CN105670981A - Serratia marcescens SPG-1 and method for producing chitosanase by adopting same - Google Patents
Serratia marcescens SPG-1 and method for producing chitosanase by adopting same Download PDFInfo
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Abstract
The invention discloses a serratia marcescens SPG-1 with the preservation number of CCTCC No. M2015784; the serratia marcescens SPG-1 is Gram negative bacteria, short rod-shaped, singly arranged, free from spores and has a size of 0.5-1.0*1.2-2.0 [mu]m. The invention further discloses a method for preparing chitosanase by adopting the serratia marcescens SPG-1. The serratia marcescens SPG-1 strain is higher in yield of chitosanase and low in medium cost, culture conditions are easy to control, and the extraction process is simple, energy-saving and environment-friendly. The method for producing the chitosanase has the advantages of being simple and practical and low in cost and is liable to popularization in and application to industrial production.
Description
Technical field
The present invention relates to a kind of microorganism, particularly a kind of serratia marcescens SPG-1, the present invention also relates to the method that this bacterial strain produces chitoanase.
Background technology
Chitoanase can be hydrolyzed deacetylated chitosan, is therefore considered as being had by linear chitosan the enzyme of single-minded hydrolysis. Chitoanase is mainly present in the biotic populations such as actinomycetes, fungi, bacterium and animals and plants, and it acts primarily on β-1.4-glucosamine glycosides key, obtains its oligopolymer with internal-cutting way degrade chitosan. Chitoanase is divided into 2 classes at first, but Fukamizo etc. propose be divided into 3 classes, namely chitoanase I, chitoanase II, chitoanase III. The first kind can be hydrolyzed Gen Gen key and Glucina Gen key; 2nd class can only be hydrolyzed Gen Gen key; 3rd class can be hydrolyzed Gen Glen key, can be hydrolyzed again Gen Cenacle key.
Research finds that the relative molecular mass of the chitoanase of different sources exists difference due to the difference of its structure and composition. Being separated the chitoanase molecular mass obtained in plant is 10000 ~ 23000u, and is separated the chitoanase molecular mass obtained from microorganism and is approximately 20000 ~ 40000u. Chitoanase major part is basic protein, derives from the chitoanase of each quasi-microorganism, and its optimum pH scope is generally 4.0 ~ 8.0. Iso-electric point (pI) variation range of enzyme is also bigger, mostly concentrates on 4.0 ~ 10.1.
Utilize chitoanase hydrolyzing chitosan to obtain the oligose being formed by connecting by β-1,4-glycosidic link by 2 ~ 10 glucosamines, especially it is that typical case represents taking oligochitosan. The main application fields of oligochitosan has:
(1) medicine
There are sizable potentiality in oligochitosan, wound can be made from bacteriological infection in medicine, also has good air permeable effect simultaneously, and the healing of wound is helpful. Can being degraded by the enzyme lysozyme and generate natural metabolite, have the features such as nontoxic, easy absorption, using it as medicinal slow release agent has very big superiority. 1997, scholar is had to carry out assisting therapy with low molecular chitosan, it has been found that lymphocyte and total white blood cells remain stable, and T cell quantity obviously increases, and this kind of low molecular chitosan is that antineoplastic research provides foundation. At present, the experiment of cell levels proves that kinds of tumor cells is had fragmentation effect by oligochitosan.
(2) food
Oligochitosan receives much concern especially in the application of field of food, and it has multinomial physiological regulation function, as the activation factor of beneficial bacteria of intestinal tract, promotes the absorption of calcium and mineral substance, absorption heavy metal of body etc. In alternative seasonings, the Chemical Preservative such as Sodium Benzoate, becomes natural green aseptic production. It also can reduce the absorption of cholesterol and fat, reduces hypertension, is widely used in the functional drinkss such as weight-reducing, toxin-expelling and face nourishing, immunomodulatory. Available it fruit and vegetable is carried out coating-film fresh-keeping, its rete has good permeability, water preventing ability, simultaneously also have anti-corrosive antibacterial effect.
(3) agricultural
Oligochitosan is as a kind of novel agrochemical, have fertilizer efficiency and the feature of drug effect dual-use function concurrently, and the advantage such as nontoxic, free from environmental pollution, it can change the bacterium group in soil, promote beneficial microorganism growth, oligochitosan also can the immunity system of induced activation plant, improve plant to the resistivity of virus, the diseases such as wheat mosaic disease, rice blast, cotton verticillium wilt, tomato late blight are had good anti-pre-and therapeutic action, it is possible to be developed as growth regulator, biological pesticide and fertilizer etc.
(4) daily-use chemical industry
Oligochitosan has good humidity-holding effect, activation body cell, suppress that skin surface harmful bacteria grows, anti-tetter and absorb the effect such as ultraviolet, important is that it derives from bio-extract, can well avoid the side effect that ordinary cosmetics brings, such that it is able to be applied in the skin care product of moisturizing, the type such as crease-resistant, sun-proof; Oligochitosan also has the permeability keeping hair surface film forming, moistening easy combing, and has anti-dandruff and itching-relieving, dust-proof, antistatic effect.
(5) biological veterinary
Utilizing its anti-microbial effect, prevention and therapy is by thin microbial Animal diseases such as streptococcus aureuses; Oligochitosan also can be utilized to promote the healing of wound, it is used to the assisting therapy of wound or fracture; Zine ion, iron ion and calcium ion etc. are all had good complexing action by carboxymethyl chitosan oligosaccharide, are expected to make new natural zinc supplementation agent, iron supplementary and calsium supplement.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, it is provided that a kind of serratia marcescens SPG-1 bacterial strain that can produce chitoanase newly.
Another technical problem to be solved by this invention there is provided the method that above-mentioned serratia marcescens SPG-1 bacterial strain produces chitoanase, to overcome the high problem of enzyme production cost that prior art exists.
Technical problem to be solved by this invention is realized by following technical scheme. The feature of the present invention comprises serratia marcescens (Serratiamarcescens) SPG-1 bacterial strain itself, and utilizes this bacterial strain to produce the method for chitoanase.
Bacterial strain involved in the present invention is serratia marcescens SPG-1 (Serratiamarcescens), hereinafter referred to as serratia marcescens SPG-1 or bacterial strain SPG-1. This bacterial strain is preserved in China typical culture collection center CCTCC on December 28th, 2015, and preserving number is CCTCCNO:M2015784. Preservation address: Wuhan Wuhan University of China, postcode 430072, phone 027-68754052.
The present invention also relates to the method that above-mentioned serratia marcescens (Serratiamarcescens) SPG-1 produces chitoanase, is characterized in, its step is as follows:
(1) preparation of bacterial classification: by serratia marcescens SPG-1 bacterial strain at the flat lining out of sterilized rich medium, in 28 DEG C of quiescent culture after 1 ~ 2 day, chooses and gets single bacterium colony and carry out rich medium slant culture and be stored in 4 DEG C of refrigerators for subsequent use after 1 day;Described rich medium is: extractum carnis 3g, peptone 10g, NaCl5g, agar 15～20g, water 1000mL, pH7.0～7.2;
(2) cultivation of cell and the collection of crude enzyme liquid: choose the bacterial classification getting slant culture, it is inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, at 25 ~ 30 DEG C, 150 ~ 250r/min, shaking table on cultivate after 18 ~ 30h to obtain seed liquor, seed liquor is inoculated in the 250mL triangular flask containing 30 ~ 50mL liquid nutrient medium by the inoculum size of 5%, 26 ~ 30 DEG C, 150 ~ 200r/min shaking table takes out after cultivating 60 ~ 75h, at 8000r/min centrifugal segregation bacterial sediment, collect supernatant liquor and it is crude enzyme liquid, be placed in 4 DEG C of refrigerators and store for subsequent use; Described liquid nutrient medium is: bean cake powder 25g, sucrose 20g, K2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, water 1000mL, pH6.0;
(3) extraction of chitoanase and purifying: crude enzyme liquid in step (2), when adopting the extraction using alcohol of 1 times of volume, obtains and lives as the thick enzyme of 24U/mgprotein precipitates than enzyme; Being that to record the chitoanase relative molecular mass that separation and purification obtains be 30000 to 30U/mgprotein, SDS-PAGE method with the work of the ratio enzyme of SephadexG75 separation and purification gained enzyme, the iso-electric point tentatively recording chitoanase with iso-electric point coagulation method is 5.98.
In the method and technology scheme of above-described product chitoanase, preferred technology feature is further: in step (2): choose the bacterial classification getting slant culture, be inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, 25 DEG C, 200r/min, obtains seed liquor after shaking table is cultivated 24h.
In the method and technology scheme of above-described product chitoanase, preferred technology feature is further: in step (2): seed liquor be inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium by the inoculum size of 5%, 28 DEG C, 180r/min shaking table takes out after cultivating 72h.
The present invention will be further elaborated below.
One, the screening method of bacterial strain serratia marcescens SPG-1 of the present invention.
This bacterial strain serratia marcescens SPG-1 is obtained by the screening of following method:
Removing the foreign material in soil sample, the sample that fetches earth adds a little 5mL enrichment medium (colloid chitosan 5, small molecules chitosan 3, extractum carnis 2, (NH is housed4)2SO410, K2HPO42, KH2PO42, NaCl5, MgSO4·7H2O0.25, water 1000mL, pH6.5) test tube in, in 30 DEG C in shaking table, shaking speed 160r/min cultivates 2d; Getting a little nutrient solution after enrichment, dilution is coated with just sieves substratum (yeast extract paste 1, colloid chitosan 10, powder chitosan (molecular weight 50,000) 5, (NH4)2SO45, K2HPO42, NaCl5, MgSO4·7H2O0.5, agar 15～20, water 1000mL, pH6.5～7.5) dull and stereotyped, it is inverted for 30 DEG C and cultivates 3d. Choose again and get single colony inoculation in liquid seed culture medium (peptone 5, yeast extract 5, glucose 5, K2HPO42, KH2PO42, NaCl5, MgSO4·7H2O0.5, water 1000mL, pH6.5～7.5), at 28 DEG C, obtain seed liquor after the shaking table of 180r/min is cultivated 24h, seed liquor be inoculated in fermention medium (powder chitosan 10, glucose 1.0, yeast extract 3, (NH by the inoculum size of 5%4)2SO45, K2HPO42, NaCl5, MgSO4·7H2O0.5, water 1000mL, pH6), in 28 DEG C, 180r/min shaking table takes out after cultivating 60h, at 8000r/min centrifugal segregation bacterial sediment, collects supernatant liquor and is crude enzyme liquid, measure chitoanase vigor by DNS method, from 50 strain Hou Xuan bacterial strains, finally select the microorganism that chitoanase is produced in a strain: serratia marcescens SPG-1.
Two, the character of serratia marcescens SPG-1 bacterial strain.
There is following character:
(1) morphological specificity: Gram-negative bacteria, rod-short, becomes single arrangement, without gemma. Size is 0.5～1.0 × 1.2～2.0 μm, do not move or mobility not strong.
(2) cultural characteristic: bacterium colony is circular, opaque, and bacterium colony is protruding, sticky thick easily choosing is got, color is red, neat in edge, and corrugationless is aerobic, liquid culture is evenly muddy, and without precipitation, bubble-free produces, and substratum turns into redness from the yellow of clarification and turns into muddy yellow again.
(3) bio-chemical characteristics result:
1. sugar fermentating test result: bacterial strain SPG-1 ferments sucrose, sorbose, ribose, fructose, sorbyl alcohol, N.F,USP MANNITOL, trehalose, rhamnosyl, glucose, but does not produce gas, nonfermented wood sugar, pectinose, lactose, semi-lactosi.
2. carbon assimilation test-results: in table 1.
Table 1 bacterial strain SPG-1 carbon assimilation test-results
3. nitrogenous source assimilation experiments result: in table 2.
Table 2 bacterial strain SPG-1 nitrogenous source assimilation experiments result
4. other physiological and biochemical test results: bacterial strain SPG-1 liquefy gelatin, urease negative, catalase is positive, and methyl red experiment is negative, and V-P experimental result is positive, and indoles experimental result is positive, and produces H2S experimental result display strong positive, milk experiment produces acid cure phenomenon, and reduction nitrate, does not reduce nitrite, and benzylpenicillin sodium is insensitive, and produce red material has faint restraining effect to subtilis, and polychrom hydrolysising experiment be the positive.
(4) ecosystem characterization
1. differing temps growth result: temperature growth scope, between 10～40 DEG C, can normally produce haematochrome, not produce haematochrome more than 37 DEG C when 15 ~ 30 DEG C.
2. different pH growth result: growth pH scope is 4 ~ 10, the most suitable growth pH is 7 ~ 7.5.
3. salt tolerance experiment: grow when NaCl concentration is 2%～8%, along with the increase of salt concn, produces hypopigmentation, and when NaCl concentration is 2%, color is the reddest.
(5) 16SrDNA sequencing result: in table 3.
Table 316SrDNA sequencing result
(6) evolutionary tree
Morphological specificity according to bacterial strain SPG-1, cultural characteristic, physiological and biochemical property and ecosystem characterization, this bacterial strain is consistent with the characteristic of division of serratia marcescens (Serratiamarcescens) in " uncle's Jie Shi determinative bacteriology handbook " most, in conjunction with 16SrDNA sequencing result, so identify that bacterial strain SPG-1 is serratia marcescens, name as serratia marcescens SPG-1 (SerratiamarcescensSPG-1). Existing Beijing three Bo Zhiyuan Bioisystech Co., Ltd checked order in June, 2013, and sequencing primer is: 27F, and order-checking is numbered: 06050080, and sequencing result is that bacterial strain SPG-1 and serratia marcescens homology degree are up to 99%. Evolutionary tree is see Fig. 1.
Three, enzyme producing method.
1, to produce the method steps of chitoanase as follows for serratia marcescens SPG-1 (SerratiamarcescensSPG-1):
(1) preparation of bacterial classification: by serratia marcescens SPG-1 bacterial strain at the flat lining out of sterilized rich medium, in 28 DEG C of quiescent culture after 1 ~ 2 day, chooses and gets single bacterium colony and carry out rich medium slant culture and be stored in 4 DEG C of refrigerators for subsequent use after 1 day; Described rich medium is: extractum carnis 3g, peptone 10g, NaCl5g, agar 15～20g, distilled water 100mL, pH7.0～7.2;
(2) cultivation of cell and the collection of crude enzyme liquid: choose the bacterial classification getting slant culture, it is inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, at 25 ~ 30 DEG C, seed liquor is obtained after the shaking table of 150 ~ 250r/min is cultivated 18 ~ 30h, seed liquor is inoculated in the 250mL triangular flask containing 30 ~ 50mL liquid nutrient medium by the inoculum size of 5%, 26 ~ 30 DEG C, 150 ~ 200r/min shaking table takes out after cultivating 60 ~ 75h, at 8000r/min centrifugal segregation bacterial sediment, collect supernatant liquor and it is crude enzyme liquid, be placed in 4 DEG C of refrigerators and store for subsequent use;Described liquid nutrient medium is: bean cake powder 25g, sucrose 20g, K2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, water 1000mL, pH6.0.
(3) extraction of chitoanase and purifying: crude enzyme liquid in step (2), when adopting the extraction using alcohol of 1 times of volume, obtains and lives as the thick enzyme of 24U/mgprotein precipitates than enzyme. Being that to record the chitoanase relative molecular mass that separation and purification obtains be 30000 to 30U/mgprotein, SDS-PAGE method with the work of the ratio enzyme of SephadexG75 separation and purification gained enzyme, the iso-electric point tentatively recording chitoanase with iso-electric point coagulation method is 5.98.
Compared with prior art, the present invention provides a kind of serratia marcescens SPG-1 bacterial strain that can produce chitoanase newly. The chitoanase output of bacterial strain of the present invention is higher, and culture medium cost is low, and culture condition is easy to control, the simple also energy-conserving and environment-protective of leaching process. The method producing chitoanase has advantage simple and practical, with low cost, it is easy to apply in industrial production.
Accompanying drawing explanation
Fig. 1 is the bacterial strain evolutionary tree of serratia marcescens SPG-1 of the present invention.
Embodiment
Hereinafter further describe the concrete technical scheme of the present invention, so that the technician of this area understands the present invention further, and do not form the restriction to its right.
Embodiment 1, a kind of serratia marcescens (Serratiamarcescens) SPG-1, the preserving number of this bacterial strain is CCTCCNO:M2015784. This bacterial strain has following feature:
(1) morphological specificity: Gram-negative bacteria, rod-short, becomes single arrangement, without gemma. Size is 0.5～1.0 × 1.2～2.0 μm, do not move or mobility not strong.
(2) cultural characteristic: bacterium colony is circular, opaque, and bacterium colony is protruding, sticky thick easily choosing is got, color is red, neat in edge, and corrugationless is aerobic, liquid culture is evenly muddy, and without precipitation, bubble-free produces, and substratum turns into redness from the yellow of clarification and turns into muddy yellow again.
(3) physiological and biochemical property: Trisodium Citrate, sucrose, glucose, maltose, semi-lactosi, trehalose, ribose, D-Fructose, sorbyl alcohol, N.F,USP MANNITOL can be utilized as sole carbon source, does not utilize Zulkovsky starch, Mierocrystalline cellulose, pectinose, L-sorbose, lactose, D-wood sugar as sole carbon source; Urea element, casein food grade, saltpetre, ammonium chloride, ammonium sulfate, glycine, aspartic acid can be utilized as only nitrogen source, do not utilize Valine as only nitrogen source. Bacterial strain SPG-1 ferments sucrose, sorbose, ribose, fructose, sorbyl alcohol, N.F,USP MANNITOL, trehalose, rhamnosyl, glucose, but does not produce gas, nonfermented wood sugar, pectinose, lactose, semi-lactosi.
Bacterial strain SPG-1 liquefy gelatin, urease negative, catalase is positive, and methyl red experiment is negative, and V-P experimental result is positive, and indoles experimental result is positive, and produces H2S experimental result is positive, milk experiment produces acid cure phenomenon, reduction nitrate, not reducing nitrite, polychrom hydrolysising experiment is positive, and benzylpenicillin sodium is insensitive, this bacterium generates a kind of red red material, this material is almost insoluble to water, but is dissolved in ethanol, and subtilis is had faint restraining effect by this red material.
Embodiment 2, a kind of serratia marcescens (Serratiamarcescens) SPG-1 as described in Example 1 produces the method for chitoanase, and its step is as follows:
(1) preparation of bacterial classification: by serratia marcescens SPG-1 bacterial strain at the flat lining out of sterilized rich medium, in 28 DEG C of quiescent culture after 1 day, chooses and gets single bacterium colony and carry out rich medium slant culture and be stored in 4 DEG C of refrigerators for subsequent use after 1 day;Described rich medium is: extractum carnis 3g, peptone 10g, NaCl5g, agar 15g, distilled water 100mL, pH7.0;
(2) cultivation of cell and the collection of crude enzyme liquid: choose the bacterial classification getting slant culture, it is inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, at 25 DEG C, 150r/min, shaking table on cultivate after 18 ~ 30h to obtain seed liquor, seed liquor is inoculated in the 250mL triangular flask containing 30mL liquid nutrient medium by the inoculum size of 5%, 26 DEG C, 150r/min shaking table takes out after cultivating 60h, at 8000r/min centrifugal segregation bacterial sediment, collect supernatant liquor and it is crude enzyme liquid, be placed in 4 DEG C of refrigerators and store for subsequent use; Described liquid nutrient medium is: bean cake powder 25g, sucrose 20g, K2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, tap water 1000mL, pH6.0.
(3) extraction of chitoanase and purifying: crude enzyme liquid in step (2), when adopting the extraction using alcohol of 1 times of volume, obtains and lives as the thick enzyme of 24U/mgprotein precipitates than enzyme. Being that to record the chitoanase relative molecular mass that separation and purification obtains be 30000 to 30U/mgprotein, SDS-PAGE method with the work of the ratio enzyme of SephadexG75 separation and purification gained enzyme, the iso-electric point tentatively recording chitoanase with iso-electric point coagulation method is 5.98.
Embodiment 3, a kind of serratia marcescens (Serratiamarcescens) SPG-1 as described in Example 1 produces the method for chitoanase, and its step is as follows:
(1) preparation of bacterial classification: by serratia marcescens SPG-1 bacterial strain at the flat lining out of sterilized rich medium, in 28 DEG C of quiescent culture after 2 days, chooses and gets single bacterium colony and carry out rich medium slant culture and be stored in 4 DEG C of refrigerators for subsequent use after 1 day; Described rich medium is: extractum carnis 3g, peptone 10g, NaCl5g, agar 20g, distilled water 100mL, pH7.2;
(2) cultivation of cell and the collection of crude enzyme liquid: choose the bacterial classification getting slant culture, it is inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, at 30 DEG C, 250r/min, shaking table on cultivate after 30h to obtain seed liquor, seed liquor is inoculated in the 250mL triangular flask containing 50mL liquid nutrient medium by the inoculum size of 5%, 30 DEG C, 200r/min shaking table takes out after cultivating 75h, at 8000r/min centrifugal segregation bacterial sediment, collect supernatant liquor and it is crude enzyme liquid, be placed in 4 DEG C of refrigerators and store for subsequent use; Described liquid nutrient medium is: bean cake powder 25g, sucrose 20g, K2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, tap water 1000mL, pH6.0.
(3) extraction of chitoanase and purifying: crude enzyme liquid in step (2), when adopting the extraction using alcohol of 1 times of volume, obtains and lives as the thick enzyme of 24U/mgprotein precipitates than enzyme. Being that to record the chitoanase relative molecular mass that separation and purification obtains be 30000 to 30U/mgprotein, SDS-PAGE method with the work of the ratio enzyme of SephadexG75 separation and purification gained enzyme, the iso-electric point tentatively recording chitoanase with iso-electric point coagulation method is 5.98.
Embodiment 4, a kind of serratia marcescens (Serratiamarcescens) SPG-1 as described in Example 1 produces in the step (2) of the method for chitoanase: choose the bacterial classification getting slant culture, it is inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, 25 DEG C, 200r/min, obtains seed liquor after shaking table is cultivated 24h; Seed liquor being inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium by the inoculum size of 5%, 28 DEG C, 180r/min shaking table takes out after cultivating 72h.All the other are identical with embodiment 1.
Embodiment 5, a kind of serratia marcescens SPG-1 (SerratiamarcescensSPG-1) as described in Example 1 produces the method for chitoanase, and its step is as follows:
(1) in the big triangular flask of volume 1L, 150mL fermention medium (bean cake powder 25g, sucrose 20g, K is loaded2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, tap water 1000mL, pH6.0), by the inoculum size access SPG-1 seed culture fluid of 5% after sterilizing, at 28 DEG C, 165r/min shaking table takes out after cultivating 69h, at 8000r/min centrifugal segregation bacterial sediment, collect supernatant liquor and it is crude enzyme liquid, be placed in 4 DEG C of refrigerators and store for subsequent use;
(2) getting the ice ethanol that supernatant liquor 100mL adds 100mL, in 4 DEG C of standing 5-10min after mixed even, obtain thick enzyme precipitation at 12000r/min frozen centrifugation, thick enzyme precipitates to live as 24U/mgprotein than enzyme; Thick enzyme precipitation was dissolved in the acetate buffer of the pH4.4 of 5mL crosses SephadexG75 gel column, with pH4.4 acetate buffer wash-out and collect flow point, the high flow point of being lived by enzyme merges, and is 69U/mgprotein with the work of the ratio enzyme of gained enzyme after ice alcohol settling.
Embodiment 6, a kind of serratia marcescens SPG-1 (SerratiamarcescensSPG-1) as described in Example 1 produces the method for chitoanase, and its step is as follows:
(1) in 5L fermentor tank, load 3.5L fermention medium (bean cake powder 25g, sucrose 20g, K2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, tap water 1000mL, pH6.0), by the inoculum size access SPG-1 seed culture fluid of 5% after sterilizing, it it is 28 DEG C in temperature, fermentation when being 0.9vvm that mixing speed is 350r/min and Ventilation Rate, controlling pH in fermenting process is 6.0, and when putting tank to 69h, in fermented supernatant fluid, chitoanase content is 3500U/L.
(2) getting the ice ethanol that supernatant liquor 300mL adds 300mL, leave standstill 5 ~ 10min in 4 DEG C after mixed even, obtain thick enzyme precipitation at 12000r/min frozen centrifugation, the ratio enzyme of thick enzyme precipitation is lived as 35U/mgprotein; Thick enzyme precipitation was dissolved in the acetate buffer of the pH4.4 of 5mL crosses SephadexG75 gel column, with pH4.4 acetate buffer wash-out and collect flow point, the high flow point of being lived by enzyme merges, and is 76U/mgprotein with the work of the ratio enzyme of gained enzyme after ice alcohol settling.
Embodiment 7. A kind of serratia marcescens SPG-1 (SerratiamarcescensSPG-1) as described in Example 1 produces the method for chitoanase hydrolyzing chitosan, and its step is as follows:
(1) in 5L fermentor tank, load 3.5L fermention medium (bean cake powder 25g, sucrose 20g, K2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, tap water 1000mL, pH6.0), by the inoculum size access SPG-1 seed culture fluid of 5% after sterilizing, it it is 28 DEG C in temperature, fermentation when being 0.9vvm that mixing speed is 350r/min and Ventilation Rate, controlling pH in fermenting process is 6.0, and when putting tank to 69h, in fermented supernatant fluid, chitoanase content is 3500U/L.
(2) getting the ice ethanol that supernatant liquor 300mL adds 300mL, leave standstill 5 ~ 10min in 4 DEG C after mixed even, obtain thick enzyme precipitation at 12000r/min frozen centrifugation, the ratio enzyme of thick enzyme precipitation is lived as 35U/mgprotein; Thick enzyme precipitation was dissolved in the acetate buffer of the pH4.4 of 5mL crosses SephadexG75 gel column, with pH4.4 acetate buffer wash-out and collect flow point, the high flow point of being lived by enzyme merges, and is 76U/mgprotein with the work of the ratio enzyme of gained enzyme after ice alcohol settling.
(3) it is 20g/L in 100mL concentration, in the colloid chitosan solution of pH6, add the chitoanase in 50mg step (2), react at 45 DEG C and terminate for 5 hours, it is mainly oligochitosan through its enzyme reaction product of silica gel plate layer chromatography, a small amount of glucosamine, all the other are the oligose of different molecular weight.
Claims (4)
1. serratia marcescens (Serratiamarcescens) SPG-1, it is characterised in that: the preserving number of this bacterial strain is CCTCCNO:M2015784.
2. the method for serratia marcescens as claimed in claim 1 (Serratiamarcescens) SPG-1 product chitoanase, it is characterised in that, its step is as follows:
(1) preparation of bacterial classification: by serratia marcescens SPG-1 bacterial strain at the flat lining out of sterilized rich medium, in 28 DEG C of quiescent culture after 1 ~ 2 day, chooses and gets single bacterium colony and carry out rich medium slant culture and be stored in 4 DEG C of refrigerators for subsequent use after 1 day; Described rich medium is: extractum carnis 3g, peptone 10g, NaCl5g, agar 15～20g, water 1000mL, pH7.0～7.2;
(2) cultivation of cell and the collection of crude enzyme liquid: choose the bacterial classification getting slant culture, it is inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, at 25 ~ 30 DEG C, 150 ~ 250r/min, shaking table on cultivate after 18 ~ 30h to obtain seed liquor, seed liquor is inoculated in the 250mL triangular flask containing 30 ~ 50mL liquid nutrient medium by the inoculum size of 5%, 26 ~ 30 DEG C, 150 ~ 200r/min shaking table takes out after cultivating 60 ~ 75h, at 8000r/min centrifugal segregation bacterial sediment, collect supernatant liquor and it is crude enzyme liquid, be placed in 4 DEG C of refrigerators and store for subsequent use; Described liquid nutrient medium is: bean cake powder 25g, sucrose 20g, K2HPO42g, KH2PO42g, NaCl5g, MgSO4·7H2O0.5g, water 1000mL, pH6.0;
(3) extraction of chitoanase and purifying: crude enzyme liquid in step (2), when adopting the extraction using alcohol of 1 times of volume, obtains and lives as the thick enzyme of 24U/mgprotein precipitates than enzyme; Being that to record the chitoanase relative molecular mass that separation and purification obtains be 30000 to 30U/mgprotein, SDS-PAGE method with the work of the ratio enzyme of SephadexG75 separation and purification gained enzyme, the iso-electric point tentatively recording chitoanase with iso-electric point coagulation method is 5.98.
3. the method for product chitoanase according to claim 2, it is characterized in that: in step (2): choose the bacterial classification getting slant culture, it is inoculated in the 250mL triangular flask that 50mL liquid nutrient medium is housed, 25 DEG C, 200r/min, obtains seed liquor after shaking table is cultivated 24h.
4. the method for product chitoanase according to Claims 2 or 3, it is characterised in that: in step (2): seed liquor be inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium by the inoculum size of 5%, 28 DEG C, 180r/min shaking table takes out after cultivating 72h.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071181A (en) * | 2010-05-14 | 2011-05-25 | 昆明理工大学 | Method for preparing alkali protease from serratieae and special culture medium thereof |
| CN105441367A (en) * | 2016-01-07 | 2016-03-30 | 福建省农业科学院土壤肥料研究所 | Serratia marcescens and application thereof |
| CN105483036A (en) * | 2015-10-30 | 2016-04-13 | 西安交通大学第一附属医院 | Serratia marcescens and preparation method and application thereof |
-
2016
- 2016-04-22 CN CN201610253272.1A patent/CN105670981B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071181A (en) * | 2010-05-14 | 2011-05-25 | 昆明理工大学 | Method for preparing alkali protease from serratieae and special culture medium thereof |
| CN105483036A (en) * | 2015-10-30 | 2016-04-13 | 西安交通大学第一附属医院 | Serratia marcescens and preparation method and application thereof |
| CN105441367A (en) * | 2016-01-07 | 2016-03-30 | 福建省农业科学院土壤肥料研究所 | Serratia marcescens and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| ANDREW T GREEN等: "Production of chitinolytic enzymes by Serratia marcescens QMB1466 using various chitinous substrates", 《JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY》 * |
| KAZUSHI SUZUKI等: "Chitinases A,B and C1 of Serratia marcescens 2170 Produced by Recombinant Escherichia coli:Enzymatic Properties and Synergism on Chitin Degradation", 《BIOSCI. BIOTECHNOL. BIOCHEM.》 * |
| MAY B. BRURBERG等: "Comparative studies of chitinases A and B from Serratia marcescens", 《MICROBIOLOG》 * |
| N.N. NAWANI等: "One-step puri®cation of chitinase from Serratia marcescens NK1, a soil isolate", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| SAN-LANG WANG等: "Purification and characterization of a chitosanase from Serratia marcescens TKU011", 《CARBOHYDRATE RESEARCH》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113930353A (en) * | 2021-08-31 | 2022-01-14 | 广西大学 | Selenite-resistant serratia marcescens and reduction characteristic identification method thereof |
| CN113930353B (en) * | 2021-08-31 | 2023-07-07 | 广西大学 | Serratia marcescens with selenite resistance and reduction characteristic identification method thereof |
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