WO2021217902A1 - 一种噬热β-葡萄糖苷酶的制备及应用 - Google Patents

一种噬热β-葡萄糖苷酶的制备及应用 Download PDF

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WO2021217902A1
WO2021217902A1 PCT/CN2020/102671 CN2020102671W WO2021217902A1 WO 2021217902 A1 WO2021217902 A1 WO 2021217902A1 CN 2020102671 W CN2020102671 W CN 2020102671W WO 2021217902 A1 WO2021217902 A1 WO 2021217902A1
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glucosidase
recombinant
glucose
enzyme
preparation
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吴敬
夏伟
徐星豪
黄燕
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江南大学
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    • 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
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • 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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    • 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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    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention relates to the preparation and application of a thermophagocytic ⁇ -glucosidase, and belongs to the technical field of genetic engineering and fermentation engineering.
  • Gentio-oligosaccharides are new functional oligosaccharides formed by the combination of glucose with ⁇ -1,6 glycosidic bonds, including gentiobiose, a small amount of trisaccharides and tetrasaccharides.
  • Gentian oligosaccharides are not degraded by human enzymes, low in calories, suitable for people with obesity, hyperlipidemia, high blood pressure, diabetes, etc.; it also has high moisture retention and hygroscopicity, which is good for keeping the water in foods.
  • the oligo-gentianose Prevents the aging of starchy foods; at the same time, the oligo-gentianose has high pH and thermal stability, which is suitable for food used under high temperature and special pH conditions; its water activity is low, which can effectively prevent food from being contaminated by microorganisms; it also has a soft It refreshes the bitterness and can be added to foods to increase the richness of the taste.
  • gentian oligosaccharides are widely used in chocolate, ice cream, coffee, condiments, baked goods and beverages.
  • Gentian oligosaccharides were first extracted from the roots of Gentian plants and obtained by acid hydrolysis of starch. These two methods have extremely low yields and the product components are complex and difficult to separate and extract. The chemical synthesis yields high but it is easy to cause pollution, so Enzymatic production has become a trend at present, and the current research is mainly focused on the use of ⁇ -glucosidase to use glucose as a substrate to polymerize products through transglycosylation. ⁇ -glucosidase is distributed in glycoside hydrolysase (GH) Among the 6 families of 1, 3, 5, 9, 30 and 116, they are widely found in plants, animals and microorganisms. Among them, the sources of microorganisms are more extensive.
  • GH glycoside hydrolysase
  • the currently reported strains produce ⁇ -glucosidase for the production of oligomers.
  • There are three main problems with gentianose one is the low yield of oligogentianose, the other is the high amount of enzyme added, and the third is the insufficient amount of enzyme produced in high-density fermentation.
  • the purpose of the present invention is not only to increase the conversion rate of oligogentianose and reduce the amount of enzyme added for production, but also to construct a genetically engineered bacteria capable of efficiently expressing recombinase in high-density fermentation.
  • ⁇ -glucosidase can hydrolyze and release the non-reducing glucosyl group of ⁇ -D-glycoside compounds. It is an important member of the cellulose degrading enzyme system. It can prevent the accumulation of cellobiose during the process of cellulose degradation, thereby effectively removing cellobiose. Strong product inhibition of upstream endo-cellulase and exo-cellulase.
  • the present invention provides a sequence of a gene encoding a mature protein of ⁇ -glucosidase TpBgl3A.
  • the nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of the mature protein of ⁇ -glucosidase TpBgl3A is shown in SEQ. ID NO.2.
  • the present invention provides a vector which carries a gene encoding ⁇ -glucosidase TpBgl3A or expresses ⁇ -glucosidase TpBgl3A.
  • the starting vector of the vector is pPIC9K.
  • the present invention provides a recombinant strain KM71/pPIC9K-TpBgl3A, which uses Pichia pastoris KM71 as a host and expresses the ⁇ -glucosidase TpBgl3A whose amino acid sequence is shown in SEQ ID NO.2.
  • the present invention provides a method for improving the yield of ⁇ -glucosidase, which is obtained by high-density fermentation of the recombinant bacteria KM71/pPIC9K-TpBgl3A.
  • the recombinant bacterium KM71/pPIC9K-TpBgl3A is inoculated into the fermentor at an inoculum of 8% to 12%, and the temperature is controlled to be 28 to 30°C, the initial rotation speed is 180 to 220 rpm, and the initial ventilation rate is 5 ⁇ 8L/min, dissolved oxygen 28 ⁇ 32%, pH 4.5 ⁇ 5.5; when the dissolved oxygen rises to 80 ⁇ 100%, feed culture with constant flow and glycerol; when the concentration of recombinant bacterial cells is at OD 600 When the temperature is between 100 and 200, use a methanol flow feeder to add methanol to induce enzyme production, and control the temperature at 15-25°C.
  • the concentration of the glycerol is 80% (v/v), and the glycerol flow acceleration rate is 15-25 mL/h.
  • the OD 600 of the initial induced bacteria is 100-200
  • the methanol concentration is 0.5%-2.0% (v/v)
  • the induction temperature is 20°C-35°C.
  • the high-density fermentation conditions when the concentration of bacterial cells in the recombinant strain OD 600 of 130 to 170, added at a concentration of 0.8 to 1.5% of enzyme production induced by methanol, the temperature control 22°C ⁇ 28°C.
  • the present invention provides a method for increasing the yield of oligogentianose, which uses 700-900 g/L glucose as a substrate and the ⁇ -glucosidase as a catalyst for reaction.
  • the ⁇ -glucosidase added enzyme amount is 200-600 U/g glucose
  • the enzyme reaction is carried out at pH 4.5-5.5, temperature 50-70°C, and reaction time is 65- 75h.
  • the ⁇ -glucosidase plus enzyme amount is 250-400 U/g glucose
  • the enzyme reaction is carried out at pH 4.8-5.2, temperature 55-65°C
  • reaction time is 70- 75h.
  • the present invention protects the application of the gene encoding the ⁇ -glucosidase TpBgl3A in the preparation of oligogentiose in the field of food and cosmetics.
  • the invention protects the application of the recombinant bacterium KM71/pPIC9K-TpBgl3A in the preparation of oligogentiose in the field of food and cosmetics.
  • the invention protects the application of the carrier in the preparation of oligogentiose in the fields of food and cosmetics.
  • the present invention protects the application of the method for improving the production of ⁇ -glucosidase in the preparation of oligogentiose in the field of food and cosmetics.
  • the present invention also protects the application of the method for improving the yield of oligogentianose in the preparation of oligogentianose in the food and cosmetic fields.
  • the beneficial effects of the present invention expresses the ⁇ -glucosidase derived from Talaromyces piceae heterologously into Pichia pastoris, using pPIC9K as the expression vector and Pichia pastoris KM71 as the expression host to obtain a strain with high yield of ⁇ -glucose
  • the genetically engineered bacteria KM71/pPIC9K-TpBgl3A of sidase, the genetically engineered bacteria KM71/pPIC9K-TpBgl3A can produce 2324U/mL in a 3.6L fermenter.
  • the ⁇ -glucosidase TpBgl3A produced is lower than the similar enzymes in the existing production, and the amount of enzyme added is lower, and the substrate conversion rate and the yield of oligogentianose are higher.
  • the amount of enzyme added is 300 U/g
  • the oligomeric dragon The highest bile sugar output can reach 125.0g/L
  • the conversion rate is 15.62%, and it has good industrial value.
  • Figure 1 is a diagram showing the relationship between the initial induced cell concentration and the fermentation enzyme activity of the recombinant engineered strain KM71-pPIC9K-TpBgl3A.
  • Figure 2 is a graph showing the relationship between the induced methanol concentration and the fermentation enzyme activity of the recombinant engineered strain KM71-pPIC9K-TpBgl3A.
  • Figure 3 is a graph showing the relationship between the induction culture temperature and the fermentation enzyme activity of the recombinant engineered strain KM71-pPIC9K-TpBgl3A.
  • Figure 4 is an SDS-PAGE electrophoresis diagram of the fermentation supernatant of recombinant bacteria in a 3.6L fermentor; 24, 48, 72, 84, 96, 108, 120, 132, and 144 in the figure indicate that the fermentation time is 24h, 48h, 72h, 84h, respectively , 96h, 108h, 120h, 132h, 144h.
  • Figure 5 shows the optimum temperature for ⁇ -glucosidase.
  • Figure 6 shows the optimal pH value of ⁇ -glucosidase.
  • Figure 7 is a graph showing the relationship between the amount of ⁇ -glucosidase added to the enzyme and the conversion rate of oligogentiose prepared with glucose as a substrate.
  • Figure 8 is a graph showing the relationship between the concentration of the substrate and the conversion rate of gentiooligosaccharide prepared with glucose and cellobiose as the substrate.
  • MD solid medium YNB 13.4g/L, biotin 4.0 ⁇ 10 -4 g/L, glucose 20g/L, agar 20g/L.
  • YPD medium peptone 20g/L, yeast extract 10g/L, glucose 20g/L, solid medium with 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
  • Fermentation seed medium yeast powder 5.0g/L, tryptone 10.0g/L, glucose 10.0g/L, glycerol 30g/L.
  • BSM medium 85% phosphoric acid 26.7mL/L, CaSO 4 0.93g/L, K 2 SO 4 18.2g/L, MgSO 4 ⁇ 7H 2 O 14.9g/L, KOH 4.13g/L, glycerol 30.0g/L , Trace element salt solution 4.32mL/L.
  • Growth phase feed medium 80% glycerol, 4.92mL/L trace element solution.
  • Induction phase feed medium 100% methanol, 12.5mL/L trace element solution; high-density fermentation uses 100% ammonia and 50% phosphoric acid to adjust the pH.
  • the enzyme activity of hydrolyzing pNPG to produce 1 ⁇ mol of p-nitrophenol per milliliter of enzyme solution per minute is one unit of enzyme activity.
  • the reaction system is 1mL, pH 5.0 acetate buffer solution 960 ⁇ L, add 20 ⁇ L of moderately diluted crude enzyme solution, then add 20 ⁇ L of 100mmol/L pNPG, react in a constant temperature water bath at 60°C for 10min, add 200 ⁇ L of 1mol/L immediately after 10min
  • the reaction was terminated by Na 2 CO 3 solution, ice bath for 5 min, and the absorbance value was measured at 405 nm.
  • the enzyme solution inactivated by heating is treated in the same way as a blank.
  • the recovery kit was purchased from Tiangen Biochemical Technology Co., Ltd.
  • Example 1 Construction of Pichia pastoris KM71/pPIC9K-TpBgl3A genetically engineered strain
  • the coding sequence of ⁇ -glucosidase TpBgl3A mature protein was chemically synthesized (the nucleotide sequence is as SEQ ID Shown in NO.1).
  • the specific method is: respectively use the coding sequence of TpBgl3A and the vector pPIC9K with EcoR I and Not I enzymes After cutting and recovering, ligate with T4 ligase to obtain the ligation product, and transfer the ligation product into E.coli.JM109 to obtain the transformation product; spread the transformation product on LB solid medium (containing 0.05mg/mL Kanamycin Inverted culture in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants.
  • LB solid medium containing 0.05mg/mL Kanamycin Inverted culture in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants.
  • the recombinant plasmid pPIC9K-TpBgl3A was electrotransformed and integrated into Pichia pastoris KM71.
  • Pichia pastoris KM71 competent The preparation and transformation steps of Pichia pastoris KM71 competent are as follows:
  • the supernatant was tested for ⁇ -glucosidase activity, and the recombinant transformant with the highest enzyme activity was selected.
  • Example 2 Enzyme production by recombinant Pichia pastoris engineering strain Pichia pastoris/KM71-pPIC9K-TpBgl3A
  • the engineered bacteria are fermented in a 3.6L fermentor
  • Feeding fermentation stage When the dissolved oxygen rises to 80-100%, feed the culture by adding glycerin at a constant flow rate, controlling the temperature at 28-30°C, dissolved oxygen at 28-32%, and pH 4.5-5.5 ;
  • the glycerol concentration is 80% (v/v), and the glycerol flow acceleration rate is 17.5-18.5mL/h;
  • the initial induced cell OD 600 in the above step (3) 100, 150, 200, the methanol concentration is 0.5%, 1.0%, 1.5%, and the induction temperature is 20, 25 , 30°C, explore the influence of different fermentation conditions on the expression of recombinant enzyme.
  • the results showed that the optimal induction culture temperature was 25°C, and the highest enzyme activity of recombinant bacteria producing ⁇ -glucosidase reached 2324U/mL ( Figure 3).
  • the ⁇ -glucosidase enzyme solution obtained by the above fermentation was qualitatively enzymatically, and the enzyme activity was measured at different temperatures with pNPG as the substrate. The results showed that the relative enzyme of ⁇ -glucosidase at 50°C and 65°C The activity was 57.04% and 50.08%, respectively, and the optimum temperature was 60°C ( Figure 5).
  • the enzyme activity of ⁇ -glucosidase was measured with different pH gradients under the optimal temperature of 60°C. The results showed that the relative enzyme activity of ⁇ -glucosidase at pH 4.5, 5.5, and 6.0 were 75.49% and 84.51%, respectively. , 70.22%, and the optimum pH is 5.0 ( Figure 6).
  • Example 3 Application of ⁇ -glucosidase in the preparation of gentio-oligosaccharides using glucose as a substrate
  • the reaction system for preparing oligogentianose with glucose as the substrate 800g/L glucose as the substrate, react at pH 5.0 and 60°C for 72 hours, and set different ⁇ -glucosidase and enzyme amounts (200-600U/g) The effect of glucose) on the enzyme reaction.
  • the experimental results are shown in Figure 7.
  • the cumulative content of oligogentianose increases with the increase in the amount of enzyme added within a certain range, but when the amount of enzyme exceeds 300 U/g glucose, the increase Instead, the amount of enzymes reduces the production of oligogentianose. Therefore, it is most suitable to choose 300U/g glucose with enzyme added. At this time, the output of oligogentianose can reach 125.0g/L, and the conversion rate is 15.62%.
  • the specific implementation mode is the same as in Example 4, the difference is that the ⁇ -glucosidase is replaced with the ⁇ -glucosidase derived from Trichoderma viride, the added amount of the enzyme is set to 300 U/g glucose, and the gentio-oligosaccharide is determined. The yield and the conversion rate of the product are 60g/L and 7.5% respectively.
  • the optimal conditions for the enzyme conversion of ⁇ -glucosidase in Trichoderma viride pH 5.0, 60°C, the amount of enzyme added is 900 U/g glucose, and the substrate glucose concentration is 800 g/L, which is low.
  • the yield of polygentianose can reach the maximum value of 130g/L, the conversion rate is 16.25%, and the high-density fermentation level is 1402U/mL.
  • Example 4 Application of ⁇ -glucosidase in the preparation of gentio-oligosaccharides using glucose and cellobiose as substrates
  • the reaction system for preparing oligogentiose with glucose and cellobiose as substrates maintain the molar concentration ratio of glucose to cellobiose at 1:2 (mass concentration ratio is 1:4), 50-150g/L glucose and 200 -600g/L cellobiose as the substrate, react for 48h under the conditions of 400U/g cellobiose with enzyme added, pH 5.0 and 60°C.
  • the results showed that the optimal substrate concentration was 75g/L glucose and 300g/L cellobiose, and the highest conversion rate of oligogentianose was 26.2% (Figure 8).
  • the specific embodiment is the same as in Example 4, the difference is that the ⁇ -glucosidase is replaced with the ⁇ -glucosidase derived from Trichoderma viride, under the optimal conditions for enzyme conversion: pH 5.0, 60°C, substrate concentration When it is 20% glucose and 40% cellobiose, the amount of ⁇ -glucosidase added to the cellobiose is 400 U/g cellobiose, and the conversion rate of oligogentianose is 19.4%.

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Abstract

一种噬热β-葡萄糖苷酶的制备及应用，属于基因工程和发酵工程技术领域。通过构建重组菌，异源表达了来源于嗜热真菌Talaromyces piceae的β-葡萄糖苷酶TpBgl3A，所述重组菌在3.6L发酵罐中产酶可达2324U/mL。生产得到的β-葡萄糖苷酶TpBgl3A能在较低加酶量下利用葡萄糖为底物高转化率制备低聚龙胆糖，显著降低生产成本；在以葡萄糖和纤维二糖为底物的反应体系中，低聚龙胆糖转化率达到26.2％，具有工业化生产低聚龙胆糖的潜力。

Description

一种噬热β-葡萄糖苷酶的制备及应用 技术领域
本发明涉及一种噬热β-葡萄糖苷酶的制备及应用，属于基因工程和发酵工程技术领域。
背景技术
低聚龙胆糖是葡萄糖以β-1,6糖苷键结合而形成的新型功能性低聚糖，包括龙胆二糖，少量的三糖和四糖。低聚龙胆糖不被人体的酶所降解，低热量，适合肥胖症、高血脂、高血压、糖尿病等人群食用；其还具有高保湿性和吸湿性，有利于保持食品中的水分，可防止淀粉类食品的老化；同时低聚龙胆糖pH和热稳定性高，适用于高温和特殊pH条件下使用的食品；其水活性低，可以有效的防止食品被微生物污染；还具有柔和的提神苦味，可以添加在食品中增加口味的丰富性。目前，低聚龙胆糖被广泛的运用于巧克力、冰淇凌、咖啡、调味品、烘烤食品和饮料中。
龙胆低聚糖最早从龙胆属植物根茎中提取、酸法水解淀粉得来，这两种方法产率极低且产物成分复杂不易分离提取，化学合成得率高但又易造成污染，所以酶法生产在目前已成趋势，目前研究主要集中在利用β-葡萄糖苷酶以葡萄糖为底物通过转糖基作用聚合形成产物，β-葡萄糖苷酶分布于糖苷水解酶(Glycoside hydrolysase,GH)1、3、5、9、30和116等6个家族中，广泛存在于植物、动物和微生物中，其中微生物来源更为广泛，目前报道的这些菌株产β-葡萄糖苷酶用于生产低聚龙胆糖主要存在三个问题，一是低聚龙胆糖产率低，二是加酶量偏高，三是高密度发酵中的产酶量不足。
因此，本发明的目的不仅在于提高低聚龙胆糖的转化率和降低生产所需的加酶量，还要构建一种能够在高密度发酵中高效表达重组酶的基因工程菌。
发明内容
β-葡萄糖苷酶可水解释放β-D-糖苷类化合物非还原端葡萄糖基，是纤维素降解酶系的重要成员，可防止纤维素降解过程中纤维二糖的积累，从而有效解除纤维二糖对上游内切纤维素酶和外切纤维素酶的强烈产物抑制。
本发明提供一种β-葡萄糖苷酶TpBgl3A成熟蛋白的编码基因的序列，所述基因的核苷酸序列如SEQ ID NO.1所示，所述β-葡萄糖苷酶TpBgl3A成熟蛋白氨基酸序列如SEQ ID NO.2所示。
本发明提供了一种载体，所述载体携带编码β-葡萄糖苷酶TpBgl3A的基因，或表达β-葡萄糖苷酶TpBgl3A。
在本发明的一种实施方式中，所述载体的出发载体为pPIC9K。
本发明提供了一种重组菌KM71/pPIC9K-TpBgl3A，所述重组菌是以毕赤酵母KM71为宿主，表达氨基酸序列如SEQ ID NO.2所示的β-葡萄糖苷酶TpBgl3A。
本发明提供了一种提高β-葡萄糖苷酶产量的方法，所述方法是将所述重组菌KM71/pPIC9K-TpBgl3A进行高密度发酵得到。
在本发明的一种实施方式中，将重组菌KM71/pPIC9K-TpBgl3A，以8％～12％接种量接种于发酵罐中，控制温度28～30℃、初始转速180～220rpm、初始通气量5～8L/min、溶氧28～32％、pH 4.5～5.5；待溶氧上升至80～100％，以恒速流加甘油的方式进行补料培养；当重组菌菌体细胞浓度在OD 600为100～200时，用甲醇流加仪流加甲醇诱导产酶，控制温度15～25℃。
在本发明的一种实施方式中，所述甘油的浓度为80％(v/v)，甘油流加速率为15～25mL/h。
在本发明的一种实施方式中，初始诱导菌体的OD 600为100～200，甲醇浓度为0.5％～2.0％(v/v)，诱导温度为20℃～35℃。
在本发明的一种实施方式中，所述高密度发酵条件为：当重组菌菌体细胞浓度在OD 600为130～170时，添加浓度为0.8％～1.5％的甲醇诱导产酶，控制温度22℃～28℃。
本发明提供了一种提高低聚龙胆糖产量的方法，所述方法是以700～900g/L葡萄糖为底物，以所述β-葡萄糖苷酶为催化剂进行反应。
在本发明的一种实施方式中，所述β-葡萄糖苷酶加酶量为200～600U/g葡萄糖，在pH4.5～5.5、温度50～70℃下进行酶反应，反应时间为65～75h。
在本发明的一种实施方式中，所述β-葡萄糖苷酶加酶量为250～400U/g葡萄糖，在pH4.8～5.2、温度55～65℃下进行酶反应，反应时间为70～75h。
本发明保护所述编码β-葡萄糖苷酶TpBgl3A的基因在食品和化妆品领域制备低聚龙胆糖中的应用。
本发明保护所述重组菌KM71/pPIC9K-TpBgl3A在食品和化妆品领域制备低聚龙胆糖中的应用。
本发明保护所述载体在食品和化妆品领域制备低聚龙胆糖中的应用。
本发明保护所述提高β-葡萄糖苷酶产量的方法在食品和化妆品领域制备低聚龙胆糖中的应用。
本发明还保护所述提高低聚龙胆糖产量的方法在食品和化妆品领域制备低聚龙胆糖中的应用。
本发明的有益效果：本发明将来源于Talaromyces piceae的β-葡萄糖苷酶异源表达到毕 赤酵母中，以pPIC9K为表达载体，以毕赤酵母KM71为表达宿主，得到一株高产β-葡萄糖苷酶的基因工程菌KM71/pPIC9K-TpBgl3A，将基因工程菌KM71/pPIC9K-TpBgl3A在3.6L发酵罐中产酶可达2324U/mL。生产得到的β-葡萄糖苷酶TpBgl3A较现有生产中的同类酶，加酶量低且底物转化率和低聚龙胆糖产量高，在加酶量为300U/g条件下，低聚龙胆糖产量最高能达到125.0g/L，转化率为15.62％，具有良好的工业价值。
附图说明
图1是初始诱导菌体浓度与重组工程菌KM71-pPIC9K-TpBgl3A发酵酶活关系图。
图2是甲醇诱导浓度与重组工程菌KM71-pPIC9K-TpBgl3A发酵酶活关系图。
图3是诱导培养温度与重组工程菌KM71-pPIC9K-TpBgl3A发酵酶活关系图。
图4是重组菌3.6L发酵罐发酵上清SDS-PAGE电泳图；图中的24、48、72、84、96、108、120、132、144分别表示发酵时间为24h、48h、72h、84h、96h、108h、120h、132h、144h。
图5是β-葡萄糖苷酶最适温度。
图6是β-葡萄糖苷酶最适pH值。
图7是β-葡萄糖苷酶加酶量与以葡萄糖为底物制备低聚龙胆糖转化率关系图。
图8是底物浓度与以葡萄糖和纤维二糖为底物制备低聚龙胆糖转化率关系图。
具体实施方式
实施例中所需培养基：
MD固体培养基：YNB 13.4g/L，生物素4.0×10 -4g/L，葡萄糖20g/L，琼脂20g/L。
YPD培养基：蛋白胨20g/L，酵母提取物10g/L，葡萄糖20g/L，固体培养基添加20g/L琼脂
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(pH6.0)，蛋白胨20g/L，酵母粉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。
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。
生长阶段补料培养基：80％甘油，4.92mL/L微量元素液。
诱导阶段补料培养基：100％甲醇，12.5mL/L微量元素液；高密度发酵用100％氨水和50％磷酸来调节pH。
β-葡萄糖苷酶酶活力分析：
(1)酶活单位定义
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。
(2)酶活力测定步骤
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，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。
回收试剂盒购自天根生化科技有限公司。
实施例1：毕赤酵母KM71/pPIC9K-TpBgl3A基因工程菌的构建
根据数据库中来源于Talaromyces piceae的β-葡萄糖苷酶氨基酸序列(NCBI登录号ATQ35964)，化学合成β-葡萄糖苷酶TpBgl3A成熟蛋白(去除信号肽序列)的编码序列(将核苷酸序列如SEQ ID NO.1所示)。
然后将TpBgl3A的编码序列插入酵母表达载体pPIC9K的α-factor信号肽(含起始密码子atg)下游进行融合表达，具体方法为：分别将TpBgl3A的编码序列和载体pPIC9K用EcoR I和Not I酶切回收后，用T4连接酶酶连，得到连接产物，将连接产物转入E.coli.JM109中，得到转化产物；将转化产物涂布在LB固体培养基(含有0.05mg/mL卡那霉素)上，于37℃恒温培养箱中倒置培养8～12h，得到转化子。
热激转化法：
(1)将E.coli.JM109感受态细胞提前放在冰上5min中，待感受态完全融化后，向其中加入10μL完整质粒或者PCR产物，轻柔吹吸均匀后，置于冰上放置45min；
(2)将感受态放置到42℃水浴锅中热激90s，热激结束后，放置在冰上5min；
(3)冰浴结束后，向感受态中加入0.8mL LB液体培养基，混匀后放到37℃摇床中震荡培养60min左右；
(4)培养结束的感受态3000rpm离心5min，弃部分上清液，留200μL左右的发酵液将菌体重新吹吸重悬，涂布到含氨苄抗生素的LB固体平板上，37℃培养箱静置培养10h左右，等待平板长出单菌落。
挑取单克隆菌落接种至含0.05mg/mL卡那霉素抗性LB液体培养基中，于37℃、120～180rpm的条件下摇瓶培养8～12h后提取质粒进行酶切验证以及测序验证，验证正确即获得重组质粒pPIC9K-TpBgl3A。
将重组质粒pPIC9K-TpBgl3A电转整合到毕赤酵母KM71。
毕赤酵母KM71感受态制备与转化步骤如下：
(1)从毕赤酵母KM71甘油管中吸取30μL菌液接种于10mL液体YPD培养基，在30℃摇床中200rpm培养24h，然后从中吸取100μL接种于100mL液体YPD培养基30℃恒温振荡培养16h；
(2)在无菌环境中，将菌液分装至三管事先预冷的50mL离心管中，4℃条件下5000rpm离心5min后弃上清收集菌体；
(3)在无菌环境中，用4mL ddH 2O重悬菌体，之后将其合并为一管，并相继加入2mL的TE缓冲液、2mL的LiAc缓冲液和0.5mL的DTT缓冲液，轻轻吹吸混匀，将离心管置于30℃水浴摇床中50rpm培养45～50min，以上试剂都要预冷处理；
(4)在无菌环境中，向离心管加入事先预冷的13.5mL ddH 2O，4℃条件下5000rpm离心5min后弃上清收集菌体；
(5)在无菌环境中，用25mL ddH 2O重悬菌体，4℃条件下5000rpm离心5min后弃上清收集菌体，之后换用25mL，1M山梨醇重复完成重悬、收集菌体操作两次；
(6)向离心管中加入1mL预冷的山梨醇并轻轻重悬菌体，按照每管80μL分装至1.5mL的EP管中并暂时存放在-80℃低温冰箱中。
(7)取10μL线性化质粒加至毕赤酵母KM71感受态中并轻敲管壁混匀，之后将感受态沿壁加入事先预冷的电极杯中，轻敲电击杯底部以排除气泡；
(8)将电击杯放入电压为1500V的电转仪进行电击；
(9)立即取出电击杯并迅速加入事先预冷的1mL，1M山梨醇轻轻吹吸混匀，随后将菌液转进EP管中，于30℃摇床孵育1～2h；
(10)从EP管中吸取100μL菌液均匀涂布于MD固体培养基，30℃培养48h。
转化子筛选：待上述MD平板长出单克隆，然后用灭菌的牙签挑取96个转化子按照编号同时转移至带有编号的装有4mL BMGY培养基的10mL规格培养管和新的MD平板上。MD平板倒置于30℃恒温培养箱培养；培养管30℃220rpm振荡培养2d后4,500rpm离心5min，弃上清，用2mL BMGY培养基重悬菌体，30℃220rpm诱导培养2d后离心取上清为粗酶液。
对上清液进行β-葡萄糖苷酶的活性检测，筛选出酶活最高的重组转化子。从相应的MD平板上挑取对应编号的转化子菌斑，接种于YPD培养基中30℃220rpm活化培养2d，保存为重组菌株毕赤酵母/KM71-pPIC9K-TpBgl3A。
实施例2：重组毕赤酵母工程菌毕赤酵母/KM71-pPIC9K-TpBgl3A发酵产酶
1、工程菌在3.6L发酵罐发酵
(1)分批发酵阶段：将种子液(重组菌株于YPD液体培养基中30℃培养36h获得)以8％-12％接种量接种于发酵罐中，控制温度28-30℃、初始转速180-220rpm、初始通气量7L/min、溶氧28-32％、pH 4.5-5.5；
(2)补料发酵阶段：待溶氧上升至80-100％，以恒速流加甘油的方式进行补料培养，控制温度28-30℃、溶氧28-32％、pH4.5-5.5；甘油的浓度为80％(v/v)，甘油流加速率为17.5-18.5mL/h；
(3)诱导培养阶段：当菌体细胞浓度在OD 600＝100-200范围内，用甲醇流加仪流加甲醇诱导产酶，甲醇浓度控制在0.5-1.5％，控制温度20-30℃、溶氧28-32％、pH 4.5-5.5，诱导96-144h，结束发酵。将发酵液离心取上清液，得到粗酶液。
2、发酵条件优化
为了提高β-葡萄糖苷酶的发酵水平，将上述步骤(3)中的初始诱导菌体OD 600＝100、150、200，甲醇浓度为0.5％、1.0％、1.5％，诱导温度为20、25、30℃，探究不同发酵条件对重组酶表达的影响。
具体优化过程如下：
(1)初始诱导菌体浓度：以17.5-18.5mL/h的速率流加甘油促使菌体浓度达到初始诱导菌体浓度为不同设定值(分别为OD 600＝100、150、200)后，开始添加甲醇诱导培养，甲醇诱导浓度为1.0％，将诱导培养温度设为25℃，诱导144h完成发酵过程。结果显示，最优初始诱导菌体浓度为OD 600＝150，工程菌产β-葡萄糖苷酶最高酶活力达2054U/mL(图1)。
表1工程菌在不同初始诱导菌体浓度时产酶量(U/mL)
Figure PCTCN2020102671-appb-000001
(2)甲醇诱导浓度：在初始菌体浓度为OD 600＝150，诱导培养温度为25℃的条件下，分别采用0.5％、1.0％、1.5％3个不同浓度梯度的甲醇进行诱导，诱导144h完成发酵过程。结果显示，最优甲醇诱导浓度为1.0％，重组菌产β-葡萄糖苷酶最高酶活力达2010U/mL(图2)。
表2工程菌在不同浓度的甲醇诱导下的产酶量(U/mL)
Figure PCTCN2020102671-appb-000002
(3)诱导培养温度：在初始菌体浓度为OD 600＝150，甲醇诱导浓度1.0％，分别在20、 25、30℃3个培养温度下诱导144h完成发酵过程。结果显示，最优诱导培养温度为25℃，重组菌产β-葡萄糖苷酶最高酶活力达2324U/mL(图3)。
表3工程菌在不同诱导培养温度下的产酶量(U/mL)
Figure PCTCN2020102671-appb-000003
经发酵条件优化，在初始诱导菌体浓度为OD 600＝150，甲醇诱导浓度1.0％，诱导温度为25℃的最佳发酵条件下，测得β-葡萄糖苷酶最高酶活为2324U/mL，重组β-葡萄糖苷酶发酵液SDS-PAGE电泳图见图4。
3、β-葡萄糖苷酶的酶学性质
(1)β-葡萄糖苷酶的最适温度
将上述发酵得到的β-葡萄糖苷酶酶液进行酶学定性，以pNPG为底物，在不同的温度下测酶活，结果表明：β-葡萄糖苷酶在50℃、65℃时的相对酶活分别为57.04％、50.08％，最适温度为60℃(图5)。
(2)β-葡萄糖苷酶的最适pH
在最适温度60℃条件下设置不同pH梯度测β-葡萄糖苷酶的酶活，结果表明：β-葡萄糖苷酶在pH为4.5、5.5、6.0时的相对酶活分别为75.49％、84.51％、70.22％，最适pH为5.0(图6)。
实施例3：β-葡萄糖苷酶在以葡萄糖为底物制备低聚龙胆糖中的应用
以葡萄糖为底物制备低聚龙胆糖的反应体系：以800g/L葡萄糖为底物，在pH 5.0、60℃下反应72h，设置不同β-葡萄糖苷酶加酶量(200-600U/g葡萄糖)对酶反应的影响，实验结果如图7所示，低聚龙胆糖累积含量在一定范围内随加酶量升高而增加，但当加酶量超过300U/g葡萄糖时，增加加酶量反而降低低聚龙胆糖的产量。所以选择加酶量300U/g葡萄糖时最合适，此时低聚龙胆糖的产量能达到125.0g/L，转化率为15.62％。
转化率计算公式：
Figure PCTCN2020102671-appb-000004
表4不同加酶量下的以葡萄糖为底物制备低聚龙胆糖的产量及转化率
Figure PCTCN2020102671-appb-000005
Figure PCTCN2020102671-appb-000006
对比例1
具体实施方式同实施例4，区别在于，将β-葡萄糖苷酶替换为来源于绿色木霉Trichoderma viride的β-葡萄糖苷酶，设置酶的添加量为300U/g葡萄糖，测定低聚龙胆糖的产量和转化率分别为60g/L、7.5％。
在此基础上，在绿色木霉Trichoderma viride的β-葡萄糖苷酶的酶转化最优条件：pH 5.0，60℃，加酶量为900U/g葡萄糖，底物葡萄糖浓度为800g/L时，低聚龙胆糖产量能达到最大值130g/L，转化率为16.25％，高密度发酵水平为1402U/mL。
实施例4：β-葡萄糖苷酶在以葡萄糖和纤维二糖为底物制备低聚龙胆糖中的应用
以葡萄糖和纤维二糖为底物制备低聚龙胆糖的反应体系：维持葡萄糖与纤维二糖摩尔浓度比为1:2(质量浓度比为1:4)，50-150g/L葡萄糖和200-600g/L纤维二糖为底物，在加酶量400U/g纤维二糖，pH 5.0、60℃条件下反应48h。结果显示，最优底物浓度为75g/L葡萄糖和300g/L纤维二糖，低聚龙胆糖转化率最高为26.2％(图8)。
表5不同底物浓度下以葡萄糖和纤维二糖为底物制备低聚龙胆糖的产量及转化率
Figure PCTCN2020102671-appb-000007
对比例2
具体实施方式同实施例4，区别在于，将β-葡萄糖苷酶替换为来源于绿色木霉Trichoderma viride的β-葡萄糖苷酶，在其酶转化最优条件：pH 5.0，60℃，底物浓度为20％葡萄糖、40％纤维二糖时，β-葡萄糖苷酶加酶量为400U/g纤维二糖，低聚龙胆糖的转化率为19.4％。
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。

Claims (19)

  1. 一种编码β-葡萄糖苷酶的基因，其特征在于，核苷酸序列如SEQ ID NO.1所示。
  2. 携带权利要求1所述基因的载体。
  3. 根据权利要求2所述的载体，其特征在于，以pPIC9K为表达载体。
  4. 一种重组菌，其特征在于，携带权利要求1所述基因。
  5. 根据权利要求4所述的重组菌，其特征在于，所述重组菌以毕赤酵母KM71为宿主。
  6. 一种重组菌，其特征在于，表达氨基酸序列如SEQ ID NO.2所示的β-葡萄糖苷酶。
  7. 根据权利要求6所述的重组菌，其特征在于，所述重组菌以毕赤酵母KM71为宿主。
  8. 一种提高β-葡萄糖苷酶产量的方法，其特征在于，所述方法是利用权利要求3或4所述重组菌发酵产酶。
  9. 根据权利要求8所述的方法，其特征在于，将所述重组菌加入发酵体系中发酵，当重组菌菌体细胞浓度在OD 600为100～200时，用甲醇诱导重组菌产酶，并控制温度在15～25℃。
  10. 根据权利要求9所述的方法，其特征在于，所述甘油的浓度为80mL/100mL，甘油流加速率为15～25mL/h。
  11. 根据权利要求10所述的方法，其特征在于，所述甲醇的浓度为0.5～2.0mL/100mL。
  12. 一种生产低聚龙胆糖的方法，其特征在于，以权利要求6或7所述重组菌表达的β-葡萄糖苷酶催化葡萄糖生成低聚龙胆糖。
  13. 根据权利要求12所述的方法，其特征在于，所述葡萄糖的浓度为500～1000g/L
  14. 根据权利要求12所述的方法，其特征在于，所述β-葡萄糖苷酶的加酶量为200～600U/g葡萄糖。
  15. 权利要求1所述基因在食品和化妆品领域制备低聚龙胆糖中的应用。
  16. 权利要求2或3所述载体在食品和化妆品领域制备低聚龙胆糖中的应用。
  17. 权利要求4～7任一所述重组菌在食品和化妆品领域制备低聚龙胆糖中的应用。
  18. 权利要求8～11任一所述方法在食品和化妆品领域制备低聚龙胆糖中的应用。
  19. 权利要求12～14任一所述方法在食品和化妆品领域制备低聚龙胆糖中的应用。
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