CN102154240A - Method for producing chitosanase by using Aspergillus usamii - Google Patents
Method for producing chitosanase by using Aspergillus usamii Download PDFInfo
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- CN102154240A CN102154240A CN2010105508731A CN201010550873A CN102154240A CN 102154240 A CN102154240 A CN 102154240A CN 2010105508731 A CN2010105508731 A CN 2010105508731A CN 201010550873 A CN201010550873 A CN 201010550873A CN 102154240 A CN102154240 A CN 102154240A
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- chitosanase
- state fermentation
- aspergillus usamii
- chitoanase
- solid state
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Abstract
The invention discloses a method for producing chitosanase by using Aspergillus usamii, which belongs to the technical field of biological engineering. In the method, agricultural and sideline products such as bran, soybean cake powder and rapeseed cake powder are used as fermentation substrates, a proper amount of inorganic nitrogen source, chitosan, inorganic salt, surfactant and the like are added, and the chitosanase is produced by the solid fermentation of an Aspergillus usamii E001 strain. The invention provides the formula and culture conditions of a solid fermentation culture medium for test production of chitosanase by the Aspergillus usamii E001 in a laboratory, and the chitosanase activity of a mature fermented grain material of the medium reaches 1,942 to 2,461U/g. The method has the advantages of simple equipment, small investment, quick effectiveness, low production cost, environmentally-friendly, high activity of chitosanase fermentation enzyme, and the like, contributes to the development and utilization of chitosanase and has high economic and practical value.
Description
Technical field
The present invention relates to a kind of method of utilizing Aspergillus usamii to produce chitoanase (Chitosanase), particularly be meant the novel process of utilizing Aspergillus usamii (Aspergillus usamii) E001 bacterial strain solid state fermentation to produce chitoanase, belong to technical field of bioengineering.
Background technology
Chitosan is chitinous deacetylation product, is a kind of macromolecule cation flocculation agent.Chitosan has good water-solubility through the oligochitosan that partial hydrolysis obtains, and nontoxic, no thermal source, no variability are easily absorbed by living organism, and has unique physiologically active, has a wide range of applications in fields such as medicine, food, agricultural.Utilize the high biological catalyst chitoanase hydrolyzing chitosan of specificity, have the reaction conditions gentleness, reaction process is easy to control, and environmentally safe, and characteristics such as the oligochitosan product is the highest, steady quality are the methods that a kind of ideal is produced oligochitosan.
Chitoanase is distributed more widely general, in bacterium (Bacillus, Myxobacter, Enterobacter), actinomycetes (Streptomyces, Nocardioides), mould (Aspergillus, Penicillium), virus (Chlorella virus PBCV-1, CVK-2) and plant, detect the existence of chitoanase at present, and from microbial fermentation solution purifying obtained chitoanase.Most microorganism chitoanases belong to inducible enzyme, and in the eubolism activity of cell, trace is synthetic or not synthetic, and in the presence of suitable substrate chitosan, chitoanase is induced synthetic.Chen Xiaoe etc. have reported and have utilized a strain aspergillus tubigensis (Aspergillus sp.) shake flask fermentation to produce chitoanase that the chitoanase activity of its fermented liquid is 197U/mL; Studied carbon source simultaneously to the influence that aspergillus mutant CJ22-326 produces enzyme, found that this mutant strain chitoanase is a kind of typical inducible enzyme, its synthetic and secretion all needs the existence of chitosan, and 2% chitosan (w/v) plays the best effect of inducing.The Microbacterium sp.OU01 bacterial strain of reports such as Sun Yuying produces the chitoanase activity and reaches 118U/mL.Yu Rong etc. have reported the new technology of utilizing Guizhou green muscardine fungus solid state fermentation to produce chitoanase in patent of invention (" chitosanase preparing process ", publication number CN1810963A), but the chitoanase activity on average only is the dried substratum of 83.1U/g.In sum, no matter be liquid fermenting or solid state fermentation, production by biological chitoanase activity is all lower at present, has caused the production and the application cost height of chitoanase, thereby has limited the widespread use of this enzyme; And do not see document or the patent report that utilizes the Aspergillus usamii solid state fermentation to produce chitoanase arranged.Microorganism solid fermentation has that equipment is simple, less investment, instant effect, production cost is low and advantage such as environmentally friendly.
Summary of the invention
The purpose of this invention is to provide agricultural byproducts such as a kind of employing wheat bran, soybean cake powder and coarse colza meal is the solid state fermentation base-material, and add an amount of inorganic nitrogen-sourced, chitosan, inorganic salt, tensio-active agent etc., on the bench scale of laboratory, utilize Aspergillus usamii E001 solid state fermentation to produce the method for chitoanase.The technical scheme of solid state fermentation of the present invention and theing contents are as follows:
1. bacterial strain: Aspergillus usamii (Aspergillus usamii) E001 bacterial strain, by Southern Yangtze University's screening and preservation, this bacterial strain is open at the Vol.6 No.2 of " biological processing " magazine Pg.38 in 2008, and the inventor promises to undertake that this bacterial strain provided to the public in 20 years.
2. inclined-plane seed culture: peeling potato 200g/L, sucrose 20g/L, agar 20g/L, pH 6.0; 121 ℃ of sterilization 20min place the test tube slant; Cooling back inoculation E001 bacterial strain is cultivated 96h for 30 ℃.This slant medium can be used for activation, preservation and the inclined-plane seed etc. of bacterial classification, switching in per 3 months 1 time.
3. wheat bran seed preparation: the bottled 10g base-material of 250mL triangle (wheat bran 10g), (NH
4)
2SO
4100mg, tap water 12mL, pH 6.0; Abundant mixing, 121 ℃ of sterilization 40min; Cooling back inoculation E001 test tube slant seed, 30 ℃ leave standstill and cultivate 96h, during respectively turn over Qu Yici respectively at 24h and 44h.
4. solid state fermentation: the bottled 10g base-material of 250mL triangle (wheat bran 8～10g, soybean cake powder 0～2g, coarse colza meal 0～1g), (NH
4)
2SO
450～100mg, chitosan 0～0.2g, MgSO
45～10mg, CaCl
25～10mg, KH
2PO
430～50mg, Tween-80 40mg, tap water 12～15mL, pH 6.0; 121 ℃ of sterilizations of substratum 40min; Cooling back inoculation one bacterium ear E001 wheat bran seed is cultivated 72～96h for 30～34 ℃, during respectively turn over Qu Yici respectively at 16～20h and 36～40h.
5. crude enzyme liquid extracts: solid state fermentation takes by weighing leaven material 1g after finishing, and adds pH 5.0,0.05mol/L citric acid-Sodium phosphate dibasic damping fluid of 9mL, soaking at room temperature 1h, and filter paper filtering, gained filtrate is the chitosan crude enzyme liquid.
6. analytical procedure:
Chitoanase determination of activity: in 25mL tool plug test tube A and B, add the mass concentration of preparing with pH 5.0, citric acid-Sodium phosphate dibasic damping fluid respectively and be 1.0% chitosan solution 2.4mL, 50 ℃ of preheating 10min, in the A pipe, add the suitably enzyme liquid of dilution of 0.1mL, 50 ℃ of accurate response 20min; Respectively add 2.5mL 3 ' immediately, 5 '-dinitrosalicylic acid (DNS) reagent, in the B pipe, add 0.1mL enzyme liquid again, A and B pipe all boil 7min; Respectively add deionized water 5mL, mixing after the cooling; 540nm sentences the B pipe and measures A pipe absorbance for blank, and finds corresponding reducing sugar (in glucosamine) content and be converted to unit of enzyme activity from the glucosamine hydrochloride typical curve.Unit of enzyme activity is defined as: under this condition determination, produce the required enzyme amount of 1 μ g reducing sugar with per minute and be defined as 1 chitoanase activity unit (U).
Reducing sugar test: adopting the DNS method, is standard with the glucosamine hydrochloride.
Bent material moisture determination: take by weighing a certain amount of solid state fermentation culture (bent material), dry to constant weight for 105 ℃.
Beneficial effect of the present invention: the present invention utilizes Aspergillus usamii E001 bacterial strain, adopts solid state fermentation to produce chitoanase, and the laboratory lab scale produces the chitoanase activity can reach 1942～2461U/g dry medium.The advantage that the present invention gives prominence to has: 1. adopt culture medium prescription and the fermentation condition of optimizing, fully excavated the potentiality that the E001 bacterial strain produces chitoanase; 2. adopt cheap agricultural byproducts such as wheat bran, soybean cake powder and coarse colza meal etc. as the main raw material of solid state fermentation, utilized natural renewable resources fully; 3. solid state fermentation is environmentally friendly, is a kind of mode of production of environmental protection; 4. compare advantage such as solid state fermentation is produced chitoanase and had that equipment is simple, less investment, production cost are low with utilizing the microbial liquid fermentation method.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, but this should be interpreted as that the scope of foregoing of the present invention only is confined to following embodiment.
Embodiment 1
Bacterial strain: Aspergillus usamii (Aspergillus usamii) E001 bacterial strain.
Solid-state fermentation culture medium: the bottled 10g base-material of 250mL triangle (wheat bran 9g+ soybean cake powder 1g), (NH
4)
2SO
4100mg, MgSO
410mg, CaCl
210mg, KH
2PO
450mg, tap water 12mL, pH 6.0.
Solid state fermentation conditions: 121 ℃ of substratum sterilization 40min, cooling back inoculation one bacterium ear E001 wheat bran seed, 30 ℃ leave standstill and cultivate 84h, during respectively turn over Qu Yici respectively at 20h and 40h.
The above-mentioned triangular flask experimental result that repeats 3 batches (every batches parallel do 3 bottles) shows that the chitoanase of the bent material of the ripe solid state fermentation of E001 produces equal enzymic activity and reaches the 1942U/g dry medium.
Embodiment 2
Bacterial strain: Aspergillus usamii (Aspergillus usamii) E001 bacterial strain.
Solid-state fermentation culture medium: the bottled 10g base-material of 250mL triangle (wheat bran 8g, soybean cake powder 1g, coarse colza meal 1g), chitosan 0.2g, (NH
4)
2SO
4100mg, Tween-80 40mg, MgSO
410mg, CaCl
210mg, KH
2PO
450mg, tap water 14mL, pH 6.0.
Solid state fermentation conditions: 121 ℃ of substratum sterilization 40min, cooling back inoculation one bacterium ear E001 wheat bran seed, 30 ℃ leave standstill and cultivate 96h, during respectively turn over Qu Yici respectively at 20h and 40h.
The above-mentioned triangular flask experimental result that repeats 3 batches (every batches parallel do 3 bottles) shows that the average enzymic activity of chitoanase of the bent material of the ripe solid state fermentation of E001 reaches the 2274U/g dry medium.
Embodiment 3
In order to help the growth of thalline, reach the purpose that shortens fermentation period and control living contaminants, present embodiment adopts the low alternating temperature master mode in the preceding high back of solid state fermentation temperature, and its fermentation strain and culture medium prescription are with embodiment 2.
Solid-state fermentation culture medium is after sterilization, cooling, inoculation, and 34 ℃ are cultured to 16h, turn over song the 1st time; 32 ℃ are cultured to 36h subsequently, turn over song the 2nd time; Last 30 ℃ are cultured to 72h.
The above-mentioned triangular flask experimental result that repeats 3 batches (every batches parallel do 3 bottles) shows that the average enzymic activity of chitoanase of the bent material of the ripe solid state fermentation of E001 reaches the 2461U/g dry medium.
Claims (5)
1. novel method that the laboratory bench scale is produced chitoanase, it is characterized in that: with agricultural byproducts such as wheat bran, soybean cake powder and coarse colza meals is the base-material of solid-state fermentation culture medium, and add an amount of chitosan, inorganic salt, tensio-active agent etc., in the 250mL triangular flask, utilize Aspergillus usamii (Aspergillus usamii) E001 bacterial strain solid state fermentation to produce chitoanase.
2. method according to claim 1 is characterized in that the bacterial classification that uses is an Aspergillus usamii E001 bacterial strain.
3. method according to claim 1 is characterized in that the zymotechnique that adopts is a solid state fermentation.
4. method according to claim 1 is characterized in that the solid-state fermentation culture medium prescription that uses is: the bottled 10g base-material of 250mL triangle (wheat bran 8～10g, soybean cake powder 0～2g, coarse colza meal 0～1g), (NH
4)
2SO
450～100mg, chitosan 0～0.2g, MgSO
45～10mg, CaCl
25～10mg, KH
2PO
430～50mg, Tween-80 40mg, tap water 12～15mL, pH 6.0.
5. method according to claim 1, it is characterized in that the solid state fermentation culture condition that adopts is: 121 ℃ of sterilizations of fermention medium 40min, cooling back inoculation one bacterium ear Aspergillus usamii E001 wheat bran seed, cultivate 72～96h for 34～30 ℃, during respectively turn over Qu Yici respectively at 16～20h and 36～40h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146587A (en) * | 2013-03-22 | 2013-06-12 | 江南大学 | Method for producing keratinase by induced fermentation of aspergillus usaanii for originally producing acidic proteinase |
-
2010
- 2010-11-19 CN CN2010105508731A patent/CN102154240A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146587A (en) * | 2013-03-22 | 2013-06-12 | 江南大学 | Method for producing keratinase by induced fermentation of aspergillus usaanii for originally producing acidic proteinase |
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Application publication date: 20110817 |