CN109486795A - A method of utilizing protease deficiency micro-organisms chitosan enzyme - Google Patents
A method of utilizing protease deficiency micro-organisms chitosan enzyme Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
The present invention provides a kind of methods using protease deficiency micro-organisms chitosan enzyme, the protease deficiency microorganism is bacillus subtilis WB800, the following steps are included: S1: the bacillus subtilis WB800 being inoculated in activation medium plate, is placed in incubator and is inverted culture with activated strains；Single colonie after S2: picking step S1 activation is inoculated in seed culture medium, is placed in the OD of shaking table shake culture to seed liquor600Reach 0.6 ~ 1.0；S3: by the seed liquor access fermentation medium in step S2, it is placed in shaking table shake culture；S4: the product after step S3 fermentation is centrifuged, supernatant is collected, obtains the crude enzyme liquid of chitosan enzyme.The safe bacterial strain that the bacillus subtilis WB800 that the present invention uses belongs to microbial fermentation and technical field of enzyme engineering is used for a long time, it is nontoxic；In addition, fermentation period is short, fermentation liquid chitosan enzyme vigor reaches 45U/mL, and the crude enzyme liquid for being centrifugated acquisition may be directly applied to the production of chitosan oligosaccharide.
Description
Technical field
The present invention relates to microbial fermentation and technical field of enzyme engineering more particularly to a kind of utilization protease deficiency microorganisms
The method for producing chitosan enzyme.
Background technique
Chitosan enzyme can be used for food industry, by chitosan hydrolyzate at chitosan oligosaccharide.Chitosan oligosaccharide is chitin, chitosan product
Upgrading products, with the incomparable superiority of chitosan.It has that molecular weight is low, good water solubility, function are big, easy quilt
The advantages such as absorption of human body, bioactivity height, before the fields such as medicine, food, agricultural, environment and water process have a wide range of applications
Scape.About the strain source for producing chitosan enzyme, domestic and international most of Screening, Mutations from nature are obtained, but enzymatic production activity
It is universal not high.The safety of some bacterial strains itself also needs to be studied.Bacillus subtilis belongs to microbial fermentation and enzyme work
The safe bacterial strain that journey technical field is used for a long time, it is nontoxic, there are some reports for fermenting and producing chitosan enzyme.But it is withered
Careless bacillus itself can a large amount of protease of secreting, expressing, to degrade to the chitosan enzyme in fermentation liquid, make its by
Gradually lose activity.
Chinese patent CN107236721A provides a kind of bacillus subtilis chitosan enzyme and its preparation method and application,
According to the Preference of Pichia pastoris codon, the chitosan enzyme encoding gene of bacillus subtilis is optimized, further
Efficient secretory expression is carried out using chitosan enzyme encoding gene of the pichia yeast expression system the optimization, obtains withered grass bud
Spore bacillus chitosan enzyme.Degradation this method avoid the protease of bacillus secreting, expressing itself to chitosan enzyme, still
It needs to be produced using transgenic microorganism.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of using protease deficiency micro-organisms chitosan enzyme
Method, the protease deficiency microorganism are bacillus subtilis WB800, are preserved in the micro- life of Southern Yangtze University's Chinese Universities ' industry
Goods and materials source and information centre, CICIM number is B0132, be the described method comprises the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, is placed in incubator and is inverted culture to activate bacterium
Strain；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, is placed in the OD of shaking table shake culture to seed liquor600
Reach 0.6 ~ 1.0；
S3: enzymatic production
By in the seed liquor access fermentation medium in step S2, it is placed in shaking table shake culture；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, supernatant is collected, obtains the crude enzyme liquid of chitosan enzyme.
Wherein, the seed liquor is accessed in the fermentation medium by the inoculum concentration of percent by volume 1.5 ~ 3%.
Wherein, the formula of activation medium described in step S1 are as follows: 8 ~ 12 g/L of tryptone, 3 ~ 7 g/ of yeast extract
L, 7 ~ 11g/L of sodium chloride, 15 ~ 20 g/L of agar powder are 7.2 ~ 7.6 with the pH that sodium hydroxide adjusts the activation medium.
Preferably, the formula of the activation medium are as follows: 10 g/L of tryptone, 5 g/L of yeast extract, sodium chloride
10g/L, 20 g/L of agar powder are 7.4 with the pH that sodium hydroxide adjusts the activation medium.
Wherein, the formula of seed culture medium described in step S2 are as follows: 8 ~ 12 g/L of tryptone, 3 ~ 7 g/ of yeast extract
L, 7 ~ 11g/L of sodium chloride adjust the pH of the seed culture medium with sodium hydroxide, reach 7.2 ~ 7.6.
Preferably, the formula of the seed culture medium are as follows: tryptone 10 g/L, yeast extract 5g/L, sodium chloride
10g/L adjusts the pH of the seed culture medium with sodium hydroxide, reaches 7.4.
Wherein, the formula of fermentation medium described in step S3 are as follows: 20 ~ 30 g/L of chitosan oligosaccharide, 9 ~ 11 g/L of tryptone,
4 ~ 6 g/L of yeast extract, 9 ~ 11 g/L of sodium chloride, the pH of the fermentation medium is adjusted with sodium hydroxide, reaches 7.2
~7.6。
Preferably, the formula of the fermentation medium are as follows: 25 g/L of chitosan oligosaccharide, 10 g/L of tryptone, yeast extract 5
G/L, 10 g/L of sodium chloride, the pH of the seed culture medium is adjusted with sodium hydroxide, reaches 7.4.
Wherein, the chitosan oligosaccharide is the product for the chitosan partial hydrolysis that the degree of polymerization is 2 ~ 10.
Wherein, in step S1, the cultivation temperature of the actication of culture is 35 ~ 39 DEG C, the time is 12 ~ for 24 hours.
Preferably,
The cultivation temperature is 36 DEG C, 37 DEG C, 38 DEG C；
The incubation time is 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h.
Wherein, in step S2, the temperature of the seed culture is 35 ~ 39 DEG C, and the time is 12 ~ 16 h, and revolving speed is 180 ~ 220
r/min。
Preferably,
The cultivation temperature is 36 DEG C, 37 DEG C, 38 DEG C；
The incubation time is 13h, 14h, 15h；
The culture revolving speed be 185 r/min, 190 r/min, 195 r/min, 200 r/min, 205 r/min, 210r/min,
215 r/min。
Wherein, in step S3, the temperature of the enzymatic production is 35 ~ 39 DEG C, and the time is 25 ~ 30 h, and revolving speed is 180 ~ 220
r/min。
Preferably,
The fermentation temperature is 36 DEG C, 37 DEG C, 38 DEG C；
The fermentation time is 26 h, 27 h, 28 h, 29 h；
The fermentation revolving speed be 185 r/min, 190 r/min, 195 r/min, 200 r/min, 205 r/min, 210r/min,
215 r/min。
Wherein, in step S4, the time of the centrifuge separation is 10 ~ 20min, and revolving speed is 3000 ~ 5000 r/min.
Preferably,
The time of the centrifuge separation be 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min,
19min；
The revolving speed of the centrifuge separation is 3500 r/min, 4000 r/min, 4500 r/min.
Beneficial effects of the present invention:
Method provided by the invention using protease deficiency micro-organisms chitosan enzyme, the protease deficiency microorganism
It is bacillus subtilis WB800, bacillus subtilis WB800 belongs to microbial fermentation and technical field of enzyme engineering is used for a long time
Safe bacterial strain, nontoxic, the chitosan enzyme of production can trust the production applied to chitosan oligosaccharide.In addition, fermentation period is short, fermentation
Liquid chitosan enzyme vigor reaches 45U/mL, and the crude enzyme liquid for being centrifugated acquisition may be directly applied to the production of chitosan oligosaccharide.
Specific embodiment
It is the preferred embodiment of the present invention below, it is noted that for those skilled in the art,
Various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as this hair
Bright protection scope.
Embodiment 1
The present invention provides a kind of method using protease deficiency micro-organisms chitosan enzyme, the protease deficiency is micro-
Biology is bacillus subtilis WB800, comprising the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, controlled at 37 DEG C, is placed in incubator
Culture 20h is set with activated strains；
The formula of the activation medium are as follows: 10 g/L of tryptone, yeast extract 5 g/L, sodium chloride 10g/L, agar powder
20 g/L are 7.4 with the pH that sodium hydroxide adjusts the activation medium；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, controlled at 37 DEG C, revolving speed be 200 r/min in
15 h of shaking table shake culture to seed liquor OD600Reach 0.8；
The formula of the seed culture medium are as follows: 10 g/L of tryptone, yeast extract 5 g/L, sodium chloride 10g/L use hydrogen-oxygen
Change the pH that sodium adjusts the seed culture medium, reaches 7.4；
S3: enzymatic production
Seed liquor in step S2 is accessed in fermentation medium by the inoculum concentration of percent by volume 2%, controlled at 37 DEG C,
Revolving speed is 200 r/min in 28 h of shaking table shake culture；
The formula of the fermentation medium are as follows: 25 g/L of chitosan oligosaccharide, 10 g/L of tryptone, 5 g/L of yeast extract, sodium chloride
10 g/L adjust the pH of the fermentation medium with sodium hydroxide, reach 7.4；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, control revolving speed is 3500 r/min, is centrifuged 15min, collects supernatant, obtains
Obtain the crude enzyme liquid of chitosan enzyme.
The vigor of the chitosan enzyme produced through this embodiment reaches 45U/mL.
Embodiment 2
The present invention provides a kind of method using protease deficiency micro-organisms chitosan enzyme, the protease deficiency is micro-
Biology is bacillus subtilis WB800, comprising the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, controlled at 37 DEG C, is placed in incubator
Culture 15h is set with activated strains；
The formula of the activation medium are as follows: 10 g/L of tryptone, yeast extract 5 g/L, sodium chloride 10g/L, agar powder
20 g/L are 7.4 with the pH that sodium hydroxide adjusts the activation medium；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, controlled at 38 DEG C, revolving speed be 220 r/min in
14 h of shaking table shake culture to seed liquor OD600Reach 0.7；
The formula of the seed culture medium are as follows: 10 g/L of tryptone, yeast extract 5 g/L, sodium chloride 10g/L use hydrogen-oxygen
Change the pH that sodium adjusts the seed culture medium, reaches 7.4；
S3: enzymatic production
Seed liquor in step S2 is accessed in fermentation medium by the inoculum concentration of percent by volume 2.5%, controlled at 36
DEG C, revolving speed is 220 r/min in 25 h of shaking table shake culture；
The formula of the fermentation medium are as follows: 25 g/L of chitosan oligosaccharide, 10 g/L of tryptone, 5 g/L of yeast extract, sodium chloride
10 g/L adjust the pH of the fermentation medium with sodium hydroxide, reach 7.4；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, control revolving speed is 4000 r/min, is centrifuged 10min, collects supernatant, obtains
Obtain the crude enzyme liquid of chitosan enzyme.
The vigor of the chitosan enzyme produced through this embodiment reaches 42U/mL.
Embodiment 3
The present invention provides a kind of method using protease deficiency micro-organisms chitosan enzyme, the protease deficiency is micro-
Biology is bacillus subtilis WB800, comprising the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, controlled at 39 DEG C, is placed in incubator
Culture 13h is set with activated strains；
The formula of the activation medium are as follows: 10 g/L of tryptone, yeast extract 5 g/L, sodium chloride 10g/L, agar powder
20 g/L are 7.4 with the pH that sodium hydroxide adjusts the activation medium；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, controlled at 38 DEG C, revolving speed be 180 r/min in
12 h of shaking table shake culture to seed liquor OD600Reach 0.9；
The formula of the seed culture medium are as follows: 10 g/L of tryptone, yeast extract 5 g/L, sodium chloride 10g/L use hydrogen-oxygen
Change the pH that sodium adjusts the seed culture medium, reaches 7.4；
S3: enzymatic production
Seed liquor in step S2 is accessed in fermentation medium by the inoculum concentration of percent by volume 3%, controlled at 37 DEG C,
Revolving speed is 210 r/min in 28 h of shaking table shake culture；
The formula of the fermentation medium are as follows: 25 g/L of chitosan oligosaccharide, 10 g/L of tryptone, 5 g/L of yeast extract, sodium chloride
10 g/L adjust the pH of the fermentation medium with sodium hydroxide, reach 7.4；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, control revolving speed is 5000 r/min, is centrifuged 12min, collects supernatant, obtains
Obtain the crude enzyme liquid of chitosan enzyme.
The vigor of the chitosan enzyme produced through this embodiment reaches 38U/mL.
Embodiment 4
The present invention provides a kind of method using protease deficiency micro-organisms chitosan enzyme, the protease deficiency is micro-
Biology is bacillus subtilis WB800, comprising the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, controlled at 37 DEG C, is placed in incubator
Culture 20h is set with activated strains；
The formula of the activation medium are as follows: 9 g/L of tryptone, yeast extract 5 g/L, sodium chloride 9g/L, agar powder 18
G/L is 7.3 with the pH that sodium hydroxide adjusts the activation medium；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, controlled at 37 DEG C, revolving speed be 200 r/min in
15 h of shaking table shake culture to seed liquor OD600Reach 0.8；
The formula of the seed culture medium are as follows: 12 g/L of tryptone, yeast extract 5 g/L, sodium chloride 10g/L use hydrogen-oxygen
Change the pH that sodium adjusts the seed culture medium, reaches 7.5；
S3: enzymatic production
Seed liquor in step S2 is accessed in fermentation medium by the inoculum concentration of percent by volume 2%, controlled at 37 DEG C,
Revolving speed is 200 r/min in 28 h of shaking table shake culture；
The formula of the fermentation medium are as follows: 30 g/L of chitosan oligosaccharide, 9 g/L of tryptone, 6 g/L of yeast extract, sodium chloride
10 g/L adjust the pH of the fermentation medium with sodium hydroxide, reach 7.4；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, control revolving speed is 3500 r/min, is centrifuged 15min, collects supernatant, obtains
Obtain the crude enzyme liquid of chitosan enzyme.
The vigor of the chitosan enzyme produced through this embodiment reaches 43U/mL.
Embodiment 5
The present invention provides a kind of method using protease deficiency micro-organisms chitosan enzyme, the protease deficiency is micro-
Biology is bacillus subtilis WB800, comprising the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, controlled at 37 DEG C, is placed in incubator
Culture 20h is set with activated strains；
The formula of the activation medium are as follows: 9 g/L of tryptone, yeast extract 5 g/L, sodium chloride 8g/L, agar powder 17
G/L is 7.5 with the pH that sodium hydroxide adjusts the activation medium；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, controlled at 37 DEG C, revolving speed be 200 r/min in
15 h of shaking table shake culture to seed liquor OD600Reach 0.8；
The formula of the seed culture medium are as follows: 8 g/L of tryptone, yeast extract 6 g/L, sodium chloride 11g/L use hydroxide
Sodium adjusts the pH of the seed culture medium, reaches 7.5；
S3: enzymatic production
Seed liquor in step S2 is accessed in fermentation medium by the inoculum concentration of percent by volume 2%, controlled at 37 DEG C,
Revolving speed is 200 r/min in 28 h of shaking table shake culture；
The formula of the fermentation medium are as follows: 23 g/L of chitosan oligosaccharide, 11 g/L of tryptone, 4 g/L of yeast extract, sodium chloride
9 g/L adjust the pH of the fermentation medium with sodium hydroxide, reach 7.5；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, control revolving speed is 3500 r/min, is centrifuged 15min, collects supernatant, obtains
Obtain the crude enzyme liquid of chitosan enzyme.
The vigor of the chitosan enzyme produced through this embodiment reaches 37 U/mL.
Embodiment 6
The present invention provides a kind of method using protease deficiency micro-organisms chitosan enzyme, the protease deficiency is micro-
Biology is bacillus subtilis WB800, comprising the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, controlled at 37 DEG C, is placed in incubator
Culture 20h is set with activated strains；
The formula of the activation medium are as follows: tryptone 12 g/L, yeast extract 6g/L, sodium chloride 8g/L, agar powder 17
G/L is 7.3 with the pH that sodium hydroxide adjusts the activation medium；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, controlled at 37 DEG C, revolving speed be 200 r/min in
15 h of shaking table shake culture to seed liquor OD600Reach 0.8；
The formula of the seed culture medium are as follows: 10 g/L of tryptone, yeast extract 4 g/L, sodium chloride 10g/L use hydrogen-oxygen
Change the pH that sodium adjusts the seed culture medium, reaches 7.5；
S3: enzymatic production
Seed liquor in step S2 is accessed in fermentation medium by the inoculum concentration of percent by volume 2%, controlled at 37 DEG C,
Revolving speed is 200 r/min in 28 h of shaking table shake culture；
The formula of the fermentation medium are as follows: 24 g/L of chitosan oligosaccharide, 10 g/L of tryptone, 4 g/L of yeast extract, sodium chloride
11 g/L adjust the pH of the fermentation medium with sodium hydroxide, reach 7.6；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, control revolving speed is 3500 r/min, is centrifuged 15min, collects supernatant, obtains
Obtain the crude enzyme liquid of chitosan enzyme.
The vigor of the chitosan enzyme produced through this embodiment reaches 41U/mL.
At present it has been reported that shake flask fermentation production chitosan enzyme vigor it is highest be Qilu University of Technology's food and life
One plant of solution keratan microbacterium of object engineering college and Jinan Haidebei Marine Organism Engineering Co., Ltd.'s joint study
(Microbacterium keratanolyticum), in optimal conditions, the bacterial strain is with 4% inoculum concentration, fermented and cultured 96
H, the enzyme activity for producing chitosan enzyme reach 124U/mL.Although enzyme activity of the invention is up to 45U/mL, but fermentation time contracts
25 ~ 30 h are short to, unit time institute's producing enzyme vigor is practical higher.And the present invention uses safe bacterial strain, and more added with
Conducive to large scale fermentation production from now on.
It should be noted that bacillus subtilis WB800 described in the embodiment of the present invention, it is high to be preserved in China, Southern Yangtze University
Gyp's industry microbial resources and information centre, CICIM number is B0132；Chitosan oligosaccharide described in the embodiment of the present invention is polymerization
The product for the chitosan partial hydrolysis that degree is 2 ~ 10.
Only several embodiments of the present invention are expressed for above embodiments, and the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these are all to belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of method using protease deficiency micro-organisms chitosan enzyme, the protease deficiency microorganism is withered grass
Bacillus WB800, is preserved in Southern Yangtze University's Chinese Universities ' industrial microorganism resource and information centre, and CICIM number is
B0132, which is characterized in that the described method comprises the following steps:
S1: actication of culture
The bacillus subtilis WB800 is inoculated in activation medium plate, is placed in incubator and is inverted culture to activate bacterium
Strain；
S2: seed culture
Single colonie after picking step S1 activation is inoculated in seed culture medium, is placed in the OD of shaking table shake culture to seed liquor600
Reach 0.6 ~ 1.0；
S3: enzymatic production
By in the seed liquor access fermentation medium in step S2, it is placed in shaking table shake culture；
S4: centrifuge separation
Product after step S3 fermentation is centrifuged, supernatant is collected, obtains the crude enzyme liquid of chitosan enzyme.
2. a kind of method using protease deficiency micro-organisms chitosan enzyme according to claim 1, feature exist
In: the seed liquor is accessed in the fermentation medium by the inoculum concentration of percent by volume 1.5 ~ 3%.
3. a kind of method using protease deficiency micro-organisms chitosan enzyme according to claim 1, feature exist
In the formula of activation medium described in step S1 are as follows: 8 ~ 12g/L of tryptone, 3 ~ 7g/L of yeast extract, sodium chloride 7 ~
11g/L, 15 ~ 20g/L of agar powder are 7.2 ~ 7.6 with the pH that sodium hydroxide adjusts the activation medium.
4. a kind of method using protease deficiency micro-organisms chitosan enzyme according to claim 1, feature exist
In the formula of seed culture medium described in step S2 are as follows: 8 ~ 12g/L of tryptone, 3 ~ 7g/L of yeast extract, sodium chloride 7 ~
11g/L adjusts the pH of the seed culture medium with sodium hydroxide, reaches 7.2 ~ 7.6.
5. a kind of method using protease deficiency micro-organisms chitosan enzyme according to claim 1, feature exist
In the formula of fermentation medium described in step S3 are as follows: 20 ~ 30g/L of chitosan oligosaccharide, 9 ~ 11g/L of tryptone, yeast extract 4 ~
6g/L, 9 ~ 11g/L of sodium chloride adjust the pH of the fermentation medium with sodium hydroxide, reach 7.2 ~ 7.6.
6. a kind of method using protease deficiency micro-organisms chitosan enzyme according to claim 5, feature exist
In: the chitosan oligosaccharide is the product for the chitosan partial hydrolysis that the degree of polymerization is 2 ~ 10.
7. a kind of poly- using protease deficiency micro-organisms shell described in claim according to claim 1 ~ any one of 6
The method of carbohydrase, it is characterised in that: in step S1, the cultivation temperature of the actication of culture is 35 ~ 39 DEG C, the time is 12 ~ for 24 hours.
8. a kind of poly- using protease deficiency micro-organisms shell described in claim according to claim 1 ~ any one of 6
The method of carbohydrase, it is characterised in that: in step S2, the temperature of the seed culture is 35 ~ 39 DEG C, and the time is 12 ~ 16h, revolving speed
For 180 ~ 220 r/min.
9. a kind of poly- using protease deficiency micro-organisms shell described in claim according to claim 1 ~ any one of 6
The method of carbohydrase, it is characterised in that: in step S3, the temperature of the enzymatic production is 35 ~ 39 DEG C, and the time is 25 ~ 30h, revolving speed
For 180 ~ 220 r/min.
10. a kind of described in claim according to claim 1 ~ any one of 6 utilize protease deficiency micro-organisms shell
The method of dextranase, it is characterised in that: in step S4, the time of the centrifuge separation is 10 ~ 20min, and revolving speed is 3000 ~ 5000
r/min。
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|---|---|---|---|---|
| CN116064619A (en) * | 2022-09-26 | 2023-05-05 | 无锡特殊食品与营养健康研究院有限公司 | Bacillus licheniformis cell capable of being stably and repeatedly used for D-psicose conversion synthesis |
| CN116064619B (en) * | 2022-09-26 | 2023-11-21 | 无锡特殊食品与营养健康研究院有限公司 | Bacillus licheniformis cell capable of being stably and repeatedly used for D-psicose conversion synthesis |
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