CN109628340B - A kind of Bacillus circulans strain producing high activity β-galactosidase and its breeding method - Google Patents

A kind of Bacillus circulans strain producing high activity β-galactosidase and its breeding method Download PDF

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CN109628340B
CN109628340B CN201811561982.6A CN201811561982A CN109628340B CN 109628340 B CN109628340 B CN 109628340B CN 201811561982 A CN201811561982 A CN 201811561982A CN 109628340 B CN109628340 B CN 109628340B
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邓宝浣
陈振鹏
陈子健
杨新球
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Abstract

The invention discloses a Bacillus circulans strain for producing high-activity beta-galactosidase and a breeding method thereof, wherein the Bacillus circulans strain for producing high-activity beta-galactosidase is Bacillus circulans QHT-310-M481(Bacillus circulans QHT-310-M481), which is preserved in China center for type culture collection in 2018, 10 and 22 months, and the preservation number is CCTCC NO: m2018699, the preservation address is Wuhan university in Wuhan City, China. The bacillus circulans strain for producing the beta-galactosidase with high activity is obtained by carrying out mutagenesis treatment on a bacillus circulans starting strain by using a 1-methyl-3-nitro-1-nitrosoguanidine mutagen, can produce beta-galactosidase liquid with high fermentation activity, and is used for industrially catalyzing galactosyl transfer reaction to produce galactooligosaccharides.

Description

一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株及其选育 方法A kind of Bacillus circulans strain producing high activity β-galactosidase and its breeding method

技术领域technical field

本发明属于发酵工程和微生物育种领域,具体涉及一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株及其选育方法。The invention belongs to the fields of fermentation engineering and microbial breeding, and in particular relates to a Bacillus circulans strain producing high-activity β-galactosidase and a breeding method thereof.

背景技术Background technique

低聚半乳糖(Galactooligosaccharides,GOS)是一种具有天然属性的功能性低聚糖。低聚半乳糖是一种低能量糖,天然存在于动物乳和人乳中。低聚半乳糖是人体肠道中双歧杆菌、嗜酸乳酸杆菌等有益菌极好的营养源和有效的增殖因子,可以改善人体肠道的消化吸收功能。此外,低聚半乳糖还可改善脂质代谢,降低血清胆固醇浓度、促进矿物质元素吸收、改善乳糖不耐症、提高机体免疫力等功能。Galactooligosaccharides (GOS) are functional oligosaccharides with natural properties. Galacto-oligosaccharide is a low-energy sugar that occurs naturally in animal and human milk. Galacto-oligosaccharide is an excellent source of nutrients and an effective proliferation factor for beneficial bacteria such as Bifidobacterium and Lactobacillus acidophilus in the human intestinal tract, which can improve the digestion and absorption function of the human intestinal tract. In addition, galactooligosaccharides can also improve lipid metabolism, reduce serum cholesterol concentration, promote mineral element absorption, improve lactose intolerance, and improve immunity.

低聚半乳糖在食品行业应用广泛,作为益生元成分应用于乳制品、焙烤食品、糖果加工及功能性食品等领域。随着法规的认可,β-半乳糖苷酶制得的低聚半乳糖批准成为食品添加剂新品种,用于婴幼儿配方食品和婴幼儿谷类辅助食品,单独或混合使用。伴随着全面二孩政策效应充分发挥,生育水平适度提高。婴配乳粉、其他乳制品以及保健品作为低聚半乳糖主要研究和应用的领域,迎来了新的发展高峰。Galacto-oligosaccharides are widely used in the food industry and are used as prebiotic ingredients in dairy products, bakery products, candy processing and functional foods. With the approval of the regulations, galacto-oligosaccharides prepared by β-galactosidase have been approved as a new variety of food additives, which can be used in infant formula and cereal complementary food for infants and young children, alone or in combination. With the full effect of the comprehensive two-child policy, the fertility level has been moderately improved. Infant milk powder, other dairy products and health care products, as the main research and application fields of galactooligosaccharides, have ushered in a new development peak.

在工业生产中,以乳糖为原料,利用微生物β-半乳糖苷酶催化半乳糖基转移反应来生产低聚半乳糖。低聚半乳糖的分子结构是在半乳糖或葡萄糖分子上通过β-(1→3)半乳糖苷键、β-(1→4)半乳糖苷键或β-(1→6)半乳糖苷键连接1-7个半乳糖基。In industrial production, galacto-oligosaccharides are produced by using lactose as raw material and catalyzing the galactosyl transfer reaction by microbial beta-galactosidase. The molecular structure of galacto-oligosaccharides is through β-(1→3) galactosidic bond, β-(1→4) galactosidic bond or β-(1→6) galactoside on galactose or glucose molecule Bonds link 1-7 galactosyl groups.

β-半乳糖苷酶广泛存在于动植物和微生物内,它的主要功能是催化乳糖水解生成葡萄糖和半乳糖,该酶还具有催化半乳糖基转移活力,生成低聚半乳糖。β-Galactosidase widely exists in animals, plants and microorganisms. Its main function is to catalyze the hydrolysis of lactose to generate glucose and galactose.

在工业生产中,β-半乳糖苷酶基本上来源于微生物,且具有易于大量制备、稳定性好等特点。但从自然界直接分离的菌种,一般而言其酶发酵活力是比较低的,不能达到工业生产的要求,因此要根据菌种的形态、生理上的特点,改良菌种。微生物的突变在自然条件下可自发进行,但是自发突变的突变率很低,获得符合要求的突变株的几率更低,其概率仅为10-6~10-10。为了使酶更适合于实际应用,采用物理化学因素等手段来提高菌株的突变率,使具有有利性状的突变株筛选的可能性大大增加。化学诱变剂主要是通过与核酸碱基作用、作为碱基类似物或移码突变剂来干扰DNA复制,使其遗传物质发生改变而突变。诱变剂1-甲基-3-硝基-1-亚硝基胍是一种能与核酸碱基作用的双功能烷化剂,可与DNA分子的许多部位发生作用,易取代DNA分子中活泼的氢原子,使DNA分子上的碱基及核算部分被烷化,DNA复制时导致碱基配对错误而引起突变,此外,其还能在DNA双链间形成共价键,阻碍DNA复制过程中双链的解开,从而引起突变,能大大提高突变频率。In industrial production, β-galactosidase is basically derived from microorganisms, and has the characteristics of easy mass preparation and good stability. However, the strains directly isolated from nature generally have low enzymatic fermentation activity and cannot meet the requirements of industrial production. Therefore, the strains should be improved according to the morphology and physiological characteristics of the strains. The mutation of microorganisms can be carried out spontaneously under natural conditions, but the mutation rate of spontaneous mutation is very low, and the probability of obtaining mutant strains that meet the requirements is even lower, and the probability is only 10 -6 to 10 -10 . In order to make the enzyme more suitable for practical application, physical and chemical factors are used to improve the mutation rate of the strain, so that the possibility of screening mutant strains with favorable characters is greatly increased. Chemical mutagens mainly interfere with DNA replication by interacting with nucleic acid bases, acting as base analogs or frameshift mutagenic agents, so that their genetic material changes and mutates. The mutagen 1-methyl-3-nitro-1-nitrosoguanidine is a bifunctional alkylating agent that can interact with nucleic acid bases. It can interact with many parts of the DNA molecule and easily replace the The active hydrogen atom causes the base and accounting part of the DNA molecule to be alkylated, which leads to base pairing errors during DNA replication and causes mutations. In addition, it can also form covalent bonds between the DNA double strands, hindering the DNA replication process. The unwinding of the double-strand, thereby causing mutation, can greatly increase the mutation frequency.

目前相关研究报导证明环状芽孢杆菌是β-半乳糖苷酶的重要来源,且所生产的β-半乳糖苷酶对乳糖具有理想的转化效果。利用环状芽孢杆菌生产β-半乳糖苷酶具有显著的应用价值。Current related research reports prove that Bacillus circulans is an important source of β-galactosidase, and the produced β-galactosidase has an ideal transformation effect on lactose. The use of Bacillus circulans to produce β-galactosidase has significant application value.

因此,有必要研究能够生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株及其选育方法。Therefore, it is necessary to study strains of Bacillus circulans capable of producing high-activity β-galactosidase and their breeding methods.

发明内容SUMMARY OF THE INVENTION

针对现有技术的不足,本发明的目的是提供一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株,该菌株能生产高活力的β-半乳糖苷酶,用于工业上催化半乳糖基转移反应来生产低聚半乳糖。本发明还提供了生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株的选育方法,该选育方法以1-甲基-3-硝基-1-亚硝基胍为诱变剂,经多轮诱变处理,检测诱变菌株的β-半乳糖苷酶酶活力,以获得生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株,选育方法简单易行,诱变筛选成功率高。In view of the deficiencies of the prior art, the object of the present invention is to provide a Bacillus circulans strain for producing high-activity β-galactosidase, which can produce high-activity β-galactosidase for industrial catalysis Galactosylation reaction to produce galactooligosaccharides. The invention also provides a breeding method for Bacillus circulans strain producing high activity β-galactosidase. The breeding method uses 1-methyl-3-nitro-1-nitrosoguanidine as a mutagen , After multiple rounds of mutagenesis, the β-galactosidase enzyme activity of the mutant strains was detected to obtain a Bacillus circulans strain that produced high-activity β-galactosidase. The breeding method is simple and feasible, and the mutation screening High success rate.

本发明采用的技术方案是:一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株,所述生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株为环状芽孢杆菌QHT-310-M481(Bacillus circulans QHT-310-M481),于2018年10月22日在中国典型培养物保藏中心保藏,保藏编号为CCTCC NO:M 2018699,保藏地址为中国武汉市武汉大学。The technical scheme adopted in the present invention is: a strain of Bacillus circulans for producing high activity β-galactosidase, and the Bacillus circulans strain for producing high activity β-galactosidase is Bacillus circulans QHT- 310-M481 (Bacillus circulans QHT-310-M481), was deposited in the China Center for Type Culture Collection on October 22, 2018, the deposit number is CCTCC NO: M 2018699, and the deposit address is Wuhan University, Wuhan, China.

具体的,所述生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株是由环状芽孢杆菌出发菌株经诱变剂进行诱变处理得到的,所述环状芽孢杆菌出发菌株为环状芽孢杆菌(Bacillus circulars),购自美国菌种保藏中心(ATCC),编号为31382。Specifically, the Bacillus circus strain producing high-activity β-galactosidase is obtained from a Bacillus circus origin strain that undergoes mutagenesis treatment with a mutagen, and the originating Bacillus circus strain is a cyclic Bacillus circus strain Bacillus circulars, purchased from the American Type Culture Collection (ATCC), No. 31382.

优选的,所述诱变剂为1-甲基-3-硝基-1-亚硝基胍溶液,1-甲基-3-硝基-1-亚硝基胍溶液为浓度为0.2-1.0mg/mL的1-甲基-3-硝基-1-亚硝基胍丙酮溶液。Preferably, the mutagen is a 1-methyl-3-nitro-1-nitrosoguanidine solution, and the 1-methyl-3-nitro-1-nitrosoguanidine solution has a concentration of 0.2-1.0 mg/mL solution of 1-methyl-3-nitro-1-nitrosoguanidine acetone.

优选的,诱变处理过程中,0.2-1.0mg/mL的1-甲基-3-硝基-1-亚硝基胍丙酮溶液的用量为0.5-1.5mL。Preferably, during the mutagenesis process, the amount of 0.2-1.0 mg/mL 1-methyl-3-nitro-1-nitrosoguanidine acetone solution is 0.5-1.5 mL.

优选的,诱变处理的时间为15-60min。Preferably, the time of the mutagenesis treatment is 15-60 min.

所述的生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株的选育方法,包括以下步骤:The method for selecting and breeding Bacillus circulans strains for producing high-activity β-galactosidase comprises the following steps:

1)将环状芽孢杆菌出发菌株接入种子培养基中,在37℃培养4-6h后,离心收集菌体,加入无菌磷酸缓冲溶液清洗过滤,调整菌悬液浓度为105-107个/mL;1) Insert the starting strain of Bacillus circulans into the seed medium, cultivate at 37°C for 4-6 hours, collect the bacteria by centrifugation, add sterile phosphate buffer solution to wash and filter, and adjust the concentration of the bacterial suspension to 10 5 -10 7 pcs/mL;

2)往步骤1)的菌悬液中加入诱变剂,进行诱变处理,得到诱变菌液,将诱变菌液涂布于固体培养基平板,在37℃培养1-2天,待长出菌落后,挑选长势好、菌落大的菌落,接种于含有发酵培养基的96孔板中,在37℃培养36-48h,得到M1诱变菌株,检测M1诱变菌株的β-半乳糖苷酶酶活力,计算M1诱变菌株的诱变效果;2) Add mutagen to the bacterial suspension in step 1), carry out mutagenesis treatment to obtain a mutagenic bacterial liquid, coat the mutagenic bacterial liquid on a solid medium plate, cultivate at 37° C. for 1-2 days, wait for After the colonies grow, select the colonies with good growth and large colonies, inoculate them in a 96-well plate containing fermentation medium, and cultivate at 37°C for 36-48 hours to obtain the M1 mutant strain, and detect the β-galactogen of the M1 mutant strain. The enzyme activity of glycosidase was calculated, and the mutagenic effect of the M1 mutant strain was calculated;

3)对步骤2)中β-半乳糖苷酶酶活力的提高幅度大于30%的M1诱变菌株进行遗传稳定分析,连续7次传代培养,检测诱变菌株的β-半乳糖苷酶酶活力,挑选遗传稳定性最好的M1诱变菌株作为诱变母本;3) Carry out genetic stability analysis on the M1 mutant strain whose β-galactosidase activity in step 2) has increased by more than 30%, subculture for 7 consecutive times, and detect the β-galactosidase activity of the mutant strain , select the M1 mutagenic strain with the best genetic stability as the mutagenic parent;

4)对步骤3)得到的诱变母本进行1-5轮诱变处理,检测诱变菌株的β-半乳糖苷酶酶活力,挑选β-半乳糖苷酶酶活力最高的诱变菌株作为生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株。4) Carry out 1-5 rounds of mutagenesis treatment on the mutagenized parent obtained in step 3), detect the β-galactosidase enzyme activity of the mutant strain, and select the mutant strain with the highest β-galactosidase enzyme activity as the A strain of Bacillus circus that produces a highly active beta-galactosidase.

优选的,一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株的选育方法,包括以下步骤:Preferably, a method for breeding Bacillus circulans strains that produce high activity β-galactosidase, comprising the following steps:

1)将环状芽孢杆菌出发菌株接入种子培养基中,在37℃培养4-6h后,离心收集菌体,加入无菌磷酸缓冲溶液清洗过滤,调整菌悬液浓度为105-107个/mL;1) Insert the starting strain of Bacillus circulans into the seed medium, cultivate at 37°C for 4-6 hours, collect the bacteria by centrifugation, add sterile phosphate buffer solution to wash and filter, and adjust the concentration of the bacterial suspension to 10 5 -10 7 pcs/mL;

2)往1mL步骤1)的菌悬液中加入1mL浓度为0.2-1.0mg/mL的1-甲基-3-硝基-1-亚硝基胍丙酮溶液,进行诱变处理30min,得到诱变菌液Ⅰ,取100μL诱变菌液Ⅰ涂布固体培养基平板,在37℃培养1-2天,进行菌落计数,计算致死率Ⅰ,确定最佳诱变浓度;2) Add 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with a concentration of 0.2-1.0 mg/mL to 1 mL of the bacterial suspension in step 1), and perform mutagenesis for 30 min to obtain a mutagenic solution. 100 μL of mutagenic bacterial solution Ⅰ was coated on solid medium plate, cultured at 37°C for 1-2 days, colony count was carried out, lethality Ⅰ was calculated, and the optimal mutagenic concentration was determined;

往1mL步骤1)的菌悬液中加入1mL步骤2)确定的最佳诱变浓度的1-甲基-3-硝基-1-亚硝基胍丙酮溶液,进行诱变处理15-60min,得到诱变菌液Ⅱ,取100μL诱变菌液Ⅱ涂布固体培养基平板,在37℃培养1-2天,进行菌落计数,计算致死率Ⅱ,确定最佳诱变时间;Add 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the optimal mutagenesis concentration determined in step 2) to 1 mL of the bacterial suspension in step 1), and carry out mutagenesis treatment for 15-60 min. To obtain the mutagenic bacterial solution II, take 100 μL of the mutagenic bacterial solution II to coat the solid medium plate, cultivate at 37°C for 1-2 days, count the colonies, calculate the lethality II, and determine the optimal mutagenesis time;

将得到的最佳诱变时间的诱变菌液Ⅱ涂布固体培养基平板,待长出菌落后,挑选长势好、菌落大的菌落,接种于含有发酵培养基的96孔板中,在37℃培养36-48h,得到M1诱变菌株,检测诱变菌株的β-半乳糖苷酶酶活力,计算M1诱变菌株的诱变率;The obtained mutagenic bacteria solution II with the best mutagenesis time was coated on a solid medium plate, and after the colonies had grown, the colonies with good growth and large colonies were selected and inoculated in a 96-well plate containing fermentation medium. Cultivated at ℃ for 36-48h to obtain the M1 mutant strain, detect the β-galactosidase enzyme activity of the mutant strain, and calculate the mutagenesis rate of the M1 mutant strain;

3)对步骤2)中β-半乳糖苷酶酶活力的提高幅度大于30%的M1诱变菌株进行遗传稳定分析,连续7次传代培养,检测诱变菌株的β-半乳糖苷酶酶活力,挑选遗传稳定性最好的M1诱变菌株作为诱变母本;3) Carry out genetic stability analysis on the M1 mutant strain whose β-galactosidase activity in step 2) has increased by more than 30%, subculture for 7 consecutive times, and detect the β-galactosidase activity of the mutant strain , select the M1 mutagenic strain with the best genetic stability as the mutagenic parent;

4)对步骤3)得到的诱变母本进行1-5轮诱变处理,检测诱变菌株的β-半乳糖苷酶酶活力,挑选β-半乳糖苷酶酶活力最高的诱变菌株作为生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株。4) Carry out 1-5 rounds of mutagenesis treatment on the mutagenized parent obtained in step 3), detect the β-galactosidase enzyme activity of the mutant strain, and select the mutant strain with the highest β-galactosidase enzyme activity as the A strain of Bacillus circus that produces a highly active beta-galactosidase.

优选的,步骤1)中,种子培养基由以下方法制备得到:往15-20g预制营养肉汤干粉中加入1000mL蒸馏水,加热溶解,pH值不需要另行调整,冷却,得到种子培养基。Preferably, in step 1), the seed culture medium is prepared by the following method: adding 1000 mL of distilled water to 15-20 g of prefabricated nutrient broth dry powder, heating to dissolve, the pH value does not need to be adjusted separately, and cooling to obtain the seed culture medium.

更优选的,步骤1)中,种子培养基由以下方法制备得到:往18g预制营养肉汤干粉中加入1000mL蒸馏水,加热溶解,pH值不需要另行调整,冷却,得到种子培养基。More preferably, in step 1), the seed medium is prepared by the following method: adding 1000 mL of distilled water to 18 g of prefabricated nutrient broth dry powder, heating and dissolving, the pH value does not need to be adjusted separately, and cooling to obtain the seed medium.

优选的,步骤1)中,无菌磷酸缓冲溶液为pH值为6的0.01mol/L磷酸缓冲溶液。Preferably, in step 1), the sterile phosphate buffer solution is a 0.01 mol/L phosphate buffer solution with a pH value of 6.

优选的,步骤2)中,固体培养基平板由以下方法制备得到:往30-35g预制营养琼脂干粉中加入1000mL蒸馏水,加热溶解,pH值不需要另行调整,倒入培养皿中,冷却,得到固体培养基平板。Preferably, in step 2), the solid medium plate is prepared by the following method: adding 1000 mL of distilled water to 30-35 g of prefabricated nutrient agar dry powder, heating to dissolve, the pH value does not need to be adjusted separately, poured into a petri dish, cooled, and obtained Solid medium plates.

更优选的,步骤2)中,固体培养基平板由以下方法制备得到:往33g预制营养琼脂干粉中加入1000mL蒸馏水,加热溶解,pH值不需要另行调整,倒入培养皿中,冷却,得到固体培养基平板。More preferably, in step 2), the solid medium plate is prepared by the following method: add 1000 mL of distilled water to 33 g of prefabricated nutrient agar dry powder, heat to dissolve, the pH value does not need to be adjusted separately, poured into a petri dish, cooled to obtain a solid medium plate.

优选的,步骤2)中,发酵培养基包括以下重量百分比的组分:0.6-1.6%大豆蛋白胨、0.35-0.65%酵母抽提物、0.2-0.5%磷酸氢二钠、0.12-0.25%碳酸钠、0.12-0.55%硫酸镁、1.0-1.5%乳糖和95.5-97.5%水,发酵培养基pH值为6.0-6.5。Preferably, in step 2), the fermentation medium comprises the following components by weight: 0.6-1.6% soy peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate , 0.12-0.55% magnesium sulfate, 1.0-1.5% lactose and 95.5-97.5% water, the pH value of the fermentation medium is 6.0-6.5.

在工业生产的应用中,我们的目的是,单位体积内发酵生产的总酶活的活力单位最大化。环状芽孢杆菌的β-半乳糖苷酶的酶活性变化,是由细菌体内DNA、RNA和蛋白质等不同层次的非线性调控网络所控制。每轮诱变导致的菌株变异,影响的只是调控网络的局部区域,例如,β-半乳糖苷酶基因表达调控元件得到增强、β-半乳糖苷酶基因的转录产物的稳定性显著提高、β-半乳糖苷酶的酶活性口袋的关键氨基酸对应的基因序列发生正向改变。一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株的选育过程,是以发酵液的总酶活力的显著提高为导向,根据每轮诱变的菌株的β-半乳糖苷酶的总酶活性提高的实际情况,灵活设定每轮菌株的筛选标准,最终获得一种当前最优化的菌株。In industrial production applications, our aim is to maximize the activity unit of total enzyme activity produced by fermentation per unit volume. The enzymatic activity changes of β-galactosidase in Bacillus circulans are controlled by different levels of non-linear regulatory networks such as DNA, RNA and protein in bacteria. The strain variation caused by each round of mutagenesis only affects the local area of the regulatory network, for example, the β-galactosidase gene expression regulatory element is enhanced, the stability of the β-galactosidase gene transcript is significantly improved, the β-galactosidase gene - Positive changes in the gene sequence corresponding to the key amino acids of the enzymatic pocket of galactosidase. A breeding process for a Bacillus circulans strain producing high-activity β-galactosidase is guided by a significant increase in the total enzyme activity of a fermentation broth, according to the β-galactosidase of the strains of each round of mutagenesis According to the actual situation that the total enzyme activity is improved, the screening criteria of each round of strains are flexibly set, and a currently optimized strain is finally obtained.

本发明与现有技术相比,具有如下有益效果:Compared with the prior art, the present invention has the following beneficial effects:

1.本发明的一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株,能生产高发酵活力的β-半乳糖苷酶液,用于工业上催化半乳糖基转移反应来生产低聚半乳糖。1. A kind of Bacillus circulans strain of producing high activity β-galactosidase of the present invention, can produce the β-galactosidase liquid of high fermentation activity, is used for industrially catalyzed galactosyl transfer reaction to produce low Polygalactose.

2.本发明的生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株的选育方法,以1-甲基-3-硝基-1-亚硝基胍为诱变剂,经多轮诱变处理,获得生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株,1-甲基-3-硝基-1-亚硝基胍诱变剂是一种能与核酸碱基作用的双功能烷化剂,可与DNA分子的许多部位发生作用,易取代DNA分子中活泼的氢原子,使DNA分子上的碱基及核酸部分被烷化,DNA复制时导致碱基配对错误而引起突变,此外,其还能在DNA双链间形成共价键,阻碍DNA复制过程中双链的解开,从而引起突变,能大大提高突变频率。2. The breeding method of the Bacillus circulans strain for producing high-activity β-galactosidase of the present invention, using 1-methyl-3-nitro-1-nitrosoguanidine as a mutagen, after several rounds of Mutagenesis treatment to obtain a strain of Bacillus circulans that produces high activity β-galactosidase, 1-methyl-3-nitro-1-nitrosoguanidine mutagen is a mutagen that can interact with nucleic acid bases Bifunctional alkylating agent can interact with many parts of DNA molecules, easily replace active hydrogen atoms in DNA molecules, and make the bases and nucleic acid parts of DNA molecules alkylated, resulting in base pairing errors during DNA replication. Mutation, in addition, it can also form a covalent bond between the DNA double strands, which hinders the unwinding of the double strands during DNA replication, thereby causing mutations, which can greatly increase the mutation frequency.

3.该选育方法是以发酵液的总酶活力的显著提高为导向,根据每轮诱变的菌株的β-半乳糖苷酶的总酶活性提高的实际情况,灵活设定每轮菌株的筛选标准,最终获得一种当前最优化的菌株,方法灵活务实,简单易行,诱变筛选成功率高,更有利于获得正向诱变结果,获得生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株。3. The breeding method is oriented to the significant improvement of the total enzyme activity of the fermentation broth, and according to the actual situation of the increase of the total enzyme activity of β-galactosidase of the strains of each round of mutagenesis, flexibly set the total enzyme activity of each round of strains. Screening criteria, and finally obtain a currently optimized strain, the method is flexible and pragmatic, simple and easy to implement, and the success rate of mutagenesis screening is high, which is more conducive to obtaining positive mutagenesis results and obtaining a ring that produces high activity β-galactosidase. Bacillus strains.

附图说明Description of drawings

图1是实施例1的M4诱变菌株β-半乳糖苷酶酶活力检测结果图。FIG. 1 is a graph showing the results of the detection of the β-galactosidase enzyme activity of the M4 mutant strain of Example 1. FIG.

图2是实施例1的M4-81诱变菌株的菌体镜检图。FIG. 2 is a microscopic view of the microbial cells of the M4-81 mutant strain of Example 1. FIG.

图3是实施例1的M4-81诱变菌株的菌落形态图。FIG. 3 is a diagram of the colony morphology of the M4-81 mutant strain of Example 1. FIG.

具体实施方式Detailed ways

下面结合实施例对本发明做进一步说明。The present invention will be further described below in conjunction with the embodiments.

β-半乳糖苷酶酶活力的计算方法:Calculation method of β-galactosidase enzyme activity:

1.试剂配制1. Reagent preparation

(1)Z-缓冲液(1) Z-buffer

在800mL水中溶解16.1g磷酸氢二钠、5.5g磷酸二氢钠、0.75g氯化钾、0.246g硫酸镁和2.7mL 2-巯基乙醇,加入2mol/L的氢氧化钠溶液,调pH至6.0±0.05,用pH仪检测。将溶液转移到1000mL容量瓶,用水定容,混匀。Dissolve 16.1g disodium hydrogen phosphate, 5.5g sodium dihydrogen phosphate, 0.75g potassium chloride, 0.246g magnesium sulfate and 2.7mL 2-mercaptoethanol in 800mL water, add 2mol/L sodium hydroxide solution, adjust pH to 6.0 ±0.05, detected by pH meter. Transfer the solution to a 1000mL volumetric flask, make up to volume with water, and mix.

(2)邻硝基苯-β-D-吡喃半乳糖苷(ONPG)溶液(2) o-nitrophenyl-β-D-galactopyranoside (ONPG) solution

用75mL Z-缓冲液溶解250.0mg的ONPG,将溶液转入100mL容量瓶,用Z-缓冲液定容,作为底物。Dissolve 250.0 mg of ONPG with 75 mL of Z-buffer, transfer the solution to a 100-mL volumetric flask, and make up to volume with Z-buffer as a substrate.

(3)终止溶液(3) Stop solution

以水为溶剂,溶解10g碳酸钠后,转入100mL容量瓶定容。Using water as solvent, dissolve 10g of sodium carbonate, transfer to a 100mL volumetric flask and dilute to volume.

(4)测试样制备(4) Test sample preparation

准备测试酶样品,使得每mL的最终溶液中含有0.05~0.25单位的β-半乳糖苷酶。Prepare test enzyme samples such that 0.05-0.25 units of β-galactosidase are contained per mL of final solution.

2.检测步骤2. Detection steps

将一系列规格20*150mm的玻璃试管置于50±0.1℃的水浴锅中,再用移液枪分别吸0.25mL的ONPG溶液到玻璃试管中,水浴恒温。A series of glass test tubes with a size of 20*150mm were placed in a water bath at 50±0.1°C, and then 0.25mL of ONPG solution was sucked into the glass test tubes with a pipette, and the water bath was kept at a constant temperature.

迅速用移液器枪加入0.25mL的待检测样品(对照试管中加入0.25mL的水),然后启动振荡,按下秒表开始计时;振荡10min后从每个试管中吸出0.5mL的反应溶液,加入到装有0.5mL终止溶液的试管中,然后搅拌混匀。Quickly add 0.25mL of the sample to be tested with a pipette gun (add 0.25mL of water to the control test tube), then start the shaking, press the stopwatch to start timing; after shaking for 10min, suck out 0.5mL of the reaction solution from each test tube, add into a tube containing 0.5 mL of stop solution, then stir to mix.

将样品移入酶标板中,检测波长为405nm,以对照试管中的溶液作为对照,检测各样品的吸光度。The samples were transferred to the ELISA plate, the detection wavelength was 405 nm, and the solution in the control test tube was used as a control to detect the absorbance of each sample.

3.标准曲线的绘制3. Drawing of the standard curve

将139.0mg邻-硝基苯酚(ONP)转进1000mL容量瓶,转之前用95%酒精溶解,用水定容,混匀。用移液枪分别吸2.5、5、12.5和25mL的溶液至100mL的容量瓶,用10wt%的碳酸钠溶液定容,混匀。这些溶液中每mL分别含有0.025、0.05、0.125、0.25微摩尔的ONP。Transfer 139.0 mg of o-nitrophenol (ONP) into a 1000 mL volumetric flask, dissolve with 95% alcohol before transferring, make up to volume with water, and mix well. Aspirate 2.5, 5, 12.5 and 25 mL of the solution into a 100 mL volumetric flask with a pipette respectively, make up to the volume with 10 wt% sodium carbonate solution, and mix well. These solutions contained 0.025, 0.05, 0.125, 0.25 micromoles of ONP per mL, respectively.

用一个1cm的石英管装上标准溶液,在波长405nm条件下测吸光度,以水为对照,ONP的浓度为横坐标,各浓度标准物的吸光度为纵坐标,可以得到一条通过原点的直线。A 1cm quartz tube was used to install the standard solution, and the absorbance was measured at a wavelength of 405nm. Taking water as the control, the concentration of ONP was the abscissa, and the absorbance of each concentration standard was the ordinate, and a straight line passing through the origin could be obtained.

4.计算4. Calculation

经计算分析得到标准曲线:Y=4.0688X+0.0152,R2=0.9991The standard curve is obtained by calculation and analysis: Y=4.0688X+0.0152, R 2 =0.9991

(Y为405nm处光吸收值,X为ONP的浓度)(Y is the light absorption value at 405nm, X is the ONP concentration)

β-半乳糖苷酶活力测定方法为:一个乳糖酶单位(LacU)被定义为在本方法条件下每分钟释放1微摩尔ONP所需的酶活量。The β-galactosidase activity assay method is as follows: one lactase unit (LacU) is defined as the amount of enzyme activity required to release 1 micromol of ONP per minute under the conditions of this method.

酶活力(U/mL)=4*DF*X/10Enzyme activity (U/mL)=4*DF*X/10

5.将0.25mL稀释酶液中的酶活折算为1mL的酶活性;5. Convert the enzyme activity in 0.25mL diluted enzyme solution to 1mL enzyme activity;

DF:稀释倍数;DF: dilution factor;

X:ONP浓度(μmol);X: ONP concentration (μmol);

10:反应时间,10min。10: reaction time, 10 min.

实施例中所用到的种子培养基由以下方法制备得到:往15-20g预制营养肉汤干粉中加入1000mL蒸馏水,加热溶解,pH值不需要另行调整,冷却,得到种子培养基。The seed culture medium used in the examples was prepared by the following method: adding 1000 mL of distilled water to 15-20 g of prefabricated nutrient broth dry powder, heating to dissolve, the pH value does not need to be adjusted separately, and cooling to obtain the seed culture medium.

无菌磷酸缓冲溶液为pH值为6的0.01mol/L磷酸缓冲溶液。The sterile phosphate buffer solution is a 0.01 mol/L phosphate buffer solution with a pH value of 6.

固体培养基平板由以下方法制备得到:往30-35g预制营养琼脂干粉中加入1000mL蒸馏水,加热溶解,pH值不需要另行调整,倒入培养皿中,冷却,得到固体培养基平板。The solid medium plate is prepared by the following method: add 1000 mL of distilled water to 30-35 g of prefabricated nutrient agar dry powder, heat to dissolve, the pH value does not need to be adjusted separately, pour it into a petri dish, and cool to obtain a solid medium plate.

发酵培养基包括以下重量百分比的组分:0.6-1.6%大豆蛋白胨、0.35-0.65%酵母抽提物、0.2-0.5%磷酸氢二钠、0.12-0.25%碳酸钠、0.12-0.55%硫酸镁、1.0-1.5%乳糖和95.5-97.5%水,发酵培养基pH值为6.0-6.5。The fermentation medium includes the following components by weight: 0.6-1.6% soy peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate, 0.12-0.55% magnesium sulfate, 1.0-1.5% lactose and 95.5-97.5% water, fermentation medium pH 6.0-6.5.

实施例1Example 1

1.生产高活力β-半乳糖苷酶的环状芽孢杆菌的初步筛选,包括以下步骤:1. Preliminary screening of Bacillus circulans producing high activity β-galactosidase, including the following steps:

1)出发菌株活化:将环状芽孢杆菌(Bacillus circulars,购自美国菌种保藏中心(ATCC,编号为31382)作为出发菌株,进行划线培养,出发菌株经过三次斜面营养琼脂培养基活化培养后,接种于100mL发酵培养基(发酵培养基的组成为:1.2wt%的大豆蛋白胨、0.65wt%的酵母抽提物、0.35wt%的磷酸氢二钠、0.25wt%的碳酸钠、0.55wt%的硫酸镁、1.5wt%的乳糖和95.50wt%的水,pH6.0-6.5),37℃下摇瓶培养48h后离心,取上清液,进行β-半乳糖苷酶酶酶活力测定,其测定结果如表1所示:1) Activation of the starting strain: Bacillus circulars (Bacillus circulars, purchased from the American Culture Collection Center (ATCC, No. 31382) was used as the starting strain for streak culture. , inoculated in 100mL fermentation medium (the composition of fermentation medium is: 1.2wt% soybean peptone, 0.65wt% yeast extract, 0.35wt% disodium hydrogen phosphate, 0.25wt% sodium carbonate, 0.55wt% of magnesium sulfate, 1.5wt% lactose and 95.50wt% water, pH 6.0-6.5), incubate the flask at 37°C for 48h, centrifuge, take the supernatant, and measure the enzyme activity of β-galactosidase. The measurement results are shown in Table 1:

表1环状芽孢杆菌出发菌株β-半乳糖苷酶酶活力Table 1 β-galactosidase enzyme activity of Bacillus circulans starting strain

Figure BDA0001913493530000061
Figure BDA0001913493530000061

2)挑取一环斜面菌种于种子培养基(预制营养肉汤培养基)中,在37℃培养6h后,10000rpm离心10min,收集菌体,重新悬浮于pH6.0磷酸缓冲溶液中,经脱脂棉过滤后置于装有玻璃珠的小三角瓶中,振荡均匀,制备成单细胞菌悬液浓度为105~107个/mL。2) Pick a ring of slanted bacteria in the seed medium (prefabricated nutrient broth medium), culture at 37°C for 6 hours, centrifuge at 10,000 rpm for 10 minutes, collect the bacterial cells, resuspend in pH 6.0 phosphate buffer solution, and pass After filtration with absorbent cotton, it was placed in a small triangular flask equipped with glass beads, shaken evenly, and prepared into a single-cell bacterial suspension with a concentration of 10 5 to 10 7 cells/mL.

3)取1mL浓度为0.25、0.5、0.75、1.0mg/mL的1-甲基-3-硝基-1-亚硝基胍丙酮溶液,分别加入到1mL菌悬液中,进行诱变处理30min,得到诱变菌液Ⅰ,取100μL诱变菌液Ⅰ涂布固体培养基平板,在37℃培养48h,进行菌落计数,计算致死率Ⅰ,确定最佳诱变浓度为0.5mg/mL。结果如表2所示。3) Take 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with concentrations of 0.25, 0.5, 0.75, and 1.0 mg/mL, add them to 1 mL of bacterial suspension, and perform mutagenesis for 30 min , get the mutagenic bacterial solution I, take 100 μL of the mutagenic bacterial solution I to coat the solid medium plate, incubate at 37 ℃ for 48 h, count the colonies, calculate the lethality I, and determine the optimal mutagenic concentration to be 0.5 mg/mL. The results are shown in Table 2.

表2 1-甲基-3-硝基-1-亚硝基胍诱变浓度Table 2 Mutagenic concentration of 1-methyl-3-nitro-1-nitrosoguanidine

诱变浓度(mg/mL)Mutagenic concentration (mg/mL) 0.250.25 0.50.5 0.750.75 11 致死率Ⅰ(%)Lethality I (%) 82.682.6 99.099.0 99.999.9 99.999.9

4)取1mL浓度为0.5mg/mL的1-甲基-3-硝基-1-亚硝基胍丙酮溶液,加入到1mL菌悬液中,37℃条件下,分别振荡15、30、45、60min,得到诱变菌液Ⅱ,取100μL诱变菌液Ⅱ涂布固体培养基平板,在37℃培养48h,计算致死率Ⅱ,确定最佳诱变时间为30min。结果如表3所示。4) Take 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with a concentration of 0.5 mg/mL, add it to 1 mL of bacterial suspension, and shake for 15, 30, and 45 minutes at 37°C, respectively. , 60min, to get the mutagenic bacteria solution II, take 100 μL of the mutagenic bacteria solution II to coat the solid medium plate, cultivate at 37 ℃ for 48h, calculate the lethality II, and determine the optimal mutagenesis time as 30min. The results are shown in Table 3.

表3 1-甲基-3-硝基-1-亚硝基胍诱变时间Table 3 Mutagenesis time of 1-methyl-3-nitro-1-nitrosoguanidine

诱变时间(min)Mutagenesis time (min) 1515 3030 4545 6060 致死率Ⅱ(%)Lethality II (%) 80.380.3 99.099.0 99.999.9 99.999.9

5)取100μL步骤4)中诱变处理30min的诱变菌液Ⅱ,涂布固体培养基平板,在37℃培养48h。5) Take 100 μL of the mutagenized bacterial solution II treated for 30 min in step 4), spread it on a solid medium plate, and cultivate at 37° C. for 48 h.

6)待长出菌落后,挑选长势好、菌落大的菌落,用无菌牙签逐个点种标记的菌落于含有发酵培养基(发酵培养基的组成与步骤1)的相同)的96孔板(2mL深孔板,每孔装液量为800uL),将96孔板置于摇床中培养,37℃、湿度80%、300r/min培养48h,得到M1诱变菌株,用酶标仪检测M1诱变菌株的β-半乳糖苷酶酶活力,计算M1诱变菌株的诱变效果。6) After the colony is grown, select the colony with good growth and large colony, and use a sterile toothpick to seed the labeled colonies one by one in a 96-well plate (the composition of the fermentation medium is the same as that of step 1). 2mL deep-well plate, the volume of each well is 800uL), place the 96-well plate in a shaker and cultivate it at 37°C, 80% humidity, 300r/min for 48h to obtain the M1 mutant strain, and use a microplate reader to detect M1 The β-galactosidase enzyme activity of the mutant strain was calculated, and the mutagenic effect of the M1 mutant strain was calculated.

7)将步骤6)挑取出来的β-半乳糖苷酶酶活力的提高幅度大于30%的M1诱变菌株进行复检,移种于固体培养基平板,培养48h,再移接于96孔板发酵培养,每株做5个平行验证,在37℃培养48h,对β-半乳糖苷酶酶活力进行测定,其中M1-38、M1-63的酶活力提高最为显著,分别比出发菌株提升30.35%和30.22%;7) Re-examine the M1 mutant strains whose β-galactosidase enzyme activity increased by more than 30% picked out in step 6), transplanted to a solid medium plate, cultured for 48 hours, and then transplanted to 96 wells The β-galactosidase enzyme activity was measured, and the enzyme activities of M1-38 and M1-63 were most significantly improved, which were higher than the starting strains respectively. 30.35% and 30.22%;

2.生产高活力β-半乳糖苷酶的环状芽孢杆菌的遗传稳定性分析2. Genetic stability analysis of Bacillus circulans producing high activity β-galactosidase

将M1-38、M1-63诱变菌株接入斜面固体培养基中,在37℃培养48h,加入9mL无菌磷酸缓冲溶液,洗脱、稀释涂布固体培养基平板,待长出菌落后,挑选长势好、菌落大的单菌落,进行连续7次传代培养,并检测菌株的β-半乳糖苷酶酶活力,确定菌株在生产高活力β-半乳糖苷酶方面的遗传稳定性。其中M1-38具有良好的遗传稳定性,选取M1-38诱变菌株作为诱变母本,具体数据见表4。The M1-38 and M1-63 mutant strains were inserted into the slanted solid medium, cultured at 37°C for 48 hours, 9 mL of sterile phosphate buffer solution was added, eluted, diluted and coated on a solid medium plate, and after the colonies grew, A single colony with good growth and large colony was selected and subcultured for 7 consecutive times, and the β-galactosidase enzyme activity of the strain was detected to determine the genetic stability of the strain in producing high activity β-galactosidase. Among them, M1-38 has good genetic stability, and the M1-38 mutant strain was selected as the mutagenic parent. The specific data are shown in Table 4.

表4 M1代遗传稳定性验证Table 4 M1 generation genetic stability verification

酶活力(U/mL)Enzyme activity (U/mL) 原代original 第1代1st generation 第2代2nd generation 第3低3rd lowest 第4代4th generation 第5代5th generation 第6代6th generation 第7代7th generation M1-38M1-38 19.9419.94 19.9219.92 19.9819.98 19.8919.89 19.9519.95 19.9419.94 19.9619.96 19.9119.91 M1-63M1-63 18.8218.82 19.4519.45 17.8817.88 15.7115.71 15.2415.24 14.9514.95 15.3615.36 14.8114.81

3.生产高活力β-半乳糖苷酶的环状芽孢杆菌多轮次诱变3. Multiple rounds of mutagenesis of Bacillus circulans producing high activity β-galactosidase

选取遗传性稳定的M1-38诱变菌株为诱变母本,挑取一环斜面菌种于新鲜的种子培养基中,37℃培养6h后,10000rpm离心10min,收集菌体,重新悬浮于pH6.0磷酸缓冲溶液中,经脱脂棉过滤后置于装有玻璃珠的小三角瓶中,振荡均匀,制备成单细胞菌悬液浓度为105~107个/mL,取1mL浓度为0.5mg/mL的1-甲基-3-硝基-1-亚硝基胍丙酮溶液,加入到1mL菌悬液中,处理30min,将诱变菌液涂布固体培养基平板培养,挑选长势好的单菌落,利用96孔板培养体系,发酵培养并检测菌株生物量以及β-半乳糖苷酶酶活力。重复诱变步骤3次,得到后代诱变菌株分别标记为M2、M3、M4。经过4轮次1-甲基-3-硝基-1-亚硝基胍诱变剂处理,最终筛选出M4-81诱变菌株为生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株,M4代生产的酶的活力见图1,横轴是菌株编号,纵轴是酶活力。Select the genetically stable M1-38 mutagenic strain as the mutagenic parent, pick a ring of slanted strains in fresh seed medium, cultivate at 37°C for 6 hours, centrifuge at 10,000 rpm for 10 minutes, collect the bacteria, and resuspend in pH 6 .0 phosphate buffer solution, filtered with absorbent cotton, placed in a small triangular flask with glass beads, shaken evenly, and prepared into a single-cell bacterial suspension with a concentration of 10 5 to 10 7 /mL, taking 1 mL of the concentration of 0.5 mg /mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution was added to 1mL of bacterial suspension, treated for 30min, the mutagenized bacterial solution was coated with solid medium for plate culture, and good growth was selected. A single colony was fermented and cultured using a 96-well plate culture system and the biomass and β-galactosidase activity of the strain were detected. Repeat the mutagenesis step 3 times to obtain progeny mutagenized strains labeled as M2, M3, and M4, respectively. After 4 rounds of 1-methyl-3-nitro-1-nitrosoguanidine mutagen treatment, the M4-81 mutant strain was finally screened out as a Bacillus circus strain that produces high activity β-galactosidase , the activity of the enzyme produced by the M4 generation is shown in Figure 1, the horizontal axis is the strain number, and the vertical axis is the enzyme activity.

由图1可知,第四代突变株M4-81,酶活力为39.86U/mL,是出发菌株所产酶的活力(环状芽孢杆菌ATCC,No.31382)的2.60倍,是当前最优化的菌株。将该菌株命名为环状芽孢杆菌QHT-310-M481(Bacillus circulans QHT-310-M481),于2018年10月22日在中国典型培养物保藏中心保藏,保藏编号为CCTCC NO:M 2018699,保藏地址为中国武汉市武汉大学。It can be seen from Figure 1 that the enzyme activity of the fourth-generation mutant M4-81 is 39.86 U/mL, which is 2.60 times that of the enzyme produced by the starting strain (Bacillus circulans ATCC, No. 31382), and is currently the most optimized. strains. The strain was named Bacillus circulans QHT-310-M481 (Bacillus circulans QHT-310-M481), and it was deposited in the China Center for Type Culture Collection on October 22, 2018, and the deposit number is CCTCC NO: M 2018699. The address is Wuhan University, Wuhan, China.

所述的一种生产高活力β-半乳糖苷酶的环状芽孢杆菌菌株的形态如图2和图3所示,菌落为亮黄色,形状规则,表面湿润,边缘整齐;菌体杆状,单个排列,芽孢端生,包囊不膨大,革兰氏阳性。The morphology of the described Bacillus circulans strain producing high activity β-galactosidase is shown in Figure 2 and Figure 3, the colony is bright yellow, the shape is regular, the surface is moist, and the edges are neat; Single arrangement, spore terminal, cyst not inflated, Gram-positive.

Claims (1)

1.一种环状芽孢杆菌(Bacillus circulans)菌株QHT-310-M481,其于2018年10月22日在中国典型培养物保藏中心保藏,保藏编号为CCTCC NO:M 2018699,保藏地址为中国武汉市武汉大学。1. A Bacillus circulans strain QHT-310-M481, which was deposited in the China Center for Type Culture Collection on October 22, 2018, with the deposit number CCTCC NO: M 2018699, and the deposit address is Wuhan, China Wuhan University.
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