CN102719370B - Escherichia coli RB3 strain and method for liquid state fermentation preparation of acetylesterase by the Escherichia coli RB3 strain - Google Patents

Escherichia coli RB3 strain and method for liquid state fermentation preparation of acetylesterase by the Escherichia coli RB3 strain Download PDF

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CN102719370B
CN102719370B CN 201110413232 CN201110413232A CN102719370B CN 102719370 B CN102719370 B CN 102719370B CN 201110413232 CN201110413232 CN 201110413232 CN 201110413232 A CN201110413232 A CN 201110413232A CN 102719370 B CN102719370 B CN 102719370B
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acetylase
escherichia coli
acetylesterase
buffer
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CN102719370A (en
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陈玉香
佟金
邢岩
梁彦龙
赵寿经
周皓芹
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Jilin University
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Abstract

The invention relates to an Escherichia coli RB3 strain and a method for liquid state fermentation preparation of acetylesterase by the Escherichia coli RB3 strain. The method is characterized in that a liquid medium containing corn stalk powder as a carbon source and having a pH value of 8.0 is inoculated with an Escherichia coli RB3 strain CGMCC No. 5252; the Escherichia coli RB3 strain CGMCC No. 5252 is fermented at a temperature of 40 DEG C under anaerobic conditions for 4 days; and fermentation products are centrifuged and a supernatant which is a crude acetylesterase liquid is obtained. The method adopts corn stalks as carbon sources of a fermentation medium and realizes utilization of abundant resources. Compared with acetylesterase disclosed by the existing published literatures, acetylesterase produced by the Escherichia coli RB3 strain has higher enzyme activity of which the maximum value is 0.59U/mL.

Description

A kind of colon bacillus RB3 bacterial strain and prepare the method for acetylase with its liquid state fermentation
Technical field
The present invention relates to the new bacterial strain of a strain that colon bacillus belongs to and the method for preparing acetylase with its liquid state fermentation.
Background technology
The thorough degraded of hemicellulose and lignocellulose resource conversion with utilize closely related.Hemicellulose is to be only second to cellulosic second largest vegetable polysaccharides, by the heterogeneity polysaccharide of the cell formations such as wood sugar, pectinose.In the most plants cell walls, hemicellulose is take xylan as skeleton, and the C-2 of xylose residues or C-3 all have in various degree acetylize, and the existence of ethanoyl is hindered the xylanase hydrolysis effect.The hemicellulose degraded relates to the synergy of plurality of enzymes, for example, and β-endo-xylanase, xylobiase, acetylase etc.Acetylase (acetylesterase, EC 3.1.1.6) is in the hemicellulose degradation process, and the acetyl ester bond of hydrolysis xylose residues is removed the part side chain hydrolysis of hemicellulose, the space steric effect of enzyme effect when reducing the xylan backbone degraded.In the lignocellulose saccharifying, acetylase and other hemicellulases have synergy, optimize the synergy of acetylase and zytase, can improve saccharification efficient.Acetylase is with a wide range of applications in biological degradation, paper-making pulping, the ruminating animal roughage processing and other fields of lignocellulose.According to bibliographical information, the microorganism that produces acetylase mainly has: Penicillium notatum [1], Neocallimastix frontalis [2], Trichoderma reesei, Aspergillus awamori, A.niger, A.oryzae, Schizophyllum commune, Fibrobacter succinogenes [3], great majority are fungi, only having minority is bacterium.Concentrate at present zytase and the β-Isosorbide-5-Nitrae xylosidase of degradation of hemicellulose main chain about the research of hemicellulase system, and for example acetylase research is less to the enzyme of degraded side chain.The method of producing at present acetylase mainly is to adopt the mode of liquid state fermentation, but owing to selecting bacterial classification different, institute's acetonyl ester enzyme activity that produces is difference all, and it is common problem that the acetylase vigor hangs down.The people such as Cao Yangchun report that rumen anaerobic fungi Neocallimastix frontalis produces the acetylase vigor and is up to 0.165U/mL [2]The high enzymatic activity of Yue et al. report rumen anaerobic fungi Neocallimastix acetylase that sp.YQ2 produces reaches 0.018U/mL [4]Take corn cob meal as the liquid state fermentation carbon source, Candida guilliermondii produces the acetylase vigor and is up to about 0.28U/mL [3]Take birch xylan or made from bracteal leaf of corn powder as carbon source liquid state fermentation, Streptomyces sp.PC22 produces the acetylase vigor and is up to 0.3U/mL [5]Corn cob meal is added in liquid state fermentation, and Bacillus pumilus produces the acetylase vigor and is up to about 0.11U/mL [6]Therefore, develop a kind of suitable industrialness batch production and have the method for the acetylase of high enzyme vigor, have significant actual application value.
Reference:
[1]Atta?S，Ali?S，Akhtar?MN，et?al.Determination?of?some?significant?batch?culture?conditions?affecting?acetyl-xylan?esterase?production?by?Penicillium?notatum?NRRL-1249.BMC?Biotechnology，2011，11：52.
[2] Cao Yangchun, Yang Hongjian, Shen Botong. screening and the evaluation of high yield fiber degradation enzyme Yak Rumen anaerobic fungi strain isolated. China Agricultural University's journal, 2010,15 (3): 70-74.
[3]Basaran?P，Hang?YD.Purification?and?characterization?of?acetyl?esterase?from?Candida?guilliermondii.Letters?in?Applied?Microbiology，2000，30：167-171.
[4]Yue?Q，Yang?HJ，Cao?YC，et?al.Feruloyl?and?acetyl?esterase?production?of?an?anaerobic?rumen?fungus?Neocallimastix?sp.YQ2?effected?by?glucose?and?soluble?nitrogen?supplementations?and?its?potential?in?the?hydrolysis?of?fibrous?feedstuffs.Animal?Feed?Science?and?Technology，2009，153：263-277.
[5]Chungool?W，Thongkam?W，Raweesri?P，et?al.Production，purification，and?characterization?of?acetyl?esterase?from?Streptomyces?sp.PC22?and?its?action?in?cooperation?with?xylanolytic?enzymes?on?xylan?degradation.World?Journal?of?Microbiology?and?Biotechnology，2008，24：549-556.
[6]Degrassi?G，Okeke?B，Bruschi?C，et?al.Purification?and?characterization?of?an?Acetyl?Xylan?Esterase?from?Bacillus?pumilus.Applied?and?Environmental?Microbiology，1998，64(2)：789-792.
Summary of the invention
The object of the invention aims to provide the new bacterial strain of a strain and prepares the method for acetylase with its liquid state fermentation, the low problem of acetylase enzyme activity that obtains to overcome present the whole bag of tricks.
The inventor studies by lot of experiments, cultivates in the following manner to obtain the new bacterial strain of a strain:
1. the sampling and processing of bovine rumen liquid: rumen fluid is taken from the fresh beef cattle of butchering of Jilin Province bright moon group, collects fresh rumen fluid and places the in advance vacuum flask of sterilization, and four layers of filtered through gauze are filled CO in packing into 2Vacuum flask in, take back rapidly the laboratory, filtrate is originated as strain screening.
2. Enrichment of bacteria is cultivated: get the above-mentioned rumen fluid of 1mL and be inoculated in the 9mL perfect medium, make 10 -1Diluent is filled with nitrogen and CO in the anaerobism bottle 2, 39 ℃ of constant temperature culture 3d in the anaerobic culture box.
Described perfect medium is constructed as follows: buffer A 165mL, buffer B 165mL, acellular rumen fluid 170mL, NaHCO 35.0g, peptone 1.0g, yeast leaches cream 1.5g, glucose 1.0g, Cys hydrochloride 1.5g (adding behind the ventilation 30min), 0.1g/L resazurin solution 1.0mL, distilled water is settled to 1000mL, regulates pH7.0,1 * 10 5The Pa 30min that sterilizes.
Described buffer A: KH 2PO 43g, (NH 4) 2SO 43g, NaCl 6g, CaCl 22H 2O 0.4g, MgSO 47H 2O0.6g, distilled water is settled to 1000mL.
Described buffer B: K 2HPO 43H 2O 4g, distilled water is settled to 1000mL.
The preparation of described acellular rumen fluid: with the resulting supernatant liquor of the centrifugal 10min of fresh rumen fluid 8000g.
3. the screening and culturing of bacterial strain: the nutrient solution of getting after the enrichment is diluted to respectively 10 with sterilized water -2, 10 -4With 10 -6Doubly, coat the screening culture medium flat board, be filled with nitrogen and CO 2, sealing places 39 ℃ of constant temperature culture 7d in the anaerobic culture box.After growing bacterium colony, choose the highest flat board of extension rate, the doubtful single bacterium colony of picking is repeatedly streak culture on screening culture medium, obtains single bacterium colony, chooses the bacterial classification that the full bacterial strain of form adopts as the present invention.In screening process, obtain the bacterial strain that acetylase is produced in 3 strains, be respectively RB1, RB3 and RB6, wherein the acetylase vigor of bacterial strain RB3 is the highest, as the bacterial strain of the present invention's employing.
Described screening culture medium is constructed as follows: buffer A 165mL, buffer B 165mL, acellular rumen fluid 170mL, NaHCO 35.0g, alkali lignin powder 1.0g, agar 20g, 0.1g/L resazurin solution 1.0mL, distilled water is settled to 1000mL, regulates pH7.0.Add in addition cycloheximide (0.05mg/mL) and nystatin (200U/mL).1 * 10 5The Pa 30min that sterilizes.
Described buffer A: KH 2PO 43g, (NH 4) 2SO 43g, NaCl 6g, CaCl 22H 2O 0.4g, MgSO 47H 2O0.6g, distilled water is settled to 1000mL.
Described buffer B: K 2HPO 43H 2O 4g, distilled water is settled to 1000mL.
The preparation of described acellular rumen fluid: with the resulting supernatant liquor of the centrifugal 10min of fresh rumen fluid 8000g.
4. the domestication of bacterial strain is cultivated: with single bacterium colony that screening obtains, be inoculated in the anaerobically fermenting bottle that 70mL domestication substratum is housed, be filled with nitrogen and CO in the bottle 2, sealing, 39 ℃ of anaerobic culture boxes are cultivated, and cultivate 3d and are forwarded in the fresh culture, and switching is a generation once, to the 12 generation, obtains bacterial isolates of the present invention.Evidence, this bacterial strain have the feature of the higher acetylase of under anaerobic fermentation preparation enzyme activity.
Described domestication substratum is constructed as follows: buffer A 165mL, buffer B 165mL, acellular rumen fluid 170mL, alkali lignin powder 1.0g, Cys hydrochloride 1.5g, 0.1g/L resazurin solution 1.0mL, distilled water is settled to 1000mL, regulates pH7.0.1 * 10 5The Pa 30min that sterilizes.
The above-mentioned bacterial isolates RB3 that the present invention obtains carries out strain identification through Sangon Biotech (Shanghai) Co., Ltd., names to be colon bacillus (Escherichia coli) RB3.
This bacterial strain is the (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City at China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica) preservation, be numbered CGMCC No.5252, preservation date on September 17th, 2011.
The colon bacillus that the present invention obtains (Escherichia coli) RB3CGMCC No.5252 bacterial strain has following Microbiological Characteristics: gramstaining is the result show, this bacterial strain is Gram-negative, rod-shaped bacterium.Citrate trianion utilizes test-results to show, this bacterial strain can not utilize Citrate trianion.The methyl red test result is positive.On the methylene blue substratum of Yihong, be characterized as purple, smooth, moistening circular bacterium colony.Above test-results is the characteristic feature of colon bacillus.
Liquid state fermentation of the present invention prepares the method for acetylase, be with above-mentioned colon bacillus (Escherichia coli) RB3CGMCC No.5252 inoculation take corn stalk powder as carbon source, the pH value is in 8.0 liquid culture medium, at 40 ℃, anaerobic condition bottom fermentation 4d, tunning is centrifugal, and supernatant liquor is the acetylase crude enzyme liquid;
Described liquid culture medium is constructed as follows: corn stalk powder 10g, and acellular rumen fluid 170mL, buffer A 165mL, buffer B 165mL, peptone 1.0g, yeast leaches cream 1.0g, Cys hydrochloride 1.5g, distilled water is settled to 1000mL.1 * 10 5The Pa 30min that sterilizes.
Described buffer A: KH 2PO 43g, (NH 4) 2SO 43g, NaCl 6g, CaCl 22H 2O 0.4g, MgSO 47H 2O0.6g, distilled water is settled to 1000mL.
Described buffer B: K 2HPO 43H 2O 4g, distilled water is settled to 1000mL.
The preparation of described acellular rumen fluid: with the resulting supernatant liquor of the centrifugal 10min of fresh rumen fluid 8000g.
The acetylase enzyme activity that obtains by the invention described above method is up to 0.59U/mL.The bacterial strain that is higher than present bibliographical information produces the acetylase vigor, has overcome present method and has produced the low shortcoming of acetylase vigor.In addition, the present invention adopts corn stalk powder as carbon source, is beneficial to realize the lignocellulose biomass efficient utilization of resource.
The inventive method has the following advantages:
1. the bacterial strain that adopts of fermentation is seminar's bacterium, produces the acetylase vigor high, and high enzymatic activity reaches 0.59U/mL, is higher than present bibliographical information bacterial classification and produces the acetylase vigor.
2. fermentation adopts corn stalk powder as carbon source, can produce acetylase by inducible strain, improves the acetylase vigor, is beneficial to and realizes the straw lignocellulose efficient utilization of resource.
3. the acetylase of producing is with a wide range of applications in fields such as the bio-transformation of lignocellulose resource and degraded, the processing of ruminating animal roughage, paper-making pulpings.
Description of drawings:
Fig. 1 is the acetylase vigor dynamic curve diagram of the inventive method preparation;
Fig. 2 be in the inventive method liquid culture medium pH value to acetylase effect of vigor graphic representation;
Fig. 3 be in the inventive method temperature to acetylase effect of vigor graphic representation.
Embodiment
By further describing the method that the present invention prepares acetylase by following examples.But be not to unique restriction of the present invention.
Embodiment 1
Adopt the liquid state fermentation of colon bacillus provided by the invention (Escherichia coli) RB3 CGMCC No.5252 bacterial strain to prepare acetylase.
With colon bacillus (Escherichia coli) RB3 inoculation to perfect medium, activation culture 3d in 40 ℃ of anaerobic culture boxes, draw the 1mL bacteria suspension, switching enters to be equipped with in the anaerobism bottle of 100mL enzymatic production liquid culture medium, 40 ℃ of ferment at constant temperature 4d in the anaerobic culture box, with fermented liquid 1000g, 4 ℃ of low-temperature centrifugation 10min, supernatant liquor is crude enzyme liquid.
Described enzymatic production liquid culture medium is constructed as follows: corn stalk powder 10g, acellular rumen fluid 170mL, buffer A 165mL, buffer B 165mL, peptone 1.0g, yeast leaches cream 1.0g, regulating pH is 8.0, Cys hydrochloride 1.5g (adding behind the ventilation 30min), distilled water is settled to 1000mL, and 1 * 10 5The Pa 30min that sterilizes.
Described buffer A: KH 2PO 43g, (NH 4) 2SO 43g, NaCl 6g, CaCl 22H 2O 0.4g, MgSO 47H 2O0.6g, distilled water is settled to 1000mL
Described buffer B: K 2HPO 43H 2O 4g, distilled water is settled to 1000mL.
The preparation of described acellular rumen fluid: with the resulting supernatant liquor of the centrifugal 10min of fresh rumen fluid 8000g.
Measure the acetylase vigor after the fermentation ends, the result shows the acetylase enzyme activity between 0.30-0.59U/mL, and the optimal pH of acetylase that this bacterial strain produces is 8.0,40 ℃ of optimum temperutures, and enzyme activity is up to 0.59U/mL.
Fig. 1 is the acetylase vigor dynamic curve diagram of the inventive method preparation, adopts the liquid state fermentation mode, and bacterial strain acetylase vigor presents ascendant trend at the fermentation initial stage, and enzyme activity reaches the highest when fermenting to 4d, for about 0.5U/mL, then begins to descend.Therefore adopt the inventive method to produce acetylase, fermentation time is that 4d is more suitable.
Fig. 2 be in the inventive method liquid culture medium pH value to acetylase effect of vigor graphic representation, as can be seen from the figure, the optimal pH that this bacterial strain produces acetylase is 8, and this moment, the acetylase vigor reached 0.59U/mL, the pH value is higher than at 8 o'clock, and the acetylase vigor descends rapidly.
Fig. 3 be in the inventive method temperature to acetylase effect of vigor graphic representation, as can be seen from the figure, the optimum temperuture that this bacterial strain produces acetylase is 40 ℃, this moment, the acetylase vigor reached 0.56U/mL, temperature is higher than 40 ℃, the acetylase vigor descends rapidly, therefore adopts the inventive method to produce acetylase, and leavening temperature should be controlled at 40 ℃.
The measuring method of acetylase enzyme activity is: 6 times of crude enzyme liquid dilutions, substrate is the p-oil of mirbane ethyl ester of 2mmol/L, the 50mmol/L sodium phosphate buffer.Add 100 μ L sodium phosphate buffers and 50 μ L substrates in the rear crude enzyme liquid of 50 μ L dilution, 40 ℃ of reaction 30min measure absorbancy at 415nm wavelength place.Calculate the acetylase vigor according to p-oil of mirbane typical curve.
The enzyme activity unit definition: 1 enzyme activity unit (U) is defined as under above enzymatic reaction condition, and 1mL enzyme liquid generates the needed enzyme amount of the p-oil of mirbane of 1 μ mol from substrate in 1min.

Claims (1)

1. a liquid state fermentation prepares the method for acetylase, it is characterized in that: with preserving number be CGMCC No.5252 colon bacillus RB3 inoculation take corn stalk powder as carbon source, the pH value is in 8.0 liquid culture medium, at 40 ℃, anaerobic condition bottom fermentation 4d, tunning is centrifugal, and supernatant liquor is the acetylase crude enzyme liquid;
Described liquid culture medium is constructed as follows: corn stalk powder 10g, and acellular rumen fluid 170mL, buffer A 165mL, buffer B 165mL, peptone 1.0g, yeast leaches cream 1.0g, Cys hydrochloride 1.5g, distilled water is settled to 1000mL; 1 * 10 5The Pa 30min that sterilizes;
Described buffer A: KH 2PO 43g, (NH 4) 2SO 43g, NaCl6g, CaCl 22H 2O0.4g, MgSO 47H 2O0.6g, distilled water is settled to 1000mL;
Described buffer B: K 2HPO 43H 2O4g, distilled water is settled to 1000mL;
The preparation of described acellular rumen fluid: with the resulting supernatant liquor of the centrifugal 10min of fresh rumen fluid 8000g.
CN 201110413232 2011-12-10 2011-12-10 Escherichia coli RB3 strain and method for liquid state fermentation preparation of acetylesterase by the Escherichia coli RB3 strain Expired - Fee Related CN102719370B (en)

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