CN1858236B - Quick screening method for laccase high yield fungus with optimized cultivation conditions - Google Patents
Quick screening method for laccase high yield fungus with optimized cultivation conditions Download PDFInfo
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- CN1858236B CN1858236B CN200610039351A CN200610039351A CN1858236B CN 1858236 B CN1858236 B CN 1858236B CN 200610039351 A CN200610039351 A CN 200610039351A CN 200610039351 A CN200610039351 A CN 200610039351A CN 1858236 B CN1858236 B CN 1858236B
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- laccase
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Abstract
The quick screening method for high yield laccase fungus includes the culture of the spawn with PDA culture medium containing guaiacol and the combined culture test after the culture medium becomes iron red. It includes the following steps: designing several PB culture medium combination based on the key factors for raising enzyme generating activity and culturing spawn; culturing for 7 days whilemeasuring the laccase activity once every day; obtaining highest laccase activity and performing variance analysis to obtain the p values of all the factors; and estimating the effects of all factorson the laccase generation based on the p values. The present invention has greatly reduced test times, can determine the laccase producing capacity under the action of different factors, and obtain high yield laccase fungus through comparing the laccase yield in optimized condition.
Description
Technical field
The present invention relates to a kind of fungi selection, be specifically related to the screening method rapidly and efficiently that a kind of laccase high yield fungus is optimized culture condition.
Background technology
Laccase (EC 1.10.3.2) is the oxydo-reductase that copper atom is contained in a class active centre, is mainly produced by higher plant and fungi.Because its effect substrate is very extensive, has been applied to paper industry, printing and dyeing industry, foodstuffs industry etc.Because laccase can act on the organic compound that some have the phenols structure, therefore very strong application potential is also arranged at environment and soil remediation field.Growth is fast because fungi has, the source extensively, is easily cultivated and laccase extracts advantages such as convenient, become the main raw that laccase is produced, but it is higher that the principal element that restricts the laccase practical application at present is a production cost, is one of important method that reduces production costs to the screening of laccase high yield fungus.
The screening method of present laccase high yield fungus, generally be at first to separate the bacterial strain that acquisition has laccase activity, determine the factor of influence of laccase high yield then by one-factor experiment, utilize multifactor experiment to determine best condition of enzyme production again, thereby identify the inulinase-producing activity of laccase.This method because the factor of influence product enzyme is a lot, often can not be measured all factors in the test on the one hand, causes result's deviation; On the other hand, because the factor is one by one tested, workload is big, and the test period is long, is unfavorable for from a large amount of candidate strain acquisition laccase high yield fungus rapidly.
Summary of the invention
Existing screening method easily causes result error and workload is big and the deficiency of cycle length in order to overcome, from a large amount of candidate strain, screen laccase high yield fungus, the present invention will provide a kind of quick method that high yield laccase fungi is optimized culture condition that screens, not only significantly reduced the number of times of test, and can determine the ability of strain enzyme-producing under the different factor effects, final by to optimizing the comparison of laccase output under the culture condition, obtain the laccase generation bacterium of high yield.
The present invention is based on the fractional factorial experiment mentality of designing, nature is had the fungi that produces the laccase activity potentiality carry out separation and Culture on special culture medium, and determine that it at the inulinase-producing activity of optimizing under the condition of enzyme production, obtains laccase high yield fungus fast.
The triage techniques scheme that the present invention adopts is that bacterial strain laccase activity flat board develops the color and identifies---the strain enzyme-producing ability under the composite test identification optimizing culture condition, and promptly laccase high yield fungus is optimized the rapid screening method of culture condition, and step is as follows:
Employing contains the PDA substratum of methyl catechol (Guaiacol) bacterial classification is cultivated;
After substratum becomes rust, make up culture experiment, testing sequence is:
Selection designs several PB substratum and makes up 1-2 representative species in the factor that improves inulinase-producing activity and have keying action or condition, and bacterial classification is cultivated;
In the culturing process, measured the laccase activity in the nutrient solution one time, carried out altogether 7 days in per 24 hours;
Each combination is tested the highest laccase activity of back acquisition and carried out variance analysis, obtain the p value of each factor;
Can estimate that according to the size of p value this factor pair bacterial strain produces the influence of laccase.
More optimize and more particularly, step of the present invention is:
Employing contains the PDA substratum of methyl catechol (Guaiacol) and bacterial classification is cultivated concrete grammar: identify on the flat board at the PDA that contains 0.02% methyl catechol and insert bacterial classification that 28 ℃ leave standstill cultivation 4-5 days.
Described PDA identifies that substratum is:
100 milliliters of 18～22% potato diffusion juices (the peeling potatoes stripping and slicing adds water, boils 30min, uses filtered through gauze, down together)
Sucrose 1.5～2.5 grams
Agar 1.2%～2.5 gram
Methyl catechol 0.01～0.03 gram.
Culture condition: constant temperature leaves standstill for 28 ℃ to be cultivated 4-5 days.
Identify substratum for this PDA, the application recommends following ratio:
100 milliliters of 20% potato diffusion juices
Sucrose 2 grams
Agar 1.5-2 gram
Methyl catechol 0.02 gram.
Through the cultivation of aforesaid method, the bacterial strain with laccase activity will make PDA identify that substratum becomes rust, thus the fungal bacterial strain of can direct viewing determining to have laccase activity.
After determining to have the bacterial strain of laccase activity, design a combination culture experiment, can determine fast bacterial strain in the laccase output of optimizing under the culture condition.By the literature search analysis, we find to improving the factor that inulinase-producing activity has keying action following a few class to be arranged in cultivating in that fungi is liquid: 1, the ratio of carbon source and nitrogenous source and concentration; 2, divalent-metal ion is as Cu
2+And Mn
2+3, the micromolecular organism that has benzene ring structure is as methyl catechol and veratryl alcohol; 4, some amino acids materials are as l-asparagine; 5, ethanol; 6, oxygen supply situation.The different bacterial strain of these factor pairs has different effects, therefore needs to investigate its product enzyme influence to specific bacterial strain comprehensively.From the cost angle, the application possibility of amino acids material in industrial production is less, does not consider.Therefore, select 1-2 representative species or condition in all the other five class factors,, design 12 PB substratum combinations altogether, as following table based on the fractional factorial experiment mentality of designing.
The potential product laccase of table 1 bacterium is cultivated assembled scheme
Wherein in " oxygen " hurdle, cultivation is left standstill in 0 representative, and 1 represents shaking culture.The pseudo-variable of A, B, the design of C, D system is used for calculating the test confidence level.Culture condition is: 28 ℃, leave standstill or (180 rev/mins) cultivation of vibrating.
In the culturing process, measured the laccase activity in the nutrient solution one time, carried out altogether 7 days in per 24 hours.Laccase activity is measured the cuvette that adopts 1 centimetre of light path, adds the B﹠amp of 1.8mL successively; The 30mM ABTS[2 of R damping fluid, 0.1mL, 2 '-Lian nitrogen-two (3-ethyl-benzothiazole-6 sulfonic acid)] aqueous solution, 0.1mL culture supernatant, mix the back 40 ℃ of incubations 5 minutes, use spectrophotometer to measure the absorption value at 420nm place every 1 minute, line taking is partly calculated.
B﹠amp; The composition of R damping fluid is: 0.1mol/L boric acid, and 0.1mol/L acetic acid, 0.1mol/L phosphoric acid uses the NaOH of 0.5mol/L to regulate pH to 4.0.
The calculation formula of laccase activity is:
In the formula, Δ A
420The changing value that refers to 420nm place light absorption value per minute.
Use suitable statistics software, each combination is tested the highest laccase activity of back acquisition and carried out variance analysis, obtain the p value of each factor.Can estimate that according to the size of p value this factor pair bacterial strain produces the influence of laccase.Cultivate in the combination for 12, the optimization culture condition that is combined as strain enzyme-producing that laccase activity is the highest, the yield of enzyme of this combination is the enzymatic productivity of this bacterial strain, the laccase that the enzymatic productivity of more a plurality of bacterial classifications can obtain high yield produces bacterial classification.
The present invention has overcome existing screening method and has easily caused the deficiency that result error and workload are big and the cycle is grown, method of the present invention has not only significantly reduced the number of times of test, and can determine the ability of strain enzyme-producing under the different factor effects, final by to optimizing the comparison of laccase output under the culture condition, obtain the laccase generation bacterium of high yield.
Description of drawings
Fig. 1 is each factor of influence effect figure in the high bacterium rapid screening method of laccase.
Among the figure ● the expression effect is not remarkable, and ■ represents that effect is remarkable.
Embodiment
Embodiment 1, to the evaluation of a strain separating obtained fungi WF-10 laccase production ability from soil:
Inoculation is identified on the flat board to the PDA that contains 0.02% methyl catechol constant temperature leaves standstill for 28 ℃ to be cultivated after 5 days, the substratum color of observing the colony growth place becomes rust by original oyster white, can determine that therefore this bacterial classification has the ability that produces laccase.
According to table 1, prepare corresponding substratum, 3 repetitions are arranged in each combination, and the data that the test back obtains are:
Obviously, the laccase output of test number 11 is the highest, gets the laccase production ability of 29.4U/L for this bacterial strain, is used for determining the comparison of superior strain.Above test-results is carried out multifactorial variance analysis, result such as following table:
As can be seen from the above table, the p value of glucose, bean cake powder, methyl catechol, ethanol and oxygen is all less than 0.05, and the output of laccase is had remarkable influence, and wherein glucose and methyl catechol embody negative effect, and ethanol, bean cake powder, oxysome reveal positive-effect.And CuSO
4And MnSO
4Generation to laccase has no significant effect.Pseudo-variable A, B, C, D all do not make significant difference, the definition that this also meets pseudo-variable, and the result that this test is described is believable.Concrete outcome such as accompanying drawing.
Claims (3)
1. a laccase high yield fungus is optimized the rapid screening method of culture condition, and step is as follows:
Employing contains the PDA substratum of methyl catechol bacterial classification is cultivated;
After substratum becomes rust, make up culture experiment, testing sequence is:
Selection designs several PB substratum and makes up 1-2 representative species in the factor that improves inulinase-producing activity and have keying action or condition, and bacterial classification is cultivated;
In the culturing process, measured the laccase activity in the nutrient solution one time, carried out altogether 7 days in per 24 hours;
Each combination is tested the highest laccase activity of back acquisition and carried out variance analysis, obtain the p value of each factor;
Can estimate that according to the size of p value this factor pair bacterial strain produces the influence of laccase;
Each combination is tested the highest laccase activity of back acquisition and carried out variance analysis, the optimization culture condition that is combined as strain enzyme-producing that laccase activity is the highest.
2. optimize the rapid screening method of culture condition according to the described laccase high yield fungus of claim 1, it is characterized in that concrete steps are:
Described usefulness contains the PDA substratum of creating wooden phenol and bacterial classification is carried out cultural method is: identify on the flat board at the PDA that contains 0.02% methyl catechol and insert bacterial classification, 28 ℃ leave standstill and cultivated 4-5 days,
Described PDA identifies that substratum is:
100 milliliters of 18～22% potato diffusion juices
Sucrose 1.5～2.5 grams
Agar 1.2%～2.5 gram
Methyl catechol 0.01～0.03 gram,
Culture condition: constant temperature leaves standstill for 28 ℃ to be cultivated 4-5 days;
Through the cultivation of aforesaid method, the bacterial strain with laccase activity will make PDA identify that substratum becomes rust, thus the fungal bacterial strain of can direct viewing determining to have laccase activity;
Making up " the having the factor of keying action to improving inulinase-producing activity " described in the culture experiment has: the ratio of carbon source and nitrogenous source and concentration; Divalent-metal ion; Micromolecular organism with benzene ring structure; The amino acids material; Ethanol; The oxygen supply situation,
Design the combination of 12 PB substratum altogether, as following table:
The potential product laccase of table 1 bacterium is cultivated assembled scheme
Wherein in " oxygen " hurdle, cultivation is left standstill in 0 representative, and 1 represents shaking culture, and the pseudo-variable of A, B, the design of C, D system is used for calculating the test confidence level, and culture condition is: 28 ℃, leave standstill or shaking culture;
In the culturing process, measured the laccase activity in the nutrient solution one time, carried out altogether 7 days in per 24 hours; Laccase activity is measured the cuvette that adopts 1 centimetre of light path, adds the B﹠amp of 1.8mL successively; The 30mM ABTS[2 of R damping fluid, 0.1mL, 2 '-Lian nitrogen-two (3-ethyl-benzothiazole-6 sulfonic acid)] aqueous solution, 0.1mL culture supernatant, mix the back 40 ℃ of incubations 5 minutes, use spectrophotometer to measure the absorption value at 420nm place every 1 minute, line taking is partly calculated;
B﹠amp; The composition of R damping fluid is: 0.1mol/L boric acid, and 0.1mol/L acetic acid, 0.1mol/L phosphoric acid uses the NaOH of 0.5mol/L to regulate pH to 4.0;
The calculation formula of laccase activity is: laccase activity
In the formula, Δ A
420The changing value that refers to 420nm place light absorption value per minute;
Each combination is tested the highest laccase activity of back acquisition and carried out variance analysis, obtain the p value of each factor;
Can estimate that according to the size of p value this factor pair bacterial strain produces the influence of laccase.
3. optimize the rapid screening method of culture condition according to the described laccase high yield fungus of claim 2, it is characterized in that, described PDA identifies that the substratum composition is:
100 milliliters of 20% potato diffusion juices
Sucrose 2 grams
Agar 1.5-2 gram
Methyl catechol 0.02 gram.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102134555A (en) * | 2010-12-03 | 2011-07-27 | 浙江大学 | Liquid fermentation method for producing laccase by using marine endogenous pestalotia |
| CN104020122B (en) * | 2014-06-20 | 2016-02-24 | 中国科学院亚热带农业生态研究所 | Laccase activity assay method in a kind of agricultural land soil |
| CN107236673A (en) * | 2017-06-10 | 2017-10-10 | 安徽科技学院 | A kind of effective ways of quick and precisely screening production laccase fungi |
| CN113388660B (en) * | 2021-06-29 | 2023-05-19 | 白银赛诺动物保健技术有限公司 | Liquid color development method for rapidly screening laccase-producing bacteria |
| CN117568195B (en) * | 2024-01-16 | 2024-06-14 | 安琪酵母（滨州）有限公司 | Method for improving activity of low-sugar yeast |
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Granted publication date: 20100512 Termination date: 20120407 |