阅读说明：本技术 一种生产角蛋白酶的方法 (Method for producing keratinase ) 是由 张娟 冒鑫哲 万云蕾 陈坚 郭荣 邓小华 李江华 周冠宇 于 2020-04-22 设计创作，主要内容包括：本发明公开了一种生产角蛋白酶的方法,属于微生物和发酵技术领域。本发明通过从发酵培养基成分(碳源、氮源、金属离子、磷酸盐)和发酵条件(温度、pH、接种量)两个方面入手,对利用枯草芽孢杆菌发酵生产角蛋白酶的工艺进行了优化。利用本发明的方法发酵生产角蛋白酶,可使得角蛋白酶的产量较对照提高3.3倍,在3L发酵罐产酶,酶活达到704400U/mL。因而本发明的方法在饲料、制革等方面角蛋白酶的制备中有着广阔的应用前景。(The invention discloses a method for producing keratinase, which belongs to the technical field of microorganisms and fermentation, and optimizes the process for producing the keratinase by fermenting bacillus subtilis by starting from two aspects of fermentation medium components (carbon source, nitrogen source, metal ions and phosphate) and fermentation conditions (temperature, pH and inoculum size).)

1. A method for increasing the production of keratinase by fermenting a keratinase-producing strain in a medium comprising: sucrose, soybean meal, yeast extract and Na₂HPO₄·12H₂O、KH₂PO₄And MgSO₄·7H₂O。

2. The method according to claim 1, wherein the keratinase fermentation strain is inoculated with an initial OD of 3-8%₆₀₀0.6 to 1.2.

3. The method of claim 1, wherein the fermentation strain of keratinase is Bacillus subtilis.

4. The method according to claim 1, wherein the sucrose content is 15-40 g/L, the soybean meal content is 35-45 g/L, and the yeast extract content is 5-15 g/L or 25-30 g/L.

5. The method of claim 1, wherein said Na is₂HPO₄·12H₂The content of O is 2-20 g/L, and the content of KH₂PO₄The content of (b) is 1-10 g/L.

6. The method of claim 1, wherein the MgSO₄·7H₂The content of O is 0.1 to 1.0 g/L.

7. The method according to claim 1, wherein the enzyme production is carried out at 36-40 ℃.

8. The method according to claim 1, wherein the enzyme production is carried out at a pH of 6 to 8.

9. A culture medium is characterized in thatThe components of the culture medium are sucrose, soybean meal, yeast extract and Na₂HPO₄·12H₂O、KH₂PO₄And MgSO₄·7H₂O, the content of the sucrose is 15-40 g/L, the content of the soybean meal is 35-45 g/L, the content of the yeast extract is 5-15 g/L or 25-30 g/L, and the Na is₂HPO₄·12H₂The content of O is 2-20 g/L, and the KH is₂PO₄The content of (1-10 g/L) of the MgSO 2₄·7H₂The content of O is 0.1 to 1.0 g/L.

10. The method according to any one of claims 1 to 8, or the use of the medium according to claim 9 for preparing keratinase or degrading keratin in the fields of feed, tanning, livestock and medicine.

Technical Field

The invention relates to a method for producing keratinase, belonging to the technical field of microorganisms and fermentation.

Background

Keratinase is a protease that specifically degrades keratin-like substrates. The keratinase-producing microorganisms are mainly bacteria, fungi and actinomycetes. At present, research aiming at keratinase fermentation optimization mainly focuses on screening of wild bacteria, optimization of fermentation medium components and optimization of fermentation conditions, the most common bacteria in the excavation of keratinase-producing strain resources have the characteristics of short fermentation period, high enzyme activity, good production safety and the like, and the keratinase has good application prospects in the fields of feed, fertilizer, detergent, leather, textile, cosmetic industry, medical treatment and the like. With the continuous deepening of the understanding of the potential of the catalytic property of keratinase in industrial application, the performance and the yield of the keratinase produced by wild fungi can not meet the market demand. The wild keratinase-producing bacteria obtained by screening at present are mostly concentrated in the bacillus subtilis, and the extracellular secreted enzymes have the defects of various types, poor substrate action specificity, unstable enzyme production and difficulty in industrial production. In recent years, more and more keratinase genes are cloned and expressed in a heterologous way, gene engineering bacteria are adopted to strengthen gene transcription and translation, high-efficiency expression and active secretion are achieved, and the production intensity of keratinase can be effectively improved. The performance of the genetically engineered bacteria and the recombinant keratinase is improved, the extracellular secretase is single, and the purification work of the downstream fermentation is simplified. However, high production costs and low yields limit the industrial production of keratinase, and thus yield increases and costs decrease by fermentation optimization techniques.

Disclosure of Invention

Aims to solve the problems of low fermentation enzyme activity and overhigh fermentation cost of keratinase production strains. The method starts from two aspects of fermentation medium components (carbon source, nitrogen source, metal ions and phosphate) and fermentation conditions (temperature, pH and inoculation amount), and further optimizes the process for producing the keratinase by fermenting the bacillus subtilis; the method for producing keratinase by fermentation can improve the yield of the keratinase and has wide application prospect in the aspects of feed, leather making and the like.

The invention optimizes the fermentation medium. Firstly, optimizing the components of a culture medium and culture conditions by utilizing a single-factor test; then, a Plackett-Burman test is designed to screen three significant factors: yeast extract, pH, temperature; and finally designing a Box-Behnken center combination test and carrying out response surface analysis. Obtaining the best process for verification and high-density fermentation.

The invention provides a culture medium, which comprises the components of cane sugar, bean pulp, yeast extract and Na₂HPO₄·12H₂O、KH₂PO₄And MgSO₄·7H₂O。

In one embodiment of the invention, the content of the sucrose is 20-40 g/L.

In one embodiment of the invention, the content of the soybean meal is 35-45 g/L.

In one embodiment of the invention, the content of the yeast extract is 5-10 g/L or 25-30 g/L.

In one embodiment of the present invention, the Na₂HPO₄·12H₂The content of O is 2-20 g/L.

In one embodiment of the present invention, the KH is₂PO₄The content of (b) is 1-10 g/L.

In one embodiment of the invention, the MgSO₄·7H₂The content of O is 0.1 to 1.0 g/L.

The invention provides a method for improving the yield of keratinase, which is to inoculate a keratinase fermentation strain into a culture medium for producing enzyme.

In one embodiment of the present invention, the amount of the keratinase fermentation strain inoculated is 4 to 7% (v/v) of the fermentation medium, and the OD of the bacterial liquid at the time of inoculation₆₀₀0.6 to 1.0.

In one embodiment of the invention, the keratinase fermenting strain is bacillus subtilis.

In one embodiment of the present invention, the Bacillus subtilis is WB600-pP43NMK-ker, and the WB600-pP43NMK-ker is described in Zheng Pen et al, "Biotransformation of ketone waste materials and active peptides based on cell-free analysis".

In one embodiment of the invention, the enzyme production is carried out at 36-40 ℃.

In one embodiment of the invention, the enzyme production is carried out at a pH of 6 to 8.

The invention also protects the application of the culture medium or the method for improving the yield of the keratinase in preparing the keratinase or degrading the keratinase in the fields of feed, tanning, livestock raising and medicine.

The invention has the following beneficial effects:

1. the optimized culture medium realizes the remarkable improvement of the keratinase yield

The fermentation culture medium (30 g/L sucrose, 40 g/L soybean meal, 5.72 g/L yeast extract and 3 g/L Na) obtained by optimization in the invention₂HPO₄·12H₂O，1.5g/L KH₂PO₄，0.3g/L MgSO₄·7H₂O); at a temperature of 37 ℃, the bacterial concentration OD of the seed liquid₆₀₀The activity of the keratin is 260480U/m L when the inoculation amount is 0.8, the pH value is 7.68, the rotation speed is 220rpm, and the liquid loading amount is 20 percent, while the activity of the keratin is only 61210U/m L compared with 3.3 times of the control.

2. The optimized culture medium realizes the obvious reduction of the keratinase fermentation cost

The raw materials of the optimized culture medium are low in price, good in fermentation repeatability and low in cost, and are reduced by 96% compared with the raw materials before optimization.

3. The optimized culture medium realizes high-density fermentation, and the keratinase yield is further improved

The optimized culture medium is utilized to carry out the amplification production of a 3L fermentation tank, the enzyme activity reaches 704400U/m L, and the industrial production potential is huge.

Drawings

FIG. 1 shows the enzyme activities of keratinase produced by fermentation in different kinds of carbon source species.

FIG. 2 shows the enzyme activities of keratinase produced by fermentation in sucrose at various concentrations.

FIG. 3 shows the enzyme activities of keratinase produced by fermentation in different kinds of first nitrogen sources.

FIG. 4 shows the enzyme activities of keratinase produced by fermentation in bean pulp with different addition amounts.

FIG. 5 shows the enzymatic activities of fermentation keratinase in different kinds of second nitrogen sources.

FIG. 6 shows the enzyme activities of keratinase produced by fermentation in yeast extract with different addition amounts.

FIG. 7 shows the enzymatic activities of fermentation keratinase in different kinds of metal ions.

FIG. 8 shows the enzyme activities of keratinase produced by fermentation in magnesium sulfate heptahydrate at various addition levels.

FIG. 9 shows the enzymatic activities of keratinase produced by fermentation in various concentrations of phosphate.

FIG. 10 shows the enzyme activities of fermentative keratinase production at different initial pH.

FIG. 11 shows the enzyme activities of keratinase produced by fermentation at different inoculum sizes.

FIG. 12 shows the enzyme activities of keratinase produced by fermentation at different temperatures.

FIG. 13 is a graph of keratinase production by fermentation of strains.

Detailed Description

1. Method for measuring enzyme activity of keratinase

Preparing a crude enzyme solution: the resulting fermentation broth was centrifuged at 7000rpm at 4 ℃ for 5 min.

Enzyme reaction, adding Gly-NaOH solution (pH 10)150 mu L, 2.5% soluble keratin 100 mu L and keratinase solution 50 mu L into a centrifugal tube of 1.5M L, mixing uniformly, reacting for 20min at 60 ℃, adding 0.5M trichloroacetic acid solution 200 mu L to terminate the reaction, centrifuging at 12000rpm for 2min, and adding 200 mu L trichloroacetic acid solution before adding the enzyme solution as a control.

And (3) performing color reaction, namely adding 200 mu L supernatant into 1m L5% sodium carbonate solution, adding 200 mu L forinophenol reagent, uniformly mixing, performing color development at 50 ℃ for 10min, and measuring the absorbance value at 660nm by using a spectrophotometer.

Definition of enzyme activity: 1U is the absorbance value increased by 0.001 unit at 660 nm.

2. L B Medium 10g tryptone, 5g yeast extract, 5g NaCl in 1L deionized water was added, pH was adjusted to 7.4 with 1 mol/L NaOH, and steam-sterilized at 121 ℃ under high pressure for 20 min.