CN106754486B

CN106754486B - Pseudomonas for high-yield trehalose synthase and fermentation enzyme production method thereof

Info

Publication number: CN106754486B
Application number: CN201611080880.3A
Authority: CN
Inventors: 王兴吉; 刘文龙; 曹世源; 张�杰
Original assignee: Shandong Lonct Enzymes Co ltd
Current assignee: Shandong Lonct Enzymes Co ltd
Priority date: 2016-11-30
Filing date: 2016-11-30
Publication date: 2020-06-23
Anticipated expiration: 2036-11-30
Also published as: CN106754486A

Abstract

The invention discloses a Pseudomonas for high-yield trehalose synthase and a fermentation enzyme production method thereof, belonging to the field of microorganisms or enzymes, wherein the strain is specifically a Pseudomonas strain (Pseudomonas sp) CSY-67 with the preservation number of CGMCC No. 13154. The strain is subjected to deep fermentation in a 50L liquid tank, fed and fermented for 80h, and the average level of enzyme production is 39575U/mL. According to the method for liquid fermentation of the trehalose synthase, the produced trehalose synthase has the most suitable pH range of 7.5-8.5, the most suitable action temperature range of 35-45 ℃, the residual enzyme activity of 88% after heat preservation at 60 ℃ for 1 hour and the residual enzyme activity of 59.5% after heat preservation at 80 ℃ for 2 hours, has good heat resistance and can be widely applied to industrial production of trehalose.

Description

Pseudomonas for high-yield trehalose synthase and fermentation enzyme production method thereof

Technical Field

The invention belongs to the field of microorganisms or enzymes, and particularly relates to pseudomonas for producing trehalose synthase in a high yield manner and a fermentation enzyme production method thereof.

Background

Trehalose is a non-reducing disaccharide in which two molecules of glucose are linked by a 1, 1-glycosidic bond, and is widely present in organisms such as bacteria, yeast, filamentous fungi, plants, insects, and invertebrates. Trehalose has very important biological significance for organisms, is a reserve of energy and carbon sources, is a stabilizer and a protective agent of protein and biomembrane molecules in severe environments such as dehydration, high temperature, oxygen free radical, low temperature and the like, is a signal sensing compound and a growth regulating factor, and is also one of components of certain bacterial cell walls. Trehalose has special properties of moderate sweetness, stable property, difficult decomposition, no reducibility and the like, and has a unique function of protecting biomacromolecules (biomembranes, proteins and DNA) from being damaged by environmental pressure such as drying, high temperature, low temperature, peroxide and the like, so the disaccharide has a very great application value and is widely applied to the food processing industry, the pharmaceutical industry, the agriculture industry, the biochemical product industry and the cosmetic industry.

Trehalose synthase (trehalase synthase) was first discovered in 1995 by the Japanese Biochemical research institute, and it was capable of directly converting maltose into trehalose, and thereafter, in the case of Bacillus stearothermophilus: (Pimelobacter sp.R48), Pseudomonas putida (B)Pseudomonas putidaH262) And some thermophilic bacteria, and it is hopeful that this enzyme specifically catalyzes the conversion of maltose to trehalose without acting on glucose, maltotriose, maltoseBud oligosaccharide, sucrose, isomaltose, isomaltooligosaccharide, etc.

However, due to the restriction of enzyme activity and enzyme application conditions, trehalose synthase in the prior art can not meet the requirements of industrial production, so that a pseudomonas strain with high trehalose synthase yield is bred, the fermented product has good heat resistance and low production cost, and the method has great significance for promoting mechanization of various industries on a large scale.

Disclosure of Invention

The invention aims to provide a pseudomonas for producing trehalose synthase with high yield and a fermentation enzyme production method thereof, which can ensure the stability of the trehalose synthase with high yield by pseudomonas fermentation and obviously improve the good heat resistance of the trehalose synthase.

The purpose of the invention is realized by the following technical scheme:

the strain is pseudomonas of high-yield trehalose synthase, and the strain is pseudomonas (pseudomonas), and (pseudomonas)Pseudomonas sp.) CSY-67, the preservation number of the strain is CGMCC number 13154.

The optimum action pH range of trehalose synthase produced by the fermentation of the pseudomonas CSY-67 is 7.5-8.5, and the optimum action temperature range is 35-45 ℃.

The invention also aims to provide the method for producing the enzyme by the fermentation of the pseudomonas CSY-67, which mainly comprises the following steps:

slant culture: selecting a ring of pseudomonas CSY-67, inoculating the ring of pseudomonas CSY-67 to a solid slant culture medium, and culturing at a constant temperature of 37 ℃ for 48 hours to obtain first-stage seeds;

and (3) shake flask culture: taking a ring of the first-stage seeds, inoculating the ring of the first-stage seeds into a seed culture medium, and culturing for 48 hours at the constant temperature of 37 ℃ and the rotating speed of a shaking table of 200r/min to obtain a second-stage seed solution;

seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 5% (v/v) of the inoculum size, and culturing for 14-18 h at the constant temperature of 37 ℃ and the rotating speed of 200-;

culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a fermentation tank culture medium according to the proportion of 5% (v/v) of the inoculation amount, keeping the temperature at 37 ℃, setting the rotation speed at 200-: 0.25 vvm for 0h to 5h, 0.5 vvm for 5h to 20h, and 1.0 vvm for 20h to the end of fermentation, wherein the pH value of the fermentation broth is controlled to be 6.5 by using a supplemented medium in the whole fermentation process, the fermentation is finished when the enzyme activity is slowly increased and the bacterial autolysis is serious, and the fermentation period is 75-85 h, so that the final fermentation broth is obtained;

and (4) extracting and refining the final fermentation liquor to obtain the trehalose synthase finished product liquid enzyme preparation.

Further, the solid slant medium (g/L): peptone 12, yeast extract 6, agar 15, sodium chloride 1 and the balance of water, the pH value is 7.0, and the sterilization is carried out for 20min at the temperature of 121 ℃;

the seed culture medium (g/L): malt syrup 40, peptone 5, yeast extract 5, sodium sulfate 0.8, magnesium sulfate 0.5, calcium chloride 0.1, disodium hydrogen phosphate 0.5, and water in balance, pH7.0, sterilizing at 121 deg.C for 20 min;

the seeding tank medium (g/L): 40 parts of maltose syrup, 10 parts of glucose, 15 parts of yeast extract, 5 parts of corn steep liquor and KH₂PO₄1.5, 0.1 of calcium chloride, 7.0 of pH, and sterilizing for 30 min at the temperature of 123 ℃;

the fermenter medium (g/L): maltose 80, peptone 10, yeast extract 5, magnesium sulfate 0.5, calcium chloride 0.1, dipotassium hydrogen phosphate 0.3, pH7.0, 121-;

the feed flask culture medium (g/L): malt syrup 450, KH₂PO₄5, 8 parts of corn steep liquor and CaCl₂5, pH 4.0, sterilization at 123 ℃ for 30 min.

The extraction and refining method of the trehalose synthase comprises the following steps:

adding 1% of toluene into the final fermentation liquor to perform cell wall breaking treatment, adding 1-5% of perlite filter aid, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;

carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using a 20000 molecular weight ultrafiltration membrane to obtain a concentrated solution;

adding 20 percent (m/v) of stabilizer and 0.45 percent (m/v) of preservative by mass into the concentrated solution, adjusting the pH to 7.5, and then carrying out filtration sterilization to obtain the trehalose synthase finished product liquid enzyme preparation.

Preferably, the stabilizer is glycerin, and the preservative is 1 by mass; 2 potassium sorbate and sodium benzoate.

Further, the trehalose synthase finished product liquid enzyme preparation product is prepared by the extraction and refining method.

Pseudomonas bacteria (I) according to the inventionPseudomonas sp.) The CSY-67 is obtained by carrying out normal temperature and pressure plasma mutagenesis on a pseudomonas original strain CAO12, wherein the pseudomonas CSY-67 is preserved in China general microbiological culture Collection center (CGMCC for short) in 2016, 10, 26 days, and has the preservation number of CGMCC No.13154 and the preservation address of: western road No.1, north chen, west road, 3, china academy of sciences, zip code: 100101, classified and named as Pseudomonas (Pseudomonas sp.）。

The high-yield trehalose synthase strain of the invention comprises the following components: the colony morphology characteristics of the pseudomonas CSY-67 are as follows: round, milky white, regular periphery, lenticular, smooth surface, colored, opaque, odorous.

Has the advantages that:

the invention breeds a mutant strain of high-yield trehalose synthase by carrying out normal-temperature normal-pressure plasma mutagenesis on an original strain CAO12 of pseudomonas, optimizes the feeding conditions in the fermentation process of pseudomonas CSY-67 to ensure that the enzyme activity in the final fermentation liquid reaches 39000U/mL to 40000U/mL, and obtains the trehalose synthase finished product liquid enzyme preparation by an extraction and refining method, wherein the enzyme activity of the trehalose synthase finished product liquid enzyme preparation reaches 200000U/mL to 210000U/mL. And the highest enzyme activity in fermentation liquor after the pseudomonas original strain CAO12 is fermented in a liquid state is 36100U/mL. Thus, the enzyme activity of the pseudomonas CSY-67 is obviously higher than that of the pseudomonas original strain CAO 12.

The components of the culture medium in the fermentation method of the strain are all derived from raw materials with lower cost, and the fermentation cost is greatly reduced while the fermentation productivity is improved and the production efficiency is improved, thereby being greatly helpful for production.

The trehalose synthase obtained by fermentation of pseudomonas CSY-67 has the optimum pH range of 7.5-8.5, the optimum action temperature range of 35-45 ℃, the residual enzyme activity of 88% after heat preservation for 1h at 60 ℃ and the residual enzyme activity of 59.5% after heat preservation for 2h at 80 ℃, has obvious heat resistance, can be widely applied to high-temperature industrial production, obviously expands the industrial application range of the trehalose synthase and improves the application value of the trehalose synthase.

Drawings

FIG. 1: relative enzyme activity at different pH;

FIG. 2: relative enzyme activity at different temperatures;

FIG. 3: keeping the temperature at 80 ℃ for 0-3 h to obtain the relative enzyme activity.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

Example 1 Pseudomonas CSY-67 fermentation enzyme production and extraction and purification method of trehalose synthase produced by the same

The optimum action pH of trehalose synthase produced by fermentation of pseudomonas CSY-67 is 7.5, and the optimum action temperature range is 35 ℃.

The fermentation enzyme production method of pseudomonas CSY-67 mainly comprises the following steps:

seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 5% (v/v) of the inoculation amount, and culturing for 14-18 h at the constant temperature of 37 ℃ and the rotation speed of 200 r/min;

culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a 50L fermentation tank culture medium according to the proportion of 5% (v/v) of the inoculation amount, keeping the temperature at 37 ℃, setting the rotation speed at 200r/min, and setting the ventilation: 0.25 vvm for 0h to 5h, 0.5 vvm for 5h to 20h, and 1.0 vvm for 20h to the end of fermentation, wherein the pH value of the fermentation liquor is controlled to be 6.5 by using a supplemented medium in the whole fermentation process, the fermentation is finished when the enzyme activity is slowly increased and the bacterial autolysis is serious, and the fermentation period is 75 h, so that the final fermentation liquor is obtained;

Solid slant medium (g/L): peptone 12, yeast extract 6, agar 15, sodium chloride 1 and the balance of water, the pH value is 7.0, and the sterilization is carried out for 20min at the temperature of 121 ℃;

seed medium (g/L): malt syrup 40, peptone 5, yeast extract 5, sodium sulfate 0.8, magnesium sulfate 0.5, calcium chloride 0.1, disodium hydrogen phosphate 0.5, and water in balance, pH7.0, sterilizing at 121 deg.C for 20 min;

seeding tank medium (g/L): 40 parts of maltose syrup, 10 parts of glucose, 15 parts of yeast extract, 5 parts of corn steep liquor and KH₂PO₄1.5, 0.1 of calcium chloride, 7.0 of pH, and sterilizing for 30 min at the temperature of 123 ℃;

fermenter Medium (g/L): maltose 80, peptone 10, yeast extract 5, magnesium sulfate 0.5, calcium chloride 0.1, dipotassium hydrogen phosphate 0.3, pH7.0, 121-;

feed flask medium (g/L): malt syrup 450, KH₂PO₄5, 8 parts of corn steep liquor and CaCl₂5, pH 4.0, sterilization at 123 ℃ for 30 min.

The extraction and refining method of trehalose synthase is as follows:

firstly, adding 1% of toluene into the final fermentation liquor to perform cell wall breaking treatment, then adding 1% of perlite filter aid, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;

adding 20 percent (m/v) of glycerol, 0.15 percent (m/v) of potassium sorbate and 0.3 percent (m/v) of sodium benzoate into the concentrated solution by mass percentage, adjusting the pH value to 7.5, and then carrying out filtration sterilization to obtain the trehalose synthase finished product liquid enzyme preparation.

The trehalose synthase finished product liquid enzyme preparation product is prepared by the extraction and refining method.

And finally, the enzyme activity of the fermentation liquor is 39865U/mL, and the enzyme activity of the trehalose synthase finished product liquid enzyme preparation is 208800U/mL by an extraction and refining method.

Example 2 Pseudomonas CSY-67 fermentation enzyme production and extraction and purification method of trehalose synthase produced by the same

The optimum action pH range of trehalose synthase produced by fermentation of pseudomonas CSY-67 is 8.5, and the optimum action temperature range is 45 ℃.

seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 5% (v/v) of the inoculum size, and culturing for 14-18 h at the constant temperature of 37 ℃ and the rotating speed of 800 r/min;

culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a 50L fermentation tank culture medium according to the proportion of 5% (v/v) of the inoculation amount, keeping the temperature at 37 ℃, setting the rotation speed at 800 r/min, and setting the ventilation: 0.25 vvm for 0h to 5h, 0.5 vvm for 5h to 20h, and 1.0 vvm for 20h to the end of fermentation, wherein the pH value of the fermentation liquor is controlled to be 6.5 by using a supplemented medium in the whole fermentation process, the fermentation is finished when the enzyme activity is slowly increased and the bacterial autolysis is serious, and the fermentation period is 85 h, so that the final fermentation liquor is obtained;

The extraction and refining method of trehalose synthase is as follows:

firstly, adding 1% of toluene into the final fermentation liquor to perform cell wall breaking treatment, then adding 5% of perlite filter aid, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;

The final enzyme activity of the fermentation broth is 39025U/mL, and the enzyme activity of the trehalose synthase finished product liquid enzyme preparation is 207540U/mL by an extraction and refining method.

Example 3 Pseudomonas CSY-67 fermentation enzyme production and extraction and purification method of trehalose synthase produced by the same

The optimum action pH range of trehalose synthase produced by fermentation of pseudomonas CSY-67 is 8, and the optimum action temperature range is 40 ℃.

seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 5% (v/v) of the inoculation amount, and culturing for 16 h at the constant temperature of 37 ℃ and the rotating speed of 500 r/min;

culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a 50L fermentation tank culture medium according to the proportion of 5% (v/v) of the inoculation amount, keeping the temperature at 37 ℃, setting the rotation speed at 500r/min, and setting the ventilation: 0.25 vvm for 0h to 5h, 0.5 vvm for 5h to 20h, and 1.0 vvm for 20h to the end of fermentation, wherein the pH value of the fermentation liquor is controlled to be 6.5 by using a supplemented medium in the whole fermentation process, the fermentation is ended when the enzyme activity is slowly increased and the bacterial autolysis is serious, and the fermentation period is 80h, so that the final fermentation liquor is obtained;

The extraction and refining method of trehalose synthase is as follows:

Finally, the enzyme activity of the fermentation liquor is 40025U/mL, and the enzyme activity of the trehalose synthase finished product liquid enzyme preparation is 210450U/mL through an extraction and refining method.

Example 4 mutagenesis selection of Pseudomonas CSY-67

Collecting fresh slant of two original strains CAO12, eluting thallus with sterile water, shaking in test tube with glass beads to disperse thallus, centrifuging to collect thallus, re-suspending thallus with 5% glycerol, and counting with blood counting plate until the concentration is 10⁷-10⁸And (4) one/mL, and taking the obtained product as a starting bacterial suspension.

Starting the normal temperature and pressure plasma system, wiping the inside and outside of the operating room with alcohol cotton, and starting the ultraviolet lamp for sterilization for 30 min. After the sterilization in the operating room of the system is finished, 10 μ L of the bacterial suspension is spotted on the rough surface of the slide, and the slide is transferred to the table top of the operating room by tweezers under the aseptic condition. And opening a helium valve, and setting the air flow and the mutagenesis time for mutagenesis. The mutagenesis time was set to 90 s, 120 s, 150 s, 180 s, and 210 s, respectively. After each mutagenesis, the slide glass is placed in an EP tube containing 990 mu L of sterile physiological saline and vortexed for 1 min. After dilution and coating, the mixture is placed in an incubator at 30 ℃ for culture.

Solid broth medium (g/L): tryptone 10, yeast extract powder: 5, sodium chloride 10, agar 20 and the balance of water, wherein the pH value is 7.0;

tube slant medium (g/L): the concentration of the glucose 20 is controlled by the concentration of the glucose,peptone 10, yeast extract 2, KCl 10, MgSO₄·7H₂0.5 of O, 20 of agar and the balance of water, and the pH value is 7.2;

hypertonic solid medium (g/L): maltose 20, peptone 5, beef extract 2, K₂HPO₄1，NaCl 50，MgSO₄·7H₂0.5 of O, 20 of agar and the balance of water, and the pH value is 7.2;

enzyme production medium (g/L): maltose 20, peptone 6, beef extract 3, K₂HPO₄1.2，MgSO₄·7H₂O0.3, the balance being water, pH 7.2;

primary screening of strains: diluting the enriched bacterial liquid into 10 in sequence^-3、10^-4、10^-5、10^-6、10^-7And spreading the suspension on solid broth culture medium and hypertonic solid culture medium plates, and culturing at 37 ℃ and 45 ℃ for 72 hours respectively.

Re-screening of strains: selecting single bacterial colonies with different sizes, shapes and colors and typical characteristics from a primary screening solid plate, streaking, separating and purifying the single bacterial colonies on a solid broth culture medium, repeating the steps for 3 times, selecting the typical single bacterial colonies, streaking the single bacterial colonies on a test tube inclined plane of the solid broth culture medium, culturing at 37 ℃ for 96 h, and storing at 4 ℃ in a refrigerator.

Strain metabolic enzyme production test (preliminary screening): inoculating the screened strain into a 500 mL triangular flask containing 100 mL enzyme-producing culture medium, performing shake culture at 37 ℃ and 200r/min for 72h, and centrifuging at 5000 r/min for 20min to collect thalli. Washed twice with 0.03mol/L sodium phosphate buffer, pH7.2, and centrifuged under the following conditions: 5000 r/min and 20 min. Then, the cells were suspended in 9.8 mL of 0.03mol/L sodium phosphate buffer solution (pH 7.2), 0.2 mL of toluene was added thereto, and the reaction was carried out at 37 ℃ and 200r/min for 2 hours. Adding 10mL of 10% maltose solution, reacting at 37 ℃ and 200r/min for 10 h, and then inactivating the enzyme in boiling water bath for 10 min. Centrifuging at 5000 r/min for 20min, and collecting supernatant. The sugar content in the supernatant was qualitatively analyzed by thin layer chromatography, and the amount of the spot was 10. mu.L.

Strain metabolic enzyme production test (rescreening): and (3) qualitatively analyzing strains containing trehalose in the primary screening, re-screening, inoculating each strain into 5 bottles, inoculating the strains into 500 mL triangular bottles containing 100 mL enzyme-producing culture medium, carrying out shake cultivation at 37 ℃ at 200r/min for 72h, centrifuging at 5000 r/min for 20min, collecting thalli, weighing wet weight, and measuring moisture. The sugar content of the supernatant was quantitatively analyzed by HPLC, and the amount of the spot was 10. mu.L.

And (3) shaking a flask for re-screening:

the secondary screening method comprises the following steps: inoculating the mutagenic strain into a fermentation shake flask for fermentation at 30 ℃ at 220 r/min, culturing for 62 h, and measuring the fermentation enzyme activity of each mutant strain by a spectrophotometry.

The list of 5 mutant strains with higher enzyme activity screened by the shake flask is as follows:

TABLE 1 comparison of enzyme activities of fermentation broths of original strains and mutant strains with higher enzyme activities

Bacterial strains	Original bacteria	CSY-02	CSY-23	CSY-67	CSY-123	CSY-231
							Enzyme activity (U/mL)	1432	1571	1592	1768	1601	1632

And after the secondary shake flask fermentation, CSY-67 is bred to be the stable strain with the highest enzyme activity.

Example 5 Pseudomonas CSY-67 fermentation Performance validation

According to the embodiment 3, a 50L fermentation tank verification experiment is carried out according to the pseudomonas CSY-67 fermentation enzyme production and the extraction and refining method of the trehalose synthase produced by the fermentation enzyme production, the fermentation period is 80h, the fermentation enzyme production condition of 6 batches of the fermentation enzyme production is the average enzyme production level, and the enzyme activity in the final fermentation liquid is 39575U/mL. Table 2 shows that the strain not only produces trehalose synthase with high yield, but also has remarkable stability in fermentation performance and enzyme activity of the produced trehalose synthase.

It should be noted that: the mixed probiotic tablet prepared in the embodiments 1-2 of the invention also has the experimental effect, and the effect difference between the embodiments and the experimental effect is not large.

TABLE 26 fermentation enzyme production of high trehalose synthase Strain by batch

Batches of	Fermentation period (h)	Final enzyme activity in fermentation broth (U/mL)	Finished product liquid enzyme preparation enzyme activity (U/mL)
				1	80	39825	207300
2	80	38560	198984
				3	80	40025	210450
4	80	39690	205940
				5	80	39400	202875
6	80	39950	209425

Example 6 determination of enzyme Activity of trehalose synthase

(1) Preparation of crude enzyme solution: centrifuging the fermentation liquid at 6000 r/min for 15min, collecting thallus, adding KH (pH 7.0) 20 mmol/L₂PO₄-Na₂HPO₄And washing the bacterial suspension with a buffer solution, centrifuging, collecting thalli, preparing bacterial suspension, performing ultrasonic treatment, wherein the ultrasonic power is 200W, the ultrasonic time is 2 s, the interval is 3 s, the total ultrasonic time is 10 min, centrifuging the obtained treatment solution at 10000 r/min for 15min, and obtaining supernatant as crude enzyme solution.

(2) Definition of enzyme activity: the amount of enzyme that catalyzes the conversion of 30% maltose per hour per mL trehalose synthase to give 1 g trehalose was 1 unit (U/mL) at pH7.4 at 50 ℃

(3) And (3) enzyme activity determination: using maltose solution as substrate, and performing HPLCThe enzyme activity of the enzyme was measured. The enzyme activity measuring system is 800 mul containing maltose with 30% (W/V) final concentration and KH with 20 mmol/L₂PO₄-Na₂HPO₄Buffer solution, pH7.4, adding 400 μ L of diluted enzyme solution at 50 deg.C, reacting for 60 min, stopping enzyme reaction in boiling water bath for 10 min, centrifuging at 10000 r/min for 20min, and measuring the content of trehalose by HPLC.

Example 7 optimal pH Range for trehalose synthase

The trehalose synthase with the enzyme activity of 39000U/mL is taken as a reference, the enzyme activity is measured under different pH values (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5) at 35 ℃, the measured relative enzyme activity change curve is shown in figure 1, and the optimum action pH range of the enzyme is 7.5-8.5.

Example 8 optimum temperature Range for trehalose synthase

With trehalose synthase with the enzyme activity of 39000U/mL as a reference, the enzyme activity is measured under the condition that the pH value is 8.0 and at different temperatures (30, 35, 40, 45, 50, 55 and 60), the measured relative enzyme activity change curve is shown in figure 2, and the optimal action temperature range of the enzyme is 35-45 ℃.

Example 9 thermostability of trehalose synthase

Based on trehalose synthase with the enzyme activity of 39000U/mL in the final fermentation broth, the residual enzyme activity is determined by keeping the temperature at 80 ℃ for 0-3 h under the condition that the pH value is 8.0, as shown in figure 3, the residual enzyme activity is 59.5% after the temperature is kept at 80 ℃ for 2h, the trehalose synthase has good heat-resistant preservation activity, can be widely applied to high-temperature industrial production, obviously expands the industrial application range of the trehalose synthase and improves the application value of the trehalose synthase.

Claims

1. The pseudomonas for high-yield production of trehalose synthase is characterized in that: the strain is pseudomonas (Pseudomonas spCSY-67, the preservation number of the strain is CGMCC No. 13154.

2. The pseudomonad of claim 1, which produces trehalose synthase in high yield, wherein: the optimum action pH range of trehalose synthase produced by the fermentation of the pseudomonas CSY-67 is 7.5-8.5, and the optimum action temperature range is 35-45 ℃.

3. The method for producing trehalose synthase from pseudomonas by fermentation according to any one of claims 1-2, which comprises the following steps:

seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the volume ratio (v/v) of 5% of the inoculum size, and culturing for 14-18 h at the constant temperature of 37 ℃ and the rotating speed of 200-;

culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a fermentation tank culture medium according to the proportion of 5% volume ratio (v/v) of the inoculation amount, keeping the temperature at 37 ℃, setting the rotation speed at 200-: 0.25 vvm for 0h to 5h, 0.5 vvm for 5h to 20h, and 1.0 vvm for 20h to the end of fermentation, wherein the pH value of the fermentation broth is controlled to be 6.5 by using a supplemented medium in the whole fermentation process, the fermentation is finished when the enzyme activity is slowly increased and the bacterial autolysis is serious, and the fermentation period is 75-85 h, so that the final fermentation broth is obtained;

4. The process of claim 3, wherein the fermentation of Pseudomonas bacteria for high production of trehalose synthase comprises:

the mass-to-volume ratio (g/L) of the solid slant culture medium is as follows: peptone 12, yeast extract 6, agar 15, sodium chloride 1 and the balance of water, the pH value is 7.0, and the sterilization is carried out for 20min at the temperature of 121 ℃;

the mass-to-volume ratio (g/L) of the seed culture medium is as follows: malt syrup 40, peptone 5, yeast extract 5, sodium sulfate 0.8, magnesium sulfate 0.5, calcium chloride 0.1, disodium hydrogen phosphate 0.5, and water in balance, pH7.0, sterilizing at 121 deg.C for 20 min;

the mass-to-volume ratio (g/L) of the culture medium in the seeding tank is as follows: 40 parts of maltose syrup, 10 parts of glucose, 15 parts of yeast extract, 5 parts of corn steep liquor and KH₂PO₄1.5, 0.1 of calcium chloride and the balance of water, and sterilizing for 30 min at the temperature of 123 ℃ and the pH value of 7.0;

the mass-to-volume ratio (g/L) of the culture medium of the fermentation tank is as follows: maltose 80, peptone 10, yeast extract 5, magnesium sulfate 0.5, calcium chloride 0.1, dipotassium hydrogen phosphate 0.3 and the balance of water, and the pH value is 7.0, and the sterilization is carried out at the temperature of 123 ℃ for 30 min;

the mass-to-volume ratio (g/L) of the culture medium in the feeding bottle is as follows: malt syrup 450, KH₂PO₄5, 8 parts of corn steep liquor and CaCl₂5, the balance being water, pH 4.0, sterilization at 123 ℃ for 30 min.

5. The process of claim 3, wherein the fermentation of Pseudomonas bacteria for high production of trehalose synthase comprises: the extraction and refining method of the trehalose synthase comprises the following steps:

and adding 20% of stabilizer and 0.45% of preservative in mass-volume ratio (m/v) into the concentrated solution, adjusting the pH to 7.5, and then carrying out filtration sterilization to obtain the trehalose synthase finished product liquid enzyme preparation.

6. The process of claim 5, wherein the fermentation of Pseudomonas bacteria for high production of trehalose synthase comprises: the stabilizer is glycerin, and the preservative is prepared from the following components in a mass ratio of 1: 2 potassium sorbate and sodium benzoate.

7. The process of claim 3, wherein the fermentation of Pseudomonas bacteria for high production of trehalose synthase comprises: