WO2021212723A1 - 一种β-半乳糖苷酶GALA及其应用 - Google Patents

一种β-半乳糖苷酶GALA及其应用 Download PDF

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WO2021212723A1
WO2021212723A1 PCT/CN2020/113644 CN2020113644W WO2021212723A1 WO 2021212723 A1 WO2021212723 A1 WO 2021212723A1 CN 2020113644 W CN2020113644 W CN 2020113644W WO 2021212723 A1 WO2021212723 A1 WO 2021212723A1
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galactosidase
gala
beta
enzyme activity
lactose
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PCT/CN2020/113644
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French (fr)
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陈豪
董娟
陈鹏
狄国虎
吴祥根
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青岛大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C7/00—Other dairy technology
    • A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
    • A23C7/043—Removing unwanted substances other than lactose or milk proteins from milk using chemicals in liquid or solid state, e.g. flocculating, adsorbing or extracting agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

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  • the invention relates to a novel beta-galactosidase GalA and its application, and belongs to the field of biotechnology.
  • ⁇ -Galactosidase (EC 3.2.1.23) is a glycoside hydrolase that can catalyze the hydrolysis and transgalactosylation of lactose.
  • ⁇ -galactosidase As a commonly used medical enzyme, ⁇ -galactosidase has been widely used in the dairy industry to decompose lactose into galactose and glucose.
  • ⁇ -galactosidase can also produce galactose oligosaccharides through transglycosylation during the hydrolysis of lactose or other structurally related galactosides.
  • ⁇ -galactosidase can be widely used in nutrition and food processing.
  • ⁇ -galactosidase The hydrolytic activity of ⁇ -galactosidase has been used in the food industry for decades to help absorb undigested lactose. All over the world, especially in East Asia, many children and adults are facing intolerance caused by lactose indigestion and lactase deficiency. The reason behind these diseases is decreased ⁇ -galactosidase activity or decreased synthesis in the small intestine. People with lactose intolerance can only consume dairy products that contain no or very little lactose. In order to overcome the limitations caused by lactose intolerance, the lactose in milk can be degraded by degrading the lactose in milk.
  • ⁇ -galactosidase has been purified and identified from a variety of organisms, such as bacteria, fungi, yeast, plants and mammals. Due to issues such as yield, stability, and degradation efficiency, ⁇ -galactosidase that can be used in commercial applications is still very small. Moreover, the ⁇ -galactosidase currently on the market is expensive and single in variety, which cannot meet the application under specific conditions. Therefore, there is an urgent need to develop new ⁇ -galactosidase.
  • the present invention provides a novel beta-galactosidase GalA and a preparation method thereof.
  • the present invention provides a recombinant expression and purification preparation method of the beta-galactosidase GalA, the enzyme activity of the fermentation broth can reach 3795.6 U/mL, the one-step purification purity is >97%, and the recovery rate is 79.5%.
  • the ⁇ -galactosidase GalA of the present invention has good thermal stability and storage stability.
  • the optimal reaction temperature is 50°C. After continuous incubation at 50°C for 24 hours, it still retains 89.7% of the enzyme activity. Normal temperature (30°C) After storing for 30 days, it can still retain 81.5% of the enzyme activity.
  • the beta-galactosidase GalA of the present invention has high activity, good stability, strong lactose degradation ability, and good application potential.
  • the present invention provides a novel ⁇ -galactosidase GalA, the amino acid sequence of which is shown in SEQ ID NO.1.
  • the present invention also provides a nucleic acid sequence corresponding to the ⁇ -galactosidase GalA, as shown in SEQ ID NO.2.
  • the present invention also provides a method for preparing and purifying the ⁇ -galactosidase GalA.
  • the present invention also provides the application of the ⁇ -galactosidase GalA in the degradation of lactose.
  • the ⁇ -galactosidase enzyme selected is GalA.
  • the reaction temperature in the degradation conditions is 0-70°C.
  • the optimum reaction temperature is 50°C.
  • the ⁇ -galactosidase GalA of the present invention is a novel ⁇ -galactosidase, and the highest amino acid sequence similarity to the ⁇ -galactosidase of the present invention in the "Genbank" database is only 88.87%, indicating The ⁇ -galactosidase of the present invention is a ⁇ -galactosidase with novel sequence and structure.
  • the present invention provides a method for preparing ⁇ -galactosidase, that is, using genetic engineering techniques to express the gene sequence of ⁇ -galactosidase GalA heterologously recombinantly into E. coli, and after fermentation, fermentation
  • the enzyme activity reached 3795.6U/mL.
  • the enzyme purification method is simple, using a nickel column for one-step affinity purification, the recovery rate is as high as 79.5%, and the protein purity is >97%.
  • the ⁇ -galactosidase GalA of the present invention has good thermal stability and storage stability. It retains high enzyme activity after continuous incubation at 50°C for 24 hours, and is suitable for large-scale continuous degradation. It is stored at room temperature for 30 days. It can still retain more than 80% of the enzyme activity, is convenient to store, and has good application potential.
  • the ⁇ -galactosidase GalA sequence of the present invention is novel, high in yield, simple in preparation, stable in properties, and has good potential for industrial application.
  • Figure 1 is a diagram of protein separation and purification of ⁇ -galactosidase GalA of the present invention (M, protein standard; 1, purified ⁇ -galactosidase GalA);
  • Figure 2 is a thermal stability diagram of ⁇ -galactosidase GalA incubated at 50°C of the present invention
  • Figure 3 is a graph of the storage stability of the ⁇ -galactosidase GalA of the present invention at room temperature 30°C;
  • FIG 4 is a diagram showing the detection of lactose degradation products of ⁇ -galactosidase GalA of the present invention in milk by thin layer chromatography (TLC) (1, glucose standard; 2, galactose standard; 3, lactose substrate; 4, Poly-oligosaccharide standard; 5, lactose degradation 10min sample, 6, lactose degradation 20min sample; 7, lactose degradation 40min sample; 8, lactose degradation 60min sample).
  • TLC thin layer chromatography
  • the enzyme-producing gene galA of ⁇ -galactosidase GalA of the present invention is an artificially synthesized sequence.
  • the inventors found that the deep-sea bacterium Q15 has a high lactose degradation activity. When the whole gene was sequenced, it was found that it contained a predicted ⁇ -galactosidase sequence and named it galA. Under the condition that the amino acid sequence is unchanged, the inventor has optimized the base sequence of the gene sequence according to the codon preference of the host (E. coli), which is conducive to its efficient expression in E. coli.
  • the ⁇ -galactosidase of the present invention contains 2007 base sequences and encodes 669 amino acid sequences.
  • the amino acid sequence similarity between "WP_053430202” and the ⁇ -galactosidase GalA of the present invention is 88.87%; the amino acid sequence similarity of "WP_134230305” and the ⁇ -galactosidase GalA of the present invention is 88.72%; The amino acid sequence similarity between WP_010899827 and the ⁇ -galactosidase GalA of the present invention is 88.42%.
  • the inventors in-depth analysis found that ⁇ -galactosidase (WP_053430202, WP_134230305 and WP_010899827) in "Genbank” is a predicted protein, and there is no prior art report on its biological function.
  • the base sequence of ⁇ -galactosidase GalA uses restriction endonucleases Nco I and Xho I as restriction enzyme sites, and the recombination primers are designed as follows (underlined are restriction enzyme sites, and italics are restriction endonuclease sites) Enzyme protection base):
  • PCR amplification conditions are: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 1 minute, total 30 cycles; 72°C extension for 2 minutes; 4°C stability for 15 minutes.
  • the DNA polymerase used in the PCR reaction was Primerstar HS and was purchased from Dalian Bao Biological Company.
  • the PCR product was double digested with restriction enzymes Nco I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis.
  • pET22b(+) plasmid DNA (Invitrogen, USA), also double-enzyme digestion with restriction enzymes Nco I and Xho I, agarose gel electrophoresis, and recovery of the digested product fragments.
  • the enzyme and substrate reaction system (temperature, time, amount of DNA, etc.) used for digestion are operated in accordance with the product instructions provided by Dalian Bio-Bio.
  • the PCR product and pET-22b(+) plasmid vector treated by double enzyme digestion were ligated according to the instructions of DNA ligase (Dalian Bao Biological Company); the ligation product was transformed into Escherichia coli DH5 ⁇ strain (Invitrogen Company, USA) and coated on Luria- Bertani (LB) medium solid plate (containing 50 ⁇ g/mL ampicillin), cultured in a 37°C incubator for 12-16 hours, pick a single clone; transfer the single clone to LB liquid medium (containing 50 ⁇ g /mL ampicillin), cultured overnight in a 37°C shaker with a rotating speed of 180 rpm.
  • DNA ligase DNA ligase
  • LB Luria- Bertani
  • the single clone was sequenced, and the positive clone was selected and named pET22b-GalA.
  • the recombinant plasmid was transformed into Escherichia coli BL21(DE3) (purchased from Dalian Bao Biological Company), the recombinant large intestine strain was named BL21(DE3)/pET22b-GalA, and it was stored at -80°C for later use.
  • the recombinant strain BL21 (DE3) / pET22b-GalA in 100mL of LB liquid medium (50 ⁇ g / mL ampicillin), shaking at 180rpm shaking at 37 °C grown to OD 600 0.6, was added to a final concentration of 0.1mM induced Isopropyl- ⁇ -D-thiogalactoside (IPTG), induced at 20°C for 20h.
  • IPTG Isopropyl- ⁇ -D-thiogalactoside
  • ONPG purchased in Sigma Alddrich, USA
  • the standard method for determining the enzyme activity of ⁇ -galactosidase GalA is as follows: 100 ⁇ L enzyme solution is added with 900 ⁇ L 0.3% ONPG substrate (20mM Tris-HCl buffer, pH 7.0), reacted at 50°C for 10min, and 1M Na 2 CO is added. 3 Reagents terminate the reaction, and the activity is determined at OD 420 nm according to the ONP standard curve.
  • the unit (U) of ⁇ -galactosidase is defined as the amount of enzyme required to release 1 ⁇ mol ONP per minute under the analytical conditions.
  • the purified ⁇ -galactosidase GalA was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). As shown in Figure 1, the molecular weight of ⁇ -galactosidase GalA was about 80kDa, which was consistent with the protein size predicted in the sequence analysis. (78.4kDa). Through gel analysis, it was found that the protein purity of the purified ⁇ -galactosidase GalA reached more than 97%.
  • the ⁇ -galactosidase GalA purified in Example 2 was subjected to enzyme activity determination under different conditions, and the effects of different temperatures and pH on enzyme activity were detected. React at different temperatures (0-70°C) for 10 minutes, test the effect of different reaction temperatures on enzyme activity, and use the highest enzyme activity as 100% to calculate the relative enzyme activity of ⁇ -galactosidase GalA at different temperatures, ⁇ -half
  • the optimal reaction temperature of lactosidase GalA is 50°C.
  • ⁇ -galactosidase GalA has good thermostability, and it retains 89.7% of the enzyme activity after continuous incubation at 50°C for 24 hours, indicating that the ⁇ -galactosidase GalA of the present invention can maximize It can continue to play a role at the right reaction temperature.
  • the ⁇ -galactosidase GalA purified in Example 2 was incubated at room temperature (30°C) for different times (10, 20, 30, 40, 50, 60 days). The enzyme activity was tested at °C), and the activity before incubation was taken as 100%. As shown in Figure 3, ⁇ -galactosidase GalA could still retain 81.5% of the enzyme activity when stored at 30°C for 30 days, indicating the present invention The ⁇ -galactosidase GalA has good storage stability.
  • the ⁇ -galactosidase GalA of the present invention has good thermal stability and storage stability, which is beneficial to its subsequent development and utilization.
  • Example 2 The ⁇ -galactosidase GalA purified in Example 2 was incubated with milk (purchased from Mengniu) at 50°C for 0, 10, 20, 40, and 60 minutes, and then tested on a high-performance thin-layer chromatography (HPTLC) plate.
  • milk purchased from Mengniu
  • HPTLC high-performance thin-layer chromatography

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Abstract

提供了一种β-半乳糖苷酶GalA及其应用。所述β-半乳糖苷酶GalA氨基酸序列如SEQ ID NO.1所示。所述β-半乳糖苷酶GalA的重组表达及纯化制备方法，发酵液酶活力可达3795.6U/mL，一步纯化纯度>97％，回收率为79.5％。所述β-半乳糖苷酶GalA具有良好的热稳定性和储存稳定性，最适反应温度为50℃，在50℃下持续孵育24h仍保留89.7％的酶活力，室温下(30℃)储存30天，仍然能保留81.5％的酶活力。所述β-半乳糖苷酶GalA活力高、稳定性好，降解乳糖能力强，具有良好的应用潜质。

Description

[根据细则37.2由ISA制定的发明名称]　一种β-半乳糖苷酶GalA及其应用 技术领域
本发明涉及一种新型β-半乳糖苷酶GalA及其应用，属于生物技术领域。
背景技术
β-半乳糖苷酶(EC 3.2.1.23)是一种糖苷水解酶，可催化乳糖水解和转半乳糖基化反应。作为一种常用的医用酶，β-半乳糖苷酶已广泛用于乳制品行业中将乳糖分解为半乳糖和葡萄糖。此外，β-半乳糖苷酶还可以在乳糖或其他与结构相关的半乳糖苷水解过程中通过转糖基化作用产生半乳糖寡糖。β-半乳糖苷酶可广泛用于营养和食品加工。
β-半乳糖苷酶的水解活性已经在食品工业中使用了数十年，以帮助吸收未消化的乳糖。全世界，特别是在东亚，许多儿童和成人正面临乳糖消化不良和乳糖酶缺乏引起的不耐受的问题。这些疾病背后的原因是小肠中β-半乳糖苷酶活性降低或合成降低。患有乳糖不耐症的人只能食用不含或很少乳糖的乳制品。为了克服乳糖不耐症带来的局限性，可以通过降解牛奶中的乳糖来使牛奶中的乳糖降解。此外，人们在食用奶制品之前服用含有β-半乳糖苷酶的药物，这也可以有效降低乳糖浓度。在食品工业中，β-半乳糖苷酶的水解活性还被用于减少冰淇淋和炼乳中的结晶。
尽管β-半乳糖苷酶已从多种生物中纯化和鉴定，例如细菌，真菌，酵母，植物和哺乳动物等。由于产量，稳定性，降解效率等问题，能够用于商品化应用的β-半乳糖苷酶仍然很少。而且，目前市场上销售的β-半乳糖苷酶价格昂贵、品种单一，无法满足特定条件下的应用，因此急需开发新的β-半乳糖苷酶。
发明内容
本发明针对现有技术的不足，提供了一种新型β-半乳糖苷酶GalA及其制备方法。本发明提供一种所述β-半乳糖苷酶GalA的重组表达及纯化制备方法，发酵液酶活力可达3795.6U/mL，一步纯化纯度>97％，回收率为79.5％。本发明所述β-半乳糖苷酶GalA具有良好的热稳定性和储存稳定性，最适反应温度为50℃，在50℃下持续孵育24h仍保留89.7％的酶活力，常温(30℃)下储存 30天，仍然能保留81.5％的酶活力。本发明所述β-半乳糖苷酶GalA活力高、稳定性好，降解乳糖能力强，具有良好的应用潜质。
一方面，本发明提供一种新型β-半乳糖苷酶GalA，其氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1：
Figure PCTCN2020113644-appb-000001
另一方面，本发明还提供一种所述β-半乳糖苷酶GalA对应的核酸序列，如SEQ ID NO.2所示。
SEQ ID NO.2：
Figure PCTCN2020113644-appb-000002
Figure PCTCN2020113644-appb-000003
Figure PCTCN2020113644-appb-000004
另一方面，本发明还提供一种所述β-半乳糖苷酶GalA的制备与纯化方法。
另一方面，本发明还提供了所述β-半乳糖苷酶GalA在降解乳糖中的应用。
另一方面，一种降解牛奶中乳糖的方法，所选用的β-半乳糖苷酶为GalA。
优选：所述降解条件中反应温度为0～70℃。最适反应温度为50℃。
有益效果：
1.本发明的β-半乳糖苷酶GalA为新颖的β-半乳糖苷酶，在《Genbank》数据库中与本发明所述β-半乳糖苷酶最高氨基酸序列相似度仅为88.87％，说明本发明的β-半乳糖苷酶为序列和结构新颖的β-半乳糖苷酶。
2.本发明提供了一种制备β-半乳糖苷酶的方法，即利用基因工程的技术方法，将β-半乳糖苷酶GalA的基因序列异源重组表达到大肠杆菌，经发酵后，发酵液酶活力达到3795.6U/mL。该酶纯化方法简单，利用镍柱对其进行一步亲和纯化，回收率高达79.5％，蛋白质纯度>97％。
3.本发明所述β-半乳糖苷酶GalA具有良好的热稳定性和储存稳定性，在50℃下持续孵育24h仍保留高的酶活力，适合大规模持续降解，常温下储存30天，仍然能保留80％以上的酶活力，保存方便，具有良好的应用潜质。
综上所述，本发明所述β-半乳糖苷酶GalA序列新颖、产量高、制备简单、性质稳定，具有良好的工业化应用潜质。
附图说明
图1为本发明β-半乳糖苷酶GalA的蛋白质分离纯化图(M，蛋白质标准品；1，纯化所得β-半乳糖苷酶GalA)；
图2为本发明β-半乳糖苷酶GalA在50℃孵育的热稳定性图；
图3为本发明β-半乳糖苷酶GalA在常温30℃下的储藏稳定性图；
图4为薄层层析(TLC)法检测本发明β-半乳糖苷酶GalA在牛奶中乳糖降解产物图(1，葡萄糖标准品；2，半乳糖标准品；3，乳糖底物；4，多聚寡糖标准品；5，乳糖降解10min样品，6，乳糖降解20min样品；7，乳糖降解40min样品；8，乳糖降解60min样品)。
具体实施方式
实施例1β-半乳糖苷酶GalA的序列分析
本发明所述β-半乳糖苷酶GalA的产酶基因galA为人工合成序列。前期研究中，发明人发现深海细菌Q15具有较高的乳糖降解活性，对其进行全基因测序时，发现其含有一个预测的β-半乳糖苷酶序列，将其命名为galA。在氨基酸序列不变的情况下，发明人将该基因序列根据宿主(大肠杆菌的)的密码子偏好性，进行了碱基序列优化，利于其在大肠杆菌中进行高效表达。本发明所述β-半乳糖苷酶包含有2007个碱基序列，编码669个氨基酸序列。
多重序列比对Basic Local Alignment Search Tool(Blast)发现，在《Genbank》数据库中，与本发明所述的β-半乳糖苷酶氨基酸序列相似度最高的为Bacillus属的蛋白(WP_053430202，WP_134230305和WP_010899827)，这三个蛋白均含有672个氨基酸序列。其中《WP_053430202》与本发明所述β-半乳糖苷酶GalA的氨基酸序列相似度为88.87％；《WP_134230305》与本发明所述β-半乳糖苷酶GalA的氨基酸序列相似度为88.72％；《WP_010899827》与本发明所述β-半乳糖苷酶GalA的氨基酸序列相似度为88.42％。发明人深入分析发现《Genbank》中β-半乳糖苷酶(WP_053430202，WP_134230305和WP_010899827)为预测蛋白，并未有现有技术报道其生物学功能。
将β-半乳糖苷酶GalA的碱基序列以限制性内切酶Nco I和Xho I为酶切位点，设计重组引物如下(下划线为限制性内切酶位点，斜体为限制性内切酶保护碱基)：
正向引物：PGalA-F：
5’-CATG CCATGGATGATCAACGAAAAACTGCC-3’(Nco I)
反向引物：PGalA-R：
5’-CCG CTCGAGAGCGGTACCTTCACCACGGT-3’(Xho I)
PCR扩增条件为：94℃预变性3min；94℃变性30秒，55℃退火30秒，72℃延伸1min，共30个循环；72℃延伸2min；4℃稳定15min。PCR反应所用DNA聚合酶为Primerstar HS购自大连宝生物公司。
PCR产物用限制性内切酶Nco I和Xho I进行双酶切，通过琼脂糖凝胶电泳回收酶切后的PCR产物。pET22b(+)质粒DNA(美国Invitrogen公司)，同样 用限制性内切酶Nco I和Xho I进行双酶切，进行琼脂糖凝胶电泳并回收酶切后的产物片段。酶切所用酶和底物反应体系(温度、时间、DNA用量等)均参照大连宝生物提供的产品说明操作。
双酶切处理的PCR产物和pET-22b(+)质粒载体参照DNA连接酶(大连宝生物公司)说明书进行连接反应；连接产物转化至大肠杆菌DH5α菌株(美国Invitrogen公司)，涂布在Luria-Bertani(LB)培养基固体平板上(含有50μg/mL氨苄青霉素的)，37℃温箱中培养12-16小时后，挑取单克隆；将单克隆转接至LB液体培养基中(含有50μg/mL氨苄青霉素)，转速为180rpm的37℃摇床中培养过夜。将单克隆进行序列测定，选择阳性克隆，并将其命名为pET22b-GalA。重组质粒转化至大肠杆菌BL21(DE3)(购自大连宝生物公司)，将重组大肠菌株命名为BL21(DE3)/pET22b-GalA，保存在-80℃备用。
实施例2 β-半乳糖苷酶GalA的制备及纯化方法
将重组菌株BL21(DE3)/pET22b-GalA在100mL的LB液体培养基中(50μg/mL氨苄青霉素)，在37℃摇床中180rpm震荡培养至OD 600＝0.6，加入终浓度为0.1mM的诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)，在20℃诱导20h。底物4-Nitrophenyl-β-d-glucopyranoside，ONPG(购置于Sigma Alddrich,USA)用于β-半乳糖苷酶活性测定。β-半乳糖苷酶GalA的酶活标准测定方法为：100μL酶液加入900μL 0.3％ONPG底物(20mM Tris-HCl缓冲液，pH 7.0)，在50℃下反应10min，加入1M的Na 2CO 3试剂终止反应，根据ONP标准曲线在OD 420nm处确定活性。β-半乳糖苷酶的单位(U)定义为在分析条件下每分钟释放1μmol ONP所需的酶量。
发酵停止后，12000rpm离心10min，弃菌体，收集上清液；发酵上清液上样于10mL镍离子亲和层析柱，上样流速为5mL/min，利用10mM咪唑洗脱，去除杂蛋白，再利用150mM的咪唑洗脱，收集洗脱组分。将活性成分透析去除咪唑，分装储存在-20℃备用。通过镍离子一步亲和纯化，蛋白回收率达到79.5％。纯化所得β-半乳糖苷酶GalA进行聚丙烯酰胺凝胶电泳(SDS-PAGE)，如图1所示，β-半乳糖苷酶GalA的分子量为80kDa左右，与序列分析中预测的蛋白大小一致(78.4kDa)。通过凝胶分析发现，纯化所得β-半乳糖苷酶GalA的蛋白纯度达到97％以上。
实施例3 β-半乳糖苷酶GalA的稳定性
将实施例2中纯化所得β-半乳糖苷酶GalA在不同条件下进行酶活力测定，检测不同温度和pH对酶活力的影响。在不同温度(0-70℃)下反应10min，检测不同反应温度对酶活力的影响，以最高酶活力为100％，计算不同温度下β-半乳糖苷酶GalA的相对酶活力，β-半乳糖苷酶GalA的最适反应温度为50℃。将β-半乳糖苷酶GalA在50℃孵育不同时间(6，12，18，24，30，36h)，在最适反应温度(50℃)下检测剩余酶活力，以孵育前的酶活力作为100％。如图2所示，β-半乳糖苷酶GalA具有良好的热稳定性，在50℃下持续孵育24h仍保留89.7％的酶活力，表明本发明所述β-半乳糖苷酶GalA能够在最适反应温度下能够持续发挥作用。
将实施例2中纯化所得β-半乳糖苷酶GalA在常温(30℃)下孵育不同时间(10，20，30，40，50，60天)，取出后，在其最适反应温度(50℃)下检测其酶活力，以孵育前的活力作为100％，如图3所示，β-半乳糖苷酶GalA在30℃下储存30天，仍然能保留81.5％的酶活力，表明本发明所述β-半乳糖苷酶GalA具有良好的储存稳定性。
因此，本发明所述β-半乳糖苷酶GalA具有良好的热稳定性和储存稳定性，利于其后续开发利用。
实施例4 β-半乳糖苷酶GalA酶解产物薄层层析分析
将实施例2中纯化所得β-半乳糖苷酶GalA与牛奶(采购自蒙牛)在50℃下分别孵育0，10，20，40，60min，然后在高效薄层层析板(HPTLC)检测。具体为：将HPTLC层析板裁剪成7cm宽合适大小的样本，将孵育前后样品点样在原点处，置于有展开剂(正丁醇/甲酸/水＝5:3:2)的展缸中30min，吹干层析板，浸入显色剂(90％乙醇和10％，v/v)中2s，取出吹干，80℃烘烤，至样品出现。如图4所示，本发明所述β-半乳糖苷酶GalA可迅速的将牛奶中乳糖降解为单糖(葡萄糖和半乳糖)。

Claims (6)

  1. 一种新型β-半乳糖苷酶GalA，其氨基酸序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的β-半乳糖苷酶GalA所对应的核苷酸序列，所述核苷酸序列如SEQ ID NO.2所示。
  3. 如权利要求1所述的β-半乳糖苷酶GalA的制备与纯化方法。
  4. 如权利要求1所述的β-半乳糖苷酶GalA在降解乳糖中的应用。
  5. 一种降解牛奶中乳糖的方法，其特征是，所选用的β-半乳糖苷酶为权利要求1所述的β-半乳糖苷酶GalA。
  6. 如权利要求5所述的方法，其特征是，降解条件中反应温度为0～70℃，最适反应温度为50℃。
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