CN108018234A - One plant of bacterial strain for producing algin catenase and its application - Google Patents
One plant of bacterial strain for producing algin catenase and its application Download PDFInfo
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Abstract
The invention discloses one plant of Hai Kebeite Salmonellas (Cobetia marina) HQZ08 for producing algin catenase and its application, the bacterial strain to be preserved in China typical culture collection center, address：Wuhan, China Wuhan University, postcode：430072, deposit number：CCTCC No：M 2017409, preservation date：On July 5th, 2017.The algin catenase that the bacterial strain produces is ectoenzyme, is isolated and purified simply, without clasmatosis；Under optimum condition, the crude enzyme liquid enzyme activity without isolating and purifying reaches 68.5U/mL；Bacterial strain HQZ08 fermentation medium components of the present invention are simple, and fermentation time is shorter, available for largely preparing algin oligosaccharide.
Description
Technical field
The invention belongs to microbial strains and its applied technical field, and in particular to the Hai Ke of one plant of production algin catenase
Shellfish spy Salmonella and its prepare the fermentation process of algin catenase and the preparation method of algin oligosaccharide.
Background technology
Algin is a kind of polysaccharide extracted from marine brown, is had generally in industries such as food, medicine, daily-use chemical industries
Application, it is water-soluble low but since its viscosity is high, be not easy to be absorbed by the body, be subject to certain restrictions in application aspect.Algin
Oligosaccharides is the oligosaccharide for the degree of polymerization 2~10 that algin is obtained through degrading, and due to good water solubility, is easily absorbed by the body, therefore
It has higher application value in field of medicaments.Research shows that algin oligosaccharide has neuroprotection, anti-asthma, antibacterial, increasing
Strong immunity, antitumor, reducing blood sugar and blood lipid, anticoagulation and growth promotion isoreactivity, and human senility is highly resistant to, it is a kind of
The oligosaccharide of great exploitation potential.
The preparation method of algin oligosaccharide mainly has enzyme edman degradation Edman, physics and chemical degradation method in the prior art.Physical
It is easy to operate, it is pollution-free, save reagent, can effectively cut off macromolecular chain, but the limiting molecular quality for gained of degrading compared with
Greatly, it is not easy to prepare the less oligosaccharides of molecular weight.Chemical degradation method includes acid degradation method and oxidation degradation method, acid hydrolyzation degraded speed
Degree is slow, it is necessary to which high temperature and pressure, degradation efficiency is low, and the higher oligosaccharide content of activity is not high, and environment can be polluted.Oxidation drop
Solution reaction process is simple, and cost is relatively low, but degradation condition is violent and has certain destruction to the reducing end of oligosaccharides.And enzyme
Solution is the biodegradation method that a kind of mild condition controllability is strong and specificity is high, special with catabolite activity height, product
One property is good, reaction condition is gentle, many advantages such as pollution-free, but the bacterial strain high due to lacking producing enzyme vigor, causes algin to split
It is low to solve production of enzyme, improves the production cost of enzymatic isolation method, limits the popularization and application of the method.
The content of the invention
It is an object of the invention to overcome prior art defect, there is provided the bacterial strain of one plant of production algin catenase.The present invention
Production algin catenase strain name be：Hai Kebeite Salmonellas (Cobetia marina) HQZ08, the bacterial strain are preserved in China
Type Tissue Collection, address：Wuhan, China Wuhan University, postcode：430072, deposit number：CCTCC No：M
2017409, preservation date：On July 5th, 2017.
Another object of the present invention provides a kind of Hai Kebeite Salmonellas (Cobetia by above-mentioned production algin catenase
Marina) the method that HQZ08 fermentations prepare algin catenase, specifically comprises the following steps：
(1) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 is inoculated in slant medium, is trained at 25~30 DEG C
24~48h is supported, is activated；
(2) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 after step (1) is activated is inoculated in liquid seeds training
Support in base, 12~24h of shaken cultivation, obtains seed liquor under 25~30 DEG C, 150~200r/min；
(3) seed liquor obtained by step (2) is inoculated in liquid culture medium, in 25~30 DEG C, 150~200r/min
24~48h of lower shaken cultivation, obtains the zymotic fluid containing algin catenase；
(4) by zymotic fluid 8000~12000r/min of the step (3) containing algin catenase, 10~15min centrifuges,
0.22 μm is filtered to remove thalline, and gained enzyme liquid is algin catenase crude enzyme liquid；
The formula of step (1) described slant medium is：15~20g/L of agar, 3~6g/L of sodium alginate, peptone 3~
5g/L, 25~30g/L of sodium chloride, bitter salt 0.5~1g/L, three hypophosphite monohydrate 1~2g/L of hydrogen dipotassium, seven hydrated sulfuric acids
Ferrous 0.01g/L, pH 7~7.5；
The formula of liquid seed culture medium described in step (2) is：3~6g/L of sodium alginate, 3~5g/L of peptone, ferment
1~2g/L of female powder, sodium chloride 25~30g/L, pH 7~7.5；
The formula of liquid culture medium described in step (3) is：5~7g/L of sodium alginate, 3~5g/L of peptone, chlorine
Change sodium 25~30g, three hypophosphite monohydrate hydrogen dipotassium 1~2g/L, pH 7~7.5.
The present invention also provides a kind of preparation method of algin oligosaccharide, its step is：
(1) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 is inoculated in slant medium, is trained at 25~30 DEG C
24~48h is supported, is activated；
(2) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 after step (1) is activated is inoculated in liquid seeds training
Support in base, 12~24h of shaken cultivation, obtains seed liquor under 25~30 DEG C, 150~200r/min；
(3) seed liquor obtained by step (2) is inoculated in liquid culture medium, in 25~30 DEG C, 150~200r/min
24~48h of lower shaken cultivation, obtains the zymotic fluid containing algin catenase；
(4) by zymotic fluid 8000~12000r/min of the step (3) containing algin catenase, 10~15min centrifuges,
0.22 μm is filtered to remove thalline, and gained enzyme liquid is algin catenase crude enzyme liquid；
(5) algin is digested with algin catenase crude enzyme liquid obtained by step (4), enzymatic hydrolysis system is as follows：It is enzyme
40~70U/g of amount, concentration of substrate 0.5~1%, the PBS buffer that enzymolysis buffer solution is pH 7~8,35~45 DEG C of hydrolysis temperature,
36~48h of enzymolysis time；
(6) after the enzymolysis product of step (5) is cooled down, centrifugal filtration removes undegradable sodium alginate, and algin is made
Oligosaccharides；
The formula of step (1) described slant medium is：15~20g/L of agar, 3~6g/L of sodium alginate, peptone 3~
5g/L, 25~30g/L of sodium chloride, bitter salt 0.5~1g/L, three hypophosphite monohydrate 1~2g/L of hydrogen dipotassium, seven hydrated sulfuric acids
Ferrous 0.01g/L, pH 7~7.5；
The formula of liquid seed culture medium described in step (2) is：3~6g/L of sodium alginate, 3~5g/L of peptone, ferment
1~2g/L of female powder, sodium chloride 25~30g/L, pH 7~7.5；
The formula of liquid culture medium described in step (3) is：5~7g/L of sodium alginate, 3~5g/L of peptone, chlorine
Change sodium 25~30g, three hypophosphite monohydrate hydrogen dipotassium 1~2g/L, pH 7~7.5.
The beneficial effects of the invention are as follows：
1st, the algin catenase that Hai Kebeite Salmonellas (Cobetia marina) HQZ08 of the invention is produced is extracellular
Enzyme, isolates and purifies simply, without clasmatosis；
2nd, Hai Kebeite Salmonellas (Cobetia marina) HQZ08 nutritional requirements of the invention are simple, and fermentation time is short；
3rd, the inulinase-producing activity of Hai Kebeite Salmonellas (Cobetia marina) HQZ08 of the invention is higher, in preferred stripe
Under part, reach 68.5U/mL without the crude enzyme liquid enzyme activity of purifying；
4th, the produced algin catenase reaction conditions of Hai Kebeite Salmonellas (Cobetia marina) HQZ08 of the invention
Gently, it is environmentally safe；
5th, the preparation method of algin oligosaccharide of the invention is simple, and cost is low, prepared by a large amount of available for algin oligosaccharide.
Brief description of the drawings
Hai Kebeite Salmonellas (Cobetia marina) HQZ08 that Fig. 1 is filtered out by the embodiment of the present invention 1 is trained in tablet
Support the colonial morphology figure on base；
Fig. 2 is the systematic growth of bacterial strain of the present invention and the 16S rDNA sequence constructs of bacterial strain similar in blast results
Tree；
Fig. 3 is the influence that medium pH produces Hai Kebeite Salmonellas (Cobetia marina) HQZ08 algin catenase
Figure；
Fig. 4 is the influence that cultivation temperature produces Hai Kebeite Salmonellas (Cobetia marina) HQZ08 algin catenase
Figure；
Fig. 5 is the influence that incubation time produces Hai Kebeite Salmonellas (Cobetia marina) HQZ08 algin catenase
Figure；
Fig. 6 is the influence figure that inoculum concentration produces Hai Kebeite Salmonellas (Cobetia marina) HQZ08 algin catenase；
Fig. 7 is the TLC chromatograms of the algin oligosaccharide obtained by the embodiment of the present invention 5.
Embodiment
Technical scheme is further detailed and described below by way of embodiment combination attached drawing.
Embodiment 1：Produce the screening of algin catenase bacterial strain
(1), 50mL enriched mediums are sub-packed in 250mL conical flasks, take a small amount of ooze to vibrate 30min with sterile water, so
The vibration liquid and the seawater collected are inoculated into enriched medium respectively afterwards, inoculum concentration is 5mL, 30 DEG C of culture 48h.
(2), primary dcreening operation culture medium is down flat plate, and the seawater of enrichment culture and ooze bacterium solution are carried out 10 respectively-1~10-7Gradient
Dilution, to 10-3~10-7The bacterium solution of dilution factor is coated, 30 DEG C of culture 48h.Periphery of bacterial colonies transparent circle size is observed, is chosen
The bacterial strain that upgrowth situation is good and transparent circle is larger is starting strain, carries out plate streaking and isolates and purifies.
(3), 50mL liquid seed culture mediums are fitted into the conical flask of 250mL, by the isolated strain of plate streaking
It is inoculated into the culture medium dispensed, after 30 DEG C of constant temperature incubation 24h, is inoculated into 2% inoculum concentration in enzymatic production culture medium,
After 30 DEG C of constant temperature incubation 24h, supernatant is collected by centrifugation, DNS methods measure thick enzyme activity.By the highest bacterial strain of enzyme activity add 40% glycerine and
1 ﹕ 1 of bacterium solution is mixed, and is stored in spare in -80 DEG C of refrigerators.
The formula of liquid seed culture medium is：3~6g/L of sodium alginate, 3~5g/L of peptone, 1~2g/L of dusty yeast, chlorine
Change sodium 25~30g/L, pH 7~7.5；
The formula of enzymatic production culture medium is：5~7g/L of sodium alginate, 3~5g/L of peptone, sodium chloride 25~30g, three
Hypophosphite monohydrate hydrogen 1~2g/L of dipotassium, pH7~7.5.
As shown in Figure 1, the colony characteristics of the bacterium are as follows：Bacterium colony is in circle after being coated on plating medium 30 DEG C of culture 24h
Shape, surface is smooth, neat in edge, and color is milky, and bacterial strain is rod-shaped, Gram-negative under the microscope.The bacterium
Physiological and biochemical property is shown in Table 1.Table 1：The physiological and biochemical property of Hai Kebeite Salmonellas (Cobetia marina) HQZ08
The enriched medium forms：3~6g/L of sodium alginate, 3~5g/L of ammonium sulfate, 25~30g/L of sodium chloride,
Bitter salt 0.5~1g/L, three hypophosphite monohydrate hydrogen 1~2g/L of dipotassium, green vitriol 0.01g/L, pH 7~
7.5。
The primary dcreening operation culture medium forms：3~6g/L of sodium alginate, 3~5g/L of ammonium sulfate, 25~30g/L of sodium chloride,
Bitter salt 0.5~1g/L, three hypophosphite monohydrate hydrogen 1~2g/L of dipotassium, green vitriol 0.01g/L, agar 15~
20g/L, pH 7~7.5.
Embodiment 2：Produce the identification of algin catenase bacterial strain
The bacterial strain that embodiment 1 is filtered out extracts the full-length genome of cell using bacterial genomes DNA extraction agents box, it
Afterwards using general 27F/1492R primers (forward primer 5 '-AGAGTTTGATCCTGGCTCAG-3 ', reverse primer 5 '-
GGTTACCTTGTTACGACTT-3 ') PCR amplification of 16S rDNA is carried out, PCR amplification condition is：95℃5min；95 DEG C of 30s,
55 DEG C of 30s, 72 DEG C of 60s, 35 circulations；72℃10min.Beijing six directions Hua Da Gene Tech. Company Limited is entrusted after PCR amplification
Guangzhou Branch is sequenced.
The picodna sequence of the 16S rDNA of bacterial strain is as shown in sequence table.By above-mentioned 16S rDNA picodnas sequence with
The base of US National Bio-Centers (National Center for Biotechnology Information USA, NCBI)
Because the 16S rDNA sequences in storehouse are compared, there is 99% homology with Cobetia marina strain JCM 21022,
Then it is Cobetia marina to identify the bacterium.The bacterium and the phylogenetic tree such as figure of the 16S rDNA sequence constructs of its nearly edge bacterial strain
Shown in 2.
Embodiment 3：The fermentation condition of Hai Kebeite Salmonella Cobetia marina HQZ08 production algin catenases is excellent
Change
(1) influence of the medium pH to Hai Kebeite Salmonella Cobetia marina HQZ08 producing enzymes
Six experimental groups are set, have investigated the initial pH of culture medium (6.0,6.5,7.0,7.5,8.0,8.5) to bacterial strain
The influence of producing enzyme, in formula is 3~6g/L of sodium alginate, 3~5g/L of peptone, 1~2g/L of dusty yeast by bacterial strain, sodium chloride 25
After cultivating 12~24h in the liquid seed culture medium of~30g/L, pH 7~7.5, it is inoculated in 2% inoculum concentration except pH is different
Outside, by 5~7g/L of sodium alginate, 3~5g/L of peptone, 25~30g of sodium chloride, three hypophosphite monohydrate hydrogen dipotassium 1~2g/L groups
Into liquid culture medium in, 30 DEG C cultivate 24h.The initial pH of culture medium can directly affect the penetrating of the cell membrane of thalline
Property, the vigor of stability and metabolite enzyme.The bacterium equal energy normal growth in the range of 6.0~8.5, at the beginning of fermentation medium
Strain enzyme-producing reaches maximum (such as Fig. 3) when beginning pH is 7.0.
(2) influence of the cultivation temperature to Hai Kebeite Salmonella Cobetia marina HQZ08 producing enzymes
Growth of the temperature on bacterium and enzymatic productivity influence it is more significant, set respectively 20 DEG C, 25 DEG C, 28 DEG C, 30 DEG C, 32
DEG C, 35 DEG C progress strain enzyme-producing ability investigations.In formula it is 3~6g/L of sodium alginate by bacterial strain, 3~5g/L of peptone, ferment
1~2g/L of female powder, 25~30g/L of sodium chloride, after cultivating 12~24h in the liquid seed culture medium of pH7~7.5, connect with 2%
Kind of amount is inoculated in by 5~7g/L of sodium alginate, 3~5g/L of peptone, sodium chloride 25~30g, and three hypophosphite monohydrate hydrogen dipotassiums 1~
In the liquid culture medium of the pH 7.0 of 2g/L compositions, 24h is cultivated at different temperatures.The results show is when temperature is 25 DEG C
The strain enzyme-producing ability reaches maximum (such as Fig. 4).
(3) influence of the fermentation time to Hai Kebeite Salmonella Cobetia marina HQZ08 producing enzymes
During producing enzyme culture, with the increase of incubation time, the growth of flora reaches stationary phase, and yield of enzyme starts to increase
Add, and the enzyme that when incubation time is long may result in generation gradually inactivates.By bacterial strain formula be 3~6g/L of sodium alginate,
3~5g/L of peptone, 1~2g/L of dusty yeast, cultivate 12 in the liquid seed culture medium of sodium chloride 25~30g/L, pH 7~7.5
After~24h, it is inoculated in 2% inoculum concentration by 5~7g/L of sodium alginate, 3~5g/L of peptone, 25~30g of sodium chloride, three water
In the liquid culture medium for closing the pH7.0 of dipotassium hydrogen phosphate 1~2g/L composition, cultivated respectively at 25 DEG C 18h, 24h, 30h,
36h, 42h, examine influence of the incubation time to strain enzyme-producing.As seen from Figure 5, the strain enzyme-producing when incubation time reaches 24h
Ability is maximum, is decreased obviously more than 24h enzymatic productivities.
(4) influence of the inoculum concentration to Hai Kebeite Salmonella Cobetia marina HQZ08 producing enzymes
In formula it is 3~6g/L of sodium alginate, 3~5g/L of peptone, 1~2g/L of dusty yeast by bacterial strain, sodium chloride 25~
After cultivating 12~24h in the liquid seed culture medium of 30g/L, pH 7~7.5, it is inoculated in by 5~7g/L of sodium alginate, peptone
In the liquid culture medium of the pH 7.0 of 3~5g/L, sodium chloride 25~30g, three 1~2g/L of hypophosphite monohydrate hydrogen dipotassium composition,
24h is cultivated at 25 DEG C, has been investigated when inoculum concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5%, 3% to the shadow of strain enzyme-producing
Ring, inoculation wild Oryza species cumulative volume is 50mL.Connect it will be appreciated from fig. 6 that 2% inoculum concentration is the optimal of bacterial strain HQZ08 enzymatic productions
Kind amount.
Embodiment 4：Algin catenase is produced using Hai Kebeite Salmonella Cobetia marina HQZ08
(1) Hai Kebeite Salmonella Cobetia marina HQZ08 strains are inoculated in slant medium, in 25~30 DEG C
24~48h of lower culture, the Hai Kebeite Salmonella strains after being activated；The formula of the slant medium is：Agar 15~
20g/L, 3~6g/L of sodium alginate, 3~5g/L of peptone, 25~30g/L of sodium chloride, bitter salt 0.5~1g/L, three
Hypophosphite monohydrate hydrogen 1~2g/L of dipotassium, green vitriol 0.01g/L, pH 7~7.5.
(2) the Hai Kebeite Salmonella Cobetia marina HQZ08 strains after step (1) activation culture are seeded to liquid
In body seed culture medium, 12~24h of shaken cultivation, obtains seed liquor under 25~30 DEG C, 150~200r/min；The liquid
The formula of body seed culture medium is：3~6g/L of sodium alginate, 3~5g/L of peptone, 0.5~1g/L of dusty yeast, sodium chloride 25~
30g/L, pH 7~7.5.
(3) by step (2) seed liquor with the inoculum concentration culture transferring of 0.5%~3% volume ratio into fluid nutrient medium, in 25~
30 DEG C, 150~200r/min vibrations, 24~48h of lower culture, obtain the zymotic fluid containing algin catenase；The liquid producing enzyme training
Support base formula be：5~7g/L of sodium alginate, 3~5g/L of peptone, sodium chloride 25~30g, three hypophosphite monohydrate hydrogen dipotassiums 1~
2g/L, pH 7~7.5.
(4) by the zymotic fluid of step (3) containing algin catenase in 8000~12000r/min, 10~15min, 0.22 μm
Under the conditions of centrifuge be filtered to remove thalline, gained enzyme liquid is algin catenase crude enzyme liquid, its vigor reaches 68.5U/mL.
Enzyme activity determination method is：100 μ L crude enzyme liquids, (25mM pH are 7.5 to the alginate solution of 900 μ L 0.3% of addition
Phosphate buffer dissolving) in, react 5min in 40 DEG C of thermostat water baths, add 2mL DNS reagents stop reaction, boiling
5min is boiled in boiling.Flowing water cools down, constant volume to 10mL, and 540nm surveys absorbance.The catalysis per minute of 1mL crude enzyme liquids produces 1 μ g reduced sugars
Required enzyme dosage is defined as an enzyme activity unit.
Embodiment 5：The preparation of algin oligosaccharide
Algin is digested with algin catenase crude enzyme liquid made from embodiment 4, enzymatic hydrolysis system is as follows：Enzyme concentration
40~70U/g, concentration of substrate 0.5~1%, the PBS buffer that enzymolysis buffer solution is pH 7~8,35~45 DEG C of hydrolysis temperature, enzyme
Solve 36~48h of the time.After enzymolysis product is cooled down, centrifugal filtration removes undegradable algin, and the algin oligosaccharide is made.
The TLC chromatograms of the obtained algin oligosaccharide and algin oligosaccharide standard items are as shown in fig. 7, wherein 0：Algin oligosaccharide standard
Product (disaccharides, trisaccharide, tetrose, six sugar)；1：Make algin oligosaccharide product by oneself.
The foregoing is only a preferred embodiment of the present invention, therefore cannot limit the scope that the present invention is implemented according to this, i.e.,
The equivalent changes and modifications done according to the scope of the claims of the present invention and description, all should still belong in the range of the present invention covers.
Sequence table
<110>Huaqiao University
<120>One plant of bacterial strain for producing algin catenase and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>(artificial synthesized)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213>(artificial synthesized)
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1436
<212> DNA
<213>(Hai Kebeite Salmonellas (Cobetia marina))
<400> 3
ggcggcagct tacacatgca gtcgagcgga aacgattcta gcttgctaga aggcgtcgag 60
cggcggacgg gtgagtaatg catgggaatc tgcccgatag tgggggacaa cctggggaaa 120
ctcaggctaa taccgcatac gtcctacggg agaaagcagg ggatcttcgg accttgcgct 180
atcggatgag cccatgtcgg attagcttgt tggtgaggta acggctcacc aaggcgacga 240
tccgtagctg gtctgagagg atgatcagcc acactgggac tgagacacgg cccagactcc 300
tacgggaggc agcagtgggg aatattggac aatgggcgaa agcctgatcc agccatgccg 360
cgtgtgtgaa gaaggccttc gggttgtaaa gcactttcag cgaggaagaa cgcttcggga 420
ttaatactcc cgaggaaaga catcactcgc agaagaagca ccggctaact ccgtgccagc 480
agccgcggta atacggaggg tgcaagcgtt aatcggaatt actgggcgta aagcgcgcgt 540
aggtggctaa gtcagccagg tgtgaaagcc ccgggctcaa cctgggaacg gcatctggaa 600
ctgcttggct agagtgcagg agaggaaggt agaattcccg gtgtagcggt gaaatgcgta 660
gagatcggga ggaataccag tggcgaaggc ggccttctgg actgacactg acactgaggt 720
gcgaaagcgt gggtagcaaa caggattaga taccctggta gtccacgccg taaacgatgt 780
caactagccg ttgggtccct tgaggactta gtggcgcagc taacgcaata agttgaccgc 840
ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctaccctt gacatccaga 960
ggactttcca gagatggatt ggtgccttcg ggaactctga gacaggtgct gcatggctgt 1020
cgtcagctcg tgttgtgaaa tgttgggtta agtcccgtaa cgagcgcaac ccctatcctt 1080
atttgccagc gagtaatgtc gggaactcta aggagactgc cggtgacaaa ccggaggaag 1140
gtggggacga cgtcaagtca tcatggccct tacgggtagg gctacacacg tgctacaatg 1200
gcaagtacaa agggttgcaa tacggcgacg tggagccaat cccataaagc ttgcctcagt 1260
ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat cgtggatcag 1320
aatgccacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg 1380
gactgcacca gaagtggtta gcctaacctt cgggagggcg atcaccacgg ttgtag 1436
Claims (3)
1. the bacterial strain of one plant of production algin catenase, the strain name：Hai Kebeite Salmonellas (Cobetia marina)
HQZ08, the bacterial strain are preserved in China typical culture collection center, address：Wuhan, China Wuhan University, postcode：
430072, deposit number：CCTCC No：M 2017409, preservation date：On July 5th, 2017.
A kind of 2. Hai Kebeite Salmonellas (Cobetia marina) HQZ08 for producing algin catenase according to claim 1
The method that fermentation prepares algin catenase, specifically comprises the following steps：
(1) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 is inoculated in slant medium, 24 is cultivated at 25~30 DEG C
~48h, is activated；
(2) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 after step (1) is activated is inoculated in liquid seed culture medium
In, 12~24h of shaken cultivation, obtains seed liquor under 25~30 DEG C, 150~200r/min；
(3) seed liquor obtained by step (2) is inoculated in liquid culture medium, shaken under 25~30 DEG C, 150~200r/min
24~48h of culture is swung, obtains the zymotic fluid containing algin catenase；
(4) by zymotic fluid 8000~12000r/min, 10~15min centrifugation of the step (3) containing algin catenase, 0.22
μm thalline is filtered to remove, gained enzyme liquid is algin catenase crude enzyme liquid；
The formula of step (1) described slant medium is：15~20g/L of agar, 3~6g/L of sodium alginate, 3~5g/ of peptone
L, 25~30g/L of sodium chloride, bitter salt 0.5~1g/L, three hypophosphite monohydrate 1~2g/L of hydrogen dipotassium, seven hydrated sulfuric acids are sub-
Iron 0.01g/L, pH 7~7.5；
The formula of liquid seed culture medium described in step (2) is：3~6g/L of sodium alginate, 3~5g/L of peptone, dusty yeast 1
~2g/L, sodium chloride 25~30g/L, pH 7~7.5；
The formula of liquid culture medium described in step (3) is：5~7g/L of sodium alginate, 3~5g/L of peptone, sodium chloride
25~30g, three hypophosphite monohydrate hydrogen dipotassium 1~2g/L, pH 7~7.5.
A kind of 3. Hai Kebeite Salmonellas (Cobetia marina) HQZ08 for producing algin catenase according to claim 1
Fermentation prepares the preparation method of algin oligosaccharide, its step is：
(1) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 is inoculated in slant medium, 24 is cultivated at 25~30 DEG C
~48h, is activated；
(2) Hai Kebeite Salmonellas (Cobetia marina) HQZ08 after step (1) is activated is inoculated in liquid seed culture medium
In, 12~24h of shaken cultivation, obtains seed liquor under 25~30 DEG C, 150~200r/min；
(3) seed liquor obtained by step (2) is inoculated in liquid culture medium, shaken under 25~30 DEG C, 150~200r/min
24~48h of culture is swung, obtains the zymotic fluid containing algin catenase；
(4) by zymotic fluid 8000~12000r/min, 10~15min centrifugation of the step (3) containing algin catenase, 0.22
μm thalline is filtered to remove, gained enzyme liquid is algin catenase crude enzyme liquid；
(5) algin is digested with algin catenase crude enzyme liquid obtained by step (4), enzymatic hydrolysis system is as follows：Enzyme concentration 40
~70U/g, concentration of substrate 0.5~1%, the PBS buffer that enzymolysis buffer solution is pH 7~8,35~45 DEG C of hydrolysis temperature, enzymolysis
36~48h of time；
(6) after the enzymolysis product of step (5) is cooled down, centrifugal filtration removes undegradable sodium alginate, and algin oligosaccharide is made；
The formula of step (1) described slant medium is：15~20g/L of agar, 3~6g/L of sodium alginate, 3~5g/ of peptone
L, 25~30g/L of sodium chloride, bitter salt 0.5~1g/L, three hypophosphite monohydrate 1~2g/L of hydrogen dipotassium, seven hydrated sulfuric acids are sub-
Iron 0.01g/L, pH 7~7.5；
The formula of liquid seed culture medium described in step (2) is：3~6g/L of sodium alginate, 3~5g/L of peptone, dusty yeast 1
~2g/L, sodium chloride 25~30g/L, pH 7~7.5；
The formula of liquid culture medium described in step (3) is：5~7g/L of sodium alginate, 3~5g/L of peptone, sodium chloride
25~30g, three hypophosphite monohydrate hydrogen dipotassium 1~2g/L, pH 7~7.5.
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| CN111100828A (en) * | 2020-02-27 | 2020-05-05 | 中国科学院烟台海岸带研究所 | Lexella capable of producing high-activity alginate lyase and application thereof |
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| CN112210513A (en) * | 2020-10-15 | 2021-01-12 | 中国热带农业科学院热带生物技术研究所 | Bacterial strain for producing algin lyase and application thereof |
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