.
  • Trichoderma reesei Trichoderma reesei QM9414 was purchased from the American Culture Collection (the deposit number is ATCC26921).
  • LB liquid medium yeast powder 5g/L, tryptone 10g/L, NaCl 10g/L.
  • MD solid medium YNB 13.4g/L, biotin 4 ⁇ 10 -4 g/L, glucose 20g/L, agar 20g/L.
  • YPD liquid medium peptone 20g/L, yeast extract 10g/L, glucose 20g/L.
  • YPD solid medium On the basis of YPD liquid medium, add 20g/L agar.
  • BMGY medium YNB 13.4g/L, glycerol 10g/L, biotin 4 ⁇ 10 -4 g/L, 0.1mol/L potassium phosphate buffer solution (pH 6.0), peptone 20g/L, yeast powder 10g/L.
  • BMMY medium YNB 13.4g/L, methanol 1%, biotin 4 ⁇ 10 -4 g/L, 0.1mol/L potassium phosphate buffer solution (pH 6.0), peptone 20g/L, yeast powder 10g/L.
  • Fermentation seed medium yeast powder 5.0g/L, tryptone 10.0g/L, glucose 10.0g/L, glycerol 30g/L.
  • BSM medium 85% phosphoric acid 26.7 mL/L, CaSO 4 0.93 g/L, K 2 SO 4 18.2 g/L, MgSO 4 7H 2 O 14.9 g/L, KOH 4.13 g/L, glycerol 30.0 g/L , trace element salt solution 4.32mL/L.
  • the enzymatic activity unit is defined as: the enzymatic activity of hydrolyzing cellulose substrate to produce 1 ⁇ mol of reducing sugar per minute is one filter paper enzymatic activity unit (FPU).
  • FPU filter paper enzymatic activity unit
  • the reaction system was 1 mL, 960 ⁇ L of pH 5.0 acetate buffer, 20 ⁇ L of moderately diluted crude enzyme solution was added, and then 20 ⁇ L of 100 mmol/L pNPG was added. After reacting in a constant temperature water bath at 60 °C for 10 min, 200 ⁇ L of 1 mol/L Na was added immediately. The reaction was terminated with 2 CO 3 solution, ice bathed for 5 min, and the absorbance value was measured at 405 nm. The heat-inactivated enzyme solution was treated in the same way as blank.
  • ⁇ -glucosidase enzymatic activity is that the enzymatic activity of hydrolyzing pNPG to produce 1 ⁇ mol of p-nitrophenol per milliliter of enzyme solution per minute is one unit of enzymatic activity.
  • the content of gentiosaccharide oligosaccharide was detected by HPLC;
  • the chromatographic conditions are as follows: Agilent 1200HPLC chromatograph, Agilent autosampler, RID differential detector; mobile phase is 75% acetonitrile, flow rate 0.8mL/min; column temperature 35°C.
  • ⁇ -glucosidase whose nucleotide sequence is shown in SEQ ID NO.2
  • primers for introducing Q34G, Q34M, and Y200F mutations were designed and synthesized, and the ⁇ -glucosidase gene was site-directed mutation, and the DNA coding was determined. It was identified that the Gln codon at position 34 becomes a Gly codon, the Gln codon at position 34 becomes a Met codon, and the Tyr codon at position 200 becomes a Phe codon.
  • the mutant gene is placed in an expression vector, and the expression vector is introduced into Pichia pastoris for expression to obtain a single mutant ⁇ -glucosidase.
  • single mutation Q34G, Q34M, Y200F and combined mutation Q34G/Y200F were constructed using expression vector pPIC9K-TpBgl3A (recorded in Chinese patent application with publication number CN111500560A) as a template.
  • the site-directed mutagenesis primers for introducing the Q34G mutation are:
  • Reverse primer 5'-TACACAAGAACCGTT CCC CCAACTTGTTCCCGT-3' (SEQ ID NO. 4)
  • the site-directed mutagenesis primers for introducing the Q34M mutation are:
  • Reverse primer 5'-GAACCGTT CAT CCAACTTGTTCCCGTCACGATG-3' (SEQ ID NO. 6)
  • the site-directed mutagenesis primers for introducing the Y200F mutation are:
  • Reverse primer 5'-ACTTTATT CCA CGAGCACATCACGCTGGCGACG-3' (SEQ ID NO. 8)
  • PCR reaction systems are: 5 ⁇ PS buffer 10 ⁇ L, dNTPs Mix (2.5mM) 4 ⁇ L, forward primer (10 ⁇ M) 1 ⁇ L, reverse primer (10 ⁇ M) 1 ⁇ L, template DNA 1 ⁇ L, Primerstar HS (5U/ ⁇ L) 0.5 ⁇ L, Add double distilled water to 50 ⁇ L.
  • PCR amplification conditions were: pre-denaturation at 94°C for 4 min; followed by 30 cycles (98°C for 10s, 55°C for 15s, 72°C for 10min); and continued extension at 72°C for 10min.
  • the product was transformed into E. coli JM109 competent cells to obtain the transformed product.
  • the transformed product was cultured in LB medium (containing 100 ⁇ g/mL ampicillin) overnight, positive clones were picked and cultured in LB liquid.
  • the base containing 100 ⁇ g/mL ampicillin
  • the plasmids were extracted, all mutant plasmids were sequenced correctly, the mutant plasmids were transformed into the expression host Pichia pastoris KM71 competent cells, and the competent cells were inoculated on MD solid culture After culturing on the substrate, 96 single colonies were picked and seeded at equal intervals on the new MD solid medium.
  • Example 2 Expression of wild-type and mutant ⁇ -glucosidase and determination of specific enzyme activity
  • Pichia strain KM71/pPIC9K-TpBgl3A capable of expressing wild-type ⁇ -glucosidase is described in the Chinese patent application with publication number CN111500560A.
  • the specific activity of the hydrolysis reaction catalyzed by the crude enzyme solution was measured respectively, and the specific activity of the hydrolysis reaction of the obtained ⁇ -glucosidase single mutant enzyme was listed in Table 1, wherein the specific activity of the hydrolysis reaction of the mutant Q34M increased slightly, and the mutant
  • the specific activity of the hydrolysis reaction of Q34G was basically the same as that of the wild type, and the specific activity of the hydrolysis reaction of the mutant Y200F and the combined mutant Q34G/Y200F decreased to 2.6% and 2.1% of the wild type, respectively.
  • the reduction of enzymatic hydrolysis activity is beneficial to the occurrence of transglycosidic reaction and the accumulation of transglycosidic products.
  • Phosphoric acid swollen cellulose is obtained by pretreating cellulose raw materials with phosphoric acid expansion.
  • PASC Phosphoric acid swollen cellulose
  • step (2) moving the solution obtained in step (1) to stand at 4°C for 12 hours to fully expand the cellulose;
  • step (3) adding 1 L of ice water to the puffed cellulose obtained in step (2), stirring while adding, white flocs appear, and a mixture is obtained;
  • step (3) centrifuge the mixture obtained in step (3) at 6000rpm for 20min, collect the white flocculent precipitate, discard the supernatant (large centrifuge cup);
  • step (6) add 1L of ice water to step (5), after fully stirring, centrifuge at 6000rpm for 20min, discard the supernatant;
  • step (6) Repeat step (6) until the pH of the solution reaches between 5-7, take the precipitate and dry it, the precipitate is phosphoric acid swollen cellulose, and the prepared phosphoric acid swollen cellulose PASC is stored at 4°C for subsequent use. .
  • Cellulase is obtained from Trichoderma reesei (Trichoderma reesei QM9414) fermentation, has endo-cellulase and cellobiohydrolase activity, and its concrete preparation steps are as follows:
  • Trichoderma reesei spores preserved on the PDA slant are washed with sterile water, and obtaining the spore concentration is 10 7 cfu/mL spore suspension;
  • PDA medium potato 200g/L, glucose 20g/L, agar 20g/L;
  • inoculation amount 2% (v/v)
  • fermentation medium lactose 18g/L, microcrystalline cellulose 10g/L, corn pulp powder 12g/L, (NH4) 2 SO 4 0.5g/L, MgSO 4 1g/L, CaCl 2 0.5g/L, Mandels trace element nutrients 1mL/L, Tween 80 2mL/L, pH 4.8.
  • step (3) Centrifuge the fermentation broth obtained in step (2) to take the supernatant, which is the crude cellulase enzyme solution, and measure the enzyme activity of the filter paper, and the enzyme activity is 456 FPU/mL.
  • PASC phosphoric acid swollen cellulose
  • FPU cellulose filter paper enzyme activity
  • the test results are shown in Table 2.
  • the compound enzymes catalyzed the preparation of oligosaccharides from cellulose and glucose.
  • the ⁇ -glucosidase mutant Q34G, ⁇ -glucosidase mutant Q34M, ⁇ -glucosidase mutant Y200F and ⁇ -glucosidase mutant were used respectively.
  • -glucosidase mutant Q34G/Y200F the total conversion rates were 17.6%, 16.0%, 16.7% and 19.1%, respectively, and the total conversion rates were 60.6%, 46.1%, and 52.7% higher than those of wild-type ⁇ -glucosidase, respectively. % and 74.7%.
  • the compound enzyme with the highest conversion rate uses cellulose and glucose as raw materials to prepare oligosaccharides with a yield of up to 114.7g/L, and cellobiose conversion The rate can reach 57.3%, which has certain application value.
  • Example 4 Influence of the source of ⁇ -glucosidase on the yield of gentiosaccharide oligosaccharides
  • the specific embodiment is the same as Example 3, except that the adjustment of ⁇ -glucosidase is: the wild enzyme of ⁇ -glucosidase derived from Trichoderma viride (recorded in CN107099565B Chinese invention patent text); the results show that the total conversion Rate: 8.7%.
  • Example 5 Influence of the compounding ratio of enzymes on the yield of gentiosaccharide oligosaccharides
  • the specific embodiment is the same as Example 3, except that the compounding ratio of cellulase and ⁇ -glucosidase is adjusted.
  • the amount of cellulase was adjusted to 10U/g cellulose filter paper enzyme activity (FPU), 15U/g cellulose filter paper enzyme activity (FPU), 25U/g cellulose filter paper enzymatic activity (FPU), the reaction was carried out for 96h, and the total conversion rates were 5.3%, 5.9% and 8.2%, respectively.
  • the addition amount of fixed cellulase was 50U/g cellulose filter paper enzyme activity (FPU)
  • the addition amount of wild-type ⁇ -glucosidase was adjusted to 100U/g glucose, 200U/g glucose, 300U/g glucose respectively, and the reaction was carried out for 96h. , and the total conversion rates were 3.7%, 4.9%, and 9.2%, respectively.

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Abstract

Provided are a β-glucosidase mutant and an encoding gene thereof, and a method for preparing gentiooligosaccharide by catalyzing cellulose by using a compound enzyme.

Description

一种复合酶催化纤维素制备低聚龙胆糖的方法及其应用A kind of method and application of compound enzyme catalyzing cellulose to prepare gentiosaccharide oligosaccharide 技术领域technical field
本发明涉及一种复合酶催化纤维素制备低聚龙胆糖的方法及其应用，属于酶工程技术领域。The invention relates to a method for preparing oligosaccharide gentisose from cellulose catalyzed by a compound enzyme and its application, and belongs to the technical field of enzyme engineering.
背景技术Background technique
低聚龙胆糖是葡萄糖以β-1,6糖苷键连接形成的低聚寡糖，主要成分是龙胆二糖，并含有少量的龙胆三糖和龙胆四糖，是一种新型功能性低聚糖。低聚龙胆糖具有极好的益肠道功效，与低聚异麦芽糖、低聚果糖、低聚半乳糖等功能性低聚糖相比，低聚龙胆糖具有低粘度、持水性好、对pH和热非常稳定等特点，可应用于一些其他低聚糖不耐受的食品中；且具备独特的提神苦味，可增加食品口味的丰富性。目前低聚龙胆糖在大众极为喜爱的食品中已有较为广泛的应用，是极具开发前景的高附加值糖类产品。Gentiose oligosaccharides are oligosaccharides formed by the connection of glucose by β-1,6 glycosidic bonds. The main component is gentiobiose, and contains a small amount of gentiotriose and gentiotetraose. It is a new type of functional Sexual oligosaccharides. Compared with functional oligosaccharides such as isomaltooligosaccharides, fructooligosaccharides, and galactooligosaccharides, gentiosaccharides have low viscosity, good water holding capacity, It is very stable to pH and heat, and can be used in some other foods that are intolerant of oligosaccharides; and has a unique refreshing bitter taste, which can increase the richness of food taste. Gentianose oligosaccharide has been widely used in the food that is very popular among the public, and it is a high value-added sugar product with great development prospects.
至今国内外仍仅有日本食品化工株式会社生产该产品(商品名Gentose#45、Gentose#80)，且产量不高，仅350吨，市场缺口较大。随着市场需求的扩大，酶法制备制备低聚龙胆糖具有反应条件温和、菌种安全、污染性小、成本低廉、易于分离等优点，开始受到国内外研究人员的关注，是目前生产低聚龙胆糖的主要发展趋势，但目前未有较为理想的案例报道。早期研究者利用β-葡萄糖苷酶催化逆水解缩合反应，以高浓度葡萄糖或酸水解淀粉所得的葡萄糖浆为底物制备低聚龙胆糖，在200-900g/L葡萄糖底物浓度下，转化率大致分布于5-15％之间。近年来逐渐出现了利用β-葡萄糖苷酶转糖苷反应，以纤维二糖为底物生产低聚龙胆糖的研究，相比目前以高浓度葡萄糖为底物的反应体系，这种转苷反应体系在催化效率和转化率上均具有明显优势，转化率约为19.6％，但底物纤维二糖成本昂贵，不适合大规模生产；如何开发得到一种能够有效利用廉价底物，且可高效制备低聚龙胆糖的方法，成为迫切需要解决的问题。So far, only Nippon Foods Chemical Co., Ltd. produces this product (trade name Gentose#45, Gentose#80) at home and abroad, and the output is not high, only 350 tons, and the market gap is large. With the expansion of market demand, the enzymatic preparation of gentiosaccharide has the advantages of mild reaction conditions, safe strains, low pollution, low cost, easy separation, etc., and has begun to attract the attention of domestic and foreign researchers. The main development trend of polygentisose, but there is no ideal case report. Early researchers used β-glucosidase to catalyze the reverse hydrolysis condensation reaction, using high concentration of glucose or glucose syrup obtained by acid hydrolysis of starch as the substrate to prepare oligosaccharides. Rates are roughly distributed between 5-15%. In recent years, the use of β-glucosidase transglycosidase reaction to produce gentiosaccharides with cellobiose as substrate has gradually appeared. Compared with the current reaction system with high concentration of glucose as substrate, this transglycosidation reaction The system has obvious advantages in catalytic efficiency and conversion rate, and the conversion rate is about 19.6%, but the substrate cellobiose is expensive and not suitable for large-scale production; how to develop a cheap substrate that can effectively utilize and efficiently The method for preparing oligogentisose has become an urgent problem to be solved.
发明内容SUMMARY OF THE INVENTION
本发明建立了一种复合酶催化纤维素转化制备低聚龙胆糖的反应体系，在一定浓度葡萄糖抑制β-葡萄糖苷酶水解活性的条件下，利用高转糖苷活性β-葡萄糖苷酶取代传统纤维素降解酶体系中的高水解活性β-葡萄糖苷酶，可将纤维二糖转化为低聚龙胆糖，消除纤维二糖的产物抑制；上游纤维素酶对纤维素的水解又可填补纤维二糖的消耗。通过协同反应体系的调控，可实现高效低聚龙胆糖制备。The present invention establishes a reaction system for preparing oligosaccharide gentisose by converting cellulose to catalyzed by a compound enzyme. Under the condition that a certain concentration of glucose inhibits the hydrolysis activity of β-glucosidase, the β-glucosidase with high transglycosidic activity is used to replace the traditional β-glucosidase. The high hydrolytic activity β-glucosidase in the cellulose degrading enzyme system can convert cellobiose into oligosaccharide gentisose and eliminate the product inhibition of cellobiose; the hydrolysis of cellulose by upstream cellulase can fill the fiber Disaccharide consumption. Through the regulation of the synergistic reaction system, high-efficiency preparation of gentiosaccharide can be achieved.
本发明首先提供了一种β-葡萄糖苷酶突变体，所述突变体是将氨基酸序列如SEQ ID  NO.1所示的β-葡萄糖苷酶的第34位和/或第200位突变得到的。The present invention first provides a β-glucosidase mutant, which is obtained by mutating the 34th and/or 200th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 .
在本发明的一种实施方式中，所述β-葡萄糖苷酶突变体为：将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第34位由谷氨酰胺突变为甘氨酸得到的，命名为Q34G；In one embodiment of the present invention, the β-glucosidase mutant is obtained by mutating the 34th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 from glutamine to glycine , named Q34G;
或将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第34位由谷氨酰胺突变为甲硫氨酸得到的，命名为Q34M；Or the 34th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is obtained by mutating glutamine into methionine, and named as Q34M;
或将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第200位由酪氨酸突变为苯丙氨酸得到的，命名为Y200F；Or the 200th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is obtained by mutating tyrosine into phenylalanine, and is named as Y200F;
或将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第34位由谷氨酰胺突变为甘氨酸，同时将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第200位由酪氨酸突变为苯丙氨酸得到的，命名为Q34G/Y200F。Or the 34th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is mutated from glutamine to glycine, and the 34th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is changed simultaneously. The 200-position was obtained by mutating tyrosine to phenylalanine and named Q34G/Y200F.
在本发明的一种实施方式中，所述β-葡萄糖苷酶突变体Q34G的氨基酸序列如SEQ ID NO.9所示，所述β-葡萄糖苷酶突变体Q34M的氨基酸序列如SEQ ID NO.10所示，所述β-葡萄糖苷酶突变体Y200F的氨基酸序列如SEQ ID NO.11所示，所述β-葡萄糖苷酶突变体Q34G/Y200F的氨基酸序列如SEQ ID NO.12所示。In one embodiment of the present invention, the amino acid sequence of the β-glucosidase mutant Q34G is shown in SEQ ID NO. 9, and the amino acid sequence of the β-glucosidase mutant Q34M is shown in SEQ ID NO. 10, the amino acid sequence of the β-glucosidase mutant Y200F is shown in SEQ ID NO.11, and the amino acid sequence of the β-glucosidase mutant Q34G/Y200F is shown in SEQ ID NO.12.
在本发明的一种实施方式中，编码所述β-葡萄糖苷酶的核苷酸序列如SEQ ID NO.2所示。In one embodiment of the present invention, the nucleotide sequence encoding the β-glucosidase is shown in SEQ ID NO.2.
本发明还提供了编码上述β-葡萄糖苷酶突变体的基因。The present invention also provides a gene encoding the above-mentioned β-glucosidase mutant.
本发明还提供了携带上述基因的表达载体。The present invention also provides an expression vector carrying the above gene.
本发明还提供了携带上述基因或者上述表达载体的微生物细胞。The present invention also provides a microbial cell carrying the above-mentioned gene or the above-mentioned expression vector.
在本发明的一种实施方式中，所述微生物细胞为细菌或真菌。In one embodiment of the present invention, the microbial cells are bacteria or fungi.
本发明还提供了一种多酶复配制备低聚龙胆糖的方法，所述方法为将纤维素酶和上述β-葡萄糖苷酶突变体添加至含有纤维素和葡萄糖的反应体系中进行反应，得到反应液；从反应液中分离得到低聚龙胆糖。The present invention also provides a method for preparing oligosaccharide gentisose by compounding multiple enzymes. The method comprises adding cellulase and the above-mentioned β-glucosidase mutant to a reaction system containing cellulose and glucose for reaction. , the reaction solution is obtained; the oligosaccharide gentisose are separated from the reaction solution.
在本发明的一种实施方式中，所述所述纤维素酶为内切纤维素酶、β-葡聚糖酶、I型纤维二糖水解酶、II型纤维二糖水解酶的一种或多种。In one embodiment of the present invention, the cellulase is one of endocellulase, β-glucanase, type I cellobiohydrolase, and type II cellobiohydrolase or variety.
在本发明的一种实施方式中，向反应体系中添加的纤维素酶的浓度为10-50U/g纤维素滤纸酶活。In one embodiment of the present invention, the concentration of the cellulase added to the reaction system is 10-50 U/g of cellulose filter paper enzymatic activity.
在本发明的一种实施方式中，向反应体系中添加的纤维素酶的浓度为25-50U/g纤维素滤纸酶活。In one embodiment of the present invention, the concentration of the cellulase added to the reaction system is 25-50 U/g of cellulose filter paper enzymatic activity.
在本发明的一种实施方式中，向反应体系中添加的β-葡萄糖苷酶突变体的浓度为100-400U/g葡萄糖。In one embodiment of the present invention, the concentration of the β-glucosidase mutant added to the reaction system is 100-400 U/g glucose.
在本发明的一种实施方式中，向反应体系中添加的β-葡萄糖苷酶突变体的浓度为300-400U/g葡萄糖。In one embodiment of the present invention, the concentration of the β-glucosidase mutant added to the reaction system is 300-400 U/g glucose.
在本发明的一种实施方式中，所述反应体系中，底物纤维素的浓度为200g/L。In an embodiment of the present invention, in the reaction system, the concentration of the substrate cellulose is 200 g/L.
在本发明的一种实施方式中，所述反应体系中，底物葡萄糖的浓度为100g/L-400g/L。In an embodiment of the present invention, in the reaction system, the concentration of the substrate glucose is 100g/L-400g/L.
在本发明的一种实施方式中，所述反应的温度为60℃。In one embodiment of the present invention, the temperature of the reaction is 60°C.
在本发明的一种实施方式中，所述反应的pH为5.0。In one embodiment of the invention, the pH of the reaction is 5.0.
在本发明的一种实施方式中，所述反应的时间为24-96h。In one embodiment of the present invention, the reaction time is 24-96 h.
在本发明的一种实施方式中，所述反应的时间为96h。In one embodiment of the present invention, the reaction time is 96h.
在本发明的一种实施方式中，所述底物纤维素的处理方式为：将纤维素原料采用磷酸膨化预处理获得磷酸溶胀纤维素。In an embodiment of the present invention, the processing method of the substrate cellulose is as follows: the cellulose raw material is subjected to phosphoric acid expansion pretreatment to obtain phosphoric acid swollen cellulose.
在本发明的一种实施方式中，所述方法为：向pH5.0的缓冲液中添加经磷酸溶胀后的纤维素至浓度为200g/L，添加葡萄糖至浓度为400g/L，得到反应体系，向反应体系中添加50U/g纤维素滤纸酶活的纤维素酶和上述400U/g葡萄糖的β-葡萄糖苷酶突变体，在60℃条件下，反应96h。In one embodiment of the present invention, the method is as follows: adding cellulose swollen with phosphoric acid to a pH 5.0 buffer to a concentration of 200 g/L, and adding glucose to a concentration of 400 g/L to obtain a reaction system , add 50U/g cellulose filter paper enzymatically active cellulase and the above-mentioned 400U/g glucose β-glucosidase mutant into the reaction system, and react at 60°C for 96h.
在本发明的一种实施方式中，所述缓冲液为柠檬酸-磷酸盐缓冲液。In one embodiment of the present invention, the buffer is a citric acid-phosphate buffer.
本发明还提供了上述突变体或上述基因或上述表达载体或上述微生物细胞或上述制备方法在制备低聚龙胆糖中的应用。The present invention also provides the application of the above mutant or the above gene or the above expression vector or the above microbial cell or the above preparation method in the preparation of gentiosaccharide oligosaccharides.
有益效果beneficial effect
(1)本发明的复合酶可有效转化纤维素生成低聚龙胆糖；当以20％的磷酸膨胀纤维素(PASC)和40％的葡萄糖为底物酶催化转化制备低聚龙胆糖，复配纤维素酶和β-葡萄糖苷酶突变体Q34G、Q34M、Y200F或Q34G/Y200F的总转化率分别为17.6％、16.0％、16.7％和19.1％，相比野生型β-葡萄糖苷酶分别提高60.6％、46.1％、52.7％和74.7％。其中，转化率最高的复合酶(纤维素酶+β-葡萄糖苷酶突变体Q34G/Y200F)以纤维素和葡萄糖为原料制备低聚龙胆糖产量最高可达114.7g/L，纤维二糖转化率可达57.3％。(1) The compound enzyme of the present invention can effectively convert cellulose to generate gentiosaccharide oligosaccharides; when 20% of phosphoric acid swollen cellulose (PASC) and 40% of glucose are used as substrates, the enzyme-catalyzed conversion to prepare gentiosaccharide oligosaccharides, The overall conversion rates of compound cellulase and β-glucosidase mutants Q34G, Q34M, Y200F or Q34G/Y200F were 17.6%, 16.0%, 16.7% and 19.1%, respectively, compared with wild-type β-glucosidase, respectively. Improvements of 60.6%, 46.1%, 52.7% and 74.7%. Among them, the compound enzyme with the highest conversion rate (cellulase + β-glucosidase mutant Q34G/Y200F) uses cellulose and glucose as raw materials to prepare oligosaccharides with a yield of up to 114.7g/L, and cellobiose conversion The rate can reach 57.3%.
(2)本发明实现了以更广泛、廉价的纤维素底物来制备低聚龙胆糖，大大降低了工业上低聚龙胆糖的生产成本，因此，本发明的技术方案具有很高的工业应用价值。(2) The present invention realizes the preparation of gentiosaccharide oligosaccharides with wider and cheaper cellulose substrates, which greatly reduces the production cost of gentisaccharide oligosaccharides in industry. Therefore, the technical solution of the present invention has a high Industrial application value.
附图说明Description of drawings
图1：复合酶催化纤维素制备低聚龙胆糖示意图。Figure 1: Schematic diagram of the synthesis of oligosaccharides from cellulose catalyzed by complex enzymes.
具体实施方式detailed description
下述实施例中涉及的纤维素、葡萄糖购自上海维塔化学试剂有限公司；大肠杆菌JM109 感受态细胞购自上海生工，巴斯德毕赤酵母KM71购自Invitrogen公司；下述实施例中所涉及里氏木霉(Trichoderma reesei QM9414)购自于美国菌种保藏中心(保藏编号为ATCC26921)。Cellulose and glucose involved in the following examples were purchased from Shanghai Vita Chemical Reagent Co., Ltd.; Escherichia coli JM109 competent cells were purchased from Shanghai Sangon, and Pichia pastoris KM71 was purchased from Invitrogen Company; in the following examples The involved Trichoderma reesei (Trichoderma reesei QM9414) was purchased from the American Culture Collection (the deposit number is ATCC26921).
下述实施例中涉及的培养基如下：The media involved in the following examples are as follows:
LB液体培养基：酵母粉5g/L，胰蛋白胨10g/L，NaCl 10g/L。LB liquid medium: yeast powder 5g/L, tryptone 10g/L, NaCl 10g/L.
MD固体培养基：YNB 13.4g/L，生物素4×10 -4g/L，葡萄糖20g/L，琼脂20g/L。 MD solid medium: YNB 13.4g/L, biotin 4×10 -4 g/L, glucose 20g/L, agar 20g/L.
YPD液体培养基：蛋白胨20g/L，酵母提取物10g/L，葡萄糖20g/L。YPD liquid medium: peptone 20g/L, yeast extract 10g/L, glucose 20g/L.
YPD固体培养基：在YPD液体培养基基础上，添加20g/L琼脂。YPD solid medium: On the basis of YPD liquid medium, add 20g/L agar.
BMGY培养基：YNB 13.4g/L，甘油10g/L，生物素4×10 -4g/L，0.1mol/L磷酸钾缓冲溶液(pH 6.0)，蛋白胨20g/L，酵母粉10g/L。 BMGY medium: YNB 13.4g/L, glycerol 10g/L, biotin 4×10 -4 g/L, 0.1mol/L potassium phosphate buffer solution (pH 6.0), peptone 20g/L, yeast powder 10g/L.
BMMY培养基：YNB 13.4g/L，甲醇1％，生物素4×10 -4g/L，0.1mol/L磷酸钾缓冲溶液(pH 6.0)，蛋白胨20g/L，酵母粉10g/L。 BMMY medium: YNB 13.4g/L, methanol 1%, biotin 4×10 -4 g/L, 0.1mol/L potassium phosphate buffer solution (pH 6.0), peptone 20g/L, yeast powder 10g/L.
发酵种子培养基：酵母粉5.0g/L，胰蛋白胨10.0g/L，葡萄糖10.0g/L，甘油30g/L。Fermentation seed medium: yeast powder 5.0g/L, tryptone 10.0g/L, glucose 10.0g/L, glycerol 30g/L.
BSM培养基：85％磷酸26.7mL/L，CaSO 4 0.93g/L，K 2SO 4 18.2g/L，MgSO 4·7H 2O 14.9g/L，KOH 4.13g/L，甘油30.0g/L，微量元素盐溶液4.32mL/L。 BSM medium: 85% phosphoric acid 26.7 mL/L, CaSO 4 0.93 g/L, K 2 SO 4 18.2 g/L, MgSO 4 7H 2 O 14.9 g/L, KOH 4.13 g/L, glycerol 30.0 g/L , trace element salt solution 4.32mL/L.
下述实施例中涉及的检测方法如下：The detection methods involved in the following examples are as follows:
纤维素酶滤纸酶活测定：Cellulase filter paper enzyme activity assay:
用打孔器在Waterman滤纸上打出半径为0.5cm的圆形滤纸小片，取10片圆形滤纸小片放置于900μL的适当pH缓冲液中，然后放置于一定温度的水浴锅中预热5min，再加入100μL一定稀释倍数的酶液，准确计时3h后加入1500μL DNS终止反应，沸水浴5min后于540nm处测吸光值，空白对照组采用热处理失活的酶液以同样的反应体系进行测定。Use a hole puncher to punch out circular filter paper pieces with a radius of 0.5 cm on the Waterman filter paper, take 10 circular filter paper pieces and place them in 900 μL of appropriate pH buffer, and then place them in a water bath at a certain temperature to preheat for 5 minutes, and then place them in a water bath. Add 100 μL of enzyme solution with a certain dilution ratio, add 1500 μL of DNS after accurate timing for 3 h to terminate the reaction, and measure the absorbance at 540 nm after 5 min of boiling water bath.
酶活单位定义为：每分钟水解纤维素底物产生1μmol的还原糖的酶活力为一个滤纸酶活单位(FPU)。The enzymatic activity unit is defined as: the enzymatic activity of hydrolyzing cellulose substrate to produce 1 μmol of reducing sugar per minute is one filter paper enzymatic activity unit (FPU).
β-葡萄糖苷酶酶活的测定：Determination of β-glucosidase enzyme activity:
反应体系为1mL，pH 5.0的醋酸缓冲液960μL，加入适度稀释的粗酶液20μL，再加入20μL 100mmol/L的pNPG，在60℃恒温水浴中反应10min后，立即加入200μL的1mol/L的Na 2CO 3溶液终止反应，冰浴5min，于405nm处测光吸收值。以加热失活的酶液按照同样的方法处理作空白。 The reaction system was 1 mL, 960 μL of pH 5.0 acetate buffer, 20 μL of moderately diluted crude enzyme solution was added, and then 20 μL of 100 mmol/L pNPG was added. After reacting in a constant temperature water bath at 60 °C for 10 min, 200 μL of 1 mol/L Na was added immediately. The reaction was terminated with 2 CO 3 solution, ice bathed for 5 min, and the absorbance value was measured at 405 nm. The heat-inactivated enzyme solution was treated in the same way as blank.
β-葡萄糖苷酶酶活的定义为：每毫升酶液每分钟水解pNPG产生1μmol的对硝基苯酚的酶活力为一个酶活单位。The definition of β-glucosidase enzymatic activity is that the enzymatic activity of hydrolyzing pNPG to produce 1 μmol of p-nitrophenol per milliliter of enzyme solution per minute is one unit of enzymatic activity.
低聚龙胆糖含量的检测：Detection of gentis-oligosaccharide content:
采用HPLC检测低聚龙胆糖的含量；The content of gentiosaccharide oligosaccharide was detected by HPLC;
色谱条件如下：Agilent 1200HPLC色谱仪，Agilent自动进样器，RID示差检测器；流动相为75％乙腈，流速0.8mL/min；柱温35℃。The chromatographic conditions are as follows: Agilent 1200HPLC chromatograph, Agilent autosampler, RID differential detector; mobile phase is 75% acetonitrile, flow rate 0.8mL/min; column temperature 35°C.
实施例1：β-葡萄糖苷酶突变体的构建Example 1: Construction of β-glucosidase mutants
根据核苷酸序列如SEQ ID NO.2所示的β-葡萄糖苷酶的基因序列，设计并合成引入Q34G、Q34M、Y200F突变的引物，对β-葡萄糖苷酶基因进行定点突变，测定DNA编码序列，鉴别出第34位的Gln密码子变成Gly密码子，第34位的Gln密码子变成Met密码子，第200位的Tyr密码子变成Phe密码子。将突变体基因置于表达载体中，并将表达载体导入巴斯德毕赤酵母进行表达，得到单突变β-葡萄糖苷酶。利用快速PCR技术，以表达载体pPIC9K-TpBgl3A(记载于公开号为CN111500560A的中国专利申请文本中)为模板，构建单突变Q34G、Q34M、Y200F及组合突变Q34G/Y200F。According to the gene sequence of β-glucosidase whose nucleotide sequence is shown in SEQ ID NO.2, primers for introducing Q34G, Q34M, and Y200F mutations were designed and synthesized, and the β-glucosidase gene was site-directed mutation, and the DNA coding was determined. It was identified that the Gln codon at position 34 becomes a Gly codon, the Gln codon at position 34 becomes a Met codon, and the Tyr codon at position 200 becomes a Phe codon. The mutant gene is placed in an expression vector, and the expression vector is introduced into Pichia pastoris for expression to obtain a single mutant β-glucosidase. Using rapid PCR technology, single mutation Q34G, Q34M, Y200F and combined mutation Q34G/Y200F were constructed using expression vector pPIC9K-TpBgl3A (recorded in Chinese patent application with publication number CN111500560A) as a template.
引入Q34G突变的定点突变引物为：The site-directed mutagenesis primers for introducing the Q34G mutation are:
正向引物：5’-ACGGGAACAAGTTGG GGGAACGGTTCTTGTGTA-3’(SEQ ID NO.3) Forward primer: 5'-ACGGGAACAAGTTGG GGG AACGGTTCTTGTGTA-3' (SEQ ID NO. 3)
反向引物：5’-TACACAAGAACCGTT CCCCCAACTTGTTCCCGT-3’(SEQ ID NO.4) Reverse primer: 5'-TACACAAGAACCGTT CCC CCAACTTGTTCCCGT-3' (SEQ ID NO. 4)
引入Q34M突变的定点突变引物为：The site-directed mutagenesis primers for introducing the Q34M mutation are:
正向引物：5’-CAAGTTGG ATGAACGGTTCTTGTGTAGGCAACA-3’(SEQ ID NO.5) Forward primer: 5'-CAAGTTGG ATG AACGGTTCTTGTGTAGGCAACA-3' (SEQ ID NO. 5)
反向引物：5’-GAACCGTT CATCCAACTTGTTCCCGTCACGATG-3’(SEQ ID NO.6) Reverse primer: 5'-GAACCGTT CAT CCAACTTGTTCCCGTCACGATG-3' (SEQ ID NO. 6)
引入Y200F突变的定点突变引物为：The site-directed mutagenesis primers for introducing the Y200F mutation are:
正向引物：5’-TGTGCTCG TGGAATAAAGTGAATGGTACTTATA-3’(SEQ ID NO.7) Forward primer: 5'-TGTGCTCG TGG AATAAAGTGAATGGTACTTATA-3' (SEQ ID NO. 7)
反向引物：5’-ACTTTATT CCACGAGCACATCACGCTGGCGACG-3’(SEQ ID NO.8) Reverse primer: 5'-ACTTTATT CCA CGAGCACATCACGCTGGCGACG-3' (SEQ ID NO. 8)
PCR反应体系均为：5×PS buffer 10μL，dNTPs Mix(2.5mM)4μL，正向引物(10μM)1μL，反向引物(10μM)1μL，模板DNA 1μL，Primerstar HS(5U/μL)0.5μL，加入双蒸水至50μL。PCR reaction systems are: 5×PS buffer 10μL, dNTPs Mix (2.5mM) 4μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, template DNA 1μL, Primerstar HS (5U/μL) 0.5μL, Add double distilled water to 50 μL.
PCR扩增条件为：94℃预变性4min；随后30个循环(98℃10s，55℃15s，72℃10min)；72℃继续延伸10min。PCR amplification conditions were: pre-denaturation at 94°C for 4 min; followed by 30 cycles (98°C for 10s, 55°C for 15s, 72°C for 10min); and continued extension at 72°C for 10min.
PCR产物经DpnⅠ消化后，将产物转化进入到大肠杆菌JM109感受态细胞，得到转化产物，将转化产物在LB培养基(含100μg/mL氨苄青霉素)培养过夜后，挑取阳性克隆于LB液体培养基(含100μg/mL氨苄青霉素)中培养后，提取质粒，所有突变质粒均测序正确，将突变质粒转化表达宿主巴斯德毕赤酵母KM71感受态细胞，并将感受态细胞接种在MD固体培养基上进行培养后，挑取96个单菌落在新的MD固体培养基上等间距点种，30℃培育1～2d后，全部转接至装有1mL BMGY培养基的深孔板中培养48h，之后将菌体离心，弃上 清后用0.5mL BMMY培养基悬浮菌体，并加1％(v/v)的甲醇诱导2d后，离心菌体，获取上清，测酶活，挑选出酶活最高的转化子于-80℃条件下保存；即得到的重组菌，分别命名为KM71/pPIC9K-Q34G、KM71/pPIC9K-Q34M、KM71/pPIC9K-Y200F和KM71/pPIC9K-Q34G/Y200F。After the PCR product was digested by DpnI, the product was transformed into E. coli JM109 competent cells to obtain the transformed product. After the transformed product was cultured in LB medium (containing 100 μg/mL ampicillin) overnight, positive clones were picked and cultured in LB liquid. After culturing in the base (containing 100 μg/mL ampicillin), the plasmids were extracted, all mutant plasmids were sequenced correctly, the mutant plasmids were transformed into the expression host Pichia pastoris KM71 competent cells, and the competent cells were inoculated on MD solid culture After culturing on the substrate, 96 single colonies were picked and seeded at equal intervals on the new MD solid medium. After culturing at 30°C for 1-2 days, all were transferred to a deep-well plate containing 1 mL of BMGY medium for 48 hours. , then centrifuge the bacteria, discard the supernatant, suspend the bacteria with 0.5 mL of BMMY medium, and add 1% (v/v) methanol for induction for 2 days, then centrifuge the bacteria to obtain the supernatant, measure the enzyme activity, and pick out The transformants with the highest enzymatic activity were stored at -80°C; the resulting recombinant bacteria were named KM71/pPIC9K-Q34G, KM71/pPIC9K-Q34M, KM71/pPIC9K-Y200F and KM71/pPIC9K-Q34G/Y200F, respectively.
实施例2：野生型和突变体β-葡萄糖苷酶的表达及比酶活的测定Example 2: Expression of wild-type and mutant β-glucosidase and determination of specific enzyme activity
能够表达野生型β-葡萄糖苷酶的重组毕赤酵母菌株KM71/pPIC9K-TpBgl3A记载于公开号为CN111500560A的中国专利申请文本中。The recombinant Pichia strain KM71/pPIC9K-TpBgl3A capable of expressing wild-type β-glucosidase is described in the Chinese patent application with publication number CN111500560A.
从冰箱中取出重组菌株KM71/pPIC9K-TpBgl3A和实施例1得到的重组菌株KM71/pPIC9K-Q34G、KM71/pPIC9K-Q34M、KM71/pPIC9K-Y200F和KM71/pPIC9K-Q34G/Y200F的甘油管，分别从中吸取30μL菌液接种于10mL YPD液体培养基中，并在30℃摇床中振荡培养24～36h，得到培养液，分别吸取2.5mL培养液转接至50mL BMGY培养基中，30℃培养24h后，4000rpm离心10min后，过滤收集菌体，用25mL含1％(v/v)甲醇的BMMY培养基悬浮菌体，每24h补加一次0.25mL的甲醇，其中甲醇的终浓度为1％(v/v)，并在30℃条件下培养120h，得到发酵液；将所得发酵液10000rpm离心15min后，取上清，即为分别含有野生酶和突变体Q34G、Q34M、Y200F和Q34G/Y200F的粗酶液。Take out the glycerol tubes of the recombinant strain KM71/pPIC9K-TpBgl3A and the recombinant strains KM71/pPIC9K-Q34G, KM71/pPIC9K-Q34M, KM71/pPIC9K-Y200F and KM71/pPIC9K-Q34G/Y200F obtained in Example 1 from the refrigerator, respectively. Inoculate 30 μL of bacterial liquid into 10 mL of YPD liquid medium, and inoculate it in a shaker at 30 °C for 24-36 h to obtain a culture solution, transfer 2.5 mL of the culture solution to 50 mL of BMGY medium, and cultivate at 30 °C for 24 hours. After centrifugation at 4000 rpm for 10 min, the cells were collected by filtration, suspended in 25 mL of BMMY medium containing 1% (v/v) methanol, and 0.25 mL of methanol was added every 24 h, where the final concentration of methanol was 1% (v/v). /v), and cultured at 30 °C for 120 h to obtain a fermentation broth; after centrifuging the obtained fermentation broth at 10,000 rpm for 15 min, take the supernatant, which is the crude enzyme containing the wild enzyme and mutants Q34G, Q34M, Y200F and Q34G/Y200F respectively. enzyme solution.
分别测定上述粗酶液催化水解反应的比酶活，得到β-葡萄糖苷酶单突变体酶的水解反应比活力列于表1中，其中突变体Q34M的水解反应比活力略有增加，突变体Q34G的水解反应比活力与野生型基本持平，突变体Y200F和组合突变Q34G/Y200F的水解反应比活分别降低至野生型的2.6％和2.1％。酶水解活力的降低有利于转苷反应的发生和转苷产物的积累。The specific activity of the hydrolysis reaction catalyzed by the crude enzyme solution was measured respectively, and the specific activity of the hydrolysis reaction of the obtained β-glucosidase single mutant enzyme was listed in Table 1, wherein the specific activity of the hydrolysis reaction of the mutant Q34M increased slightly, and the mutant The specific activity of the hydrolysis reaction of Q34G was basically the same as that of the wild type, and the specific activity of the hydrolysis reaction of the mutant Y200F and the combined mutant Q34G/Y200F decreased to 2.6% and 2.1% of the wild type, respectively. The reduction of enzymatic hydrolysis activity is beneficial to the occurrence of transglycosidic reaction and the accumulation of transglycosidic products.
表1 粗酶液当中不同的β-葡萄糖苷酶的酶活Table 1 Enzymatic activities of different β-glucosidases in crude enzyme solution
Figure PCTCN2021101171-appb-000001
Figure PCTCN2021101171-appb-000001
实施例3：复合酶催化纤维素制备低聚龙胆糖Example 3: Compound Enzyme Catalyzes Cellulose to Prepare Gentiose Oligosaccharides
具体步骤如下：Specific steps are as follows:
以预处理的纤维素和葡萄糖为原料，多酶复合催化转换制备低聚龙胆糖(如图1所示)。Using pretreated cellulose and glucose as raw materials, multi-enzyme complex catalyzed conversion to prepare oligogentisose (as shown in Figure 1).
1、纤维素预处理条件如下：采用磷酸膨化预处理纤维素原料，获得磷酸溶胀纤维素(Phosphoric acid swollen cellulose,PASC)，配置方法参考Schiilein等(Reference：Schulein.M.Enzymatic properties of cellulases from Humicola insolens[J].Biotechnol,1997,57(1-3):71-81)，有适当调整。1. Cellulose pretreatment conditions are as follows: Phosphoric acid swollen cellulose (PASC) is obtained by pretreating cellulose raw materials with phosphoric acid expansion. For the configuration method, please refer to Schiilein et al. insolens [J]. Biotechnol, 1997, 57(1-3):71-81), with appropriate adjustments.
具体步骤如下：Specific steps are as follows:
(1)称取10g微晶纤维素Avicel，加入30mL去离子水，充分搅拌；缓慢加入500mL预冷的86.2％磷酸溶液，边加边搅拌；在冰浴条件下充分搅拌，直至溶液呈透明粘稠状，无颗粒；a Thick, without particles;
(2)将步骤(1)得到的溶液移至4℃条件下静置12h，使纤维素充分膨化；(2) moving the solution obtained in step (1) to stand at 4°C for 12 hours to fully expand the cellulose;
(3)向步骤(2)中得到的膨化后的纤维素中加入1L的冰水，边加边搅拌，出现白色絮状物，得到混合物；(3) adding 1 L of ice water to the puffed cellulose obtained in step (2), stirring while adding, white flocs appear, and a mixture is obtained;
(4)将步骤(3)得到的混合物于6000rpm离心20min，收集白色絮状沉淀，弃掉上清(大离心杯)；(4) centrifuge the mixture obtained in step (3) at 6000rpm for 20min, collect the white flocculent precipitate, discard the supernatant (large centrifuge cup);
(5)重复步骤(3)、(4)后；向得到的白色絮状沉淀中加入10mL 2M Na 2CO 3，充分搅拌，中和剩余的磷酸； (5) After repeating steps (3) and (4); add 10 mL of 2M Na 2 CO 3 to the obtained white flocculent precipitate, stir well, and neutralize the remaining phosphoric acid;
(6)向步骤(5)中加入1L的冰水，充分搅拌后于6000rpm离心20min，弃上清；(6) add 1L of ice water to step (5), after fully stirring, centrifuge at 6000rpm for 20min, discard the supernatant;
(7)重复步骤(6)，直至溶液的pH达到5-7之间，取沉淀烘干，该沉淀即为磷酸溶胀纤维素，将制备的磷酸溶胀纤维素PASC保存于4℃条件下，备用。(7) Repeat step (6) until the pH of the solution reaches between 5-7, take the precipitate and dry it, the precipitate is phosphoric acid swollen cellulose, and the prepared phosphoric acid swollen cellulose PASC is stored at 4°C for subsequent use. .
2、纤维素酶的制备2. Preparation of cellulase
纤维素酶为里氏木霉(Trichoderma reesei QM9414)发酵获得，具有内切纤维素酶和纤维二糖水解酶活性，其具体制备步骤如下：Cellulase is obtained from Trichoderma reesei (Trichoderma reesei QM9414) fermentation, has endo-cellulase and cellobiohydrolase activity, and its concrete preparation steps are as follows:
(1)将PDA斜面保存的里氏木霉孢子用无菌水洗下，得到孢子浓度为10 7cfu/mL的孢子悬液；其中，PDA培养基：土豆200g/L，葡萄糖20g/L，琼脂20g/L； (1) the Trichoderma reesei spores preserved on the PDA slant are washed with sterile water, and obtaining the spore concentration is 10 7 cfu/mL spore suspension; Wherein, PDA medium: potato 200g/L, glucose 20g/L, agar 20g/L;
(2)按照2％(v/v)的接种量将孢子悬液接种于装有200mL发酵培养基的1L摇瓶中，搅拌转速200r/min，pH 5.0，30℃培养6d，得到发酵液；其中，发酵培养基：乳糖18g/L，微晶纤维素10g/L，玉米桨粉12g/L，(NH4) 2SO 4 0.5g/L，MgSO 4 1g/L，CaCl 2 0.5g/L，Mandels微量元素营养盐1mL/L，吐温80 2mL/L，pH 4.8。 (2) according to the inoculation amount of 2% (v/v), inoculate the spore suspension in the 1L shake flask that is equipped with 200mL fermentation medium, stir at 200r/min, pH 5.0, and cultivate 6d at 30 DEG C to obtain fermentation broth; Among them, fermentation medium: lactose 18g/L, microcrystalline cellulose 10g/L, corn pulp powder 12g/L, (NH4) 2 SO 4 0.5g/L, MgSO 4 1g/L, CaCl 2 0.5g/L, Mandels trace element nutrients 1mL/L, Tween 80 2mL/L, pH 4.8.
(3)将步骤(2)得到的发酵液离心取上清，即为纤维素酶粗酶液，测定其滤纸酶活，酶活为456FPU/mL。(3) Centrifuge the fermentation broth obtained in step (2) to take the supernatant, which is the crude cellulase enzyme solution, and measure the enzyme activity of the filter paper, and the enzyme activity is 456 FPU/mL.
3、复合酶制备低聚龙胆糖3. Compound enzyme preparation of gentiosaccharide oligosaccharides
向50mM的pH 5.0柠檬酸-磷酸盐缓冲液中添加步骤1制备得到的磷酸溶胀纤维素(PASC)至浓度为200g/L，添加葡萄糖至浓度为400g/L，添加步骤2得到的纤维素酶粗酶液，浓度为50U/g纤维素滤纸酶活(FPU)；添加实施例2得到的β-葡萄糖苷酶原始酶和突变体的粗酶液，添加的浓度为400U/g葡萄糖，得到反应体系；将反应体系置于60℃，150rpm的水浴摇床中反应96h，每隔24h取一次样，并将样品12000rpm离心10min，取上清液适 度稀释后用0.45μm超滤膜过滤，并进行HPLC分析。To 50mM pH 5.0 citric acid-phosphate buffer, add the phosphoric acid swollen cellulose (PASC) prepared in step 1 to a concentration of 200g/L, add glucose to a concentration of 400g/L, add the cellulase obtained in step 2 Crude enzyme solution, concentration is 50U/g cellulose filter paper enzyme activity (FPU); Add the crude enzyme solution of β-glucosidase original enzyme and mutant obtained in Example 2, the added concentration is 400U/g glucose, obtains the reaction The reaction system was placed in a water bath shaker at 60 °C and 150 rpm for 96 h, samples were taken every 24 h, and the samples were centrifuged at 12,000 rpm for 10 min. HPLC analysis.
检测结果见表2，复合酶催化纤维素和葡萄糖制备低聚龙胆糖，分别采用β-葡萄糖苷酶突变体Q34G、β-葡萄糖苷酶突变体Q34M、β-葡萄糖苷酶突变体Y200F和β-葡萄糖苷酶突变体Q34G/Y200F时，总转化率分别为17.6％、16.0％、16.7％和19.1％，其总转化率相比野生型β-葡萄糖苷酶分别提高60.6％、46.1％、52.7％和74.7％。The test results are shown in Table 2. The compound enzymes catalyzed the preparation of oligosaccharides from cellulose and glucose. The β-glucosidase mutant Q34G, β-glucosidase mutant Q34M, β-glucosidase mutant Y200F and β-glucosidase mutant were used respectively. -glucosidase mutant Q34G/Y200F, the total conversion rates were 17.6%, 16.0%, 16.7% and 19.1%, respectively, and the total conversion rates were 60.6%, 46.1%, and 52.7% higher than those of wild-type β-glucosidase, respectively. % and 74.7%.
其中，转化率最高的复合酶(纤维素酶+β-葡萄糖苷酶突变体Q34G/Y200F)以纤维素和葡萄糖为原料制备低聚龙胆糖产量最高可达114.7g/L，纤维二糖转化率可达57.3％，具有一定的应用价值。Among them, the compound enzyme with the highest conversion rate (cellulase + β-glucosidase mutant Q34G/Y200F) uses cellulose and glucose as raw materials to prepare oligosaccharides with a yield of up to 114.7g/L, and cellobiose conversion The rate can reach 57.3%, which has certain application value.
表2 野生酶以及突变体生产低聚龙胆糖的产量Table 2 Yields of wild enzymes and mutants producing gentiosaccharide oligosaccharides
Figure PCTCN2021101171-appb-000002
Figure PCTCN2021101171-appb-000002
实施例4：β-葡萄糖苷酶的来源对低聚龙胆糖产率的影响Example 4: Influence of the source of β-glucosidase on the yield of gentiosaccharide oligosaccharides
具体实施方式同实施例3，区别在于，调整β-葡萄糖苷酶为：来源于绿色木霉的β-葡萄糖苷酶的野生酶(记载于CN107099565B中国发明专利文本中)；结果显示，其总转化率为：8.7％。The specific embodiment is the same as Example 3, except that the adjustment of β-glucosidase is: the wild enzyme of β-glucosidase derived from Trichoderma viride (recorded in CN107099565B Chinese invention patent text); the results show that the total conversion Rate: 8.7%.
实施例5：酶的复配比例对低聚龙胆糖产率的影响Example 5: Influence of the compounding ratio of enzymes on the yield of gentiosaccharide oligosaccharides
具体实施方式同实施例3，区别在于，调整纤维素酶和β-葡萄糖苷酶的复配比例。The specific embodiment is the same as Example 3, except that the compounding ratio of cellulase and β-glucosidase is adjusted.
当固定野生型β-葡萄糖苷酶添加量为400U/g葡萄糖时，分别调整纤维素酶添加量为10U/g纤维素滤纸酶活(FPU)、15U/g纤维素滤纸酶活(FPU)、25U/g纤维素滤纸酶活(FPU)，反应96h，总转化率分别为5.3％、5.9％、8.2％。When the fixed amount of wild-type β-glucosidase was 400U/g glucose, the amount of cellulase was adjusted to 10U/g cellulose filter paper enzyme activity (FPU), 15U/g cellulose filter paper enzyme activity (FPU), 25U/g cellulose filter paper enzymatic activity (FPU), the reaction was carried out for 96h, and the total conversion rates were 5.3%, 5.9% and 8.2%, respectively.
当固定纤维素酶添加量为50U/g纤维素滤纸酶活(FPU)时，分别调整野生型β-葡萄糖苷酶添加量为100U/g葡萄糖、200U/g葡萄糖、300U/g葡萄糖，反应96h，总转化率分别为3.7％、4.9％、9.2％。When the addition amount of fixed cellulase was 50U/g cellulose filter paper enzyme activity (FPU), the addition amount of wild-type β-glucosidase was adjusted to 100U/g glucose, 200U/g glucose, 300U/g glucose respectively, and the reaction was carried out for 96h. , and the total conversion rates were 3.7%, 4.9%, and 9.2%, respectively.
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以 权利要求书所界定的为准。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (15)

  1. 一种β-葡萄糖苷酶突变体，其特征在于，所述突变体是将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第34位和/或第200位突变得到的。A β-glucosidase mutant, characterized in that the mutant is obtained by mutating the 34th and/or 200th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1.
  2. 如权利要求1所述的一种β-葡萄糖苷酶突变体，其特征在于，所述β-葡萄糖苷酶突变体为：A β-glucosidase mutant according to claim 1, wherein the β-glucosidase mutant is:
    将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第34位由谷氨酰胺突变为甘氨酸得到的；The amino acid sequence is obtained by mutating glutamine into glycine at the 34th position of the β-glucosidase shown in SEQ ID NO.1;
    或将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第34位由谷氨酰胺突变为甲硫氨酸得到的；Or the 34th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is obtained by mutating glutamine into methionine;
    或将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第200位由酪氨酸突变为苯丙氨酸得到的；Or the 200th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is obtained by mutating tyrosine into phenylalanine;
    或将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第34位由谷氨酰胺突变为甘氨酸，同时将氨基酸序列如SEQ ID NO.1所示的β-葡萄糖苷酶的第200位由酪氨酸突变为苯丙氨酸得到的。Or the 34th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is mutated from glutamine to glycine, and the 34th position of the β-glucosidase whose amino acid sequence is shown in SEQ ID NO.1 is changed simultaneously. The 200th position is obtained by mutation of tyrosine to phenylalanine.
  3. 编码权利要求1或2所述β-葡萄糖苷酶突变体的基因。A gene encoding the β-glucosidase mutant of claim 1 or 2.
  4. 一种多酶复配制备低聚龙胆糖的方法，其特征在于，所述方法为将纤维素酶和权利要求1或2所述的β-葡萄糖苷酶突变体添加至含有纤维素和葡萄糖的反应体系中进行反应，得到反应液；从反应液中分离得到低聚龙胆糖。A method for compounding multi-enzymes for preparing gentiosaccharide oligosaccharides, characterized in that the method is to add cellulase and the β-glucosidase mutant described in claim 1 or 2 to a compound containing cellulose and glucose The reaction is carried out in the prepared reaction system to obtain a reaction solution; the oligogentisose are separated from the reaction solution.
  5. 如权利要求4所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，所述纤维素酶为内切纤维素酶、β-葡聚糖酶、I型纤维二糖水解酶、II型纤维二糖水解酶的一种或多种。The method for preparing oligosaccharides by compounding multiple enzymes according to claim 4, wherein the cellulase is endocellulase, beta-glucanase, type I cellobiose One or more of hydrolase, type II cellobiohydrolase.
  6. 如权利要求5所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，向反应体系中添加的纤维素酶的浓度为10-50U/g纤维素滤纸酶活。The method for preparing gentiosaccharide oligosaccharides by compounding multiple enzymes as claimed in claim 5, wherein the concentration of the cellulase added to the reaction system is 10-50U/g cellulose filter paper enzymatic activity.
  7. 如权利要求6所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，向反应体系中添加的纤维素酶的浓度为25-50U/g纤维素滤纸酶活。The method for preparing gentiosaccharide oligosaccharide by compounding multiple enzymes as claimed in claim 6, wherein the concentration of the cellulase added to the reaction system is 25-50U/g cellulose filter paper enzymatic activity.
  8. 如权利要求7所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，向反应体系中添加的β-葡萄糖苷酶突变体的浓度为100-400U/g葡萄糖。The method for preparing gentiosaccharide oligosaccharide by compounding multiple enzymes according to claim 7, wherein the concentration of the β-glucosidase mutant added to the reaction system is 100-400 U/g glucose.
  9. 如权利要求8所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，向反应体系中添加的β-葡萄糖苷酶突变体的浓度为300-400U/g葡萄糖。The method for preparing gentiosaccharide oligosaccharides by compounding multiple enzymes according to claim 8, wherein the concentration of the β-glucosidase mutant added to the reaction system is 300-400 U/g glucose.
  10. 如权利要求9所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，所述反应体系中，底物纤维素的浓度为200g/L。The method for preparing gentiosaccharide oligosaccharide by compounding multiple enzymes according to claim 9, characterized in that, in the reaction system, the concentration of the substrate cellulose is 200 g/L.
  11. 如权利要求10所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，底物葡萄糖的浓度为100g/L-400g/L。The method for preparing gentiosaccharide oligosaccharide by compounding multiple enzymes as claimed in claim 10, wherein the concentration of the substrate glucose is 100g/L-400g/L.
  12. 如权利要求11所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，所述底物纤维素的处理方式为：将纤维素原料采用磷酸膨化预处理获得磷酸溶胀纤维素。The method for preparing gentiosaccharide oligosaccharide by compounding multiple enzymes as claimed in claim 11, wherein the substrate cellulose is processed as follows: the cellulose raw material is subjected to phosphoric acid expansion pretreatment to obtain phosphoric acid swollen fibers White.
  13. 如权利要求12所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，所述反应体系中，反应温度为：60℃；反应时间为24-96h；反应pH为5.0。The method of claim 12, wherein in the reaction system, the reaction temperature is: 60°C; the reaction time is 24-96h; the reaction pH is 5.0 .
  14. 如权利要求13所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，所述方法为：向pH5.0的缓冲液中添加经磷酸溶胀后的纤维素至浓度为200g/L，添加葡萄糖至浓度为400g/L，得到反应体系，向反应体系中添加50U/g纤维素滤纸酶活的纤维素酶和上述400U/g葡萄糖的β-葡萄糖苷酶突变体，在60℃条件下，反应96h。A method for preparing gentiosaccharide oligosaccharide by compounding multiple enzymes as claimed in claim 13, wherein the method is: adding cellulose swollen by phosphoric acid to the buffer of pH 5.0 to a concentration of 200g/L, adding glucose to a concentration of 400g/L to obtain a reaction system, adding 50U/g cellulose filter paper enzymatically active cellulase and the β-glucosidase mutant of the above-mentioned 400U/g glucose in the reaction system, Under the condition of 60℃, the reaction was carried out for 96h.
  15. 如权利要求14所述的一种多酶复配制备低聚龙胆糖的方法，其特征在于，所述缓冲液为柠檬酸-磷酸盐缓冲液。The method for preparing gentiosaccharide oligosaccharides by compounding multiple enzymes according to claim 14, wherein the buffer is a citric acid-phosphate buffer.
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