阅读说明：本技术 一种高产脂肪酶酶活菌株的筛选方法 (Screening method of high-yield lipase live strains ) 是由 蔡志强 张宇 纪元 郭静 朱孝霖 杨广花 于 2019-10-16 设计创作，主要内容包括：本发明属于生物发酵工程领域,具体公开了一种高产脂肪酶酶活菌株的筛选方法。采用甲基磺酸乙酯(EMS)诱变菌株,在致死率为70-90％的条件下成功筛选出一株酶活高达73.33U/g的产酶菌株。并通过后续优化试验确定菌株最适的培养基组分以及生长条件：豆粕20g,初始含水量68％,葡萄糖含量3％,蛋白胨含量1％,猪油含量5％,CaCl<Sub>2</Sub> 0.04％,FeCl<Sub>3</Sub> 0.04％,接种量4％,培养温度28℃,培养3天后酶活高达93.33U/g,大大提高了菌株产酶能力,达到理想目标。(The invention belongs to the field of biological fermentation engineering, and particularly discloses a screening method of a high-yield lipase live strain. An enzyme-producing strain with the enzyme activity as high as 73.33U/g is successfully screened out by adopting an Ethyl Methane Sulfonate (EMS) mutagenesis strain under the condition that the lethality is 70-90%. And determining the optimal culture medium components and growth conditions of the strain through subsequent optimization experiments: 20g of soybean meal, 68 percent of initial water content, 3 percent of glucose content, 1 percent of peptone content, 5 percent of lard oil content and CaCl 2 0.04％，FeCl 3 0.04 percent, 4 percent of inoculation amount, 28 ℃ of culture temperature, and the enzyme activity after 3 days of culture is as high as 93.33U/g, thereby greatly improving the enzyme production capability of the strain and achieving the ideal target.)

1. A high-yield lipase live strain is classified and named as Penicillium sp.Y-21, the preservation number is CGMCC NO.18556, and the enzyme activity is 93.33U/g.

2. The method for screening the live high-yield lipase strains according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

(1) 2ml of spore suspension and Ethyl Methanesulfonate (EMS) are uniformly mixed to obtain a mixed solution, and oscillation mutagenesis reaction is carried out on the mixed solution;

(2) addition of Na after mutagenesis₂S₂O₃Eliminating mutagen, terminating reaction, standing, centrifuging to obtain treating liquid, diluting the treating liquid and spreading the diluted treating liquid onto plate for culture;

(3) and selecting the strains, and carrying out re-screening and solid fermentation culture to obtain 1 strain with high lipase enzyme activity.

3. The method for screening a live high-yield lipase strain according to claim 2, wherein the method comprises the steps of: the volume concentration of the ethyl methanesulfonate in the mixed solution in the step (1) is 1-5%, and the mutagenesis reaction time is 0-60 min.

4. The method for screening a live high-yield lipase strain according to claim 2, wherein the method comprises the steps of: adding Na as described in step (2)₂S₂O₃The volume of (b) is the same as the amount of EMS added.

5. The method for screening a live high-yield lipase strain according to claim 2, wherein the method comprises the steps of: and (3) diluting the treatment solution in the step (2) by 100 times by using sterile normal saline.

6. The method for screening a live high-yield lipase strain according to claim 2, wherein the method comprises the steps of: in the step (3), the bacterial strains with 70-90% of fatality rate need to be screened again.

7. The method for screening a live high-yield lipase strain according to claim 2, wherein the method comprises the steps of: the culture medium for solid fermentation culture in the step (3) comprises the following components: taking 20g of rice bran, bean cake powder, bran, bean pulp, cottonseed cake powder or rapeseed cake powder as a solid matrix, taking the initial water content as 60-75% of the total mass of the culture medium, taking glucose, fructose, lactose or maltose as a single carbon source, taking the mass content as 1-8% of the solid matrix, taking beef extract, peptone and NH₄Cl、KNO₃As fermentation nitrogen source, 1% by mass of solid matrix, 5% by mass of induction oil (olive oil, soybean oil, coconut oil, grease, lard oil, MgSO 5%₄、CaCl₂、FeCl₃、K₂HPO₄、BaCl₂As metal ions, the mass content of the metal ions is 0.02-0.1% of that of the solid matrix; the conditions of fermentation culture are as follows: the inoculation amount is 2-10%, the culture temperature is 28 ℃, and the culture time is 3 days.

8. The method for screening a live high-yield lipase strain according to claim 2, wherein the method comprises the steps of: the culture medium for solid fermentation culture in the step (3) comprises the following components: taking 20g of bean pulp as a solid substrate, wherein the initial water content is 68 percent of the total mass of the culture medium, the glucose content is 3 percent of the solid substrate, the peptone content is 1 percent of the solid substrate, and the lard oilThe content of CaCl is 5% of the solid matrix₂0.04% of solid matrix, FeCl₃Is 0.04 percent of the solid matrix, and the growth conditions of the fermentation culture are as follows: the inoculation amount is 4 percent, the culture temperature is 28 ℃, and the culture time is 3 days.

Technical Field

The invention belongs to the field of biological fermentation engineering, and particularly relates to a screening method of a high-yield lipase live strain.

Background

Lipase is one of important industrial enzyme preparations, can catalyze reactions such as lipolysis, ester exchange, ester synthesis and the like, and is widely applied to the industrial fields such as energy, oil processing, food, medicine and the like. Lipases are ubiquitous in nature in plant seeds, animal tissues and microorganisms, with industrial lipases being sourced from microorganisms. Lipase activity was first detected by Eberer in rabbit pancreas at the beginning of 1834. The lipase in the microorganism has the advantages of rich content, various varieties, wide distribution, adaptability superior to that of animals and plants and the like, so that the microorganism is a main mode for industrial enzyme production at present. In the microbial fermentation industry, strains are critical factors for directly determining whether product quality and yield reach the standard, and strains generally separated from nature have very low product accumulation capacity and cannot meet the requirement of large-scale production, so that the strains need to be modified or improved, namely breeding. Three main mutagenesis methods are used, namely physical, chemical and composite mutagenesis.

In the modern society, due to the rapid development of food industry, consumer awareness of food safety and health is increasing, and the demand for flour and bean products is increasing. If good flour is produced, high-quality raw materials are needed, so that catalytic enzyme needs to be added in food treatment, the catalytic efficiency of the enzyme is particularly high and is more than 100 times higher than that of a common catalyst, the baking quality of bread can be improved, and the quality guarantee period of products can be prolonged.

In addition, with the reduction of social energy reserves, people are continuously developing alternative fuels such as engine oil, gasoline and the like, wherein the biodiesel is a clean ideal alternative fuel. Biodiesel is a fatty acid ester prepared from animal or vegetable fats and oils and short-chain alcohols. Up to now, the preparation of diesel oil still has many defects, which cause many bad influences on the environment, lipase can catalyze the reaction, and the reaction condition is mild, and a series of side reactions can not occur. Therefore, it is very important to develop a process for preparing biodiesel by using lipase.

Disclosure of Invention

The invention aims to solve the problems that the lipase activity is generally low in China at present and the industrial production of an enzyme preparation cannot be carried out, so that a set of mutagenesis method aiming at Penicillium sp.Y-21 and optimization of solid state fermentation conditions are worked out, and finally the enzyme activity of the strain is improved by 91.76% to 93.33U/g compared with 48.67U/g of the original strain.

In order to achieve the above objects, the present invention employs Ethyl Methanesulfonate (EMS) chemical mutagenesis method to mutate strains, and the mutagenesis technology is performed by the following steps:

(1) 2ml of spore suspension is evenly mixed with Ethyl Methane Sulfonate (EMS) and then oscillation mutagenesis reaction is carried out;

(2) addition of Na after mutagenesis₂S₂O₃Eliminating mutagen, terminating reaction, diluting certain amount of treated liquid with sterile physiological saline solution by 100 times, and spreading onto plate for culture.

(3) Selecting the strains to carry out re-screening solid fermentation culture to obtain 1 strain Penicillium sp.Y-21 with high lipase activity.

The preparation method of the spore suspension in the step (1) comprises the following steps:

culturing Penicillium sp in seed culture medium for 2 days, adding 2ml seed liquid into centrifuge tube, centrifuging at 3,000rpm for 5min, discarding supernatant, adding physiological saline, washing for 2 times, centrifuging at 3,000rpm for 5min, discarding supernatant, and adding 5ml phosphate buffer to obtain spore suspension.

Wherein, the composition of the seed culture medium is as follows: peptone 5g/L, glucose 10g/L, MgSO₄ 0.1g/L，K₂HPO₄1g/L and 20g/L of agar powder.

The composition of the phosphate buffer (pH 7.5) was as follows: KH (Perkin Elmer)₂PO₄ 3.92g/L，Na₂HPO₄·12H₂O 79.24g/L。

The volume concentration of the ethyl methanesulfonate in the mixed solution is 1-5%, the reaction time is 0-60min, and NaS added for stopping the reaction₂O₃The volume amount is the same as the added amount of EMS.

The solid fermentation culture medium in the step (2) comprises the following components: 20g of solid matrix (rice bran, bean cake powder, bran, bean pulp, cottonseed cake powder),Rapeseed cake powder), the initial water content is 60-75% of the total mass of the culture medium, the content of single carbon sources (glucose, fructose, lactose and maltose) is 1-8% of the total mass of the solid substrate, and fermentation nitrogen sources (beef extract, peptone and NH)₄Cl、KNO₃) The content of inducing oil (oleum Olivarum, soybean oil, oleum Cocois, oil and lard) is 1% of the total mass of the solid matrix, and the content of metal ion (MgSO 2)₄、CaCl₂、FeCl₃、K₂HPO₄、BaCl₂) The content is 0.02-0.1% of the total mass of the solid matrix.

The conditions of solid fermentation culture are as follows: the inoculation amount is 2-10%, the culture temperature is 28 ℃, and the culture time is 3 days.

The optimal solid fermentation culture medium comprises the following components: taking 20g of bean pulp as a solid substrate, wherein the initial water content is 68 percent of the total mass of the culture medium, the glucose content is 3 percent of the solid substrate, the peptone content is 1 percent of the solid substrate, the lard content is 5 percent of the solid substrate, and CaCl₂0.04% of solid matrix, FeCl₃Is 0.04 percent of the solid matrix, and the growth conditions of the fermentation culture are as follows: the inoculation amount is 4 percent, the culture temperature is 28 ℃, and the culture time is 3 days.

The invention also provides an optimization scheme for the strain fermentation medium after mutagenesis is finished. And establishing a single-factor test and an orthogonal test by selecting a solid matrix, a carbon-nitrogen source and the concentration, the water content, the inoculation amount, metal ions and the concentration thereof and an oil inducer in the culture medium components so as to obtain the culture medium components with the highest enzyme production of the strains. Meanwhile, the invention adopts solid fermentation to culture the strains, so that the method has the advantages of simple and convenient operation, low energy consumption, easily controlled fermentation process, relatively low requirement on sterility, difficult occurrence of large-area pollution and the like in the operation process.

Drawings

FIG. 1 is a fermentation growth graph of Penicillium sp Y-21 on different solid substrates;

FIG. 2 is a graph showing the effect of inoculum size on enzyme production by a strain;

FIG. 3 is a graph showing the effect of initial water content on enzyme production by a strain;

FIG. 4 is a graph showing the effect of carbon source on enzyme production by a strain;

FIG. 5 is a graph showing the effect of carbon source concentration on enzyme production by a strain;

FIG. 6 is a graph showing the effect of nitrogen source on enzyme production by a strain;

FIG. 7 is a graph showing the effect of nitrogen source concentration on enzyme production by a strain;

FIG. 8 is a graph showing the effect of metal ions on enzyme production by a strain;

FIG. 9 shows the metal ion concentration (Ca)²⁺Concentration) on the enzyme production of the strain;

FIG. 10 shows the metal ion concentration (Fe)³⁺Concentration) on the enzyme production of the strain;

FIG. 11 shows the effect of oil-and-fat inducers on enzyme production by the strains.

Detailed Description

The technical solutions of the present invention are further described in detail with reference to the drawings and specific examples, which are provided for illustrative purposes only and do not limit the present invention in any way. The reagents adopted by the invention are all commercially available analytical purifications or more, and the strains used in the test are laboratory preservation strains of Changzhou university. The method for determining the lipase activity (fresh starter activity) adopts an indicator titration method, and the specific method is published in GB/T23535-one 2009 issued by the national standard of the people's republic of China.