CN103343099B - 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(be separated from marine site, Lianyun Harbour, Jiangsu Province, China
arthrobactersp.) CGMCC N0.7385(is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC 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 Dextranase.
Background technology
Sucrose in fermentation using bacteria food in oral cavity, generates dextran (α-D-glucan).Dextran is by α-1; 4, α-1; 3 and α-1; 6 glycosidic links are formed; wherein α-1; the side chain that 6 glycosidic links are formed determines its adhesivity; dextran is cross-linked with each other by its adhesivity; form protectiveness reticulated structure; the suis that microorganism particularly has a carious tooth Forming ability is amount reproduction under reticulated structure protection, and in dental surface or gap, form the soft and bacterial plaque structure of thickness, this structure claims plaque; the acidic metabolite of these microorganism secretions destroys enamel, thus causes carious tooth.Therefore in order to prevention and corntrol carious tooth and periodontopathy, the generation of plaque and development is suppressed to be vital.
Dextran (acid anhydride) enzyme (dextranase, EC3.2.1.11) is the lytic enzyme of α-1,6 glycosidic link in single-minded cutting dextran (Dextean).Dextranase can be hydrolyzed the dextran in plaque, thus removes plaque.As far back as nineteen sixty-eight, the fitzgerald reported first possibility of Dextranase in preventing decayed tooth, causes the great interest of people.Within 1969, Gibbons and keyes adds Dextranase in drinking-water, successfully inhibits the formation of hamster carious tooth.Nineteen eighty-two, Japanese scholars reported effect (Sun Jinwu, 1986) with dextran enzyme level Sum decomposition artifical plaque with Tian Zhensan.Nineteen ninety and 1992 China Zhao precious flourish etc. to Paecilomyces lilacinus (
paecilomyces lilacinus) the prevention of dental caries effect of bacterial strain 8523 Dextranase is studied, result shows that this enzyme has the aobvious effect (Zhao Baorong etc., 1990 that suppress plaque generally; Li Yujing etc., 1991).Within 2002, Marotta utilizes external plaque dextran pseudostructure building-up process model, find in plaque forming process, Dextranase can reduce dextran formation volume, and the dextran formed does not have the ability (Marotta et al., 2002) of Adherent bacteria cell.Utilize dextran enzyme level plaque, be subject to the attention of various countries' stomatology research in recent years, Dextranase is a kind of biotechnological formulation, uses and preserves simple and easy to do.Nontoxicity, has no drug resistance, do not affect intraoral biological and ecological balance, to the infant that can not brush teeth and do not possess wash one's face and rinse one's mouth condition when, be a kind of preparation of prevention tooth carious tooth spot well.And everyone after meal, especially after sugar, application is added with the mouth wash shua of this enzyme, is a good anti-dental plaque, the effective measure of preventing dental caries and periodontopathy.The Dextranase with degraded plaque ability has been applied to the exploitation (by patent protection) of following commodity by the country such as the current U.S., Canada: toothpaste, chewing gum, collutory, ointment, food, beer, atomizing of liquids cleaning of teeth instrument, and the domestic report of the research about Dextranase is few, rarely seen mould produces the report of Dextranase, mould (the Sun Jinwu etc. such as Paecilomyces lilacinus, Aspergillus ustus of Institute of Microorganism, Academia Sinica's report, 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 of preventing the Dextranase of oral plaque to there is security with mould, and the product enzyme time is long, if be used in toothpaste, also deposits problem (Sun Jinwu, 1986) unstable in the basic conditions.The exploitation of marine bacteria Dextranase will overcome these problems.Ocean has the extreme environments such as high salt, low temperature, alkalescence, Enzymes from Marine Microorganisms is had unique catalytic character that land microbial enzyme do not have and application potential.From marine microorganism, screen the bacterial strain producing Dextranase be at present mainly pseudoalteromonas genus and Vibrio (patent of invention, ZL200910029584.4, patent of invention ZL 201010534675.6), the Dextranase operative temperature that these bacterium produce is low, produce the enzyme time short (12-30h), stable under alkaline conditions, good restraining effect is had to Dental plaque biofilm, the sweet enzyme of dextrose of being originated by this marine bacteria is used for oral cavity nursing agent (as collutory, toothpaste, chewing gum) in, alternative chemical composition is main oral cavity nursing agent, good economic and social benefit will be had.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, provides a kind of Arthrobacter YJ34 from ocean that can produce Dextranase newly.
Another technical problem to be solved by this invention is to provide the method for above-mentioned Arthrobacter YJ34 fermentative production Dextranase.
Feature of the present invention comprises Arthrobacter YJ34(
arthrobactersp.) CGMCC N0.7385 bacterial strain itself (hereinafter referred to as bacterial strain YJ34), and utilize this strain fermentation to produce the method for Dextranase.The method steps that Arthrobacter YJ34 produces low temperature Dextranase is as follows: be inoculated in 2216E substratum by Arthrobacter YJ34 bacterial strain inclined-plane seed, 25 DEG C, 180 r/min, and liquid amount 20%, cultivates 16 h, obtain seed liquor; By seed liquor with 2% inoculum size be inoculated in culture medium, 180r/min, cultivates 21h, 10000r/min centrifugal 5min, gets supernatant liquor and be the thick enzyme of Dextranase for 25 DEG C; Described culture medium 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(be separated in the ooze in marine site, Lianyun Harbour, Jiangsu Province, China
arthrobactersp.), this Arthrobacter YJ34 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC on March 29th, 2013, and deposit number is CGMCC N0.7385.Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
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 specificitys:
Bacterial strain YJ34 is gram negative bacillus (see figure 1), and without pod membrane, cultivate 48h in solid medium after, bacterium colony is light yellow, moistening, the smooth of the edge, center protrusion, easily picking, the transparent circle (see figure 2) that primary dcreening operation flat board is formed.
1.2 physiological and biochemical properties:
The biochemical reactions of bacterial strain YJ34 the results are shown in Table 1, bacterial strain VP tests as the positive, can utilize glucose, sucrose, rhamnosyl, melibiose, pectinose, N.F,USP MANNITOL, inositol, sorbyl alcohol and amygdaloside, energy liquefy gelatin, hydrogen sulfide can be produced, 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 | Result | Experimental project | Result |
| Colony colour | Yellow | Pectinose utilizes | + |
| 4 DEG C of growths | + | N.F,USP MANNITOL utilizes | + |
| 37 DEG C of growths | + | Inositol utilizes | + |
| Non-sodium chloride grows | + | Sorbyl alcohol utilizes | + |
| The suitableeest sodium-chlor growth concentration | 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 utility | + | Tryptophane desaminase | - |
Note :+: positive; : it is negative.
The molecular biology identification of 1.3 bacterial strain YJ34:
Utilize Takara test kit to extract strain gene group and carry out PCR, primer selects prokaryotic micro-organisms 16S rDNA universal primer, upstream primer: 5 '-agagtttgatcctggctag-3 ', downstream primer: 5 '-ggttaccttgttacgactt-3 ', glue reclaims PCR primer, is connected with pMD 19-T carrier, be converted into DH5 α competent cell, blue hickie filters out positive colony, and bacterium colony PCR verifies, delivers to the order-checking of Shanghai Sheng Gong biotech firm.The 16S rDNA of amplification bacterial strain YJ34, its length is 1429 bp, and the accession number in GenBank is JN426846.Sequence in this sequence and GenBank database is carried out tetraploid rice, 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, by this Strain Designation 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, substantially finds out the growing state of this bacterium under different condition.
The substratum related in 2.1 the present invention:
2216E substratum: peptone 0.5%, yeast powder 0.1%, agar 2%, Chen Haishui is prepared, pH8.
Primary dcreening operation substratum: peptone 0.5% yeast powder 0.1%, blue dextran 2,000 0.2%, dextran T20 0.8%, agar 2%, Chen Haishui is prepared, pH 8.0;
Sieve substratum again: peptone 0.5%, yeast powder 0.1%, dextran 1.0%, agar 2%, Chen Haishui is prepared, pH8.
Culture medium: peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T20 1%, pH7.0.
The preparation of seed liquor: be inoculated in 2216E substratum by bacterial strain YJ34 inclined-plane seed, 25 DEG C, 180 r/min, liquid amount 20%, cultivates 16 h.
The impact that 2.3 temperature grow bacterial strain YJ34:
By seed liquor with 2% inoculum size in 2216E substratum, pH 7.5, rotating speed 180r/min, liquid amount 20%, cultivates 24 h respectively at different temperatures, selects to measure OD value under 600nm wavelength, its growth temperature range is 4-37 DEG C, and optimum growth temperature is 25 DEG C, sees Fig. 4.
The impact that 2.4 pH grow bacterial strain YJ34:
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, the most suitable growth pH is 7.0, sees Fig. 5.
The impact that 2.5 NaCl grow bacterial strain KQ11:
Substratum tap water is prepared, and NaCl scope 0%-12%, cultivates 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 the most suitable growth is 2 %, sees Fig. 6.
Three, the method for bacterial strain YJ34 fermentative production Dextranase
Contriver is to bacterial strain YJ34(
arthrobactersp. YJ34) method of fermentative production Dextranase is studied.
3.1 fermentation times produce the impact of enzyme to bacterial strain YJ34: after fermentation 12h, yield of enzyme lift velocity is very fast, and the highest to yield of enzyme during fermentation 21h, increase in time, after 24h, enzyme activity declines gradually, sees Fig. 7.
3.2 leavening temperatures produce enzyme impact to bacterial strain YJ34: carry out fermentation culture at different temperatures, and it is good that this bacterial strain produces enzyme at 15 DEG C-30 DEG C, and when 25 DEG C, bacterial strain YJ34 enzymatic production reaches the highest, sees Fig. 8.
3.3 medium pHs produce the impact of enzyme to bacterial strain YJ34: under the initial pH6.0-8.5 condition of substratum, growth produces enzyme well, and during pH7.0, bacterial strain YJ34 enzymatic production is best, sees Fig. 9.
3.4 NaCl concentration produce the impact of enzyme to bacterial strain YJ34: this bacterium can grow when NaCl 0 – 5% and produce enzyme, and when substratum NaCl concentration rises to 2%, it is the highest that strain fermentation produces enzyme, sees Figure 10.
3.5 liquid amounts are on the impact of bacterial strain YJ34 enzymatic production: 20% liquid amount condition is issued to most high enzymatic activity, sees Figure 11.
3.6 inducer concentrations produce the impact of enzyme to bacterial strain YJ34: Dextranase is a kind of inducible enzyme, dextran T20 is inductor, enzyme situation is produced by being configured to different final concentration research induction, result shows when substratum exists without inductor, bacterial strain YJ34 does not produce enzyme, most high enzymatic activity can be reached under the dextran acid anhydride T20 condition of 1% concentration, increase inductor concentration and produce enzyme activity without obvious increase, see Figure 12.
3.7 dextran enzyme activity determinations: after the dextran T70 of 10 μ L enzyme liquid and 190 μ L 3% (50 mmol/L, pH 5.5 sodium acetate buffer prepare) 35 ° of C water-bath 15 min, DNS method measures reducing sugar content.Enzyme activity unit definition (U/mL): under above-mentioned reaction conditions, the per minute catalysis enzyme amount discharged needed for 1 μm of oL maltose is a unit of activity.
Four, the character of bacterial strain YJ34 Dextranase
The preparation of 4.1 crude enzyme liquids: by Arthrobacter YJ34(
arthrobactersp. YJ34) bacterial strain inclined-plane seed is inoculated in 2216E substratum, and 25 DEG C, 180 r/min, liquid amount 20%, cultivates 16 h, obtains seed liquor; By seed liquor with 2% inoculum size be inoculated in culture medium (peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T20 1%, pH7.0), in, 180r/min, cultivates 21h for 25 DEG C, the centrifugal 5min of 10000r/min, gets supernatant liquor and is the thick enzyme of Dextranase.
4.2 enzyme operative temperatures are on the impact of enzymic activity:
React with substrate under Dextranase being placed in differing temps, measure enzyme activity, the results are shown in Figure 13, the optimum temperature of enzyme is 35 DEG C.
The action pH of 4.3 enzymes is on the impact of enzymic activity:
Enzyme liquid is carried out enzyme activity determination from the dextran solution of different pH at 35 DEG C, the results are shown in Figure 14, the activity of this enzyme of pH7.0 is the highest.
Usually, the pH in oral cavity is 6.6 ~ 7.1, and temperature is 36.3 DEG C-37.2 DEG C.The pH of the Dextranase that Arthrobacter YJ34 bacterial strain of the present invention produces is 7.0, and temperature is 35 DEG C, is suitable as oral cavity nursing agent.
Accompanying drawing explanation
Fig. 1 is electromicroscopic photograph form (× 13000) figure of bacterial strain YJ34;
Fig. 2 is the transparent loop graph that bacterial strain YJ34 is formed on primary dcreening operation flat board;
Fig. 3 is bacterial strain YJ34 systematic evolution tree;
Fig. 4 is the effect diagram that temperature grows bacterial strain YJ34; In figure: ◆ 15 ° of C, ■, 20 ° of C, ▲ 25 ° of C, × 30 ° of C, ● 35 ° of C;
Fig. 5 is the effect diagram that pH grows bacterial strain YJ34;
Fig. 6 is the effect diagram that NaCl concentration grows bacterial strain YJ34;
Fig. 7 is the effect diagram of fermentation time to bacterial strain YJ34 enzymatic production;
Fig. 8 is the effect diagram of temperature to bacterial strain YJ34 enzymatic production;
Fig. 9 is the effect diagram of pH to bacterial strain YJ34 enzymatic production;
Figure 10 is the effect diagram of NaCl concentration to bacterial strain YJ34 enzymatic production;
Figure 11 is the effect diagram of liquid amount to bacterial strain YJ34 enzymatic production;
Figure 12 is the effect diagram of inducer concentrations to bacterial strain YJ34 enzymatic production;
Figure 13 is the effect diagram of temperature to YJ34 Dextranase;
Figure 14 is the effect diagram of pH to YJ34 Dextranase;
Arthrobacter YJ34(of the present invention
arthrobactersp.) oneself is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC on March 29th, 2013, and deposit number is CGMCC N0.7385.Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
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 form the restriction to its right.
Embodiment 1, a kind of Arthrobacter YJ34(dissolved from sea
arthrobactersp.) CGMCC N0.7385.This bacterial strain has following characteristics: bacterial strain YJ34 is gram negative bacillus, without pod membrane.VP experiment is the positive, can utilizes glucose, sucrose, rhamnosyl, melibiose, pectinose, N.F,USP MANNITOL, inositol, sorbyl alcohol and amygdaloside, energy liquefy gelatin, hydrogen sulfide can be produced, edwardsiella hoshinae, does not produce beta galactosidase enzyme, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophane desaminase.Bacterial strain YJ34 growth temperature range is 4-37 DEG C, and optimum growth temperature is 25 DEG C; Growth pH scope is 5-11, the most suitable growth pH is 7.0; The NaCl concentration of growth is 0%-10%, and the NaCl concentration of the most suitable growth is 2%.
Embodiment 2, a kind of Arthrobacter YJ34 CGMCC N0.7385 as described in Example 1 ferments and produces the method for Dextranase, its step is as follows: the method for Arthrobacter YJ34 being produced low temperature Dextranase, it is characterized in that, its step is as follows: be inoculated in 2216E substratum by Arthrobacter YJ34 bacterial strain inclined-plane seed, 25 DEG C, 180 r/min, liquid amount 20%, cultivates 16 h, obtains seed liquor; By seed liquor with 2% inoculum size be inoculated in culture medium (peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T20 1%, pH7.0), in, 180r/min, cultivates 21h for 25 DEG C, the centrifugal 5min of 10000r/min, gets supernatant liquor and is the thick enzyme of Dextranase.
Claims (1)
1. Arthrobacter YJ34 produces a method for low temperature Dextranase, and it is characterized in that, its step is as follows: by CGMCC N0.7385 Arthrobacter (
arthrobactersp.) YJ34 bacterial strain inclined-plane seed is inoculated in 2216E substratum, 25 DEG C, 180r/min, liquid amount 20%, cultivates 16h, obtains seed liquor; By seed liquor with 2% inoculum size be inoculated in culture medium, 180r/min, cultivates 21h, 10000r/min centrifugal 5min, gets supernatant liquor and be the thick enzyme of Dextranase for 25 DEG C; Described culture medium is: peptone 0.5%, yeast powder 0.5%, NaCl 2%, dextran T201%, pH7.0.
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| CN110343647B (en) * | 2019-08-12 | 2021-11-02 | 江苏海洋大学 | Bacillus marinus SP-5 and dextranase thereof |
| 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 |
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