CN1793352A - Process for increasing stability of elastase of lichen spore bacillus ZJUEL31410 - Google Patents
Process for increasing stability of elastase of lichen spore bacillus ZJUEL31410 Download PDFInfo
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- CN1793352A CN1793352A CN 200510061376 CN200510061376A CN1793352A CN 1793352 A CN1793352 A CN 1793352A CN 200510061376 CN200510061376 CN 200510061376 CN 200510061376 A CN200510061376 A CN 200510061376A CN 1793352 A CN1793352 A CN 1793352A
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- elastase
- enzyme liquid
- elastoser
- spore bacillus
- crude enzyme
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Abstract
The invention discloses a method uses to increase Bacillus licheniformis ZJUEL31410 elastase stability. It includes the following steps: adding bovine serum albumin, gelatin, and hydration calcium chloride into CGMCC 1397 lichen bacillus ZJUEL31410 elastase coarse enzyme liquid; and their weight-to-volume ratio with the coarse enzyme liquid are respectively 1-3g/100ml, 1-3g/100ml, and 1-3mmol/L. The method can increase lichen bacillus ZJUEL31410 elastase thermal stability and pH tolerance.
Description
Technical field
The present invention relates to the biological method of a kind of raising, improvement stability of elastase of lichen spore bacillus ZJUEL 31410.
Background technology
Elastoser is a kind of to decompose the wide spectrum proteolytic ferment that insoluble elastin is a feature, has great economic worth and using value.In recent years, elastoser is mainly used among the industry such as medicine, food and daily-use chemical industry.Elastoser has the treatment hyperlipidemia, prevents arteriosclerosis, improves effects such as lipid protein metabolism.
China is silk big producing country, and silkworm chrysalis is the main byproduct of silk industry, and 1 ton of raw silk of every production can obtain about 1 ton dried pupa.Because dried pupa has rich nutrient contents, therefore have higher health care and be worth, can be used for industries such as food, medicine after the separation and Extraction, have high economic worth.
But the utilization of relevant silkworm chrysalis can not well be resolved, and majority is in elementary processing, and quality product is inferior.And the silkworm chrysalis of employing enzyme engineering technology hydrolysis can be directly used in the production of food, makeup, will produce tangible economic benefit.
The remarkable difference of elastoser and other protease function is that elastin is had specificity, and when multiple substrate coexists, the selective hydrolysis elastin, really play the function that makes tenderization not change local flavor and taste again, rather than, be the agent of ideal tenderization therefore merely to the in addition non-selective part digestion of food protein.Elastoser many animal/vegetable proteins of can also degrading simultaneously particularly can be handled some and be difficult to handle edible as albumen byproducts such as ligament, Aorta blood vessel, muscle tendons, therefore can be used for carrying out the deep processing of agricultural-food.
Elastoser still is good daily-use chemical industry raw material, is mainly used in the production of cosmetics.The makeup that added elastoser can improve the metabolic function of skin, and elastoser is that a kind of novel cosmetics process raw material thus, has good beauty and health care functions.
At present, the main source of China's elastoser is to utilize pig pancreas, dirty extraction.Because the restriction of internal organs resource, therefore elastoser is very in short supply, supply falls short of demand, cause domestic and international valuable product, therefore the use of elastoser only limits to medicine and minute quantity makeup, before the application in other field was taken a step not, particularly the applied research wretched insufficiency of elastoser in the transformation of tenderization and aged meat had a strong impact on the research and development and the application of elastoser.This shows, utilize microbe fermentation method industrialization, mass-producing ground production elastoser that wide application development prospect is arranged.The patent of invention that the applicant has applied for " a kind of Bacillus strain and uses thereof " on September 20th, 2005, application number is 200510060829.1.It discloses a kind of Bacillus strain---the bacterial strain of Bacillus licheniformis (Bacilluslicheniformis), the preservation strain name is called: lichen spore bacillus ZJUEL 31410, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on 06 23rd, 2005, preserving number: CGMCC 1397.
But the problem of present microbial source elastoser ubiquity poor stability, especially in actual application, functioning efficiency is too low, can't reach good action effect.Existing investigator attempts to adopt chemical process such as trehalose to carry out aglucon to modify reconstruction animal source elastoser, and has obtained certain effect, has obviously improved the thermostability of elastoser and to the tolerance of low pH.But consider elastoser in medicine and Application in Food Industry, security is the huge difficult problem that the chemically modified elastoser faces.Because will use bromine cyanic acid in the chemically modified process, it is a kind of highly toxic substance, and carcinogenesis is arranged; Bring huge safety issue can for the application of actually operating and product.Still be faced with problem on many technology and the cost and adopt novel molecular biology method that elastoser is carried out molecular modification, be difficult to obtain mass-producing and use.Therefore carry out economy, effectively the exploitation of elastin enzymatic protective reagent has important Research Significance and marketable value for the commercial application of microbial source elastoser.
Summary of the invention
At the deficiencies in the prior art part, the invention provides a kind of employing external source and add composite protectant, with the thermostability of raising elastase of lichen spore bacillus ZJUEL 31410 and the method for pH tolerance.
The present invention is to realize by such technical scheme for reaching above purpose: a kind of method that improves stability of elastase of lichen spore bacillus ZJUEL 31410 is provided: add bovine serum albumin (PA), gelatin (PB) and hydration calcium chloride in described preserving number is the crude enzyme liquid of elastase of lichen spore bacillus ZJUEL 31410 of CGMCC 1397; The weightmeasurement ratio of bovine serum albumin and crude enzyme liquid is 1～3g/100ml (being the bovine serum albumin of crude enzyme liquid adding 1～3g of every 100ml), the weightmeasurement ratio of gelatin and crude enzyme liquid is 1～3g/100ml (being the gelatin of crude enzyme liquid adding 1～3g of every 100ml), and the feed ratio of hydration calcium chloride and crude enzyme liquid is 1～3mmol/L (being the hydration calcium chloride of crude enzyme liquid adding 1～3mmol of every 1L).
A kind of improvement as the method for raising stability of elastase of lichen spore bacillus ZJUEL 31410 of the present invention: the weightmeasurement ratio of bovine serum albumin and crude enzyme liquid is 2g/100ml, the weightmeasurement ratio of gelatin and crude enzyme liquid is 1.5g/100ml, and the feed ratio of hydration calcium chloride and crude enzyme liquid is 1mmol/L.
Do not need in the above-mentioned interpolation process to stir, temperature is that room temperature gets final product.
The method of raising stability of elastase of lichen spore bacillus ZJUEL 31410 of the present invention; on following basis, obtain: at first filter out and to improve and to prolong the protective material of elastase of lichen spore bacillus ZJUEL 31410 thermostability; and the composite protectant that obtains is optimized, finally obtained greatly to improve the best protection agent proportioning of stability of elastase of lichen spore bacillus ZJUEL 31410.So the method for raising stability of elastase of lichen spore bacillus ZJUEL 31410 of the present invention, safety, effective, and also used protective material raw material sources are wide, therefore are easy to extensive use.Adopt method of the present invention, make the heat resisting temperature of elastase of lichen spore bacillus ZJUEL 31410 improve 10 ℃, and can make this enzyme in the scope of pH 6～9, all keep satisfactory stability, improve the maximum effect efficient of elastoser, helped enlarging the range of application of elastoser.The elastase of lichen spore bacillus ZJUEL 31410 that adopts method of the present invention to preserve, safe, economical, can be used as food grade additives.
In order to prove creativeness of the present invention, the contriver has done 4 groups of following contrast experiments and (has annotated 1, tests the employed elastase of lichen spore bacillus ZJUEL 31410 that preserving number is CGMCC 1397 that is, hereinafter to be referred as elastoser; Annotate 2, in following each table, the thermal treatment of not doing of 4 ℃ of placements does not add protectant enzyme retention rate of living and is 100%; ):
Experiment one, to the live influence of retention rate of elastoser enzyme:
Add following protective material in the crude enzyme liquid of identical elastoser respectively, protectant consumption is as described in Table 1, has measured enzyme retention rate alive behind 50 ℃ of water bath heat preservation 60min.The thermal treatment of not doing with 4 ℃ of placements does not add protectant enzyme activity as 100%, to be treated to reference group with protective material under same experimental conditions, calculates and comparison enzyme activity retention rate (%).Concrete numerical value sees Table 1.
The different protectant interpolations of table 1 are to the influence of elastoser enzyme retention rate alive
| The protective material type of adding | Protectant consumption | Enzyme retention rate (%) alive |
| PA | 0.1g/100ml | 53.6 |
| PA | 2g/100ml | 85.2 |
| PB | 0.05g/100ml | 30.5 |
| PB | 1g/100ml | 70.9 |
| Xanthan gum | 0.05g/100ml | 43.2 |
| Xanthan gum | 0.3g/100ml | 61.4 |
| Trehalose | 0.05g/100ml | 25.7 |
| Trehalose | 0.3g/100ml | 26.6 |
| Glycerine | 10g/100ml | 54.6 |
| Glycerine | 20g/100ml | 57.4 |
| N.F,USP MANNITOL | 0.2g/100ml | 27.4 |
| N.F,USP MANNITOL | 1g/100ml | 39.7 |
| Hydration calcium chloride | 0.04mmol/L | 48.7 |
| Hydration calcium chloride | 2mmol/L | 70.0 |
| Potassium sorbate | 0.1g/100ml | 34.1 |
| Potassium sorbate | 0.5g/100ml | 51.0 |
| Sodium-chlor | 0.4mmol/L | 26.4 |
| Sodium-chlor | 2mmol/L | 27.9 |
| Xitix | 0.04mmol/L | 21.6 |
| Xitix | 0.4mmol/L | 21.0 |
| With protective material (reference group) | 0 | 47.0 |
And under identical experiment condition, adopt the method enzyme of the present invention high energy of retention rate of living to reach 98.8%.Therefore above experiment shows that the present invention can make PA, PB and hydration calcium chloride performance synergy, further improves the thermostability of enzyme.
Experiment two, to the influence of elastin enzyme heat stability:
Set and do not add any protectantly for reference group in crude enzyme liquid, the present invention has been done contrast with the enzyme work retention rate of reference group gained after the different time of thermal treatment under the different temperature, concrete outcome is shown in Table 2.
The protectant interpolation of table 2 is to the influence of elastin enzyme heat stability
| Temperature (℃) | Enzyme retention rate (%) alive behind the insulation 30min | Enzyme retention rate (%) alive behind the insulation 60min | ||
| Reference group | The present invention | Reference group | The present invention | |
| 30 | 100 | 100 | 100 | 100 |
| 40 | 100 | 100 | 90.1 | 100 |
| 50 | 56.3 | 99.4 | 28.4 | 94 |
| 60 | 5 | 60.5 | 0 | 30.2 |
| 70 | 0 | 9.6 | 0 | 5.3 |
Experiment shows that under identical heat treatment time, under the prerequisite that the enzyme that obtains retention rate alive equates substantially, the heat resisting temperature of elastase of lichen spore bacillus ZJUEL 31410 has improved 10 ℃ at last.
Experiment three, to the influence of elastoser pH stability:
In like manner, set and do not add any protectantly for reference group in crude enzyme liquid, the present invention has been done contrast with the enzyme work retention rate of reference group gained under different pH, concrete outcome is shown in Table 3.
Table 3 protective material adds influence and the comparison thereof to elastoser pH stability
| pH | Enzyme retention rate (%) alive | |
| Reference group | The present invention | |
| 5 | 22.3 | 35.0 |
| 6 | 80.0 | 98.0 |
| 7 | 88.5 | 99.5 |
| 8 | 90.5 | 99.7 |
| 9 | 80.2 | 88.9 |
Originally experiment showed, that under identical pH value enzyme of the present invention retention rate alive will be significantly higher than reference group.The present invention makes elastase of lichen spore bacillus ZJUEL 31410 keep satisfactory stability in pH 6～9 scopes, helps enlarging the range of application of this elastoser.
Experiment four, utilization accelerated test have been estimated the storage life of elastoser, and promptly when 25 ℃, 20 ℃, 4 ℃ and 0 ℃, the time that loss of activity 50% needs is respectively:
Reference group is: 36,91,2000 and 4000 days;
The present invention is: 140,330,6500 and 16000 days.
The contriver is that the security of the lichen spore bacillus ZJUEL 31410 of CGMCC 1397 is studied to the used preserving number of the present invention also: product that this strain fermentation is produced and bacterium liquid thereof detect to oral acute nontoxic through mouse through Zhejiang Center For Disease Control and Prevention, medial lethal dose is more than the 10g/kg, thereby proof application on food is safe.
Embodiment
Improve the method for stability of elastase of lichen spore bacillus ZJUEL 31410: in preserving number is the crude enzyme liquid of elastase of lichen spore bacillus ZJUEL 31410 of CGMCC 1397, add bovine serum albumin (PA), gelatin (PB) and hydration calcium chloride (CaCl
2.2H
2O); PA, PB, CaCl
2.2H
2The feed ratio of O and crude enzyme liquid shown in the embodiment 1～embodiment 10 in the table 1, does not add protectant reference group that is respectively; Measured enzyme retention rate alive behind 50 ℃ of water bath heat preservation 60min, concrete numerical value sees Table 4.
Table 4
| PA (g/100ml) | PB (g/100ml) | CaCl 2.2H 2O (mmol/L) | Enzyme retention rate (%) alive | |
| Embodiment 1 | 1 | 1 | 1 | 71.3 |
| Embodiment 2 | 1 | 3 | 1.5 | 85.1 |
| Embodiment 3 | 1 | 1.5 | 3 | 86.6 |
| Embodiment 4 | 1.5 | 1 | 1.5 | 82.7 |
| Embodiment 5 | 1.5 | 1 | 2 | 88.1 |
| Embodiment 6 | 1.5 | 1.5 | 1 | 83.6 |
| Embodiment 7 | 3 | 1 | 2 | 90.1 |
| Embodiment 8 | 3 | 1 | 1 | 89.1 |
| Embodiment 9 | 3 | 1.5 | 1.5 | 91.3 |
| Embodiment 10 | 2 | 1.5 | 1 | 98.8 |
| Reference group | 0 | 0 | 0 | 35.0 |
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (2)
1, a kind of method that improves stability of elastase of lichen spore bacillus ZJUEL 31410 is characterized in that: add bovine serum albumin, gelatin and hydration calcium chloride in described preserving number is the crude enzyme liquid of elastase of lichen spore bacillus ZJUEL 31410 of CGMCC 1397; The weightmeasurement ratio of described bovine serum albumin and crude enzyme liquid is 1～3g/100ml, and the weightmeasurement ratio of gelatin and crude enzyme liquid is 1～3g/100ml, and the feed ratio of hydration calcium chloride and crude enzyme liquid is 1～3mmol/L.
2, the method for raising stability of elastase of lichen spore bacillus ZJUEL 31410 according to claim 1, it is characterized in that: the weightmeasurement ratio of described bovine serum albumin and crude enzyme liquid is 2g/100ml, the weightmeasurement ratio of described gelatin and crude enzyme liquid is 1.5g/100ml, and the feed ratio of hydration calcium chloride and crude enzyme liquid is 1mmol/L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955923A (en) * | 2010-09-30 | 2011-01-26 | 江南大学 | Method for improving storage stability of catalase |
| CN101113422B (en) * | 2007-06-28 | 2011-06-15 | 浙江大学 | Bacillus licheniformis fibrinolytic enzyme and production method thereof |
| CN102634503A (en) * | 2011-02-14 | 2012-08-15 | 广州市开恒生物科技有限公司 | Beta-glucanase stabilizer and preparation method thereof |
| CN103509742A (en) * | 2013-09-05 | 2014-01-15 | 徐州工程学院 | Baclicus lincheniformis strain for generating elastase and screening method thereof |
| CN103627689A (en) * | 2013-09-05 | 2014-03-12 | 徐州工程学院 | Method for producing elastase preparation by utilizing liquid-state fermentation of microbes |
| CN106119236A (en) * | 2016-07-05 | 2016-11-16 | 山东大学 | A kind of composite protectant improving deep-sea stability of elastase and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ520201A (en) * | 2000-02-08 | 2004-04-30 | Allergan Inc | Botulinum toxin pharmaceutical compositions comprising a polysaccharide |
| CN1168503C (en) * | 2001-12-21 | 2004-09-29 | 卫广森 | Veterinarian virus kind biological product heat resisting freeze drying protective agent and its preparation technique |
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2005
- 2005-11-02 CN CNB2005100613764A patent/CN100384993C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113422B (en) * | 2007-06-28 | 2011-06-15 | 浙江大学 | Bacillus licheniformis fibrinolytic enzyme and production method thereof |
| CN101955923A (en) * | 2010-09-30 | 2011-01-26 | 江南大学 | Method for improving storage stability of catalase |
| CN101955923B (en) * | 2010-09-30 | 2012-05-23 | 江南大学 | Method for improving storage stability of catalase |
| CN102634503A (en) * | 2011-02-14 | 2012-08-15 | 广州市开恒生物科技有限公司 | Beta-glucanase stabilizer and preparation method thereof |
| CN103509742A (en) * | 2013-09-05 | 2014-01-15 | 徐州工程学院 | Baclicus lincheniformis strain for generating elastase and screening method thereof |
| CN103627689A (en) * | 2013-09-05 | 2014-03-12 | 徐州工程学院 | Method for producing elastase preparation by utilizing liquid-state fermentation of microbes |
| CN106119236A (en) * | 2016-07-05 | 2016-11-16 | 山东大学 | A kind of composite protectant improving deep-sea stability of elastase and preparation method thereof |
| CN106119236B (en) * | 2016-07-05 | 2019-01-04 | 山东大学 | A kind of composite protectant and preparation method thereof improving deep-sea stability of elastase |
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| CN100384993C (en) | 2008-04-30 |
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