CN112391324B - Strain for producing acid-resistant high-temperature alpha-amylase and application thereof - Google Patents

Strain for producing acid-resistant high-temperature alpha-amylase and application thereof Download PDF

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CN112391324B
CN112391324B CN202011427101.9A CN202011427101A CN112391324B CN 112391324 B CN112391324 B CN 112391324B CN 202011427101 A CN202011427101 A CN 202011427101A CN 112391324 B CN112391324 B CN 112391324B
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amylase
acid
temperature
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刘文龙
佟新伟
王兴吉
钱娟娟
刘胜利
王克芬
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Shandong Lonct Enzymes Co ltd
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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a bacterial strain for producing acid-resistant high-temperature amylase and application thereof. The Bacillus licheniformis is specifically Bacillus licheniformis (Bacillus licheniformis) YBG-90036, and the strain preservation numbers are as follows: CGMCC NO. 20672. The enzyme activity of the fermentation liquor for producing the acid-resistant high-temperature alpha-amylase by liquid fermentation of the bacillus licheniformis mutant strain CGMCC NO.20672 is 8.9-9.2U/mL; the produced acid-resistant high-temperature alpha-amylase is subjected to heat preservation for 3 hours under the conditions of pH4.0 and 98 ℃, and the residual enzyme activity is 98%; the optimum pH value is 3.5-4.4; the optimal temperature reaction is 98-100 ℃; has good high temperature resistance and acid resistance, and can be widely applied to starch processing, beer brewing, fermentation, textile and other industries.

Description

Strain for producing acid-resistant high-temperature alpha-amylase and application thereof
The technical field is as follows:
the invention belongs to the technical field of bioengineering, and particularly relates to a bacterial strain for producing acid-resistant high-temperature amylase and a liquid fermentation method thereof.
Background art:
alpha-amylase is one of the earliest enzyme preparations which are produced commercially, applied most widely and used in the largest amount. Is indispensable in the starch sugar manufacturing and fermentation industries.
The deep processing of starch products generally comprises two processes: liquefaction and saccharification. Firstly, the pH value of starch slurry is adjusted to 5.8-6.2 from natural about 4.5, high-temperature resistant amylase is added for even mixing, then steam with the temperature of 105 ℃ is used for heating to 90 ℃, and enzymolysis is carried out for 1-1.5 hours to form starch paste. Then reducing the pH value of the formed starch paste to about 4.0-4.5, cooling to 50-60 ℃, and adding glucoamylase for saccharification; the two enzymes added above have obvious difference in action pH besides different action temperatures.
The most suitable pH value of the commercial acid-resistant high-temperature alpha-amylase widely used in domestic industry at present is about 5.0-6.0, most of the commercial acid-resistant high-temperature alpha-amylase has the defects of poor thermal stability and narrow pH range, and the commercial acid-resistant high-temperature alpha-amylase has limitation on the starch deep processing technology under the acidic condition. Since glucoamylase used for saccharification in the subsequent saccharification process has an optimum pH of about 4.0-4.5, pH adjustment in these two steps increases costs, and thus development of a novel acid-resistant high-temperature alpha-amylase having a low cost, which can efficiently liquefy starch at a low pH, is of great industrial application value.
In addition, due to the enzymological characteristics of the acid-resistant high-temperature alpha-amylase under the acidic and high-temperature conditions, the acid-resistant high-temperature alpha-amylase with fixed cost can also be applied to multiple industries such as food processing, wine brewing, textile and the like, and the process improvement and upgrading of other industries are promoted.
Chinese patent CN201610670173.3 discloses a gene of acid-resistant high-temperature alpha-amylase, engineering bacteria and a preparation method thereof, the acid-resistant high-temperature alpha-amylase has higher thermal stability than a mutant reported in the literature under the conditions of pH4.2-4.6 and 95 ℃, and the expression amount reaches 2 g/L.
The amylase mutants IG 181-182/C363G and IG 181-182/N463T described in Chinese patent CN201610542505.X have half-lives at 85 ℃ increased from 5.3min of control (before mutation) to 77.0min and 82.5min, respectively.
Chinese patent CN201210532851.1 discloses an acid-resistant amylase mutant and a preparation method thereof. The bacillus subtilis amylase is used as a female parent, the bacillus subtilis amylase sequence is subjected to site-directed mutagenesis, the optimum reaction pH of the bacillus subtilis amylase is changed from 7.0 to 4.5, and the amylase catalysis efficiency is improved by 5 times under the condition of pH4.5 after mutagenesis.
Chinese patent CN201210246909.6 discloses an acid-and high-temperature-resistant alpha-amylase, a gene, an engineering bacterium and a preparation method thereof, and the technical scheme is that a high-temperature-resistant alpha-amylase gene of bacillus licheniformis is subjected to site-specific mutagenesis by utilizing a recombinant DNA technology to obtain the acid-and high-temperature-resistant mutant gene of the high-temperature-resistant alpha-amylase, namely the acid-and high-temperature-resistant alpha-amylase gene, and a bacillus subtilis expression system is utilized to secrete and express the acid-and high-temperature-resistant alpha-amylase. The acid-resistant high-temperature alpha-amylase disclosed by the invention still keeps the residual enzyme activity of more than 40% after heat preservation for 60min at the conditions of pH4.5 and 90 ℃. Under the condition of pH4.0-5.5, the enzyme activity level of the recombinant expression strain is 1120U/mL-2170U/mL, which is higher than that of the control strain 130U/mL-1130U/mL.
Chinese patent CN201010219720.9 discloses an acid amylase AMYA4 and a gene and application thereof. AmyA4 has optimum pH value of 4.2 and optimum temperature of 75 deg.C, has high activity in 55-75 deg.C range, and has strong raw starch degrading ability.
Chinese patent CN201510677368.6 discloses a bacillus licheniformis for producing acid-resistant high-temperature alpha-amylase in high yield and a liquid fermentation method thereof. The enzyme activity level of the acid-resistant high-temperature alpha-amylase produced by the liquid fermentation of the bacillus licheniformis mutagenic strain CGMCC NO.10785 is 3.8-4.2 ten thousand u/mL. The produced acid-resistant high-temperature alpha-amylase has the residual enzyme activity of 95 percent after heat preservation for 3 hours at 95 ℃, the optimal reaction temperature range of 96-100 ℃ and the optimal reaction pH of 4.0-4.5, has good high temperature resistance and acid resistance, and can be widely applied to a plurality of industries such as starch processing, beer brewing, fermentation, textile and the like.
In order to reduce the production cost, expand the action conditions and range and meet the market demand, a production strain with high enzyme production activity, good production stability and strong adaptability needs to be developed or optimized.
The invention content is as follows:
the invention aims to provide a strain of high-yield acid-resistant high-temperature alpha-amylase and a liquid fermentation method thereof.
The bacterial strain of the high-yield acid-resistant high-temperature alpha-amylase provided by the invention is specifically a Bacillus licheniformis (Bacillus licheniformis) mutant strain YBG-90036. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 18 days 9 and 18 months 2020, and the address is No. 3 of Xilu No.1 of Beijing north Shangyang, the Zeno. (postal code 100101), and the preservation number is CGMCC NO. 20672.
The bacillus licheniformis mutant strain YBG-90036 is obtained by performing Nitrosoguanidine (NTG) mutagenesis breeding on bacillus licheniformis CGMCC NO. 10785. The highest liquid fermentation enzyme activity of the bacillus licheniformis CGMCC NO.10785 is 42470U/mL, the highest liquid fermentation enzyme activity of the mutant strain YBG-90036 can reach 92040U/mL, the liquid fermentation activity is about 8.9-9.2 ten thousand U/mL through the optimization of fermentation conditions, and the average fermentation enzyme activity is 90933U/mL.
The method for producing the acid-resistant high-temperature alpha-amylase by liquid fermentation of the bacillus licheniformis YBG-90036 comprises the following steps:
and (3) streaking seeds to separate single colonies: freezing and storing the glycerinum pipe seeds of the bacillus licheniformis mutant strain YBG-90036, streaking and separating single colony in a seed slant culture medium, and culturing at 37-42 ℃ for 30-35 h.
The formula of the seed slant culture medium is as follows: 3-6% of corn starch, 2-4% of yeast powder, 1-4% of soybean peptone, 1-3% of maltose syrup, 0.2-0.5% of calcium chloride, 1.0-2.5% of ammonium chloride, 1.5-2.0% of potassium dihydrogen phosphate, 1-1.5% of agar powder and the balance of water, wherein the pH value is 4.6-5.8.
Liquid seed culture: on the seed slant culture medium, picking single colony with high HC value (HC value is hydrolysis circle diameter/colony diameter), inoculating into 200mL liquid seed culture medium (using 500mL triangular flask), culturing at 37-42 deg.C with shaking table rotation speed of 300 r/min to OD600nmThe light absorption value is about 1.8-2.8.
Liquid seed culture medium: 3-6% of corn starch, 2-4% of yeast powder, 1-4% of soybean peptone, 1-3% of maltose syrup, 0.2-0.5% of calcium chloride, 1.0-2.5% of ammonium chloride, 1.5-2.0% of potassium dihydrogen phosphate and the balance of water, wherein the pH value is 4.6-5.8.
Seed tank culture: inoculating cultured seeds into a seed tank according to the inoculation amount of 10-12%, fermenting at 37-42 deg.C under 0.08MPa, controlling dissolved oxygen at 40-50% by increasing rotation speed and ventilation amount, and culturing for 10-12 h.
Seeding tank culture medium: 4-6% of corn starch, 3-4.5% of yeast powder, 3-4% of soybean peptone, 2.1-3.2% of malt syrup, 0.4-0.6% of calcium chloride, 1-2.5% of ammonium chloride, 2.1-2.9% of potassium dihydrogen phosphate, 1.2-2.1% of dipotassium hydrogen phosphate, 0.5-2.5% of corn steep liquor and the balance of water, wherein the pH value is 4.6-5.8.
Culturing in a fermentation tank: inoculating according to 5-9% of culture medium volume of fermentation tank, culturing at 37-42 deg.C under 0.08MPa, controlling dissolved oxygen content to be greater than 10%, maximum rotation speed of 900rpm, and maximum ventilation amount of 7.0m3And/h, after the maximum rotating speed and the ventilation rate are reached, if the dissolved oxygen is lower than 10%, the dissolved oxygen is not controlled any more. Feeding materials when the pH value is higher than 5.7 in the fermentation process, controlling the pH value to be 5.5-5.8, ending the fermentation when the thallus autolysis is serious, and culturing for 110-120 h.
Fermentation medium: 6-8% of corn starch, 3-6% of soybean protein powder, 1-4% of yeast powder, 4-7% of corn steep liquor, 0.1-0.5% of calcium chloride, 1.8-2.6% of monopotassium phosphate, 0.8-2.7% of diammonium phosphate, 1.1-2.3% of magnesium sulfate, 3.6-5.4% of disodium hydrogen phosphate, 0.15-0.56% of trisodium citrate and the balance of water, wherein the pH value is 4.6-5.8.
The formula of a supplemented medium is as follows: liquefied corn starch 20-30% (liquefied starch preparation method, dissolving starch in water, adjusting pH to 5.0-6.0, adding moderate temperature amylase, liquefying at 55-60 deg.C for 1-2h), corn steep liquor 5-9%, soybean peptone 2-5%, dipotassium hydrogen phosphate 3.1-4.5%, trisodium citrate 0.2-0.4%, and water in balance, pH 3.5-4.0.
The acid-resistant high-temperature alpha-amylase produced by the strain YBG-90036 has the following enzymological properties:
(1) the optimum pH value is 3.5-4.4;
(2) the optimal temperature reaction is 98-100 ℃;
(3) keeping the temperature at 98 ℃ for 3h, wherein the residual enzyme activity is 98%.
The starting strain CGMCC NO.10785 produces the acid-resistant high-temperature alpha-amylase with the enzymological properties as follows:
(1) the optimum pH value is 4.0-5.0;
(2) the optimal temperature reaction is 96-100 ℃;
(3) keeping the temperature at 95 ℃ for 3h, wherein the residual enzyme activity is 95%.
Has the advantages that:
the invention carries out nitrosoguanidine mutagenesis on a high-temperature alpha-amylase high-yield strain CGMCC NO.10785, and obtains a mutagenized strain with the number of YBG-90036 by shake flask directional screening. The mutagenic strain YBG-90036 can grow in an acid environment (the fermentation pH is 4.6-5.8), and a new method for producing novel acid-resistant high-temperature alpha-amylase by liquid fermentation is established through further optimization of a fermentation culture medium and optimization of a fermentation process, wherein the activity of the fermentation enzyme is 8.9-9.2 ten thousand U/ml. The optimum pH of the acid-resistant high-temperature alpha-amylase produced by the mutagenic strain YBG-90036 is 3.5-4.4; the optimal temperature reaction is 98-100 ℃; the temperature is kept for 3h under the conditions of pH4.0 and 98 ℃, the residual enzyme activity is 98 percent, and the method has stable acid resistance and high temperature resistance and can be widely used in starch processing, beer brewing, alcohol, fermentation, textile and other industries.
Description of the drawings:
FIG. 1 relative enzyme activity at different pH;
FIG. 2 relative enzyme activities at different temperatures;
FIG. 398 ℃ relative enzyme activity of incubation treatment.
The specific implementation mode is as follows:
the present invention will be described in detail below with reference to specific embodiments, which are only illustrative and are not intended to limit the scope of the present invention.
Example 1 mutagenesis and directed selection of strains
The activity level of the liquid fermentation enzyme of the bacillus licheniformis CGMCC NO.10785 needs to be further improved, in order to obtain a strain of high-yield acid-resistant high-temperature alpha-amylase, nitrosoguanidine mutagenesis is carried out on the strain until a strain which has obviously faster growth, can be cultured at high density and further improves the activity level of the acid-resistant high-temperature alpha-amylase is bred.
1. Preparation of the bacterial suspension
300mL of bacillus licheniformis CGMCC NO.10785 logarithmic phase seed liquid is sucked into 6 centrifuge tubes with 50mL, centrifuged for 5min at 8000rpm, supernatant is discarded, the precipitated thalli are washed for 2 times by 600mL of normal saline, and then 100 plus 200mL of normal saline is used for resuspension to obtain the bacterial suspension.
2. Nitrosoguanidine mutagenesis and coating screening flat plate
Respectively sucking 10mL of bacterial suspension into 5 large test tubes, adding nitrosoguanidine to make the final concentration of nitrosoguanidine about 20 μ g/mL, then placing the test tubes in a shaking table at 37 ℃, rotating at 300 r/min, and starting timing, wherein the mutagenesis time is about 30min, and the lethality rate is 78%. After mutagenesis, the mutagenic bacteria suspension is placed in a 50mL centrifuge tube, centrifuged for 5min at 8000rpm, the supernatant is discarded, the precipitated bacteria are cleaned by 200mL seed culture medium for 4 times, then 30mL seed culture medium is added, 100 microliter is taken and coated on a mutagenic seed screening culture medium, the culture is carried out for 30h at 39 ℃, and after single bacteria grow out, a strain with high bacterial colony HC value (HC value is the diameter of hydrolysis ring/bacterial colony diameter) is picked.
The mutagenic seed screening culture medium comprises the following components in percentage by mass and volume: corn starch 4%, yeast powder 3%, soybean peptone 2%, maltose syrup 2%, calcium chloride 0.4%, ammonium chloride 1.5%, potassium dihydrogen phosphate 1.8%, dipotassium hydrogen phosphate 1.3%, trisodium citrate 0.5%, agar powder 1%, and water in balance, and the pH value is 5.5.
3. Directed screening of mutagenized strains in a shake flask acidic environment
The mutagenized strains screened from the mutagenized seed screening medium were subjected to shake flask fermentation. Inoculating the selected colony to 20mL acidic shake flask seed culture medium (cultured in 50mL triangular flask), placing in 37 deg.C shaking table, culturing at 300 rpm to OD600nmThe light absorption value is about 2.0, and 1mL of the strain is collected to be preserved as a glycerin tube. Inoculating 3mL of the bacterial liquid into 100mL of acid shake flask screening medium (screening by using a 500mL triangular flask) according to the inoculation amount of 3%, shaking the medium at 37 ℃ for 300 r/min, and culturing for 5 days. Three shake flasks were made for each selected mutagenized strain and the enzyme activity was measured every day from day 3 to determine the highest level of enzyme activity of the selected strain and the shake flask fermentation period. A total of 3 shake flask screens were performed for each mutagenized strain to determine simply the genetic stability of the mutagenized strain.
The shake flask seed culture medium comprises the following components in percentage by mass and volume: corn starch 4%, yeast powder 3%, soybean peptone 2%, maltose syrup 2%, calcium chloride 0.4%, ammonium chloride 1.5%, potassium dihydrogen phosphate 1.8%, agar powder 1%, and water in balance, and the pH value is 5.5.
The composition of the shake flask screening culture medium in mass-volume ratio is as follows: 7% of corn starch, 4% of bean cake powder, 1.5% of yeast powder, 3.5% of corn steep liquor, 3% of malt syrup, 0.2% of calcium chloride, 1.2% of sodium dihydrogen phosphate, 1.3% of diammonium hydrogen phosphate, 1.7% of magnesium sulfate, 3.4% of dipotassium hydrogen phosphate, 0.25% of trisodium citrate and the balance of water, wherein the pH value is 5.5.
And carrying out shake flask directional screening for multiple times to obtain a mutagenic strain with high yield of acid-resistant alpha-amylase, wherein the number is YBG-90036, the average enzyme activity level in shake flask fermentation is 8510U/mL, and the fermentation period is 120 h.
The average enzyme activity level of the starting strain CGMCC NO.10785 before mutagenesis in shaking flask fermentation is 5427U/mL, and the fermentation period is 120 h. The comparison shows that the strains screened by mutagenesis have high enzyme production capability.
EXAMPLE 2 liquid fermentation production of acid-and high temperature-resistant alpha-amylase
And (3) streaking seeds to separate single colonies: freezing and storing the glycerinum pipe seeds of the bacillus licheniformis mutant strain YBG-90036, streaking and separating single colony in a seed slant culture medium, and culturing for 30h at 37 ℃.
The formula of the seed slant culture medium is as follows: corn starch 4%, yeast powder 3%, soybean peptone 2%, maltose syrup 2%, calcium chloride 0.4%, ammonium chloride 1.5%, potassium dihydrogen phosphate 1.8%, agar powder 1%, pH 5.5%.
Liquid seed culture: on the seed slant culture medium, single colony with high HC value (HC value is hydrolysis circle diameter/colony diameter) is selected, inoculated into 200mL liquid seed culture medium (using 500mL triangular flask), cultured at 37 deg.C with shaking table rotation speed of 300 r/min to OD600nmThe light absorption value is about 2.0.
Liquid seed culture medium: corn starch 4%, yeast powder 3%, soybean peptone 2%, maltose syrup 2%, calcium chloride 0.4%, ammonium chloride 1.5%, potassium dihydrogen phosphate 1.8%, pH 5.5%.
Seed tank culture: inoculating cultured seeds into a seed tank according to 12% of inoculation amount, fermenting at 38 deg.C under 0.08MPa, controlling dissolved oxygen to be more than 40% by increasing rotation speed and ventilation, and culturing for 10 hr.
The culture medium formula of the seeding tank comprises: corn starch 5%, yeast powder 4%, soybean peptone 3%, maltose syrup 3%, calcium chloride 0.4%, ammonium chloride 2%, potassium dihydrogen phosphate 2.8%, dipotassium hydrogen phosphate 1.7%, corn steep liquor 1%, and pH 5.5.
Culturing in a fermentation tank: inoculating according to 8% of the volume of the culture medium in the fermentation tank, at a culture temperature of 39 deg.C and a tank pressure of 0.08MPa, controlling dissolved oxygen content to be greater than 10%, maximum rotation speed of 900rpm, and maximum ventilation amount of 7.0m3And/h, after the maximum rotating speed and the ventilation rate are reached, if the dissolved oxygen is lower than 10%, the dissolved oxygen is not controlled any more. Feeding materials when the pH is higher than 5.7 in the fermentation process, controlling the pH to be 5.5, and finishing the fermentation when the thalli are seriously autolyzed, wherein the culture period is about 118 h.
The fermentation medium formula comprises: 7% of corn starch, 4% of soybean protein powder, 2% of yeast powder, 6% of corn steep liquor, 0.2% of calcium chloride, 2.2% of potassium dihydrogen phosphate, 1.3% of diammonium hydrogen phosphate, 1.7% of magnesium sulfate, 4.4% of disodium hydrogen phosphate, 0.25% of trisodium citrate and pH5.5
The formula of a supplemented medium is as follows: 30% of liquefied corn starch (the preparation method of the liquefied starch is that the starch is dissolved in water and adjusted to pH5.5, medium temperature amylase is added, and the mixture is liquefied for 1.5h at 55 ℃), 8% of corn steep liquor, 3% of soybean peptone, 4.0% of dipotassium phosphate, 0.25% of trisodium citrate and 3.8 of pH.
The case of performing 3 batches of fermentations was as follows, with the average fermentation enzyme activity: 9.0933 ten thousand U/mL.
Batches of Fermentation period (h) Ferment enzyme activity (Wan U/mL)
1 115 9.2040
2 116 8.9800
3 119 9.0960
Example 3 basic Properties of acid-and high-temperature resistant alpha-Amylase
The enzyme activity is measured under the conditions of 70 ℃ and pH4.0, an acid-resistant high-temperature alpha-amylase sample with the enzyme activity of 92040U/mL is taken as a reference, and the sample is respectively taken to carry out enzyme activity measurement experiments under different temperatures and different pH values. The optimum pH judgment standard is that the enzyme activity measured in different pH buffer solutions is more than 80% of the enzyme activity of a sample at 70 ℃, namely the optimum pH range of the enzyme. The determination standard of the optimal temperature range is that under the condition of pH4.0, the enzyme activity measured at different temperatures is more than 120% of the enzyme activity of the sample, namely the optimal temperature range of the enzyme. The optimum pH of the acid-resistant high-temperature alpha-amylase produced by the mutagenic strain YBG-90036 is 3.5-4.4; the optimal temperature reaction is 98-100 ℃; keeping the temperature at pH4.0 and 98 deg.C for 3h, and keeping the residual enzyme activity at 98%, as shown in FIGS. 1-3.
In addition, the enzyme activity definition and determination method of the acid-resistant high-temperature alpha-amylase comprises the following steps:
(1) definition of enzyme activity:
under the conditions of 70 ℃ and pH4.0, the enzyme amount required for liquefying 1mg of soluble starch in 1min is an enzyme activity unit and is expressed by U/mL (U/g).
(2) Reagents and solutions:
raw iodine solution: 22.0g of potassium iodide and 11.0g of iodine were weighed, dissolved completely in a small amount of water, and the volume was adjusted to 500mL and stored in a brown bottle.
Diluting iodine solution: 2.00mL of the original iodine solution is sucked, 20.0g of potassium iodide is added, the solution is dissolved by water and the volume is adjusted to 500mL, and the solution is stored in a brown bottle.
20g/L soluble starch solution: 2.000g of soluble starch (in absolute dry basis) was weighed to 0.0001g, slurried with water, slowly poured into 70mL of boiling water with stirring, then the beaker containing the starch was rinsed with water in portions, the rinse was incorporated therein, heated with stirring until completely clear, and cooled to a volume of 100 mL. The solution is ready for use.
Phosphate buffer (pH 4.0): 771mL of 0.2mol/L disodium hydrogen phosphate was added to 1229mL of 0.1mol/L citric acid, and the mixture was mixed and corrected with a pH meter.
Hydrochloric acid solution [ c (hcl) ═ 0.1mol/L ] was prepared according to GB 601.
(3) Preparation of enzyme solution to be tested
Accurately sucking 1mL of enzyme solution, fully dissolving with a small amount of phosphate buffer (pH4.0), transferring all samples into a volumetric flask, fixing the volume to scale with the phosphate buffer (pH4.0), shaking up, filtering with four layers of gauze, and standing filtrate. The concentration of the diluted enzyme solution is in the range of 60U/mL-65U/mL.
(4) The determination method comprises the following steps:
20g/L of a soluble starch solution 20.0m L and 5.0mL of phosphate buffer (pH4.0) were pipetted into a test tube, mixed well, and equilibrated in a thermostatic water bath at 70 ℃ for 8 min. Adding 1.00mL of diluted enzyme solution to be detected, timing immediately, shaking up, and reacting for 5min accurately. After the reaction, 1.00mL of the reaction solution was immediately aspirated, and the solution was added to a test tube previously containing 0.5mL of 0.1mol/L hydrochloric acid and 5.00mL of a dilute iodine solution, followed by shaking. A mixed solution of 0.5mL of 0.1mol/L hydrochloric acid and 5.00mL of diluted iodine solution is used as a reagent blank, and the absorbance (A) of the reagent blank is rapidly measured by using a 10mm cuvette under the wavelength of 660 nm. The concentration of the test enzyme solution was determined from a look-up table of absorbance (see GBT 2441-2009 for details).
The calculation formula of the acid-resistant high-temperature alpha-amylase activity is as follows:
X＝C×N×16.67
in the formula:
x is sample enzyme activity, u/mL;
c-concentration of test enzyme sample, u/mL;
n is sample dilution times;
16.67-conversion factor calculated according to enzyme activity definition.

Claims (2)

1. The Bacillus licheniformis for producing the acid-resistant high-temperature alpha-amylase is characterized in that the strain is Bacillus licheniformis (Bacillus licheniformis) YBG-90036, and the preservation number of the strain is CGMCC NO. 20672.
2. Use of bacillus licheniformis YBG-90036 according to claim 1 for producing acid and high temperature resistant alpha-amylase.
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