CN103667150A - Bacillus subtilis capable of producing neutral protease with strong heat stability and application of bacillus subtilis - Google Patents
Bacillus subtilis capable of producing neutral protease with strong heat stability and application of bacillus subtilis Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis capable of producing a neutral protease with strong heat stability and application of the bacillus subtilis, and belongs to the technical field of preparation of enzyme preparations. The bacillus subtilis 1398-2-12 capable of producing the neutral protease with strong heat stability is used as an original strain, culture medium optimization and fermentation process improvement are performed, the neutral protease prepared through liquid submerged and mixed fermentation under special fermentation conditions is 5500-7000 U/mL in enzyme activity, 30-75 DEG C in applicable temperature range, 70 DEG C in optimum reaction temperature, completely stable in enzyme activity at the temperature of 75 DEG C, 4.5-8.5 in applicable reaction pH value and is 7.2 in optimum reaction pH value. Compared with the conventional neutral protease, the neutral protease produced by the bacillus subtilis has stronger heat stability and higher enzyme activity, and meets industrial demands.
Description
Technical field
The invention belongs to microbial fermentation technology field, specifically a kind of subtilis that produces heat-flash stability neutral protease.
Background technology
Proteolytic enzyme is the fermentoid (enzyme) in organism, and they can decomposing protein.Decomposition method is to interrupt those amino acid to be connected to the peptide bond of polypeptide chain.The micromolecular compound of arrestin enzymic activity is called as proteinase inhibitor.The inhibitor of many virus proteases is effectively antiviral drugs.Proteolytic enzyme is the general name of the class of enzymes of protein hydrolysate peptide bond.Mode by its hydrolyzed peptide, can be divided into endopeptidase and exopeptidase two classes.Endopeptidase, by protein molecule inner cut-out, forms molecular weight Shi and peptone.Exopeptidase from the free amine group of protein molecule or the end of carboxyl one by one by peptide bond hydrolysis, and the amino acid that dissociates, the former for aminopeptidase the latter be carboxypeptidase.By its active centre and optimum pH, proteolytic enzyme can be divided into serine protease, thiol proteinase, metalloprotease and aspartate protease again.Optimum pH by its reaction, is divided into aspartic protease, neutral protease and Sumizyme MP.
Proteolytic enzyme is extensively present in pluck, plant stem-leaf, fruit and microorganism.Microbial protease, mainly by mould, bacterium, is secondly produced by yeast, actinomycetes.
Existing thousand the above history of application of enzyme, although neutral protease is to find and be applied to one of protease preparation of suitability for industrialized production for the mankind the earliest, widespread use or the recently thing of decades.Along with scientific and technical development, along with the development of the zymotechnics such as preparation, enzyme and the cell fixation of enzyme, enzyme molecular modification, the range of application of neutral protease also constantly expands.Since the last century, countries in the world researcher is all devoted to research and the applied research work that neutral protease produces screening, enzyme extraction, purifying and the zymologic property of bacterium always, has obtained certain achievement.
From current domestic neutral protease development and research report, the general study level of China's neutral protease is still in a starting stage.Large quantity research shows, microorganism fermentative production and gene engineering enter the gene clone of synthetic specific amino acids in microorganism cells plasmid, thereby by certain micro-organisms propagation, produce etc., neutral protease is widespread use, and these methods have that output is higher, with short production cycle, low cost and other advantages.Adopting genetically engineered to be combined to improve bacterial classification yield of enzyme with traditional Microbial Breeding mode is an evolutionary path that is worth continuing exploration.The comprehensive achievement having obtained in the past and the problem existing under study for action, the direction of Future Development mainly contains the following aspects: 1. continue further to improve microorganism enzymatic productivity by genetically engineered and traditional Microbial Breeding technology.2. adopt the in-vitro directed technology of enzyme to change substrate specificity and the adaptability of enzyme, as substrate avidity, pH adaptability, thermotolerance and scale resistance etc., for opening up the new Application Areas of enzyme, contribute.3. continue to find product neutral protease new bacterial classification, particularly extreme microorganism source neutral protease and seem particularly important.4. in application aspect, the Application Areas of continual exploitation enzyme, simultaneously, actively develop theory and application research, how enzyme makes larger contribution in the development of food and other industry for this reason, by the key areas that is following nutrient research, will be efficient for food and other industry, continue, steady progression opens up new bright prospects.
The domestic zymin enterprise that is engaged at present neutral protease production and sales seldom, mainly because adopt production bacterial classification is mostly the mutagenic strain that last century, the eighties was developed, enzymatic productivity decline, less stable, part good character occur that part is degenerated or forfeiture, and be vulnerable to the pollution of yeast and phage, on producing, cause impact to a certain degree.In addition, China's neutral protease fermentation unit is relatively low at present, and concentrated cost is high, and inconvenience is preserved and transportation, and generally, enzyme Techniques of preserving belongs to trade secret, and therefore, the research of relevant neutral protease enzyme preservation is still a field that is worth research.How to obtain new product neutral protease bacterial strain, improve strain enzyme-producing ability and how preservation enzyme will become a developing direction.
Chinese patent CN1289659C discloses a kind of technique and high temperature neutral protease that produces the Bacillus licheniformis of high temperature neutral protease and prepare high temperature neutral protease, the applicable temperature of reaction of described neutral protease is 50-70 ℃, optimal reactive temperature is 65 ℃, applicable pH value is 5-8, and optimum pH is 7.0-7.2.At 1 hour enzyme activity of 50 ℃ of insulations, decline 10%, 45 minutes transformation period of enzyme at 60 ℃.
Neutral protease is energy catalytic proteins peptide bond hydrolysis, and Optimun pH is at the proteolytic enzyme of 7.0 left and right.Neutral protease is fast owing to having catalytic, without industrial pollution, character and the advantages such as catalytic reaction condition adaptability is wider, be applied to food, pharmacy, makeup, washing composition, silk spinning, the industries such as fur softening, but due to neutral protease poor heat stability, produce with working conditions complicated, limited its further scope of application, the suitability for industrialized production tool of further optimizing the strain excellent centering proteolytic enzyme of seed selection product neutral protease is of great significance, especially the thermostability of neutral protease, the enzymatic properties such as resistance to acids and bases are still one of problem that industry technician pays close attention to the most.
Summary of the invention
The object of this invention is to provide a kind of subtilis (Bacillus subtilis) 1398-2-12 that produces heat-flash stability neutral protease.
Subtilis 1398-2-12 provided by the invention is that subtilis (Bacillus subtilis) 1398-2 that produces neutral protease by a strain of laboratory preservation obtains through UV-LiCl-ethyl sulfate Mutation screening.
The bacterial strain of product heat-flash stability neutral protease provided by the invention is specially subtilis 1398-2-12.This bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013 and (is called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College postcode: 430072), preserving number is CCTCC NO:M2013539, systematic name subtilis (Bacillus subtilis).
Described subtilis (Bacillus subtilis) 1398-2-12 bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is oyster white, and surface drying is opaque, and neat in edge, for having the aerobic bacteria of mobility.Microscopy is elongated rod shape, and gramstaining is positive.This bacterium can utilize Citrate trianion, and nitrate reductase, V-P test into positive.
Subtilis 1398-2-12 provided by the invention has that produced neutral protease tolerable temperature is high, the feature of applicable pH value wide scope, 75 ℃ of enzymes of fermented liquid crude enzyme liquid complete stability alive, 70 ℃ of optimal reactive temperatures, pH value 4.5-8.5 enzyme is lived stable, optimal reaction pH value 7.2.This bacterial strain the most suitable growth pH value 7.0-7.2, optimum growth temperature 30-36 ℃, the suitableeest product enzyme temperature 32-35 ℃.
Press mutagenesis screening scheme, mutant strain step-sizing is eliminated, finally to strain excellent through leavening property test screen, obtain a strain and produce the bacterial strain subtilis bacterium 1398-2-12 of heat-flash stability neutral protease, the work of fermented liquid neutral protease enzyme can reach 5500-7000U/mL, thermostability to enzyme is analyzed, and crude enzyme liquid is placed in respectively at 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, every 10 minutes sampling and measuring enzymes, lives.At 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 minutes enzymes are lived and are not declined.At 60 ℃ and 65 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 95%, 60 minute.At 70 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minute.At 75 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 70% in 80%, 60 minute.
Beneficial effect: subtilis 1398-2-12 provided by the invention has and produces that neutral protease tolerable temperature is high, the feature of applicable pH value wide scope, through screening process medium optimization, compare growth with starting strain rapidly, the work of fermented liquid neutral protease enzyme can reach 5500-7000U/mL, and than setting out, strain enzyme activity improves 78.5%.The neutral protease of this bacterial strain secretion can withstand higher temperatures, and at 40 ℃, 45 ℃, 50 ℃, 55 ℃ of crude enzyme liquids, 60 minutes enzymes are lived and do not declined.At 60 ℃ and 65 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 95%, 60 minute.At 70 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minute.At 75 ℃, what within 30 minutes, drop to that constitutive enzyme lives drops to 70% in 80%, 60 minute, and similarity condition is issued to the same enzyme tolerable temperature ratio bacterium that sets out that lives and has on average improved 5-10 ℃.It is alive stable that this bacterial strain produces neutral protease pH value 4.5-8.5 enzyme, than the pH value 5-8 wide scope of neutral protease that starting strain produces.
Embodiment
Below by concrete embodiment narration the present invention.Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.
Embodiment 1
The bacillus subtilis strain that the present invention produces neutral protease is specially 1398-2-12.This bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013 and (is called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College postcode: 430072), preserving number is CCTCC NO:M2013539, and Classification And Nomenclature is subtilis (Bacillus subtilis) 1398-2-12.
The subtilis 1398-2 that subtilis 1398-2-12 provided by the invention produces neutral protease by a strain of laboratory preservation obtains through UV-LiCl-ethyl sulfate Mutation screening.
Embodiment 2 produces the screening method of heat-flash stability neutral protease bacterial strain, comprises the following steps
(1) preparation of bacteria suspension
The single bacterium colony access seed culture 100r/min that subtilis 1398-2 slant strains is grown after plate streaking separation, cultivates after 12h for 35 ℃, uses physiological saline washed twice after getting 1mL medium centrifugal, and is resuspended in 9mL physiological saline.
(2) UV-LiCl-ethyl sulfate complex mutation
Bacteria suspension is placed in to aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.To after gradient dilution, coat lithium chloride flat board through the bacterium liquid irradiating, and contrast with the bacterium liquid dilution painting flat board without uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, with cloth or the newspaper of black, wrap, put 35 ℃ and cultivate 48h, on the flat board of bacterium colony, filter out hydrolysis circle and choose to inclined-plane and preserve with colony diameter ratio the maximum growing, after purifying, be mixed with bacteria suspension, after gradient dilution, fully mix with ethyl sulfate stoste, and process 40min in 35 ℃ of concussions, the bacterium liquid of processing is coated to lithium chloride flat board after gradient dilution.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, put 35 ℃ and cultivate 48h, go out hydrolysis circle and choose to inclined-plane and preserve with colony diameter ratio the greater growing on the flat board of bacterium colony primary dcreening operation, after purifying, obtain three strain bacterium 1398-2-12,1398-2-2,1398-2-3;
(4) shake flask fermentation sieves again
By the three strain bacterium 1398-2-12 that obtain, 1398-2-2,1398-2-3 carries out shake flask fermentation in the 250mL shaking flask that contains 30mL fermention medium, seed inoculum size 10% (V/V), 35 ℃, 100r/min are cultivated 72h, and centrifuging and taking fermented supernatant fluid makes crude enzyme liquid.
(5) enzyme activity determination
Enzyme activity unit definition: 1mL crude enzyme liquid, under 70 ℃, pH7.2 condition, 1min caseinhydrolysate produces 1 μ g tyrosine, is 1 enzyme activity unit, represents with U/mL.
After measured, bacterial strain 1398-2-12 is stable superior strain, and enzyme work reaches 6000U/mL.
This bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013, and (be called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College postcode: 430072), preserving number is CCTCC NO:M2013539.
Described lithium chloride plate culture medium consists of: starch 10g, and peptone 10g, (NH)
2sO
44g, K
2hPO
48g, CaCl
22g, lithium chloride 9g, agar 20g, distilled water 1000mL, 7.2,121 ℃ of sterilizing 20min of pH value;
Described seed culture medium consists of: extractum carnis 10g, peptone 10g, Zulkovsky starch 10g, NaCl10g, distilled water 1000 mL, 7.2,121 ℃ of sterilizing 20min of pH value;
Described fermention medium: Semen Maydis powder 50-150g, soybean cake powder 40-100g, (NH)
2sO
44g, K
2hPO
48g, CaCl
22g, distilled water 1000mL, 7.2,121 ℃ of sterilizing 20min of pH value;
Embodiment 3: the optimization of producing heat-flash stability neutral protease starting strain screening culture medium
(1) bacterial strain
What starting strain was selected is the 1398-2-12 bacterial strain of the selection result the best, and the neutral protease after 72 hours that ferments can reach 6000U/mL.
(2) medium optimization
Lithium chloride plate culture medium, seed culture medium and fermention medium in bacterium process are carried out to medium optimization, and to optimize front identical fermentation condition fermentation 72 hours, after testing, fermented liquid crude enzyme liquid neutral protease can reach 7000U/mL.
Lithium chloride is optimized plate culture medium and is consisted of: starch 10g, and peptone 10g, (NH)
2sO
44g, K
2hPO
48g, CaCl
22g, lithium chloride 9g, Chinese herbal medicine powder 5-20g, agar 20g, distilled water 1000mL, 7.2,121 ℃ of sterilizing 20min of pH value;
Seed Optimal Medium consists of: extractum carnis 10g, peptone 10g, Zulkovsky starch 10g, NaCl10g, Chinese herbal medicine powder 15-20g, distilled water 1000mL, 7.2,121 ℃ of sterilizing 20min of pH value; Fermentation optimization substratum: Semen Maydis powder 50-150g, soybean cake powder 40-100g, (NH)
2sO
44g, K
2hPO
48g, CaCl
22g, Chinese herbal medicine powder 15-25g, distilled water 1000mL, 7.2,121 ℃ of sterilizing 20min of pH value;
The preparation method of described Chinese herbal medicine powder is as follows:
Take Radix Astragali 20-30 part; Radix Codonopsis 10-18 part; Radix bupleuri 10-15 part; Root of large-flowered skullcap 10-15 part; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, add the mixture of 2-3 times of weight ethanol of mixture and propyl alcohol, control temperature to 60 ℃～78 ℃ and keep 3～4h, filter; Filtrate vacuum concentration postlyophilization obtains Chinese herbal medicine powder;
The mass ratio of described ethanol and propyl alcohol is 1:1-1.5.
Embodiment 4: the thermal stability analysis of neutral protease
Thermostability to enzyme is analyzed, and embodiment 3 crude enzyme liquids are placed in respectively at 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, every 10 minutes sampling and measuring enzymes, lives.At 40 ℃, 45 ℃, 50 ℃, 55 ℃ of crude enzyme liquids, 60 minutes enzymes are lived and are not declined.At 60 ℃ and 65 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 95%, 60 minute.At 70 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minute.At 75 ℃, what within 30 minutes, drop to that constitutive enzyme lives drops to 70% in 80%, 60 minute, and similarity condition is issued to the same enzyme tolerable temperature ratio bacterium that sets out that lives and has on average improved 5-10 ℃, than the bacterium before Optimal Medium, has improved 3-5 ℃.
Embodiment 5: neutral protease pH stability analysis
PH stability to enzyme is analyzed, and embodiment 3 crude enzyme liquids are placed in respectively to pH value 3.5,4.5,5.5,6.5,7.5,8.5 times, every 10 minutes sampling and measuring enzymes, lives.Crude enzyme liquid pH value 5.5,6.5,7.5 times, 60 minutes enzymes are lived and are not declined.PH value 4.5 and 8.5 times, what within 30 minutes, drop to that constitutive enzyme lives drops to 85% in 95%, 60 minute.PH value 3.5 times, what within 30 minutes, drop to constitutive enzyme work drops to 70% in 80%, 60 minute.It is obviously more wide in range than the bacterium setting out before bacterium and Optimal Medium that similarity condition is issued to same enzyme resistance to pH value alive.
Claims (7)
1.
?subtilis (Bacillus subtilis) 1398-2-12 who produces heat-flash stability neutral protease, this bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013, and preserving number is CCTCC NO:M2013539.
2. the application of subtilis (Bacillus subtilis) 1398-2-12 in neutral protease fermentative production as claimed in claim 1.
3. the application of subtilis (Bacillus subtilis) 1398-2-12 in neutral protease fermentative production as claimed in claim 2, is characterized in that, lithium chloride is optimized plate culture medium and consisted of: starch 10g, and peptone 10g, (NH)
2sO
44g, K
2hPO
48g, CaCl
22g, lithium chloride 9g, Chinese herbal medicine powder 5-20g, agar 20g, distilled water 1000mL, pH value 7.2.
4. the application of subtilis (Bacillus subtilis) 1398-2-12 in neutral protease fermentative production as claimed in claim 2, it is characterized in that, seed Optimal Medium consists of: extractum carnis 10g, peptone 10g, Zulkovsky starch 10g, NaCl10g, Chinese herbal medicine powder 15-20g, distilled water 1000mL, pH value 7.2.
5. the application of subtilis (Bacillus subtilis) 1398-2-12 in neutral protease fermentative production as claimed in claim 2, is characterized in that, fermentation optimization substratum consists of: Semen Maydis powder 50-150g, and soybean cake powder 40-100g, (NH)
2sO
44g, K
2hPO
48g, CaCl
22g, Chinese herbal medicine powder 15-25g, distilled water 1000mL, pH value 7.2.
6. the application of subtilis (Bacillus subtilis) 1398-2-12 in neutral protease fermentative production as described in as arbitrary in claim 3-5, is characterized in that, the preparation method of described Chinese herbal medicine powder is as follows:
Take Radix Astragali 20-30 part; Radix Codonopsis 10-18 part; Radix bupleuri 10-15 part; Root of large-flowered skullcap 10-15 part; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, add the mixture of 2-3 times of weight ethanol of mixture and propyl alcohol, control temperature to 60 ℃～78 ℃ and keep 3～4h, filter; Filtrate vacuum concentration postlyophilization obtains Chinese herbal medicine powder.
7. the application of subtilis (Bacillus subtilis) 1398-2-12 in neutral protease fermentative production as claimed in claim 6, is characterized in that, the mass ratio of described ethanol and propyl alcohol is 1:1-1.5.
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Cited By (6)
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| CN104041708A (en) * | 2014-06-16 | 2014-09-17 | 邵素英 | Preparation method for compound premix in egg producing period |
| CN105087446A (en) * | 2015-09-14 | 2015-11-25 | 青岛蔚蓝生物集团有限公司 | Bacillus amyloliquefaciens for high production of neutral protease |
| CN107201354A (en) * | 2017-07-04 | 2017-09-26 | 北京科为博生物科技有限公司 | A kind of neutral proteinase and its gene and application |
| CN110656101A (en) * | 2019-11-18 | 2020-01-07 | 河北省微生物研究所 | Method for preparing neutral protease by fermenting bacillus subtilis |
| CN110760466A (en) * | 2019-11-18 | 2020-02-07 | 山东隆科特酶制剂有限公司 | Bacillus subtilis for producing high-temperature-resistant neutral protease and application thereof |
| CN112746090A (en) * | 2020-12-29 | 2021-05-04 | 南宁东恒华道生物科技有限责任公司 | Kitchen waste enzymolysis treatment process |
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| CN106676023A (en) * | 2015-11-05 | 2017-05-17 | 中国科学院天津工业生物技术研究所 | Bacillus amyloliquefaciens highly yielding neutral protease and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104041708A (en) * | 2014-06-16 | 2014-09-17 | 邵素英 | Preparation method for compound premix in egg producing period |
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| CN112746090A (en) * | 2020-12-29 | 2021-05-04 | 南宁东恒华道生物科技有限责任公司 | Kitchen waste enzymolysis treatment process |
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