CN102093991A - Method for producing low temperature cellulase through marine microbial fermentation - Google Patents
Method for producing low temperature cellulase through marine microbial fermentation Download PDFInfo
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Abstract
The invention relates to a method for producing low temperature cellulase through marine microbial fermentation, comprising the following steps of: domesticating marine microbes generating the cellulase at low temperature step by step so that the marine microbes well grow in a low temperature environment; carrying out amplification culture on cellulase producing bacteria domesticated at the low temperature at 18-24 DEG C step by step; inoculating into an enzyme producing medium according to the inoculation amount of 4-10 percent of the volume of fermentation liquid; culturing at 18-24 DEG C for 72-132 hours so as to finish producing the low temperature cellulase through the microbial fermentation; centrifugally collecting liquid when the fermentation liquid is 4000-8000 rpm, wherein the liquid is crude enzyme liquid; and further concentrating, separating and purifying the crude enzyme liquid according to different requirements and different using objects to prepare enzymatic preparations with different activities, purities and dosage forms.
Description
Technical field
The present invention relates to fields such as microbiology, enzyme engineering, fermentation engineering, biological chemistry, be specifically related to a kind of low-temperature cellulase marine microorganism fermentation method for producing.The low-temperature cellulase that this invention is produced is mainly used in feedstuff industry, food-processing, Chinese herbal medicine extracting, papermaking and weaving, Oil extraction, printing and dyeing and washing and other cellulase Application Areas.It can improve utilization ratio of raw materials, transformation efficiency and productivity, reduces production costs, and improves and improves the quality of products.
Background technology
Cellulase is meant that the energy degraded cellulose generates the general name of one group of enzyme of small-molecule substances such as cellobiose and glucose, is a kind of compound inducible enzyme, comprises multiple lytic enzyme (Zhang Qixian, microorganism journal, 1974).Cellulase has been widely used in feed, food-processing as biotechnological formulation, Chinese herbal medicine extracting, and Oil extraction, in the industries such as papermaking and weaving, printing and dyeing, but warm cellulase during the cellulase of use is mainly at present.Year surplus though domestic and international research to cellulase has 40, warm cellulase in mainly concentrating on; The research of relevant low-temperature cellulase still is in the starting stage, mainly studies (Wang Fen etc., Shandong College of Education journal and ocean science, 2003 such as strain improvement, fermentation condition optimization, zymologic property and genes involved clone; Ceng Yinxin etc., hi-tech communication, 2005; You Yinwei, microorganism journal, 2005; Hou Yunhua, Shandong University, 2006; Lv Mingsheng etc., Food science, 2007; Liu Xiuhua, Shandong University, 2007; Zhang Shuhong, JOURNAL OF MICROBIOLOGY, 2009).Low-temperature cellulase industrialization, large-scale production and application yet there are no report.Low-temperature cellulase is compared with high temperature, middle temperature cellulase has the huge applications advantage.Because low-temperature cellulase has high enzymatic activity and high catalytic efficiency at low temperatures, can reduce complete processing, shorten the time of the course of processing and save expensive heating or refrigeration costs; Aspect energy-conservation, sizable advantage is arranged; Can make the vigor forfeiture of cold-adapted enzyme through gentle thermal treatment, and low temperature or thermophilic are handled the quality that can not influence product (grandson is quiet etc., marine fishery research, 2002), its above-mentioned characteristic will help its promotion and application.The application of low-temperature cellulase will be to traditional agricultural, food, medicine, chemical industry, field such as the energy and environmental protection produces far-reaching influence, utilize the low-temperature cellulase of marine microorganism fermentative production, amount by raw material weight 0.01 ‰～0.5 ‰ makes an addition to ruminating animal cellulosic roughage, in the field such as deep processing of farm products and Chinese herbal medicine extracting, handle 0.25～1h at 6～20 ℃, can improve conversion of raw material 12.8%～19.2%, reduced production cost, improvement is also improved the quality of products, thereby is fundamentally saving middle temperature, the heating of high temperature enzyme, cooling apparatus and technology.Low-temperature cellulase will provide actual opportunity such as predicaments such as hunger, resource, energy dilemma and environmental pollutions for what the mankind broke away from the survival and development to be faced, and it has boundless potentiality to be exploited and application prospect.
Summary of the invention
The method that the purpose of this invention is to provide a kind of marine microorganism fermentative production low-temperature cellulase.This method mainly is that microorganism is through after the domestication by low temperature, at 18～24 ℃ of liquid fermentings production low-temperature cellulases.The low-temperature cellulase crude enzyme liquid activity that this production method obtains reaches 200U/mL, through separating and purifying, will obtain the zymin of different concns and purity as again.When this zymin activity is 2000U/mL or 2000U/g, addition with 0.01 ‰～0.5 ‰ is applied in the fields such as ruminating animal cellulosic roughage, deep processing of farm products and Chinese herbal medicine extracting, handle 0.25～1h at 6～20 ℃, can improve conversion of raw material 12.8%～19.2%.This zymin application operating is simple, convenient, fast, cost is low, and can fundamentally avoid heating, cooling apparatus and the technology of middle temperature, high temperature enzyme.
The method of a kind of marine microorganism fermentative production low-temperature cellulase of the present invention specifically may further comprise the steps:
(1) microorganism that will produce cellulase domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃, makes its well-grown in low temperature environment from high to low;
(2) according to a conventional method with the cellulase producing bacteria after the domestication by low temperature 18～24 ℃ of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed;
(3) with liquid first order seed or secondary seed, 4～10% inoculum sizes of pressing fermentating liquid volume insert produces in the enzyme substratum, and when cultivating 72～132h for 18～24 ℃, promptly marine microorganism fermentative production low-temperature cellulase finishes;
(4) with the fermented liquid of (3) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid;
(5) different with the use object according to different needs, the crude enzyme liquid that (4) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
The bacterial classification that uses among the present invention derives from China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and initial stage activation and growth conditions are undertaken by the explanation that culture presevation unit provides.(the CGMCC bacterium numbering: 3.3002 or 3.5455 or 3.2754 etc.), bacterial strain is activation earlier, through domestication by low temperature step by step, cultivates by the inventive method during the fermentative production low-temperature cellulase again for cellulase producing bacteria.Bacterial strain after the domestication by low temperature can be preserved 2 months in 4 ℃ of environment, bacteria suspension preservation for a long time under subzero 80 ℃ of conditions made from 25～50% glycerine.
Embodiment:
Embodiment one:
(1), medium preparation
1. strain activation and culture base: potato 200.0g, glucose 20.0g, agar 20.0g, distilled water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
2. bacterial classification domestication by low temperature substratum: potato 50.0～100.0g, the filter paper powder 5.0～8.0g of Xinhua, wheat bran 2.0～6.0g, agar 20.0g, distilled water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
3. liquid seed culture medium: rice straw powder 3.0～5.0g, peptone 0.5～1.0g, Tween800.1～0.3g, (NH
4)
2SO
40.8～1.2g, KH
2PO
41.2～1.8g, urea 0.2～0.4g, MgSO
40.2～0.3g, CaCl
20.1～0.2g, ZnSO
47H
2O 1.2mg, MnSO
4H
2O 1.6mg, CoCl
21.8mg, tap water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
4. produce the enzyme substratum: rice straw powder 7.0～12.0g, wheat bran 1.0～5.0g, Tween800.1～0.4g, (NH
4)
2SO
41.0～1.5g, KH
2PO
41.4～2.0g, urea 0.2～0.6g, MgSO
40.1～0.4g, CaCl
20.2～0.3g, ZnSO
47H
2O 2.4mg, MnSO
4H
2O 3.2mg, CoCl
23.6mg, tap water 1.0L, 6.0,121 ℃ of autoclaving 30min of pH value.
(2) will produce the bacterial classification of cellulase, carry out initial activation by the bacterial strain explanation that China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) provides;
(3) (2) are activated good bacterial classification domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃, makes its well-grown in low temperature environment from high to low;
(4) according to a conventional method the cellulase producing bacteria after the domestication by low temperature is cultivated 48～72h at 18～20 ℃, the inoculum size by 5% is carried out enlarged culturing step by step, is prepared into liquid first order seed and secondary seed;
(5) inoculum size of the secondary seed of (4) preparation being pressed fermentating liquid volume 4～6% inserts in the product enzyme substratum of 10L, and when cultivating 120～132h for 18～20 ℃, promptly marine microorganism fermentative production low-temperature cellulase finishes;
(6) with the fermented liquid of (5) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid;
(7) different with the use object according to different needs, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the low-temperature cellulase preparation of different activities, purity and formulation.
Embodiment two:
(1), medium preparation
1. strain activation and culture base: potato 200.0g, glucose 20.0g, agar 20.0g, distilled water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
2. bacterial classification domestication by low temperature substratum: potato 50.0～100.0g, the filter paper powder 5.0～8.0g of Xinhua, wheat bran 2.0～6.0g, agar 20.0g, distilled water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
3. liquid seed culture medium: rice straw powder 3.0～5.0g, peptone 0.5～1.0g, Tween800.1～0.3g, (NH
4)
2SO
40.8～1.2g, KH
2PO
41.2～1.8g, urea 0.2～0.4g, MgSO
40.2～0.3g, CaCl
20.1～0.2g, ZnSO
47H
2O 1.2mg, MnSO
4H
2O 1.6mg, CoCl
21.8mg, tap water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
4. produce the enzyme substratum: rice straw powder 7.0～12.0g, wheat bran 1.0～5.0g, Tween800.1～0.4g, (NH
4)
2SO
41.0～1.5g, KH
2PO
41.4～2.0g, urea 0.2～0.6g, MgSO
40.1～0.4g, CaCl
20.2～0.3g, ZnSO
47H
2O 2.4mg, MnSO
4H
2O 3.2mg, CoCl
23.6mg, tap water 1.0L, 6.0,121 ℃ of autoclaving 30min of pH value.
(2) will produce the bacterial classification of cellulase, carry out initial activation by the bacterial strain explanation that China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) provides;
(3) (2) are activated good bacterial classification domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃, makes its well-grown in low temperature environment from high to low;
(4) according to a conventional method the cellulase producing bacteria after the domestication by low temperature is cultivated 48～72h at 20～22 ℃, the inoculum size by 5% is carried out enlarged culturing step by step, is prepared into liquid first order seed and secondary seed;
(5) inoculum size of the secondary seed of (4) preparation being pressed fermentating liquid volume 6～8% inserts in the product enzyme substratum of 50L, and when cultivating 96～120h for 20～22 ℃, promptly marine microorganism fermentative production low-temperature cellulase finishes;
(6) with the fermented liquid of (5) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid;
(7) different with the use object according to different needs, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the low-temperature cellulase preparation of different activities, purity and formulation.
Embodiment three:
(1), medium preparation
1. strain activation and culture base: potato 200.0g, glucose 20.0g, agar 20.0g, distilled water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
2. bacterial classification domestication by low temperature substratum: potato 50.0～100.0g, the filter paper powder 5.0～8.0g of Xinhua, wheat bran 2.0～6.0g, agar 20.0g, distilled water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
3. liquid seed culture medium: rice straw powder 3.0～5.0g, peptone 0.5～1.0g, Tween800.1～0.3g, (NH
4)
2SO
40.8～1.2g, KH
2PO
41.2～1.8g, urea 0.2～0.4g, MgSO
40.2～0.3g, CaCl
20.1～0.2g, ZnSO
47H
2O 1.2mg, MnSO
4H
2O 1.6mg, CoCl
21.8mg, tap water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
4. produce the enzyme substratum: rice straw powder 7.0～12.0g, wheat bran 1.0～5.0g, Tween800.1～0.4g, (NH
4)
2SO
41.0～1.5g, KH
2PO
41.4～2.0g, urea 0.2～0.6g, MgSO
40.1～0.4g, CaCl
20.2～0.3g, ZnSO
47H
2O 2.4mg, MnSO
4H
2O 3.2mg, CoCl
23.6mg, tap water 1.0L, 6.0,121 ℃ of autoclaving 30min of pH value.
(2) will produce the bacterial classification of cellulase, carry out initial activation by the bacterial strain explanation that China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) provides;
(3) (2) are activated good bacterial classification domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃, makes its well-grown in low temperature environment from high to low;
(4) according to a conventional method the cellulase producing bacteria after the domestication by low temperature is cultivated 48～72h at 22～24 ℃, the inoculum size by 5% is carried out enlarged culturing step by step, is prepared into liquid first order seed and secondary seed;
(5) inoculum size of the secondary seed of (4) preparation being pressed fermentating liquid volume 8～10% inserts in the product enzyme substratum of 100L, and when cultivating 72～96h for 22～24 ℃, promptly marine microorganism fermentative production low-temperature cellulase finishes;
(6) with the fermented liquid of (5) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid:
(7) different with the use object according to different needs, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the low-temperature cellulase preparation of different activities, purity and formulation.
Embodiment four:
Press the low-temperature cellulase preparation that embodiment one, two and three methods are produced, average enzymic activity is 2000U/mL.With activity is the low-temperature cellulase preparation of 2000U/mL or 2000U/g, amount by raw material weight 0.01 ‰～0.5 ‰ makes an addition in the fields such as ruminating animal cellulosic roughage, deep processing of farm products and Chinese herbal medicine extracting, handle 0.25～1h at 6～20 ℃, can improve utilization ratio of raw materials, transformation efficiency and productivity, reduce production costs, improve and improve the quality of products.Low-temperature cellulase applying portion experimental result sees the following form.
| The experiment material | Low-temperature cellulase addition (‰) | Treatment time (h) | Transformation efficiency (%) | Treatment temp (℃) |
| The silkization maize straw | 0.2～0.5 | 1 | +16.3 | 6～18 |
| The silkization rice straw | 0.1～0.3 | 0.5 | +17.6 | 6～18 |
| Soybean stalk | 0.1～0.3 | 0.5 | +14.6 | 6～18 |
| Sucus Mali pumilae processing | 0.01～0.1 | 0.25 | +14.1 | 14～18 |
| Sucus Vitis viniferae processing | 0.01～0.1 | 0.25 | +12.8 | 14～18 |
| Orange juice processing | 0.01～0.1 | 0.25 | +14.3 | 14～18 |
| Chinese herbal medicine extracting | 0.3～0.5 | 1 | +19.2 | 12～20 |
Experimental result shows: low-temperature cellulase is applied to can improve utilization ratio of raw materials, transformation efficiency and productivity in the fields such as ruminating animal cellulosic roughage, deep processing of farm products and Chinese herbal medicine extracting, reduces production costs, and improves and improves the quality of products; Simultaneously illustrate that also low-temperature cellulase has high enzymatic activity and high catalytic efficiency at low temperatures, can shorten the time for the treatment of processes greatly and save expensive heating or refrigeration costs; Aspect energy-conservation, sizable advantage is arranged; This zymin application operating is simple, convenient, fast, cost is low; Can fundamentally avoid heating, cooling apparatus and the technology of middle temperature, high temperature enzyme.
Claims (2)
1. the method for a marine microorganism fermentative production low-temperature cellulase, it may further comprise the steps:
(1) marine microorganism that will produce cellulase domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃, makes its well-grown in low temperature environment from high to low;
(2) according to a conventional method with the cellulase producing bacteria after the domestication by low temperature 18～24 ℃ of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed;
(3) with liquid first order seed or secondary seed, 3～9% inoculum sizes of pressing fermentating liquid volume insert in the liquid fermentation medium, and when cultivating 72～132h for 18～24 ℃, promptly producing low temperature cellulase using microbe fermentation finishes;
(4) with the fermented liquid of (3) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid;
(5) different with the use object according to different needs, the crude enzyme liquid that (4) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
2. method according to claim 1, wherein bacterial classification domestication by low temperature substratum, liquid seed culture medium, product enzyme substratum are respectively:
(1) bacterial classification domestication by low temperature substratum: potato 50.0～100.0g, the filter paper powder 5.0～8.0g of Xinhua, wheat bran 2.0～6.0g, agar 20.0g, distilled water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
(2) liquid seed culture medium: rice straw powder 3.0～5.0g, peptone 0.5～1.0g, Tween80 0.1～0.3g, (NH
4)
2SO
40.8～1.2g, KH
2PO
41.2～1.8g, urea 0.2～0.4g, MgSO
40.2～0.3g, CaCl
20.1～0.2g, ZnSO
47H
2O 1.2mg, MnSO
4H
2O 1.6mg, CoCl
21.8mg, tap water 1.0L, pH value nature, 121 ℃ of autoclaving 30min.
(3) produce the enzyme substratum: rice straw powder 7.0～12.0g, wheat bran 1.0～5.0g, Tween80 0.1～0.4g, (NH
4)
2SO
41.0～1.5g, KH
2PO
41.4～2.0g, urea 0.2～0.6g, MgSO
40.1～0.4g, CaCl
20.2～0.3g, ZnSO
47H
2O 2.4mg, MnSO
4H
2O 3.2mg, CoCl
23.6mg, tap water 1.0L, 6.0,121 ℃ of autoclaving 30min of pH value.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653740A (en) * | 2012-04-12 | 2012-09-05 | 大连大学 | Fermenting production method of acetylcholinesterase by microorganisms |
| CN104630188A (en) * | 2015-02-16 | 2015-05-20 | 大连大学 | Method for producing low-temperature glucomannanase by fermentation of marine microorganisms |
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| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
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| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653740A (en) * | 2012-04-12 | 2012-09-05 | 大连大学 | Fermenting production method of acetylcholinesterase by microorganisms |
| CN104630188A (en) * | 2015-02-16 | 2015-05-20 | 大连大学 | Method for producing low-temperature glucomannanase by fermentation of marine microorganisms |
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Application publication date: 20110615 |