CN103343099A - Marine Arthrobactersp. and method for producing low-temperature D-glucosidase through marine Arthrobactersp. - Google Patents
Marine Arthrobactersp. and method for producing low-temperature D-glucosidase through marine Arthrobactersp. Download PDFInfo
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Abstract
The invention discloses marine Arthrobactersp. YJ34 with CGMCC (China General Microbiological Culture Collection Center) NO.7385. The growth temperature range of the strain 4-37 DEG C, and the optimal growth temperature is 25 DEG C; the growth pH range is 5-11, and the optimal pH is 7; and the concentration of NaCl for growth is 0-10%, and the optimal concentration of the NaCl for growth is 2%. The invention further discloses a method for producing low-temperature D-glucosidase through the marine arthrobactersp. The pH of the D-glucosidase produced by the marine arthrobactersp YJ34 strain disclosed by the invention is 7.0 and the temperature is 35 DEG C, and therefore the marine Arthrobactersp. YJ34 is suitable for serving as an oral care agent.
Description
Technical field
The present invention relates to a kind of microorganism, particularly a kind of Arthrobacter YJ34(that separates from marine site, Lianyun Harbour, Jiangsu Province, China
ArthrobacterSp.) CGMCC N0.7385(is deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on March 29th, 2013, and deposit number is CGMCC N0.7385); The invention still further relates to the method that this bacterial strain produces low temperature dextran enzyme.
Background technology
Sucrose in the fermentation using bacteria food in the oral cavity, and the generation dextran (α-D-glucan).Dextran is by α-1; 4, α-1; 3 and α-1; 6 glycosidic links constitute; α-1 wherein; the side chain that 6 glycosidic links form determines its adhesivity; dextran is cross-linked with each other by its adhesivity; form the protectiveness reticulated structure; microorganism particularly has suis breeding in a large number under the reticulated structure protection that carious tooth forms ability, forms soft in dental surface or slit and the bacterial plaque structure of thickness, and this structure claims plaque; the acidic metabolite of these microorganism secretions destroys enamel, thereby causes carious tooth.Therefore in order to prevent and control carious tooth and periodontopathy, the generation and the development that suppress plaque are vital.
(dextranase EC3.2.1.11), is the lytic enzyme of α-1,6 glycosidic link in the single-minded cutting dextran (Dextean) to dextran (acid anhydride) enzyme.The dextran of dextran enzyme in can the hydrolysis plaque, thus remove plaque.As far back as nineteen sixty-eight, the fitzgerald reported first possibility of dextran enzyme aspect preventing decayed tooth, caused people's very big interest.Gibbons in 1969 and keyes add the dextran enzyme in drinking-water, successfully suppressed the formation of hamster carious tooth.Nineteen eighty-two, the Japan scholar also reported the effect (Sun Jinwu, 1986) that suppresses and decompose artificial bacterial plaque with the dextran enzyme with Tian Zhensan.The precious honor of nineteen ninety and 1992 China Zhao etc. to Paecilomyces lilacinus (
Paecilomyces lilacinus) the prevention of dental caries effect of bacterial strain 8523 dextran enzymes studies, the result shows that this enzyme has and shows effect (Zhao Baorong etc., 1990 that suppress plaque generally; Li Yujing etc., 1991).Marotta utilized external plaque dextran pseudostructure building-up process model in 2002, discovery is in the plaque forming process, the dextran enzyme can reduce dextran formation amount, and the dextran that forms does not adhere to the ability (Marotta et al., 2002) of bacterial cell.Utilize the dextran enzyme to suppress plaque, be subjected to the attention of various countries' stomatology research in recent years, the dextran enzyme is a kind of biotechnological formulation, uses and preserves simple and easy to do.Nontoxicity has no drug resistance, and does not influence intraoral biological and ecological balance, under the infant that can not brush teeth and the situation that do not possess the condition of washing one's face and rinsing one's mouth, is a kind of preparation of good preventing tooth carious tooth spot.And everyone after meal, especially uses the mouth wash shua that is added with this enzyme after the sugar, is that a good restraining bacterial plaque forms the effective measure of preventing dental caries and periodontopathy.At present countries such as the U.S., the Canada dextran enzyme that will have the plaque ability of degrading is applied to the exploitation (being subjected to patent protection) of following commodity: toothpaste, chewing gum, collutory, ointment, food, beer, atomizing of liquids cleaning of teeth instrument, and domestic research report about the dextran enzyme is few, rarely seen mould produces the report of dextran enzyme, mould (Sun Jinwu etc. such as the Paecilomyces lilacinus of Institute of Microorganism, Academia Sinica's report, Aspergillus ustus, 1988,28 (1); Cheng Xiulan etc., 1992), HeFei University of Technology's report penicillium funiculosum and sour jujube spore mould (Zhu Huixia etc., 2010; Zhang Hongbin etc., 2011) and University of Anhui's report Penicillium citreo-viride (Yue Xiaojing etc., 2011), the product enzyme time is long, mostly is 6-8 days.Produce the problem that there is security in the dextran enzyme that prevents oral plaque with mould, and the product enzyme time is long, if be used in the toothpaste, also has problem of unstable under the alkaline condition (Sun Jinwu, 1986).The exploitation of marine bacteria dextran enzyme will overcome these problems.The ocean has extreme environments such as high salt, low temperature, alkalescence, makes the marine microorganism enzyme have unique catalytic property and application potential that the land microbial enzyme does not have.From marine microorganism, screen at present the bacterial strain that produces the dextran enzyme and be mainly pseudoalteromonas genus and Vibrio (patent of invention, ZL200910029584.4, patent of invention ZL 201010534675.6), the dextran enzyme operative temperature that these bacterium produce is low, produce the enzyme time short (12-30h), stable under the alkaline condition, the plaque microbial film there is the good restraining effect, the sweet enzyme of dextrose in this marine bacteria source is used for oral cavity nursing agent (as collutory, toothpaste, chewing gum) in, the instead of chemical composition is main oral cavity nursing agent, will have better economic and social benefit.
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 Arthrobacter YJ34 from the ocean that can produce the dextran enzyme is provided.
Another technical problem to be solved by this invention provides the method for above-mentioned Arthrobacter YJ34 fermentative production dextran enzyme.
Feature of the present invention comprises Arthrobacter YJ34(
ArthrobacterSp.) CGMCC N0.7385 bacterial strain (to call bacterial strain YJ34 in the following text), and the method for utilizing this strain fermentation production dextran enzyme itself.The method steps of Arthrobacter YJ34 product low temperature dextran enzyme is as follows: Arthrobacter YJ34 bacterial strain inclined-plane seed is inoculated in the 2216E substratum, and 25 ℃, 180 r/min, liquid amount 20% is cultivated 16 h, gets seed liquor; Seed liquor is inoculated in 2% inoculum size produces in the enzyme substratum, 180r/min cultivates 21h for 25 ℃, and the centrifugal 5min of 10000r/min gets supernatant liquor and is the thick enzyme of dextran enzyme; Described product enzyme substratum is: peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T20 1%, pH7.0.
Bacterial strain YJ34 involved in the present invention is the Arthrobacter YJ34(that is separated in the ooze in marine site, Lianyun Harbour, Jiangsu Province, China
ArthrobacterSp.), this Arthrobacter YJ34 is deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on March 29th, 2013, and deposit number is CGMCC N0.7385.Depositary institution address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Below the present invention is explained in detail.
One, the morphological specificity of bacterial strain YJ34 of the present invention and physiological and biochemical property.
1.1 morphological specificity:
Bacterial strain YJ34 is the gram negative bacillus (see figure 1), no pod membrane, in solid medium, cultivate 48h after, that bacterium colony is is light yellow, moistening, the edge is smooth, center protrusion, easy picking, the transparent circle (see figure 2) that forms at the primary dcreening operation flat board.
1.2 physiological and biochemical property:
The biochemical reactions of bacterial strain YJ34 the results are shown in Table 1, bacterial strain VP experiment is positive, can utilize glucose, sucrose, rhamnosyl, melibiose, pectinose, N.F,USP MANNITOL, inositol, sorbyl alcohol and amygdaloside, the energy liquefy gelatin, can produce hydrogen sulfide, edwardsiella hoshinae does not produce beta galactosidase enzyme, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophane desaminase.
Table 1 bacterial strain YJ34 physiological and biochemical property
| Experimental project | The result | Experimental project | The result |
| Colony colour | Yellow | Pectinose utilizes | + |
| 4 ℃ of growths | + | N.F,USP MANNITOL utilizes | + |
| 37 ℃ of growths | + | Inositol utilizes | + |
| The non-sodium chloride growth | + | Sorbyl alcohol utilizes | + |
| The suitableeest sodium- |
2% | Amygdaloside utilizes | - |
| VP | + | Gelatinase | + |
| Indoles produces | - | Beta galactosidase enzyme | - |
| NO 2Produce | - | Arginine dihydrolase | - |
| H 2S | + | Lysine decarboxylase | - |
| Glucose utilization | + | Ornithine decarboxylase | - |
| Sucrose utilizes | + | Citric acid utilizes | - |
| Rhamnosyl utilizes | + | Urase | + |
| Melibiose utilizes | + | The tryptophane desaminase | - |
Annotate :+: the positive; : feminine gender.
1.3 the molecular biology identification of bacterial strain YJ34:
Utilize the Takara test kit to extract the strain gene group and carry out PCR, primer is selected prokaryotic micro-organisms 16S rDNA universal primer for use, upstream primer: 5 '-agagtttgatcctggctag-3 ', downstream primer: 5 '-ggttaccttgttacgactt-3 ', glue reclaims the PCR product, is connected with pMD 19-T carrier, be converted into DH5 α competent cell, blue hickie filters out positive colony, and bacterium colony PCR checking is delivered to Shanghai and given birth to the order-checking of worker biotech firm.The 16S rDNA of amplification bacterial strain YJ34, its length is 1429 bp, the accession number in GenBank is JN426846.Sequence in this sequence and the GenBank database is carried out homology relatively, find YJ34 with
ArthrobacterSp.(GU220063.1) in same branch, homology reaches 99.9% (Fig. 3).In conjunction with the physiological and biochemical property of bacterial strain, with this bacterial strain name genus arthrobacter (
ArthrobacterSp. YJ34).
Two,The growth characteristics of bacterial strain YJ34 of the present invention
Bacterial strain YJ34 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 the substratum that relates among the present invention:
2216E substratum: peptone 0.5%, yeast powder 0.1%, agar 2%, Chen Haishui preparation, pH8.
The primary dcreening operation substratum: peptone 0.5% yeast powder 0.1%, blue dextran 2,000 0.2%, dextran T20 0.8%, agar 2%, the Chen Haishui preparation, pH 8.0;
Sieve substratum again: peptone 0.5%, yeast powder 0.1%, dextran 1.0%, agar 2%, Chen Haishui preparation, pH8.
Produce the enzyme substratum: peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T20 1%, pH7.0.
The preparation of seed liquor: bacterial strain YJ34 inclined-plane seed is inoculated in the 2216E substratum, 25 ℃, 180 r/min, liquid amount 20% is cultivated 16 h.
2.3 the influence that temperature is grown to bacterial strain YJ34:
With seed liquor with 2% inoculum size in the 2216E substratum, pH 7.5, rotating speed 180r/min, liquid amount 20% is cultivated 24 h respectively under differing temps, be chosen in and measure the OD value under the 600nm wavelength, its growth temperature range is 4-37 ℃, and optimum growth temperature is 25 ℃, sees Fig. 4.
2.4 the influence of the bacterial strain YJ34 growth of pH:
PH scope 4.0 11.0, optimum temperuture is cultivated, and all the other conditions are with 2.3, and growth pH scope is 5-11, and the suitableeest growth pH is 7.0, sees Fig. 5.
2.5 the influence of the bacterial strain KQ11 growth of NaCl:
Substratum is prepared with tap water, and NaCl scope 0%-12% is cultivated under optimum temperuture and pH, and all the other conditions are with 2.3, and the NaCl concentration of growth is 0%-10%, and the NaCl concentration of suitable growth is 2 %, sees Fig. 6.
Three, the method for bacterial strain YJ34 fermentative production dextran enzyme
The contriver is to bacterial strain YJ34(
ArthrobacterSp. YJ34) method of fermentative production dextran enzyme is studied.
3.1 fermentation time produces the influence of enzyme to bacterial strain YJ34: behind the fermentation 12h, the yield of enzyme lift velocity is very fast, and yield of enzyme is the highest during to fermentation 21h, increases in time, and enzyme activity descends gradually behind the 24h, sees Fig. 7.
3.2 leavening temperature produces the enzyme influence to bacterial strain YJ34: carry out fermentation culture under differing temps, it is good that this bacterial strain produces enzyme down at 15 ℃-30 ℃, and bacterial strain YJ34 enzymatic production reaches the highest in the time of 25 ℃, sees Fig. 8.
3.3 medium pH produces the influence of enzyme to bacterial strain YJ34: growth product enzyme is good under the initial pH6.0-8.5 condition of substratum, and during pH7.0, bacterial strain YJ34 enzymatic production is best, sees Fig. 9.
3.4 NaCl concentration is produced the influence of enzyme to bacterial strain YJ34: this bacterium can grow and produce enzyme when NaCl 0 – 5%, when substratum NaCl concentration rose to 2%, it is the highest that strain fermentation produces enzyme, sees Figure 10.
3.5 liquid amount is to the influence of bacterial strain YJ34 enzymatic production: 20% liquid amount condition is issued to high enzymatic activity, sees Figure 11.
3.6 inductor concentration is produced the influence of enzyme to bacterial strain YJ34: the dextran enzyme is a kind of inducible enzyme, dextran T20 is inductor, induce the enzyme situation of producing by being configured to different final concentration researchs, the result shows when substratum does not have inductor and exists, bacterial strain YJ34 does not produce enzyme, can reach high enzymatic activity under the dextran acid anhydride T20 condition of 1% concentration, increase inductor concentration and produce enzyme activity and does not have obvious increase, see Figure 12.
3.7 dextran enzyme activity determination: behind 35 ° of C water-bath 15 min of dextran T70 (50 mmol/L, the preparation of pH 5.5 sodium acetate buffers) with 10 μ L enzyme liquid and 190 μ L 3%, the DNS method is measured reducing sugar content.Enzyme activity unit definition (U/mL): under the above-mentioned reaction conditions, it is a unit of activity that per minute catalysis discharges the required enzyme amount of 1 μ moL maltose.
Four, the character of bacterial strain YJ34 dextran enzyme
4.1 the preparation of crude enzyme liquid: with Arthrobacter YJ34(
ArthrobacterSp. YJ34) bacterial strain inclined-plane seed is inoculated in the 2216E substratum, and 25 ℃, 180 r/min, liquid amount 20% is cultivated 16 h, gets seed liquor; Seed liquor is inoculated in 2% inoculum size produces enzyme substratum (peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T20 1%, pH7.0) in, 180r/min cultivates 21h for 25 ℃, the centrifugal 5min of 10000r/min gets supernatant liquor and is the thick enzyme of dextran enzyme.
4.2 the enzyme operative temperature is to the influence of enzymic activity:
The dextran enzyme placed under the differing temps react with substrate, measure enzyme activity, the results are shown in Figure 13, the optimum temperature of enzyme is 35 ℃.
4.3 the action pH of enzyme is to the influence of enzymic activity:
With enzyme liquid with in the dextran solution of different pH, under 35 ℃, carry out enzyme activity determination, the results are shown in Figure 14, the activity of this enzyme of pH7.0 is the highest.
Usually, the pH in the oral cavity is 6.6 ~ 7.1, and temperature is 36.3 ℃-37.2 ℃.The pH of the dextran enzyme that Arthrobacter YJ34 bacterial strain of the present invention produces is 7.0, and temperature is 35 ℃, is suitable as oral cavity nursing agent.
Description of drawings
Fig. 1 is electromicroscopic photograph form (* 13000) figure of bacterial strain YJ34;
The transparent loop graph that Fig. 2 forms at the primary dcreening operation flat board for bacterial strain YJ34;
Fig. 3 is bacterial strain YJ34 systematic evolution tree;
Fig. 4 is that temperature is to the figure that influences of bacterial strain YJ34 growth; Among the figure: ◆ 15 ° of C, 20 ° of C of ■, ▲ 25 ° of C, * 30 ° of C, ● 35 ° of C;
Fig. 5 is the figure that influences of the bacterial strain YJ34 growth of pH;
Fig. 6 is that NaCl concentration is to the figure that influences of bacterial strain YJ34 growth;
Fig. 7 is that fermentation time is to the figure that influences of bacterial strain YJ34 enzymatic production;
Fig. 8 is that temperature is to the figure that influences of bacterial strain YJ34 enzymatic production;
Fig. 9 is the figure that influences of the bacterial strain YJ34 of pH enzymatic production;
Figure 10 is that NaCl concentration is to the figure that influences of bacterial strain YJ34 enzymatic production;
Figure 11 is that liquid amount is to the figure that influences of bacterial strain YJ34 enzymatic production;
Figure 12 is that inductor concentration is to the figure that influences of bacterial strain YJ34 enzymatic production;
Figure 13 is that temperature is to the figure that influences of YJ34 dextran enzyme;
Figure 14 is the figure that influences of the YJ34 dextran of pH enzyme;
Arthrobacter YJ34(of the present invention
ArthrobacterSp.) oneself is deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on March 29th, 2013, and deposit number is CGMCC N0.7385.Depositary institution address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment
Below further describe concrete technical scheme of the present invention, so that those skilled in the art understands the present invention further, and do not constitute the restriction to its right.
Claims (2)
- Arthrobacter from the ocean ( ArthrobacterSp.) YJ34 CGMCC N0.7385.
- 2. the method that Arthrobacter YJ34 as claimed in claim 1 produces low temperature dextran enzyme is characterized in that its step is as follows: Arthrobacter YJ34 bacterial strain inclined-plane seed is inoculated in the 2216E substratum, 25 ℃, 180 r/min, liquid amount 20%, cultivate 16 h, get seed liquor; Seed liquor is inoculated in 2% inoculum size produces in the enzyme substratum, 180r/min cultivates 21h for 25 ℃, and the centrifugal 5min of 10000r/min gets supernatant liquor and is the thick enzyme of dextran enzyme; Described product enzyme substratum is: peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T20 1%, pH7.0.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679974A (en) * | 2019-02-03 | 2019-04-26 | 淮海工学院 | Marine microorganism arthrobacterium YJ34 produces dextranase genes and its recombination engineering |
| CN110343647A (en) * | 2019-08-12 | 2019-10-18 | 江苏海洋大学 | Bacillus marinus SP-5 and its dextranase |
| CN111826317A (en) * | 2020-07-28 | 2020-10-27 | 江苏海洋大学 | Malassezia G-1, method for producing endo-dextranase by using same, product and application |
| CN115786308A (en) * | 2022-11-24 | 2023-03-14 | 江苏海洋大学 | Method for improving heat stability of dextranase and mutant |
| CN115786308B (en) * | 2022-11-24 | 2024-05-31 | 江苏海洋大学 | Method for improving thermal stability of dextranase and mutant |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679974A (en) * | 2019-02-03 | 2019-04-26 | 淮海工学院 | Marine microorganism arthrobacterium YJ34 produces dextranase genes and its recombination engineering |
| CN110343647A (en) * | 2019-08-12 | 2019-10-18 | 江苏海洋大学 | Bacillus marinus SP-5 and its dextranase |
| CN110343647B (en) * | 2019-08-12 | 2021-11-02 | 江苏海洋大学 | Bacillus marinus SP-5 and dextranase thereof |
| CN111826317A (en) * | 2020-07-28 | 2020-10-27 | 江苏海洋大学 | Malassezia G-1, method for producing endo-dextranase by using same, product and application |
| CN111826317B (en) * | 2020-07-28 | 2022-05-20 | 江苏海洋大学 | Marfan bacillus G-1, method for producing endo-dextranase by using same, product and application |
| CN115786308A (en) * | 2022-11-24 | 2023-03-14 | 江苏海洋大学 | Method for improving heat stability of dextranase and mutant |
| CN115786308B (en) * | 2022-11-24 | 2024-05-31 | 江苏海洋大学 | Method for improving thermal stability of dextranase and mutant |
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