CN1230527C - Hemicellulase and its application in preparing chitoligose - Google Patents
Hemicellulase and its application in preparing chitoligose Download PDFInfo
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- CN1230527C CN1230527C CN 01114352 CN01114352A CN1230527C CN 1230527 C CN1230527 C CN 1230527C CN 01114352 CN01114352 CN 01114352 CN 01114352 A CN01114352 A CN 01114352A CN 1230527 C CN1230527 C CN 1230527C
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- enzyme
- hemicellulase
- enzymolysis
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- bacillus pumilus
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Abstract
The present invention relates to hemicellulase and application thereof in chitosan oligosaccharide preparation. The hemicellulase is a fermented product of bacillus pumilus hemicellulase A1 CCTCC M201023. The method for preparing chitosan oligosaccharide of the present invention comprises chitosan enzymolysis, centrifugal filtration of enzymolysis liquid, flocculation clarification, vacuum concentration, impurity filtration, decolorization, smell removal, ultra-filtration grading and drying. The method is characterized in that the method adopts the hemicellulase for chitosan enzymolysis. The present invention has advantage that bacterial strains for efficiently producing enzyme are provided. The enzyme activity is greatly improved to 300 u/ml. The enzymolysis technology has high efficiency, and the enzymolysis only takes 8 to 10 hours. The product has high content of effective components. The content of chitose of 3 to 10 is more than 70%.
Description
Technical field
The invention relates to a kind of hemicellulase and the application in the preparation oligochitosan thereof.
Background technology
Chitin is that shrimp, the crab shell of Yi Jiang, river, lake, marine a large amount of existence is the natural animal dietary fiber that raw material carries out process for processing.Chitosan is to slough the ethanoyl in the chitin molecule and a kind of natural high moleculer eompound of obtaining under the effect of concentrated base, is only second to Mierocrystalline cellulose at the content of occurring in nature.Chitin mainly is divided into the series derivatives product of following three levels with the difference of its working method: A. chitin (chitin), and molecular weight reaches more than 1,000,000, and its receptivity is strong, being fit to industry and environmental protection uses, but be insoluble to soda acid, also water insoluble, be difficult to be absorbed by the body.B. chitosan (chitosan), molecular weight generally only is dissolved in organic monoacid about 100,000, water insoluble, can be absorbed by the body, and can be used as foodstuff additive and healthcare products raw material etc., and promotional value is extremely widely arranged.C. oligochitosan, molecular weight is lower than 5000, and tool is water-soluble, can directly be absorbed by the body, and speed is fast, and effect is remarkable.Verified, by the oligochitosan that degradation of chitosan generates, have more unique physiologically active and physico-chemical property, be the best product of application performance in the chitin series product.
At present, the domestic and international method for preparing oligochitosan of degrading is broadly divided into enzyme liberating method, oxidation degradation method and acid degradation method three major types.Utilize the process of biological enzyme degraded preparation oligochitosan obviously to be better than two kinds of chemical degradation methods in addition, this is because the enzymic degradation process makes the easier Be Controlled of molecular weight distribution of degraded product, can monitor degradation process, produce active princlple (shell 3-10 sugar) the higher oligochitosan of content, and degradation condition is relatively gentleer, need not to add a large amount of reaction reagents, environmental pollution is less.The second phase in 1999 " Chinese marine drug " publishes " screening of high yield chitoanase bacterial classification and degrade chitosan reaction pre-test thereof " articles and opinions and has stated the biochemical chamber of Chinese Academy of Sciences's Dalian chemical physics by the effective ultraviolet mutagenesis of two-wheeled, obtained the mutant strain that 2 strains have the high yield chitoanase, has stability preferably, the thick enzymic activity of its fermented liquid reaches 0.2u/ml, although it has improved 8 times than original preservation mutant strain enzymic activity, and obtain a certain amount of oligochitosan, but its enzymic activity is still lower, can not realize suitability for industrialized production.Degradation of chitosan enzyme One's name is legion, the chitoanase ubiquity enzymic activity of degraded is low, side reaction many and problems such as enzymolysis time length, energy consumption height.
Summary of the invention
The object of the present invention is to provide a kind of hemicellulase that produces by microbial fermentation and utilize this hemicellulase to prepare the processing method of oligochitosan, make this hemicellulase have higher activity, improve enzymolysis efficiency, and making effective ingredient in the product is that the content of shell 3-10 sugar improves greatly, uses hemicellulase and realizes that the suitability for industrialized production oligosaccharide becomes possibility thereby make.
The microorganism of using
Cross fast dull and stereotyped identify and by shaking bottle, enzyme survey screened a strain rapidly degrade chitosan be the bacillus pumilus (Bacillus Pumilus) half fine enzyme A1 number (hereinafter to be referred as fine enzyme A1 bacterial strain partly) of oligochitosan, this bacterial strain in July 8 calendar year 2001 in China's typical culture collection center preservation, deposit number is CCTCCM201023, this bacterial strain is cultivated through optimizing, and fermentation reaches half above cellulase activity of 300 μ/ml.Can effectively degrade in 5～10 hours chitosan of 10% concentration of 1ml crude enzyme liquid is that 3～10 sugar are main oligosaccharides, and through mass spectrum and Analysis and Identification confirmation, makes the suitability for industrialized production oligochitosan become possibility.
The character of the half fine enzyme A1 bacterial strain that the present invention uses
Belong to short and small type bacillus, the tool gemma is gram positive bacterium;
Colonial morphology, round, white, translucent, glossy, more easily growth on polyprotein peptone substratum, 36 ℃ of the suitableeest culture temperature.
Content of the present invention comprises the product enzymatic process of producing hemicellulase and enzymolysis process two aspects that produce oligochitosan.
Producing the enzymatic process process is: half a fine enzyme A1 bacterial strain → last jar fermentation → low-temperature centrifugation → decon → cryopreservation enzyme liquid.
Use orthogonal experiment method (L4 (23) i.e. is four orthogonal experiments of three factors, two levels) and carry out culture medium prescription optimization, show that producing enzyme stablizes, average enzyme activity reaches 308u/ml.Suitable product enzyme pH value is 6.5, and producing the enzyme peak period is 12～24 hours.The suitable pH value of enzyme effect is 5.5-6.5, and the optimum pH value is 6.0, and enzyme effect optimal temperature is 45 ℃-48 ℃, optimum temperuture is 48 ℃, and pH value is that 6.7 o'clock stability is better, and the purification process of enzyme is generally: saltout, organic solvent deposit, ion-exchange, adsorption chromatography, gel-filtration, affinity chromatography, membrane sepn, low-temperature and high-speed is centrifugal etc., when producing, adopt continuous flow centrifugation to get final product, its good separating effect, convenience, the enzyme yield is more than 84%.
The generation condition of enzyme
(1) the substratum composition is to producing the influence of enzyme
Application orthogonal experiment centering dextrin, Tryptones, yeast extract are optimized with 3 kinds of main compositions, and the result is as surface 1.His-and-hers watches 1 test result analysis calculates, and find that each factor to the primary and secondary of enzyme activity influence is: B＞C＞A, its optimization of C is: middle dextrin 10%, Tryptones 1%, yeast extract 0.5%.Show that by optimizing enzyme side's playback experiment producing enzyme stablizes, average enzyme activity reaches 308u/ml.
L4 (23) the culture medium prescription prioritization scheme table that comes to the surface
(L4 (23) i.e. is four orthogonal tests of three factors, two levels)
| Tryptones (%) | Middle dextrin (%) | Yeast extract (%) | Enzymic activity (U/ML) | |
| 1 | 1(0.6) | 1(6) | 1(0.5) | 216 |
| 2 | 2(1) | 1 | 2(0.8) | 210 |
| 3 | 1 | 2(10) | 2 | 270 |
| 4 | 2 | 2 | 1 | 308 |
| Primary and secondary number | 3 | 1 | 2 |
. zymotechnique is to producing the influence of enzyme
The A.PH value is to producing the influence of enzyme: play pH value by 0.618 pair of substratum of shake flask test application and carry out preferably.The results are shown in Table two.Show that suitable product enzyme pH value is 6.5.
Table 2 substratum PH is to producing the influence of enzyme
| The substratum initial ph value | 6.0 | 6.5 | 7.5 | 8.0 | 9.0 |
| Enzyme activity | 289 | 310 | 274 | 260 | 207 |
B. produce the enzyme time course: in different incubation time samplings, the tested enzyme activity the results are shown in Table 3
Table 3 produces the enzyme time course
| Incubation time Um | 4 | 8 | 12 | 16 | 20 | 24 | 28 |
| Enzymic activity U/ml | 30 | 65 | 113 | 200 | 278 | 309 | 295 |
Analysis revealed 12-24h reaches highest level for producing the enzyme peak period at 24h, and postpone the back enzymic activity and descend, may be nutrient consumption, the thalline self-dissolving.
C. dissolved oxygen: bacillus pumilus is an aerobic bacteria, needs to consume a large amount of dissolved oxygens during the fermentation; Improve mixing speed, be added in ventilation and all can effectively improve dissolved oxygen.Through test, selected mixing speed is 350-500r/min (along with the prolongation of fermentation time improves rotating speed gradually), and ventilation: during the fermentation, ventilation is uninterrupted: at 0-16h, air flow is 0.8m
3/ min; At the 16-24h air flow is 0.6m
3/ min, the oxygen dissolving value of whole process is controlled between 65%～85%.
D. defoamer: produce in the enzymic fermentation process and can produce a large amount of foams,, therefore want timely froth breaking, in jar, add the polyethers defoamer automatically by peristaltic pump according to the fermentation situation because the foamy surface tension can make the vigor of enzyme reduce.
The general aspects of hemicellulase of the present invention
A. enzyme effect optimum pH: under the different PH condition, carry out enzyme reaction.The suitable pH value of enzyme effect is 5.5～6.5, and optimum pH is 6.0.
B. enzyme effect optimum temperuture: under condition of different temperatures, carry out enzyme reaction.Enzyme effect thermophilic degree is 45～48 ℃, and optimum temperuture is 50 ℃.
The C.PH value is to the influence of enzyme stability: enzyme is preserved 15min in the different PH buffer system, surveys enzyme work and shows: stability better during pH value 6.7.
The D.PH value is to the influence of enzyme stable range: enzyme is in 0.05mol/L, and in the phosphoric acid buffer of PH6.0, at differing temps insulation 15min, the result shows that this enzyme heat stability is relatively poor, 60 ℃ of insulation 15min, and enzyme is lived and is only remained 22%, so should preserve at low temperatures.
The purifying of enzyme
Normal and other enzymes of hemicellulase are present in the fermentation system jointly, and self also have multiple molecule-type, thereby purifying is difficult.The general method that adopts has: saltout, organic solvent deposit, ion-exchange, adsorption chromatography, gel-filtration, affinity chromatography, membrane sepn, low-temperature and high-speed are centrifugal etc., consider the requirement of production, can adopt continuous flow centrifugation to get final product, its good separating effect, convenient, the enzyme yield is more than 84%.
The enzymolysis process process that the present invention produces oligochitosan is: chitosan glacial acetic acid solution → go into a jar enzymolysis → centrifugal, flocculation, filtration, decon → decolour, take off flavor, classification → freeze-drying (spray is done) → oligochitosan
Enzymolysis process is according to the solvability difference of chitosan raw material, and the amount difference of corresponding chitosan generally is that 0.2ml glacial acetic acid (95%) can make chitosan fully dissolve than 1g chitosan and inexcessive.The concentration of glacial acetic acid solution is regulated by the variation of concentration of substrate, such as: if require to reach 10% concentration of substrate, the glacial acetic acid of the corresponding 0.2ml95% of every g chitosan is joined 100ml solution needs: chitosan 10g, glacial acetic acid 2ml, distilled water 100ml.The proterties difference of chitosan raw material, then enzyme concentration is also different.In order to obtain the product that specified molecular weight distributes, can regulate and control by the change of concentration of substrate, enzyme concentration, enzymolysis time.
The present invention compared with prior art has following outstanding feature:
1. efficient bacterium producing multi enzyme preparation
At present, general strain fermentation produces the cycle of chitoanase 6-7 days (consulting " Chinese marine drug " second phase in 1999 " screening of high yield chitoanase bacterial strain and degrade chitosan reaction pre-test " literary composition), the bacterial strain that the present invention adopts then is by mutagenesis, the efficient bacterial strain that filters out, fermentation period only needs 18-24 hour, produces enzyme efficient and improves greatly.
2. the activity of enzyme improves greatly
The enzymic activity of existing chitoanase only is 0.2u/m1, and enzyme provided by the invention then is the high reactivity hemicellulase, and enzymic activity reaches 300u/ml.(mensuration of enzyme activity: with 1% glacial acetic acid (95%) solution allocation concentration is 1% chitosan solution, and molten abundant back is in 48 ℃, and pH value is under 5.4 conditions, and the required enzyme amount of reducing sugar that enzymolysis substrate per minute discharges 1umol is a unit of activity U.)
3. enzymolysis process efficient height
The hydrolysis efficiency height is in general chitosan enzyme process technology, and hydrolysis substrate needs 2-3 days time.And in the hydrolysis process of the present invention, hydrolysis time only needs 8-10 hour.
4. active princlple content height in the product
Chemical method prepares oligochitosan, and active princlple in its product (shell 3-10 sugar) only accounts for about 20%.And hydrolysate of the present invention, by mass spectroscopy, its active princlple (shell 3-10 sugar) is more than 70%, far above the chemical method yield of domestic and international report, be entitled as in " preparation of water-soluble chitosan " literary composition the effective yield of report chemical method less than 20% as what publish in a 999 year " second chitin chemistry with use the Conference Papers collection ".
This project basic mechanical design feature index and similar products at home and abroad index are relatively
| Product basic mechanical design feature index name of the present invention | Index | Existing like product index |
| Half cellulase vigor | ≥300μ/ml | 0.2u/ml |
| The enzyme yield | ＞80％ | |
| The oligochitosan yield | ≥70％ | ≤20％ |
| Effective component in the product (shell 3 sugar-shell 10 sugar) accounts for the ratio (range of application and effect are seen attached list) of total reducing sugar | ≥60％ | About 20% (chemical method) |
| Other physics and chemistry, sanitary index | Meet | Meet |
| Outward appearance | White or buff powder | White or buff powder |
| Ash (%) | 0.1 | 0.2 |
| Heavy metal (mg/kg) | 0.1 | 0.3 |
| Molecular-weight average (Dalton) | 800～1200 | 800～1200 |
| Deacetylation % | ＞80％ | |
| Moisture content % | ≤2 | |
| Solubleness w/v | ≥10％ |
Hemicellulase activity height provided by the invention, it is stable to produce enzyme, and enzymatic hydrolysis condition is comparatively gentle, product yield height, purity height, few, the good stability of impurity.Produce enzyme and enzymolysis process maturation, the enzymolysis cost is low.
Description of drawings
Fig. 1 produces enzyme technological process of production figure for the present invention.
Fig. 2 is an enzymolysis production process schema of the present invention.
Fig. 3, Fig. 4, Fig. 5 are the oligochitosan check and analysis result (mass spectroscopy) of Wuhan University test analysis center to the inventive method preparation.
Embodiment
Embodiment 1: the preparation of hemicellulase of the present invention
1. general's half fine enzyme A1 bacterial strain is seeded in method of scoring and contains Tryptones (Tryptone) 1% yeast extract (Yeast extract) 0.5% sodium-chlor (Nacl) 1%, on the plate culture medium of agar 1%, cultivates after 18 hours under 37 ℃ of temperature and uses;
With the triangle of 1000ml shake bottled 100ml contain in dextrin 10%, the liquid nutrient medium of Tryptones (Tryptone) 1% yeast extract (Yeast extract) 0.5%Nacl 1%, sterilized 20 minutes for 10 pounds, inoculation one ring, vibration (250r/min) was cultivated 18 hours on 36 ℃ of shaking tables;
3. in 10 liters of fermentor tanks, load onto 8 liters and contain middle dextrin, Tryptones, the liquid nutrient medium that yeast extract is formed meets shake-flask seed suspension 100ml and cultivated 24 hours in 36 ℃ of following aeration-agitations after the same sterilization.
4. low-temperature centrifugation separates enzyme liquid: fermented liquid is packed in the centrifugal barrel of cryogenic freezing whizzer, after lid is tight, under+4 ℃ of conditions, with the 6000r/min rotating speed centrifugal 20 minutes, remove remnants such as thalline, take out supernatant liquor and get hemicellulase enzyme liquid of the present invention, it is standby to put 0-4 ℃ of cryopreservation.
Embodiment 2: utilize hemicellulase provided by the invention to produce oligochitosan
1. technical process: chitosan glacial acetic acid solution → go into a jar enzymolysis → centrifugal, flocculation, filter decon → decolour, take off peculiar smell → ultrafiltration classification → vacuum concentration → spraying drying → oligochitosan
2. working specification: (1) enzymolysis process is mixed with 2% solution with glacial acetic acid, adds chitosan intensification stirring and dissolving, and temperature is controlled at 48 ℃, and the envelope-bulk to weight ratio of chitosan and glacial acetic acid solution (W/V) is 10%.Add hemicellulase liquid degraded of the present invention, 48 ℃-50 ℃ of temperature of reaction, 8 hours time is stirred in insulation; (2) centrifuging is reacted enzymolysis solution centrifuging, rotating speed 3000r/min, 15 minutes time; (3) flocculation, clarification, secondary filtration add centrifugate a small amount of flocculate with chitosan 4-5 hour centrifuging again after the clarification, rotating speed 3000r/min, 15 minutes time; (4) decolour, take off peculiar smell with the secondary centrifuging clear liquid by the macroporous adsorbent resin taste removal of decolouring, flow velocity 1500ml/min, post is pressed 0.15mPa, 30 ℃ of temperature; (5) the ultrafiltration classification is carried out molecular cut-off by ultra-filtration membrane, and the film model is selected 1K film, 3K film, polysulfones material for use; The thick poly oligosaccharide liquid that decolouring is good carries out molecular cut-off by the 1K film earlier and removes monose and disaccharide, leaves and takes concentrated solution (containing the above chitooligosaccharide-of shell 3 sugar); Again concentrated solution is carried out molecular cut-off by the 3K film, leave and take through liquid (containing shell 3-10 sugar) standby.(6) the oligochitosan solution that vacuum concentration is good with ultrafiltration carries out vacuum concentration by the film under vacuum vaporizer, vacuum tightness-0.08mPa, and flow velocity 150-200L/hr, temperature 60-65 ℃, the concentrated solution degree of enrichment is controlled at 35%.(7) spraying drying selects for use 25 type high-speed centrifugal spray-driers to carry out spraying drying concentrated solution, 150 ℃ of import wind-warm syndrome, 85 ℃ of outlet wind-warm syndrome, vacuum tightness-0.03mPa, heating power 36KW, inlet velocity 25L/hr collects oligochitosan dry powder by rotoclone collector.(8) packaging fibre drum lined with polyethylene bag packing.
Claims (4)
1. the screening of a bacillus pumilus (Bacillus Pumilus) is cultivated product bacillus pumilus (Bacillus Pumilus) partly fine enzyme A1 number, and its preserving number is CCTCC M201023.
2. hemicellulase, the enzyme liquid that it is characterized in that it to be bacillus pumilus (Bacillus Pumilus) half fine enzyme A1 CCTCC M201023 obtain after impurity is removed in fermentation culture and separation and purification.
3. a method for preparing hemicellulase comprises fermentation culture of microorganism and the separation and purification of removing the thalline remnant, it is characterized in that fermentation culture bacillus pumilus (Bacillus Pumilus) half fine enzyme A1 CCTCC M201023.
4. the preparation method of an oligochitosan, comprise centrifuging, flocculate and clarify, vacuum concentration, filtration decon, decolour, take off flavor, ultrafiltration classification, drying, it is characterized in that the chitosan enzymolysis has been used the hemicellulase of producing with the described method of claim 3 chitosan enzymolysis, enzymolysis solution.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 01114352 CN1230527C (en) | 2001-07-13 | 2001-07-13 | Hemicellulase and its application in preparing chitoligose |
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|---|---|---|---|
| CN 01114352 CN1230527C (en) | 2001-07-13 | 2001-07-13 | Hemicellulase and its application in preparing chitoligose |
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| CN1230527C true CN1230527C (en) | 2005-12-07 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240305B (en) * | 2007-09-06 | 2010-12-08 | 武汉东方天琪生物工程有限公司 | Method for producing Chitosan oligosaccharide by using enzyme method to degrade chitosan |
| CN102217794B (en) * | 2011-02-23 | 2013-02-13 | 红云红河烟草(集团)有限责任公司 | Tobacco leaf biochemical additive and preparation method and application thereof |
| CN103468618B (en) * | 2013-09-25 | 2015-04-01 | 上海交通大学 | Cellulose-degrading bacteria with phosphate-dissolving capability and application thereof |
| CN104911125B (en) * | 2015-05-28 | 2019-09-13 | 迪沙药业集团有限公司 | A kind of chitosan enzyme-producing bacteria and its application |
| CN104975057B (en) * | 2015-07-15 | 2018-09-21 | 湖北工业大学 | A method of preparing chitin oligosaccharide using citric acid fermented waste mycelia |
| CN105803019A (en) * | 2015-12-31 | 2016-07-27 | 珠海市金隆生物科技有限公司 | Method for producing chitosan oligosaccharide from home-made enzyme solution |
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