阅读说明：本技术 高产果胶酶酶活菌株的诱变方法及其固态发酵条件的优化 (Mutagenesis method of high-yield pectinase enzyme activity strain and optimization of solid state fermentation conditions thereof ) 是由 蔡志强 邢新荣 胡卫华 于 2021-03-12 设计创作，主要内容包括：本发明公开了高产果胶酶酶活菌株的诱变方法及其固态发酵条件的优化,采用甲基磺酸乙酯(EMS)化学诱变法来突变菌株,其特征在于：该诱变方法包括以下步骤：将一定量的孢子悬液与EMS混合振荡反应一段时间后,NaS-(2)O-(3)终止反应,取一定量处理液稀释涂布到平板中进行培养；该高产果胶酶酶活菌株的诱变方法及其固态发酵条件的优化,在致死率为85.8％的条件下成功筛选出一株酶活高达19000U/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％,CaCl-(2)0.04％,FeCl-(3)0.04％,接种量4％,培养温度38℃,培养5天后酶活高达2200U/g,大大提高了菌株产酶能力,达到理想目标。(The invention discloses a mutagenesis method of a high-yield pectinase enzyme activity strain and optimization of solid state fermentation conditions thereof, and the strain is mutated by adopting an Ethyl Methane Sulfonate (EMS) chemical mutagenesis method, which is characterized in that: the mutagenesis method comprises the following steps: after a certain amount of spore suspension is mixed with EMS and subjected to oscillation reaction for a period of time, NaS 2 O 3 Stopping reaction, diluting a certain amount of treatment solution, and coating the diluted treatment solution on a flat plate for culture; the mutagenesis method of the high-yield pectinase enzyme activity strain and the optimization of the solid state fermentation condition successfully screen out the enzyme activity strain under the condition that the lethality rate is 85.8 percentUp to 19000U/g of enzyme-producing strain. 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 and CaCl 2 0.04％，FeCl 3 0.04 percent, 4 percent of inoculation amount, 38 ℃ of culture temperature, and the enzyme activity after 5 days of culture is as high as 2200U/g, thereby greatly improving the enzyme production capability of the strain and achieving the ideal target.)

1. The mutagenesis method of the high-yield pectinase enzyme activity strain adopts an Ethyl Methane Sulfonate (EMS) chemical mutagenesis method to mutate the strain, and is characterized in that: the mutagenesis method comprises the following steps:

step one, mixing a certain amount of spore suspension with EMS and oscillatingAfter a certain period of time, NaS₂O₃Stopping reaction, diluting a certain amount of treatment solution, and coating the diluted treatment solution on a flat plate for culture;

and step two, selecting 23 strains with 70-90% of lethality rate for secondary screening culture to obtain 1 strain Peniciliums sp.Y-21 with high pectinase enzyme activity.

2. The mutagenesis method of the high pectinase enzyme activity producing strain according to claim 1, which comprises the following steps: the EMS concentration in the step one is 1-5%, the reaction time is 0-60min, and the added NaS2O3 volume amount for terminating the reaction is the same as the added EMS amount.

3. The mutagenesis method of the high pectinase enzyme activity producing strain according to claim 1, which comprises the following steps: in the second step, the bacterial strain with 70-90% of fatality rate needs to be screened again.

4. The optimization of the solid state fermentation conditions of the high pectinase enzyme activity strain according to claim 1, through single factor test and orthogonal test optimization, an optimal enzyme production system is obtained, and the optimization process uses a solid fermentation culture medium, which is characterized in that: the solid fermentation medium takes 20g of bean pulp and 15g of bran as solid substrates, the initial water content is 50%, the glucose content is 2%, and CaCl is added₂0.04％，FeCl₃0.04 percent, the inoculation amount is 4 percent, the culture temperature is 38 ℃, and the culture time is 5 days.

Technical Field

The invention relates to the field of biological fermentation engineering, in particular to a mutagenesis method of a high-yield pectinase enzyme activity strain and optimization of solid state fermentation conditions thereof.

Background

Pectin is a high molecular weight polysaccharide compound that is present in almost all plants as part of the cellular structure. It is mainly prepared by condensation of galacturonic acid and methyl ester thereof, and also contains rhamnose, arabinose, galactose and the like. Depending on the way of extraction from plant cell walls, pectins can be classified into three categories: water-soluble pectin, chelating agent soluble pectin and protopectin. Pectinase, as one of four enzyme preparations in the world, can catalyze reactions such as hydrolysis and the like, is widely applied to food industries such as fruit juice clarification, juice extraction, wine brewing, oil extraction and the like, is developed in the fields of spinning, medicines, paper making, environment, biotechnology, feeds and the like, and is widely applied to the fields of food, medicines, feeds and the like. Pectinase for food industry is sourced from microorganisms, in recent years, the research on pectinase at home and abroad enters the molecular biology level, pectinase genes are cloned and sequenced from microorganisms of various genera, and the structure, function, regulation and control of the pectinase genes and the structure and performance of expressed products of the pectinase genes are explored. China has a great demand on pectinase, but the development of the pectinase in China is slow, and the pectinase is usually used singly, so that the application range and the effect of the pectinase are limited.

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.

Therefore, we propose a mutagenesis method of the high-yield pectinase enzyme activity strain and optimization of the solid state fermentation conditions thereof.

Disclosure of Invention

Aiming at the defects of the background technology, the invention provides a mutagenesis method of a high-yield pectinase enzyme activity strain and optimization of solid state fermentation conditions thereof, and solves the problems brought forward by the background technology.

The invention provides the following technical scheme: the mutagenesis method of the high-yield pectinase enzyme activity strain and the optimization of the solid state fermentation condition thereof adopt an Ethyl Methane Sulfonate (EMS) chemical mutagenesis method to mutate the strain, and are characterized in that: the mutagenesis method comprises the following steps:

step one, mixing a certain amount of spore suspension with EMS, oscillating and reacting for a period of time, and then NaS₂O₃Stopping reaction, diluting a certain amount of treatment solution, and coating the diluted treatment solution on a flat plate for culture;

and step two, selecting 23 strains with 70-90% of lethality rate for secondary screening culture to obtain 1 strain Peniciliums sp.Y-21 with high pectinase enzyme activity.

Preferably, the EMS concentration in the step one is 1-5%, the reaction time is 0-60min, and the added NaS2O3 volume amount for terminating the reaction is the same as the added EMS amount.

Preferably, in the second step, the strain with 70-90% of fatality rate needs to be screened again.

Optimizing the solid-state fermentation conditions of the high-yield pectinase enzyme activity strain, and optimizing through a single-factor test and an orthogonal test to obtain an optimal enzyme production system, wherein a solid fermentation culture medium is used in the optimization process, takes 20g of bean pulp and 15g of bran as solid matrixes, and has the initial water content of 50%, the glucose content of 2%, CaCl and the like₂0.04％，FeCl₃0.04 percent, the inoculation amount is 4 percent, the culture temperature is 38 ℃, and the culture time is 5 days.

The invention has the following beneficial effects: the mutagenesis method of the high-yield pectinase enzyme activity strain and the optimization of the solid state fermentation condition successfully screen out an enzyme-producing strain with the enzyme activity as high as 19000U/g under the condition that the lethality is 85.8 percent. And determining the optimal culture medium components and growth conditions of the strain through subsequent optimization experiments: 20g of soybean meal, 68% of initial water content, 3% of glucose, 1% of peptone, 20.04% of CaCl, 30.04% of FeCl30, 4% of inoculation amount, and 38 ℃ of culture temperature, wherein the enzyme activity is as high as 2200U/g after 5 days of culture, so that the enzyme production capacity of the strain is greatly improved, and an ideal target is achieved;

meanwhile, a single-factor test and an orthogonal test are established by selecting a solid matrix, a carbon-nitrogen source and the concentration, the water content, the inoculation amount, a metal ion and the concentration thereof and an oil inducer in the culture medium components, so that the culture medium component with the highest enzyme production of the strain is obtained.

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 schematic diagram showing the effect of inoculum size on enzyme production by a strain;

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

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

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

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

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

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

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The drawings in the embodiments of the invention: the different types of hatching in the figures are not given the national standards, do not require the material of the elements, and distinguish between cross-sectional views of the elements in the figures.

Referring to fig. 1-8, the mutagenesis method of the high-yield pectinase enzyme activity strain and the optimization of the solid state fermentation condition adopt Ethyl Methane Sulfonate (EMS) chemical mutagenesis method to mutate the strain, which is characterized in that: the mutagenesis method comprises the following steps:

step one, mixing a certain amount of spore suspension with EMS, oscillating and reacting for a period of time, and then NaS₂O₃Stopping reaction, diluting a certain amount of treatment solution, and coating the diluted treatment solution on a flat plate for culture;

and step two, selecting 23 strains with 70-90% of lethality rate for secondary screening culture to obtain 1 strain Peniciliums sp.Y-21 with high pectinase enzyme activity.

The EMS concentration in the step one is 1-5%, the reaction time is 0-60min, and the added NaS2O3 volume amount for terminating the reaction is the same as the added EMS amount.

In the second step, the bacterial strain with 70-90% of fatality rate needs to be screened again.

Optimizing the solid-state fermentation conditions of the high-yield pectinase enzyme activity strain, and optimizing through a single-factor test and an orthogonal test to obtain an optimal enzyme production system, wherein a solid fermentation culture medium is used in the optimization process, takes 20g of bean pulp and 15g of bran as solid matrixes, and has the initial water content of 50%, the glucose content of 2%, CaCl and the like₂0.04％，FeCl₃0.04 percent, the inoculation amount is 4 percent, the culture temperature is 38 ℃, and the culture time is 5 days.

The specific method for determining the enzyme activity of the pectinase is an indicator titration method, and is published in GB/T23535-2009 by the national standard of the people's republic of China.

Example 1

Mutagenesis of Penicillium sp Y-21

(1) Primary screening of Penicillium sp Y-21

Taking out from the strain storage room of Changzhou universityThe Penicillium sp is put into a seed culture medium to be cultured for 2 days, 10mL of seed liquid is taken and added into a centrifuge tube to be centrifuged for 5min at 3,000rpm, the supernatant is discarded and then is added with physiological saline to be washed for 2 times, the supernatant is discarded and then is centrifuged again for 5min at 3,000rpm, the supernatant is discarded and 10mL of phosphate buffer is added to prepare spore suspension. Mixing spore suspension with Ethyl Methanesulfonate (EMS) to obtain 1-5% mother solution, oscillating for 0-60min, adding physiological saline with the same volume as EMS, and adding 2% Na with the same volume as EMS after mutagenesis₂S₂O₃The mixture was allowed to stand for 10min, centrifuged at 3,000rpm for 5min, and 1mL of the treated solution was retained. Then diluting the bacterial liquid to 10 degrees by using sterile normal saline^-2Taking 100 mu L of bacterial liquid to coat on a solid plate culture medium, and re-screening after single bacterial grows on the culture medium.

The composition of the seed medium was as follows: k₂HPO₄1g/L, peptone 5g/L, glucose 10g/L, MgSO₄0.1g/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。

(2) Re-screening of Penicillium sp Y-21

Selecting 23 mutant strains (shown in Table 1) with different shapes, sizes and colors from plates with 70-90% lethality, and performing solid fermentation culture at 28 deg.C for 3 d. After the fermentation is finished, the enzyme activity of the pectinase produced by the 23 strains is determined according to a national standard method, and the strain (#21) with the highest enzyme activity is selected for subsequent optimization.

The composition of the fermentation medium was as follows: 20g of soybean meal, 70% of initial water content, 4% of glucose, 1% of peptone and MgSO₄0.02％，CaCl₂ 0.02％。

TABLE 1 Ethyl Methylsulfonate (EMS) mutagenesis rescreening table

This example shows that a strain (#21) with highest enzyme production activity is screened out after Ethyl Methanesulfonate (EMS) mutagenesis, the enzyme activity can reach 19000U/g, and the enzyme activity is improved by 37.7% compared with that of the original strain.

Example 2

By selecting the optimal solid substrate for Penicillium sp.Y-21 growth, rice bran, bean cake powder, bran, bean pulp, cottonseed cake powder and rapeseed cake powder fermentation strains are adopted, 3 controls are made in each group of experiments, and the specific experiment operation is as follows:

in an ultraclean workbench, a small amount of bacteria are selected from a flat plate by using an inoculating loop and are inoculated into a sterilized seed culture medium, after the culture is carried out until the logarithmic growth phase, the bacteria are inoculated into the fermentation culture medium which takes rice bran, bean cake powder, wheat bran, bean pulp, cottonseed cake powder and rapeseed cake powder as solid substrates according to the inoculum size of 2 percent, the fermentation culture medium is cultured for 3 days in a constant temperature incubator at 38 ℃, and the sampling is carried out to determine the enzyme activity produced by the strain, wherein the result is shown in figure 1.

This example illustrates that, when soybean meal is used as a fermentation substrate and fermented on day 3, the enzyme activity of pectinase produced by the strain is highest and reaches 13200U/g; when bran and rice bran are used as fermentation substrates, the enzyme produced by the strain is very little. Therefore, the bean pulp is finally selected as a fermentation substrate of the strain to optimize the components of the subsequent culture medium.

Example 3

By determining the optimal inoculation amount of the Penicillium sp.Y-21 strain in the fermentation process, 2%, 4%, 6%, 8% and 10% of the inoculation amount is selected to be inoculated into a fermentation medium, 3 times of comparison is carried out in each group of experiments, the specific experiment operation is shown in example 2, and the result is shown in figure 2.

This example illustrates that when bean pulp is used as a fermentation substrate and inoculated into a fermentation medium according to a seed inoculation amount of 4% for culture until 3 days, the enzyme activity of pectinase produced by the strain is the highest and reaches 22400U/g. Therefore we finally chose 4% as the optimal inoculum size for the enzyme production of the strain.

Example 4

By determining the optimum initial water content of the Penicillium sp.Y-21 strain during fermentation, we chose to add 60%, 65%, 70%, 75% of the initial water content to the fermentation medium, and each set of experiments was compared 3 times, the specific experimental procedure is shown in example 2, and the results are shown in FIG. 3.

This example shows that when bean pulp is used as fermentation substrate and inoculated into a fermentation medium with an initial water content of 70% according to an inoculum size of 4% and cultured until 3 days, the enzyme activity of pectinase produced by the strain is the highest and reaches 22600U/g.

Example 5

By determining the optimal carbon source and the concentration thereof required by the Penicillium sp.Y-21 strain in the fermentation process, glucose, fructose, lactose and maltose are selected as fermentation carbon sources, 1%, 2%, 4%, 6% and 8% of the carbon source concentration is selected to be added into a fermentation medium, each group of experiments are contrasted for 3 times, and the specific experiment operation is as follows:

selecting glucose, fructose, lactose and maltose as single carbon sources in the preparation process of the fermentation medium, ensuring other variables to be unchanged, and sterilizing at 115 ℃ for 30min after the preparation of the culture medium is finished. In a clean bench, 2mL of seed liquid is absorbed by a sterilized gun head and inoculated into a fermentation medium, the medium is stirred uniformly by a sterile glass rod and put into a constant temperature incubator at 38 ℃ for 3 days, and then sampling is carried out to measure the enzyme activity. The optimal carbon source suitable for the enzyme production of the strain is selected, and the carbon source concentration is further optimized, and the results are shown in FIGS. 4 and 5.

This example shows that the most suitable carbon source for the bacterial strain to produce enzyme is glucose, the enzyme activity of the pectinase fermented by using glucose as the carbon source can reach 22500U/g, and further, when the optimized glucose concentration is 4%, the enzyme activity produced by the bacterial strain is the highest, which reaches 23500U/g.

Example 6

By determining the optimum nitrogen source and the concentration thereof required by the Penicillium sp.Y-21 strain in the fermentation process, beef extract, peptone and NH are selected under the condition of ensuring that other components in the culture medium are unchanged₄Cl、KNO₃To ferment the nitrogen source and select 1%, 2%, 4%, 6%, 8% nitrogen source concentrations to add to the fermentation medium, each set of experiments was compared 3 times, the specific experimental procedure is shown in example 4, and the results are shown in fig. 6, 7.

This example illustrates that the best nitrogen source suitable for the production of enzyme by the strain is peptone, and the enzyme activity of the fermentation strain pectinase can reach 23550U/g by using peptone as the nitrogen source, and further, when the concentration of the optimized peptone is 1%, the enzyme activity produced by the strain is the highest, and reaches 23500U/g.

Example 7

By determining the optimum metal ion and concentration of Penicillium sp.Y-21 strain in fermentation process, we select a certain amount of MgSO (MgSO-containing medium) under the condition of ensuring that other components in the medium are not changed₄、CaCl₂、FeCl₃、K₂HPO₄、BaCl₂Adding into fermentation medium, then selecting metal ion concentration of 0.02%, 0.04%, 0.06%, 0.08%, 0.1% to add into fermentation medium again, determining the optimum metal ion concentration suitable for the strain to produce enzyme, each group of experiment makes 3 times of control, the specific experiment operation is shown in example 4, the result is shown in figure 8.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.