CN101200700B - Ocean low-temperature cellulase and enzyme producing method as well as producing strain pseudoalteromonas thereof - Google Patents
Ocean low-temperature cellulase and enzyme producing method as well as producing strain pseudoalteromonas thereof Download PDFInfo
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- CN101200700B CN101200700B CN2007101904060A CN200710190406A CN101200700B CN 101200700 B CN101200700 B CN 101200700B CN 2007101904060 A CN2007101904060 A CN 2007101904060A CN 200710190406 A CN200710190406 A CN 200710190406A CN 101200700 B CN101200700 B CN 101200700B
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Abstract
The present invention relates to a Pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6) CGMCC NO.2155. The growth temperature range of the strain is between 0 DEG C and 35 DEG C; the best growth temperatureis 25 DEG C; the growth pH range is between 4 and 11; the best growth pH is 8; the growth NaCl concentration range is between 0 percent and 11 percent; the best growth NaCl concentration is 3 percent. The present invention also discloses a method of using the Pseudoalteromonas to produce low-temperature cellulase and a low-temperature cellulase product obtained by the method. The most suitable temperature for the reaction of the low-temperature cellulase is quite low, so the low-temperature cellulase not only can react efficiently under the condition of low temperature, but the thermal treatment of quite low temperature during a production technology can also ensure that enzyme is deactivated, which saves energy and expense. The low-temperature cellulase has wide application prospect at the fields of medicine, food, cotton weaving, environmental protection, regenerative source utilization, etc.
Description
Technical field
The present invention relates to a kind of microorganism, particularly a kind of separation is from the pseudoalteromonas Z6 in marine site, Lianyun Harbour (Pseudoalteromonas sp.Z6) CGMCC N0.2155 (be deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on September 26th, 2007, deposit number is CGMCC N0.2155); The invention still further relates to this bacterial strain and produce method of low-temperature cellulase and products thereof.
Background technology
Cellulase (EC 3.2.1) is the zymin that a class can be widely used in fields such as medicine, food, cotton spinning, environmental protection and renewable resources utilization.The optimum temperature of the plain enzyme of general fibre mostly is 45～65 ℃.The low-temperature cellulase optimum temperature can be low to moderate 20 ℃.Because temperature of reaction is lower, not only can highly effective reaction under cold condition, and in production technique, can make enzyme deactivation by the thermal treatment of lesser temps, conserve energy and expense become the focus of people's research and development.
In over half a century in the past, people mainly develop and obtain low-temperature cellulase from the land of varying environments such as frigid zone and glacier.But along with deepening continuously of research, the chance of developing low-temperature cellulase from terrestrial life reduces greatly, does not satisfy people's needs.Therefore, scientific workers invest eye the resourceful ocean of taking up an area of ball surface-area 3/4.Ocean environment is very unique, and has covered low temperature environment, and extremely huge, the miscellaneous psychrophile of the quantity that distributing helps obtaining new low-temperature cellulase and produces bacterium.Novel sea cold-adapted enzyme research not only provides new foundation for fundamental researchs such as the classification of biogenesis and evolution, microorganism, enzyme catalysis mechanism, and promotes the widespread use of enzyme in industry.
The optimum temperuture of the suitable cold cellulase that the pseudoalteromonas of having reported at present, produces is respectively 40 ℃ and 35 ℃.It is 40 ℃ that the ocean psychrophilic bacteria that other screening obtains produces the low-temperature cellulase optimum temperuture.Bacterial strain Z6 institute cellulase-producing optimum temperature is lower, and the catalytic activity height is more suitable for industrial production at normal temperatures.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, and a kind of new pseudoalteromonas that can produce low-temperature cellulase is provided.
Another technical problem to be solved by this invention is the growth characteristics of research pseudoalteromonas bacterial strain, and produces the method and the product of low-temperature cellulase.
Technical problem to be solved by this invention is to realize by following technical scheme.
Feature of the present invention comprises pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6) bacterial strain itself, and the method for utilizing this bacterial strain to produce low-temperature cellulase, and the low-temperature cellulase product that utilizes this bacterial strain to produce.
Bacterial strain involved in the present invention is the pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6) that is separated in the seawater in marine site, Yellow Sea of China Lianyun Harbour, this pseudoalteromonas Z6 is deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on September 26th, 2007, and deposit number is CGMCC N0.2155.
One, the morphological specificity of bacterial strain of the present invention and physiological and biochemical property
1.1 morphological specificity:
Bacterial strain is the Gram-negative rod-shaped bacterium, has pod membrane, no gemma, size to be about 1～1.8 μ m * 0.5～0.8 μ m (Fig. 1, Fig. 2).Bacterial strain after 25 ℃ of cellulose solids substratum are cultivated 2d, colony diameter 3mm-4mm, circular white, moistening, the edge is smooth, median rise, translucent, easy picking, can produce tangible Mierocrystalline cellulose transparent circle (Fig. 3).
1.2 the physiological and biochemical property of bacterial strain
The physiological and biochemical property of bacterial strain Z6 sees Table 1, bacterial strain Z6 energy hydrolyzed starch, do not produce hydrogen sulfide, liquefy gelatin, chromogenesis not, can not utilize Citrate trianion, indole test, catalase test and V-P test are positive, MR tests negative, can utilize lactose, maltose, fructose, semi-lactosi, seminose, rhamnosyl, glycogen, wood sugar and starch to be carbon source, sucrose, cellobiose, D-glucose, glycerine and sodium acetate can not be utilized, ammonium sulfate and tyrosine, L-Ala, glycine, Sodium.alpha.-ketopropionate, methionine(Met) can be utilized.Pass through Bergey ' s Manual of Systematic Bacteriology (second edition) and analyze relatively, this bacterial strain of preliminary judgement is that Pseudoalteromonas belongs to.
The physiological and biochemical property of table 1 bacterial strain Z6
Annotate: "+": positive findings; "-": negative findings
1.3 the molecular biology identification of bacterial strain Z6
Pcr amplification and sequential analysis: adopt the Takara test kit to propose the genomic dna of high purity bacterium.Forward primer P1:5 '-GAGAGTTTGATCCTGGCTCAG-3 ', reverse primer P2:5 '-CGGCTACCTTGTTACGAC-3 '.Reaction system 50 μ l, Taq enzyme 2U.Reaction conditions is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, and 54 ℃ of annealing 40s, 72 ℃ are extended 1min, circulate 35 times, and 72 ℃ are extended 7min.The purifying of PCR product, clone, order-checking are finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, the row that check order submit to GenBank to carry out homology relatively, adopt adjacent method to construct evolutionary tree (Fig. 4), evolutionary tree shows that bacterial strain Z6 and Pseudoalteromonascarrageenovora (AM69100800) sibship are nearest, and homology is more than 99%.This bacterial strain 16S rRNA sequence is seen specification sheets final section.
Two, the growth characteristics of bacterial strain of the present invention
Pseudoalteromonas Z6 provided by the invention has carried out careful research to its growth characteristics, finds out the growing state of this bacterium under the different condition substantially.
2.1 about the substratum among the present invention
2216E substratum (g/L): peptone 5, yeast powder 1, the seawater preparation, pH 7.2;
Cellulose solids substratum (g/L): sodium carboxymethyl-cellulose (CMC-Na) 10, peptone 10, yeast extract paste 5, KH
2PO
41, agar 20, the Chen Haishui preparation, pH 8.0.
Fermention medium (g/L): sodium carboxymethyl-cellulose (CMC-Na) 10, peptone 10, yeast extract paste 5, KH
2 PO
41, the Chen Haishui preparation, pH 8.0.
Trace quantity mineral salt (every liter): CuSO
45H
2O, 0.01g; ZnSO
47H
2O, 0.1g; CoCl
26H
2O, 0.005g; MnCl
24H
2O, 0.2g; Na
2MoO
42H
2O, 0.1g; KBr, 0.05g; KI, 0.05g; H
3BO
3, 0.1g; NaF, 0.05g; LiCl, 0.05g; Al
2(SO4)
3, 0.05g; NiCl
26H
2O, 0.01g; VoSO
42H
2O, 0.005g; H
2WO
42H
2O, 0.002g; Na
2SeO
4, 0.005g; SrCl6H
2O, 0.005g; BaCl
2, 0.005g.
2.2 the preparation of seed liquor: the inclined-plane inoculation that will be deposited in the 2216E substratum in fermention medium, rotating speed 180r/min, 20%, 25 ℃ of liquid amount is cultivated 16h.
2.3 the influence of temperature to growing: seed liquor 2% is inoculated in fermention medium, the pH nature, and rotating speed 180r/min, liquid amount 20% is cultivated under differing temps respectively, measures cell concn.The results are shown in Figure 5, the growth temperature range of bacterium is 0-35 ℃, and optimum growth temperature is 25 ℃.
2.4pH the influence to growth: adding final concentration in fermention medium is the phosphoric acid of 10mmol/L: acetic acid: boric acid (1: 1: 1) is as damping fluid, the pH of fermention medium is respectively between the 3.0-11.0, cultivated 24 hours at 25 ℃, other conditions are measured cell concn with 2.2.The results are shown in Figure 6, growth pH scope is 4-11, and the suitableeest growth pH is 8.
2.5NaCl the influence to growth: (will use the trace quantity mineral salts solution instead with the seawater preparation replaces at fermention medium, every liter of substratum adds 10ml) in add NaCl, making it is 0% to 15% NaCl, cultivates 24 hours at 25 ℃, other conditions are measured cell concn with 2.2.The results are shown in Figure 7, the NaCl concentration range of growth is 0-11%, and the suitableeest NaCl concentration is 3%.
2.6 the mensuration of cell concn: be chosen in and measure light absorption value under the 600nm wavelength.
Three, bacterial strain produces the method for low-temperature cellulase
Seed liquor is inserted in the fermention medium by 2% inoculum size, and 180r/min, cultivates 24h by 20 ℃; With cultured fermented liquid 10000r/min, centrifugal 10min, get supernatant liquor, add 65% ammonium sulfate, leave standstill 2h after, the centrifugal 25min of 10000r/min, use an amount of 25mM, pH is 8.0 Tris-HCL damping fluid dissolution precipitation, and 4 ℃ of dialysis 12h obtain low-temperature cellulase through DEAE-sepharose Flat Flow and Sephacryl S-200 method more then.
Four, the character of low-temperature cellulase
4.1 the enzyme operative temperature is to the influence of enzymic activity: with crude enzyme liquid respectively under 4 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃ conditions the measuring method enzyme live, the results are shown in Figure 8, the optimum temperature of cellulase is 30 ℃, still have 22% remnant enzyme activity at 4 ℃, 20 ℃ still have 80% remnant enzyme activity, are a kind of low-temperature cellulases.
4.2 the thermostability of enzyme: crude enzyme liquid is measured the residual enzyme vigor at 30 ℃ after being incubated 0.5h, 1h, 1.5h, 2h, 2.5h respectively under 4 ℃, 25 ℃, 30 ℃, the 50 ℃ conditions.This enzyme stability in the time of 4 ℃ is best, along with temperature raises, and stability decreases, 25 ℃ transformation period is 1.5h, 30 ℃ transformation period is 1h.(see figure 9).
4.3 the enzyme action pH is to the influence of enzymic activity: prepare different pH damping fluids: Na
2HPO
4-NaH
2PO
4(pH6-8), Tris-HCl (pH8-9), Gly-NaOH (pH9-11).Under condition of different pH, measure enzyme activity.The suitableeest action pH of cellulase is 8～9, and this enzyme still has 68% remnant enzyme activity at pH10, and this enzyme still has 41% remnant enzyme activity at pH11, is the low-temperature cellulase (see figure 10) of alkalescence.
4.4 enzyme pH stability: enzyme is measured the residual enzyme vigor under optimum temperature behind insulation 1h under 30 ℃ of different pH, some stability better has alkaline resistance properties to this enzyme in pH7～10.(seeing Figure 11).
5, cellulase activity is measured.
5.1 cellulase activity measuring method: it is that substrate is measured that cellulase activity adopts CMC.
5.2 enzyme activity unit definition: the per minute hydrocellulose produces the needed enzyme amount of 1 μ g glucose and is defined as 1 enzyme activity unit (U) under 30 ℃, the condition of pH8.0.
Five, the application of low-temperature cellulase product.
Low-temperature cellulase of the present invention can be used for fields such as washing, medicine, food, cotton spinning, environmental protection and renewable resources utilization.
Description of drawings
Fig. 1 is the Gram-stained opticmicroscope microscopy of bacterial strain figure.
Fig. 2 is the opticmicroscope microscopy figure of the capsule stain of bacterial strain.
Fig. 3 is the transparent loop graph of bacterial strain on the cellulose solids substratum.
Fig. 4 is a bacterial strain 16S rRNA evolutionary tree analysis chart.
Fig. 5 is the influence figure of culture temperature to strain growth, 10 ℃ (◆), 15 ℃ (■), 20 ℃ (▲), 25 ℃ (◇), 30 ℃ (mouth), 35 ℃ (△).
Fig. 6 is the influence figure of pH to strain growth.
Fig. 7 is the influence figure of NaCl to strain growth.
Fig. 8 is the influence figure of temperature to cellulase activity.
Fig. 9 is the influence figure of temperature to cellulase stability, 4 ℃ (◆), 25 ℃ (■), 30 ℃ (▲), 50 ℃ (●).
Figure 10 is the influence figure of pH to cellulase activity, pH6.0-8.0 (◆), pH8.0-9.0 (■), pH 9.0-11.0 (▲).
Figure 11 is the influence figure of pH to cellulase stability, pH6.0-8.0 (◆), pH8.0-9.0 (■)), pH 9.0-11.0 (▲).
The pseudoalteromonas Z6 that the present invention relates to (Pseudoalteromonas sp.Z6) has been deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on September 26th, 2007, the preservation address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCC N0.2155.
Embodiment
Embodiment 1.A kind of pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6) CGMCCN0.2155.This bacterial strain has following feature: bacterial strain is the Gram-negative rod-shaped bacterium, has pod membrane, no gemma, size to be about 1～1.8 μ m * 0.5～0.8 μ m; Bacterial strain after 25 ℃ of cellulose solids substratum are cultivated 2d, colony diameter 3mm-4mm, circular white, moistening, the edge is smooth, median rise, translucent, easy picking, can produce tangible Mierocrystalline cellulose transparent circle; Bacterial strain energy hydrolyzed starch, do not produce hydrogen sulfide, liquefy gelatin, chromogenesis can not utilize Citrate trianion, and indole test, catalase test and V-P test are positive, MR tests negative, can utilize lactose, maltose, fructose, semi-lactosi, seminose, rhamnosyl, glycogen, wood sugar and starch to be carbon source, can not utilize sucrose, cellobiose, D-glucose, glycerine and sodium acetate, can utilize ammonium sulfate, tyrosine, L-Ala, glycine, Sodium.alpha.-ketopropionate and methionine(Met).
Embodiment 2.Embodiment 1 described a kind of pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6) CGMCC N0.2155.Its growth characteristics are: the growth temperature range of this bacterium is 0-35 ℃; Growth pH scope is 4-11; Growth NaCl concentration range is 0-11%.
Embodiment 3.Embodiment 1 described a kind of pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6) CGMCC N0.2155.Its growth characteristics are: the suitable growth temperature of bacterial strain is 25 ℃; Being fit to growth pH is 8; Being fit to growth NaCl concentration is 3%.
Embodiment 4.The method that a kind of embodiment 1 or 2 or 3 any one described pseudoalteromonas Z6CGMCC N0.2155 produce low-temperature cellulase, its step is as follows:
(1) seed liquor is inserted in the fermention medium by 2% inoculum size, 180r/min, cultivates 24h by 20 ℃;
(2) with cultured fermented liquid 10000r/min, centrifugal 10min, get supernatant liquor, add 65% ammonium sulfate, leave standstill 2h after, the centrifugal 25min of 10000r/min, use an amount of 25mM, pH is 8.0 Tris-HCL damping fluid dissolution precipitation, and 4 ℃ of dialysis 12h obtain low-temperature cellulase through DEAE-sepharose Flat Flow and Sephacryl S-200 method more then.
Embodiment 5.A kind of embodiment 4 low-temperature cellulases that described method is produced, this low-temperature cellulase has following feature:
(1) the suitable operative temperature of cellulase is 30 ℃, at 4 ℃ 22% remnant enzyme activity is arranged, and 20 ℃ have 80% remnant enzyme activity, is a kind of low-temperature cellulase; This enzyme is stable in the time of 4 ℃, along with temperature raises, and stability decreases, 25 ℃ transformation period is 1.5h, 30 ℃ transformation period is 1h;
(2) the suitable action pH of cellulase is 8～9, and this enzyme has 68% remnant enzyme activity at pH 10, and this enzyme has 41% remnant enzyme activity at pH11, is the low-temperature cellulase of alkalescence; This enzyme is stable in pH7～10 o'clock, has alkaline resistance properties.
＜110〉Huaihai Institute of Technology
＜120〉ocean low-temperature cellulase and enzyme producing method and generation bacterium pseudoalteromonas thereof
<160>1
<210>1
<211>1439
<212>RNA
＜213〉pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6)
<400>1
tagctagtag?cgatgagagt?ttgatcctgg?ctccggtcga?aaaaggtagc?cgaaggaaat?60
ttgacccccg?gaggacgggt?gagtaatgct?tgggaacatg?ccttgaggtg?ggggacaaca?120
gttggaaacg?actgctaata?ccgcataatg?tctacggacc?aaagggggct?tcggctctcg?180
cctttagatt?ggcccaagtg?ggattagcta?gttggtgagg?taatggctca?ccaaggcgac?240
gatccctagc?tggtttgaga?ggatgatcag?ccacactggg?actgagacac?ggcccacact?300
ccaacgggag?gcagcagtgg?ggaatattgc?acaatgggcg?caagcctgat?gcagccatgc?360
cgcgtgtgtg?aagaaggcct?tcgggttgta?aagcactttc?agtcaggagg?aaaggttagt?420
agttaatacc?tgctagctgt?gacgttactg?acagaagaag?caccggctaa?ctccgtgcca?480
gcagccgcgg?taatacggag?ggtgcgagcg?ttaatcggaa?ttactgggcg?taaagcgtac?540
gcaggcggtt?tgttaagcga?gatgtgaaag?ccccgggctc?aacctgggaa?ctgcatttcg?600
aactggcaaa?ctagagtgtg?atagagggtg?gtagaatttc?aggtgtagcg?gtgaaatgcg?660
tagagatctg?aaggaatacc?gatggcgaag?gcagccacct?gggtcaacac?tgacgctcat?720
gtacgaaagc?gtggggagca?aacgggatta?gataccccgg?tagtccacgc?cgtaaacgat?780
gtctactaga?agctcggagc?ctcggttctg?tttttcaaag?ctaacgcatt?aagtagaccg?840
cctggggagt?acggccgcaa?ggttaaaact?caaatgaatt?gacgggggcc?cgcacaagcg?900
gtggagcatg?tggtttaatt?cgatgcaacg?cgaagaacct?tacctacact?tgacatacag?960
agaacttacc?agagatggtt?tggtgccttc?gggaactctg?atacaggtgc?tgcatggctg?1020
tcgtcagctc?gtgttgtgag?atgttgggtt?aagtcccgca?acgagcgcaa?cccctatcct?1080
tagttgctag?caggtaatgc?tgagaactct?aaggagactg?ccggtgataa?accggaggaa?1140
ggtggggacg?acgtcaagtc?atcatggccc?ttacgtgtag?ggctacacac?gtgctacaat?1200
ggcgcataca?gagtgctgcg?aactcgcgag?agtaagcgaa?tcacttaaag?tgcgtcgtag?1260
tccggattgg?agtctgcaac?tcgactccat?gaagtcggaa?tcgctagtaa?tcgcgtatca?1320
gaatgacgcg?gtgaatacgt?tcccgggcct?tgtacactcc?tcccttccca?gttggggcca?1380
aggttgcccc?tgaagttttt?tcctggccca?ggtcgtaaca?aggagccggg?agagtagca 1439
Claims (6)
1. a pseudoalteromonas Z6 (Pseudoalteromonas sp.Z6) CGMCC N0.2155.
2. according to the described pseudoalteromonas Z6 of claim 1 CGMCC N0.2155, it is characterized in that this bacterial strain has following feature:
Bacterial strain is the Gram-negative rod-shaped bacterium, has pod membrane, no gemma, size to be about 1～1.8 μ m * 0.5～0.8 μ m; Bacterial strain after 25 ℃ of cellulose solids substratum are cultivated 2d, colony diameter 3mm-4mm, circular white, moistening, the edge is smooth, median rise, translucent, easy picking, can produce tangible Mierocrystalline cellulose transparent circle; Bacterial strain energy hydrolyzed starch, do not produce hydrogen sulfide, liquefy gelatin, chromogenesis can not utilize Citrate trianion, and indole test, catalase test and V-P test are positive, MR tests negative, can utilize lactose, maltose, fructose, semi-lactosi, seminose, rhamnosyl, glycogen, wood sugar and starch to be carbon source, can not utilize sucrose, cellobiose, D-glucose, glycerine and sodium acetate, can utilize ammonium sulfate, tyrosine, L-Ala, glycine, Sodium.alpha.-ketopropionate and methionine(Met).
3. according to the described pseudoalteromonas Z6 of claim 1 CGMCC N0.2155, it is characterized in that its growth characteristics are: the growth temperature range of this bacterium is 0-35 ℃; Growth pH scope is 4-11; Growth NaCl concentration range is 0-11%.
4. according to the described pseudoalteromonas Z6 of claim 1 CGMCC N0.2155, it is characterized in that its growth characteristics are: the suitable growth temperature of bacterial strain is 25 ℃; Being fit to growth pH is 8; Being fit to growth NaCl concentration is 3%.
5. according to the described pseudoalteromonas Z6 of claim 1 CGMCC N0.2155, it is characterized in that this bacterial strain 16S rRNA sequence is as follows:
tagctagtag?cgatgagagt?ttgatcctgg?ctccggtcga?aaaaggtagc?cgaaggaaat?60
ttgacccccg?gaggacgggt?gagtaatgct?tgggaacatg?ccttgaggtg?ggggacaaca 120
gttggaaacg?actgctaata?ccgcataatg?tctacggacc?aaagggggct?tcggctctcg 180
cctttagatt?ggcccaagtg?ggattagcta?gttggtgagg?taatggctca?ccaaggcgac 240
gatccctagc?tggtttgaga?ggatgatcag?ccacactggg?actgagacac?ggcccacact 300
ccaacgggag?gcagcagtgg?ggaatattgc?acaatgggcg?caagcctgat?gcagccatgc 360
cgcgtgtgtg?aagaaggcct?tcgggttgta?aagcactttc?agtcaggagg?aaaggttagt 420
agttaatacc?tgctagctgt?gacgttactg?acagaagaag?caccggctaa?ctccgtgcca 480
gcagccgcgg?taatacggag?ggtgcgagcg?ttaatcggaa?ttactgggcg?taaagcgtac 540
gcaggcggtt?tgttaagcga?gatgtgaaag?ccccgggctc?aacctgggaa?ctgcatttcg 600
aactggcaaa?ctagagtgtg?atagagggtg?gtagaatttc?aggtgtagcg?gtgaaatgcg 660
tagagatctg?aaggaatacc?gatggcgaag?gcagccacct?gggtcaacac?tgacgctcat 720
gtacgaaagc?gtggggagca?aacgggatta?gataccccgg?tagtccacgc?cgtaaacgat 780
gtctactaga?agctcggagc?ctcggttctg?tttttcaaag?ctaacgcatt?aagtagaccg 840
cctggggagt?acggccgcaa?ggttaaaact?caaatgaatt?gacgggggcc?cgcacaagcg 900
gtggagcatg?tggtttaatt?cgatgcaacg?cgaagaacct?tacctacact?tgacatacag 960
agaacttacc?agagatggtt?tggtgccttc?gggaactctg?atacaggtgc?tgcatggctg 1020
tcgtcagctc?gtgttgtgag?atgttgggtt?aagtcccgca?acgagcgcaa?cccctatcct 1080
tagttgctag?caggtaatgc?tgagaactct?aaggagactg?ccggtgataa?accggaggaa 1140
ggtggggacg?acgtcaagtc?atcatggccc?ttacgtgtag?ggctacacac?gtgctacaat 1200
ggcgcataca?gagtgctgcg?aactcgcgag?agtaagcgaa?tcacttaaag?tgcgtcgtag 1260
tccggattgg?agtctgcaac?tcgactccat?gaagtcggaa?tcgctagtaa?tcgcgtatca 1320
gaatgacgcg?gtgaatacgt?tcccgggcct?tgtacactcc?tcccttccca?gttggggcca 1380
aggttgcccc?tgaagttttt?tcctggccca?ggtcgtaaca?aggagccggg?agagtagca 1439
6. method of producing low-temperature cellulase as any one described pseudoalteromonas Z6 CGMCCN0.2155 among the claim 1-5 is characterized in that its step is as follows:
(1) seed liquor is inserted in the fermention medium by 2% inoculum size, 180r/min, cultivates 24h by 20 ℃;
(2) with cultured fermented liquid 10000r/min, centrifugal 10min, get supernatant liquor, add 65% ammonium sulfate, leave standstill 2h after, the centrifugal 25min of 10000r/min, use an amount of 25mM, pH is 8.0 Tris-HCL damping fluid dissolution precipitation, and 4 ℃ of dialysis 12h obtain low-temperature cellulase through DEAE-sepharose Fast Flow and Sephacryl S-200 method more then.
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| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
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