CN112795553A - Method for continuously producing xylanase by using clostridium - Google Patents
Method for continuously producing xylanase by using clostridium Download PDFInfo
- Publication number
- CN112795553A CN112795553A CN202110016847.9A CN202110016847A CN112795553A CN 112795553 A CN112795553 A CN 112795553A CN 202110016847 A CN202110016847 A CN 202110016847A CN 112795553 A CN112795553 A CN 112795553A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- clostridium
- xylanase
- carrier
- producing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 28
- 241000193403 Clostridium Species 0.000 title claims abstract description 22
- 238000000855 fermentation Methods 0.000 claims abstract description 57
- 230000004151 fermentation Effects 0.000 claims abstract description 57
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 29
- 229920000742 Cotton Polymers 0.000 claims description 18
- 241000193401 Clostridium acetobutylicum Species 0.000 claims description 17
- 229910052799 carbon Inorganic materials 0.000 claims description 15
- 239000004698 Polyethylene Substances 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- -1 polyethylene Polymers 0.000 claims description 10
- 229920000573 polyethylene Polymers 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 8
- 239000004927 clay Substances 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 6
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 5
- 239000000919 ceramic Substances 0.000 claims description 5
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 5
- 239000004744 fabric Substances 0.000 claims description 4
- 239000000835 fiber Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 229920001778 nylon Polymers 0.000 claims description 4
- 229920003023 plastic Polymers 0.000 claims description 4
- 239000004033 plastic Substances 0.000 claims description 4
- 239000002985 plastic film Substances 0.000 claims description 4
- 229920000747 poly(lactic acid) Polymers 0.000 claims description 4
- 229920000728 polyester Polymers 0.000 claims description 4
- 239000004626 polylactic acid Substances 0.000 claims description 4
- 229920002635 polyurethane Polymers 0.000 claims description 4
- 239000004814 polyurethane Substances 0.000 claims description 4
- 235000003301 Ceiba pentandra Nutrition 0.000 claims description 3
- 244000146553 Ceiba pentandra Species 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 31
- 108090000790 Enzymes Proteins 0.000 abstract description 31
- 230000000694 effects Effects 0.000 abstract description 19
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 5
- 230000007547 defect Effects 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 229920002488 Hemicellulose Polymers 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 4
- 241000423302 Clostridium acetobutylicum ATCC 824 Species 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 239000012876 carrier material Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 108050001049 Extracellular proteins Proteins 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 2
- 229960004050 aminobenzoic acid Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- DNZWLJIKNWYXJP-UHFFFAOYSA-N butan-1-ol;propan-2-one Chemical compound CC(C)=O.CCCCO DNZWLJIKNWYXJP-UHFFFAOYSA-N 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 229910052564 epsomite Inorganic materials 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 229910052603 melanterite Inorganic materials 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- 235000019157 thiamine Nutrition 0.000 description 2
- 239000011721 thiamine Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 229920001221 xylan Polymers 0.000 description 2
- 150000004823 xylans Chemical class 0.000 description 2
- XMTQQYYKAHVGBJ-UHFFFAOYSA-N 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA Chemical compound CN(C)C(=O)NC1=CC=C(Cl)C(Cl)=C1 XMTQQYYKAHVGBJ-UHFFFAOYSA-N 0.000 description 1
- 241001112696 Clostridia Species 0.000 description 1
- 229920001131 Pulp (paper) Polymers 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000005293 duran Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000006529 extracellular process Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- QARBMVPHQWIHKH-UHFFFAOYSA-N methanesulfonyl chloride Chemical compound CS(Cl)(=O)=O QARBMVPHQWIHKH-UHFFFAOYSA-N 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920013716 polyethylene resin Polymers 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for continuously producing xylanase by using clostridium, which comprises the step of continuously fermenting clostridium capable of producing xylanase in a fermentation medium containing a carrier to obtain fermentation liquor containing xylanase. The method realizes continuous enzyme production; meanwhile, the obtained xylanase has high enzyme activity, and the enzyme activity is stable in the fermentation process and is not lower than that of single batch fermentation; in addition, the method has the advantages of easy separation of enzyme products and low cost, and solves the defects of complicated separation and purification process, high cost, low efficiency and the like in the enzyme production in the prior art.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for continuously producing xylanase by using clostridium.
Background
Liquid fermentation is used as one of microbial fermentation methods, and is widely applied due to factors such as easy separation of products, single type, good stability, easy regulation and control of the fermentation process and the like. The enzyme products obtained by microbial fermentation generally have both intracellular and extracellular types. If extracellular, it is easy to mix with other proteins because it exists in the fermentation broth, and it is common to first obtain a crude enzyme solution by centrifugation and filtration of the culture medium and cells in the fermentation broth, then concentrate the target protein by salting out, ultrafiltration or the like, and further separate and purify the crude enzyme solution by ion exchange chromatography, affinity chromatography or the like. If intracellular, the cells must be disrupted before the extracellular process is repeated. The process has the disadvantages of high cost, low efficiency and the like.
The clostridium can adhere to the carrier under the condition of standing fermentation, aggregate and form a biofilm taking extracellular protein as a main component. According to the previous research in the laboratory and the reports of related documents, the clostridium not only has extremely strong protein secretion capacity (successfully expresses a plurality of foreign proteins), but also can be adsorbed, fixed and formed into a film on the surfaces of a plurality of medium carriers. When the clostridium forms a biofilm on the solid surface, the biofilm not only protects internal cells, but also is beneficial to continuous fermentation. Especially, cells secrete a large amount of extracellular proteins to form a stable biofilm, so that the surface immobilized continuous fermentation technology is widely used.
However, the current people focus on the chemical production of clostridia, and neglect the production of proteins with high biological activity, such as enzymes. Therefore, the invention provides a method for continuously producing xylanase by using clostridium.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of the prior art and provides a method for continuously producing xylanase by using clostridium.
In order to solve the technical problem, the invention discloses a method for continuously producing xylanase by using clostridium, which comprises the step of continuously fermenting the clostridium capable of producing the xylanase in a fermentation culture medium containing a carrier to obtain fermentation liquor containing the xylanase.
Wherein the clostridium capable of producing xylanase is clostridium acetobutylicum.
Preferably, the clostridium acetobutylicum is any one or two combinations of clostridium acetobutylicum B3 and clostridium acetobutylicum ATCC 824.
The clostridium acetobutylicum B3(C.acetobutylicum B3) has the preservation number of CGMCC No.5234, and the information of the strain is disclosed in detail in Chinese patent with the application number of 201210075094. X.
Among them, clostridium acetobutylicum ATCC824(c. acetobutylicum ATCC824), which is also disclosed in the prior art, is disclosed.
Preferably, the clostridium capable of producing xylanase is a recombinant clostridium acetobutylicum which is obtained by genetically modifying clostridium acetobutylicum.
Further preferably, the recombinant clostridium acetobutylicum is B3-XynB and 824-XynB; the construction method of the recombinant clostridium acetobutylicum is disclosed in the Chinese invention application: CN 202010085119.9A construction method of recombinant acetone butanol clostridium and application thereof in preparing butanol by fermenting hemicellulose.
Still more preferably, the recombinant Clostridium acetobutylicum is B3-XynB.
The carrier is made of any one or a combination of more of polyester fiber, polylactic acid, cotton fiber, nylon fiber, wood pulp cotton, activated carbon, polyethylene, polyvinyl alcohol, polyurethane, clay, metal and ceramic.
Wherein the carrier is made of the material or any one of cloth strips, resin, sponge analogs, plastic sheets, plastic springs and glass slides obtained by processing the carrier.
Wherein the shape of the carrier includes, but is not limited to, a strip, a hole, a sheet, a net and a sphere.
Wherein the dosage of the carrier is 5-60 g/L.
Wherein the continuous fermentation mode is that a fermentation solution is released in the fermentation process and a fresh fermentation culture medium is supplemented; alternatively, the fermentation broth is discharged at the end of each batch and simultaneously supplemented with fresh fermentation medium.
Wherein, in the continuous fermentation process, the concentration of the carbon source is 1-100 g/L.
Preferably, in the continuous fermentation, the initial concentration of the carbon source is 50-60 g/L.
Further preferably, in the continuous fermentation, the initial concentration of the carbon source is 60 g/L.
Still more preferably, the carbon source is glucose or hemicellulose.
Still further preferably, the carbon source is glucose.
Most preferably, the content of each component in the fermentation medium is 60g/L of carbon source, CH3COONH4 2.2g/L，K2HPO4 0.5g/L，KH2PO4 0.5g/L，FeSO4·7H2O 0.2g/L，MgSO4·7H2O 0.2g/L，MnSO4·H20.01g/L of O, 0.01g/L of NaCl, 0.001g/L of p-aminobenzoic acid, 0.001g/L of thiamine and 0.0001g/L of biotin.
Wherein the fermentation temperature is 30-40 ℃.
Preferably, the temperature of the fermentation is 37 ℃.
Wherein the fermentation time is 10-150 h.
Wherein, the xylanase produced by the method has relatively stable enzyme activity.
Wherein, the relatively stable enzyme activity means that the enzyme activity fluctuates up and down in a small range.
Wherein the enzyme activity is defined as that 1 enzyme activity unit U is required for degrading and releasing 1mmol of reducing sugar from a xylan solution with the concentration of 5mg/mL per minute at the temperature of 65 ℃ and the pH value of 6.0.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention provides a method for producing xylanase by immobilized continuous fermentation on the surface of a medium based on clostridium, which realizes continuous enzyme production; meanwhile, the obtained xylanase has high enzyme activity, and the enzyme activity is stable in the fermentation process and is not lower than that of single batch fermentation; in addition, the method has the advantages of easy separation of enzyme products and low cost, and solves the defects of complicated separation and purification process, high cost, low efficiency and the like in the enzyme production in the prior art.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 shows an immobilized fermentation carrier (not entering fermentation). 1 is polyester fiber, 2 is polylactic acid, 3 is cloth, 4 is cotton fiber (spherical), 5 is cotton fiber (towel), 6 is polyethylene (small), 7 is polyethylene (large), 8 is resin (porous), 9 is activated carbon (sheet), 10 is activated carbon (net), 11 is kapok (white), 12 is PVA, 13 is square cotton, 14 is sponge analogue (large pore), 15 is polyurethane, 16 is clay material, 17 is metal pipe net, 18 is plastic sheet, 19 is ceramic, 20 is nylon fiber, 21 is plastic spring, 22 is glass slide.
FIG. 2 shows the growth of bacterial cells on a carrier after completion of culture. 1 is polyester fiber, 2 is polylactic acid, 3 is cloth, 4 is cotton fiber (spherical), 5 is cotton fiber (towel), 6 is polyethylene (small), 7 is polyethylene (large), 8 is resin (porous), 9 is activated carbon (sheet), 10 is activated carbon (net), 11 is kapok (white), 12 is PVA, 13 is square cotton, 14 is sponge analogue (large pore), 15 is polyurethane, 16 is clay material, 17 is metal pipe net, 18 is plastic sheet, 19 is ceramic, 20 is nylon fiber, 21 is plastic spring, 22 is glass slide.
FIG. 3 shows the cotton towels cultured for different days, namely, 1 day of non-culture, 2 days of culture for 5 days, and 3 days of culture for 10 days.
FIG. 4 shows the enzyme activity and growth density of extracellular xylanase by using B3 as host strain in culture with glucose as carbon source.
FIG. 5 shows the enzyme activity and growth density of extracellular xylanase by using 824 as a host strain in culture with glucose as a carbon source.
FIG. 6 shows the enzyme activity of extracellular xylanase in the case of hemicellulose as carbon source, using B3 as host strain.
Detailed Description
The meaning of the present invention will be more readily understood by those skilled in the art from the following examples. The contents described in the examples are only for illustrating the present invention, and should not be limiting the present invention described in detail in the claims, and the use of the strain for immobilized expression of enzyme protein is also included in the scope of the present invention.
The technical scheme of the invention is implemented on the premise of the embodiment, and the embodiment provides a detailed implementation mode and a specific operation process. The reagents used in the examples below are all commercially available.
The following examples illustrate the process of the invention in detail by means of carrier selection and surface-immobilized continuous fermentation using xylanase as the target product.
The preservation number of the clostridium acetobutylicum (c.acetobutylicum B3) used in the following examples is CGMCC No.5234, which is deposited in the china general microbiological culture collection center; the information of the strain is disclosed in detail in the chinese patent application No. 201210075094. X.
The culture media required in the following examples are conventional media and are not described in detail.
The recombinant bacteria B3-XynB and 824-XynB related in the following examples are respectively clostridium acetobutylicum B3 and clostridium acetobutylicum ATCC824 which are used as starting bacteria, and CA _ P0053 which is used as xylanase gene, and the recombinant bacteria are applied according to Chinese invention: CN 202010085119.9A construction method of recombinant acetone butanol clostridium and application thereof in preparing butanol by fermenting hemicellulose.
Example 1: continuous culture vector selection
(1) Carrier material: the vectors are referred to the relevant fermentation articles, the specific names of which are shown in Table 1, and correspond to FIG. 1 respectively.
TABLE 1 Carrier materials and numbering
Remarking: the polyethylene (small) is circular polyethylene with the diameter of 1.3 cm; the polyethylene (big) is circular polyethylene with the diameter of 2.4 cm; the diameter of the resin (porous) was 2.5cm, and the pores were not uniform, 0.3-0.5 cm; the diameter of the clay material is 2.2 cm; the resin is polyethylene resin; the sponge analogue (macropores) is in the shape of honeycomb-shaped pores of 0.2-0.5 cm.
(2) Immobilized culture:
(i) cutting all carrier materials into proper sizes, and sterilizing at high temperature and high pressure for later use;
(ii) transferring the plate-activated Clostridium acetobutylicum B3 bacterial mud into P2 seed culture medium (containing 20 μ g/L methylsulfonylchloride), and culturing overnight at 37 deg.C in anaerobic environment to obtain seed solution (OD600＝2.2)；
(iii) The support material (50g/L) obtained in step (i) was placed in parallel in a Duran bottle of 200mL mixed with 45mL of the fermentation broth and 5mL of the seed liquid system obtained in step (ii), respectively. Standing and culturing each batch in an anaerobic box at 37 ℃ for 24 hours, then sampling and changing the liquid (2mL of sample is stored in-20 ℃), and ensuring that the carrier is completely reserved in the next batch of fermentation liquid as far as possible; the fermentation is repeated for 4 batches, and the fermentation is finished after 96 hours.
Wherein the content of each component in the fermentation medium is as follows: glucose 60g/L, CH3COONH4 2.2g/L，K2HPO4 0.5g/L，KH2PO4 0.5g/L，FeSO4·7H2O 0.2g/L，MgSO4·7H2O 0.2g/L，MnSO4·H20.01g/L of O, 0.01g/L of NaCl, 0.001g/L of p-aminobenzoic acid, 0.001g/L of thiamine and 0.0001g/L of biotin.
As can be seen from fig. 2, after the fermentation was completed, the concentrated materials having the largest biofilm adsorption amount were ceramic (19), cotton fiber (towel) (5), activated carbon (sheet) (9), cotton fiber (sphere) (4), PVA (12), and clay material (16), respectively. Considering the economic principle, cotton fiber (towel) (5) is selected as the carrier for immobilized culture.
Example 2: continuous culture of cotton towel carrier
Based on example 1, only cotton fiber towel (5) was selected as a carrier for surface immobilized continuous fermentation, and the experiment was expanded to a 3L fermenter experiment. In step (iii), the sampling and medium exchange time was changed to 12 hours, and the culture time was changed to one week.
The dry weight of each batch of the fermented cotton towel carriers was weighed, and the change of the dry weight is shown in table 2. The attachment of the cells to the cotton towel during the culture was shown in FIG. 3. The above results all indicate that the cotton towel carrier is beneficial to biofilm formation.
TABLE 2 Dry weight Change of biofilm
Example 3: xylanase enzyme activity determination-DNS method.
(1) Adding 25 mu L of enzyme solution diluted to a certain concentration, 500 mu L of xylan solution with the concentration of 5mg/mL and 225 mu L of phosphate buffer solution into each 10mL centrifuge tube;
(2) the system is placed in a constant-temperature water bath kettle at 65 ℃ for reaction for 15min, then immediately placed on ice, and 1mL of prepared DNS solution is added into each reaction system;
(3) reacting in a boiling water bath for 5min, immediately placing in an ice water bath, cooling to room temperature, and adding pure water to a constant volume of 5 mL;
(4) the absorbance at 540nm was measured using an ultraviolet spectrophotometer.
Example 4: continuous fermentation using glucose as carbon source
Based on the example 2, glucose is used as a carbon source, the initial concentration is 60g/L, and continuous fermentation is carried out by taking B3 and 824 as initial bacteria and B3-XynB and 824-XynB as recombinant bacteria.
As shown in FIGS. 4 and 5, the recombinant B3-XynB strain was cultured at OD for 240 hours600The value is not clearThe enzyme activity fluctuates up and down between 3.1-4.9U while the decrease is obvious, the average level is 10 times higher than that of the original strain B3, and the highest enzyme activity can reach 4.9U. 824-XynB recombinant bacteria are cultured for 240h continuously and then cultured at OD600The value is not obviously reduced, the enzyme activity fluctuates up and down between 1.3 and 2.3U, the average level is 5.1 times higher than that of the original strain 824, and the highest enzyme activity can reach 2.3U.
Example 5: continuous fermentation using hemicellulose as carbon source
Based on the example 2, the B3 was used as the starting bacterium and the B3-XynB was used as the recombinant bacterium, and the fermentation was continued with hemicellulose as the carbon source at an initial concentration of 60 g/L.
The result is shown in figure 6, after the B3-XynB recombinant bacteria are continuously cultured for 480 hours, the enzyme activity fluctuates up and down between 1.1 and 2.4U, the average level is 6.7 times higher than that of the original strain B3, and the highest enzyme activity can reach 2.4U.
The invention provides a method for continuously producing xylanase by using clostridium, and a method and a way for realizing the technical scheme are many, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and decorations can be made without departing from the principle of the invention, and the improvements and decorations should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (10)
1. A method for continuously producing xylanase by using clostridium is characterized in that clostridium capable of producing xylanase is continuously fermented in a fermentation culture medium containing a carrier to obtain fermentation liquor containing xylanase.
2. The method of claim 1, wherein the xylanase-producing clostridium is clostridium acetobutylicum.
3. The method according to claim 1, wherein the xylanase-producing clostridium is a recombinant clostridium acetobutylicum genetically engineered with clostridium acetobutylicum.
4. The method according to claim 1, wherein the carrier is made of one or more of polyester fiber, polylactic acid, cotton fiber, nylon fiber, kapok, activated carbon, polyethylene, polyvinyl alcohol, polyurethane, clay, metal and ceramic.
5. The method according to claim 4, wherein the carrier is present in the form of the material itself, or any one of a cloth, a resin, a sponge-like material, a plastic sheet, a plastic spring, and a slide glass obtained by processing the material.
6. The method according to claim 1, wherein the carrier is contained in an amount of 5 to 60 g/L.
7. The method according to claim 1, wherein the continuous fermentation mode is that a fermentation solution is released and a fresh fermentation medium is supplemented during the fermentation process; alternatively, the fermentation broth is discharged at the end of each batch and simultaneously supplemented with fresh fermentation medium.
8. The method of claim 1, wherein the concentration of the carbon source is 1-100g/L during the continuous fermentation.
9. The method of claim 1, wherein the initial concentration of carbon source in the continuous fermentation is 50-60 g/L.
10. The method according to claim 1, wherein the temperature of the fermentation is 30-40 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110016847.9A CN112795553A (en) | 2021-01-07 | 2021-01-07 | Method for continuously producing xylanase by using clostridium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110016847.9A CN112795553A (en) | 2021-01-07 | 2021-01-07 | Method for continuously producing xylanase by using clostridium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112795553A true CN112795553A (en) | 2021-05-14 |
Family
ID=75808777
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110016847.9A Pending CN112795553A (en) | 2021-01-07 | 2021-01-07 | Method for continuously producing xylanase by using clostridium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112795553A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008237101A (en) * | 2007-03-27 | 2008-10-09 | Toray Ind Inc | Method for producing chemical by continuous fermentation |
| US20100298611A1 (en) * | 2009-03-09 | 2010-11-25 | Qteros, Inc. | PRODUCTION OF FERMENTIVE END PRODUCTSFROM CLOSTRIDIUM sp. |
| CN103320335A (en) * | 2012-03-20 | 2013-09-25 | 南京工业大学 | Clostridium acetobutylicum and applications thereof |
| CN104894096A (en) * | 2015-06-10 | 2015-09-09 | 南京工业大学 | Method for immobilizing clostridium acetobutylicum by use of bacterial cellulose membrane |
| CN107955797A (en) * | 2017-11-15 | 2018-04-24 | 浙江惠嘉生物科技股份有限公司 | The zonal cooling zymotechnique of clostridium butyricum |
| WO2020064900A1 (en) * | 2018-09-28 | 2020-04-02 | IFP Energies Nouvelles | Process for producing alcohols with clostridium on a solid support |
| CN111269869A (en) * | 2020-02-10 | 2020-06-12 | 南京工业大学 | Construction method of recombinant clostridium acetobutylicum and application of recombinant clostridium acetobutylicum in preparation of butanol through semi-fiber fermentation |
-
2021
- 2021-01-07 CN CN202110016847.9A patent/CN112795553A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008237101A (en) * | 2007-03-27 | 2008-10-09 | Toray Ind Inc | Method for producing chemical by continuous fermentation |
| US20100298611A1 (en) * | 2009-03-09 | 2010-11-25 | Qteros, Inc. | PRODUCTION OF FERMENTIVE END PRODUCTSFROM CLOSTRIDIUM sp. |
| CN103320335A (en) * | 2012-03-20 | 2013-09-25 | 南京工业大学 | Clostridium acetobutylicum and applications thereof |
| CN104894096A (en) * | 2015-06-10 | 2015-09-09 | 南京工业大学 | Method for immobilizing clostridium acetobutylicum by use of bacterial cellulose membrane |
| CN107955797A (en) * | 2017-11-15 | 2018-04-24 | 浙江惠嘉生物科技股份有限公司 | The zonal cooling zymotechnique of clostridium butyricum |
| WO2020064900A1 (en) * | 2018-09-28 | 2020-04-02 | IFP Energies Nouvelles | Process for producing alcohols with clostridium on a solid support |
| CN111269869A (en) * | 2020-02-10 | 2020-06-12 | 南京工业大学 | Construction method of recombinant clostridium acetobutylicum and application of recombinant clostridium acetobutylicum in preparation of butanol through semi-fiber fermentation |
Non-Patent Citations (4)
| Title |
|---|
| 刘明华 等: "《发酵与酿造技术 第2版》", 31 January 2020, 武汉理工大学出版社, pages: 63 - 66 * |
| 县永平: "丙酮丁醇梭菌固定化技术用于丙酮丁醇发酵的研究", 西北师范大学学报(自然科学版), no. 03, 21 August 2001 (2001-08-21), pages 111 - 118 * |
| 县永平: "丙酮丁醇梭菌固定化技术用于丙酮丁醇发酵的研究", 西北师范大学学报(自然科学版), no. 03, pages 70 - 73 * |
| 杨静 等: "丙酮丁醇梭菌生物膜结构与组成分析", 高校化学工程学报, vol. 31, no. 1, pages 111 - 118 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Klein et al. | Immobilization of microbial cells by adsorption | |
| Yokoi et al. | Hydrogen production by immobilized cells of aciduric Enterobacter aerogenes strain HO-39 | |
| Krischke et al. | Continuous production of L-lactic acid from whey permeate by immobilized Lactobacillus casei subsp. casei | |
| Akin | Biocatalysis with immobilized cells | |
| Grootjen et al. | Cofermentation of glucose and xylose with immobilized Pichia stipitis and Saccharomyces cerevisiae | |
| CA1211729A (en) | Production of organic acids by a continuous fermentation process | |
| Zheng et al. | Scaling up of ethanol production from sugar molasses using yeast immobilized with alginate-based MCM-41 mesoporous zeolite composite carrier | |
| Tanyıldızı et al. | Optimization of lactic acid production with immobilized Rhizopus oryzae | |
| Ogawa et al. | Production of kojic acid by membrane-surface liquid culture of Aspergillus oryzae NRRL484 | |
| Ogawa et al. | Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704 | |
| Saucedo-Castañeda et al. | Potential of using a single fermenter for biomass build-up, starch hydrolysis, and ethanol production: Solid state fermentation system involving Schwanniomyces castellii | |
| Kiy et al. | Continuous high-cell-density fermentation of the ciliated protozoon Tetrahymena in a perfused bioreactor | |
| Wakisaka et al. | Development of a cylindrical apparatus for membrane-surface liquid culture and production of kojic acid using Aspergillus oryzae NRRL484 | |
| Nigam et al. | Continuous ethanol production by thermotolerant Kluyveromyces marxianus IMB3 immobilized on mineral Kissiris at 45 C | |
| Kamoshita et al. | Improvement of filtration performance of stirred ceramic membrane reactor and its application to rapid fermentation of lactic acid by dense cell culture of Lactococcus lactis | |
| CN116731933B (en) | Corynebacterium glutamicum and application thereof in valine production | |
| Gunasekaran et al. | High ethanol productivity from lactose by immobilized cells of Kluyveromyces fragilis and Zymomonas mobilis | |
| CN112795553A (en) | Method for continuously producing xylanase by using clostridium | |
| Love et al. | Continuous ethanol fermentation at 45 C using Kluyveromyces marxianus IMB3 immobilized in calcium alginate and kissiris | |
| CN114437976B (en) | Composite microbial agent and application thereof in kitchen waste biological reduction | |
| CN1219071C (en) | Method for producing yeast extracellular trehalose by two step fermentation method | |
| CN106967708B (en) | Method for secondary immobilization of debaryomyces hansenii by using composite material | |
| Ahmed et al. | Production of Bacillus licheniformis ATCC 21415 alkaline protease in batch, repeated batch and continuous culture | |
| Abu‐Shady et al. | Studies on rifamycin production by Amycolatopsis mediterranei cells immobilized on glass wool | |
| CN114015594B (en) | Bacillus tokyo and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |