CN103289923B - Paenibacillus alginolyticus for producing 2(3H)-Naphthalenone - Google Patents
Paenibacillus alginolyticus for producing 2(3H)-Naphthalenone Download PDFInfo
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- CN103289923B CN103289923B CN201310192221.9A CN201310192221A CN103289923B CN 103289923 B CN103289923 B CN 103289923B CN 201310192221 A CN201310192221 A CN 201310192221A CN 103289923 B CN103289923 B CN 103289923B
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Abstract
The invention provides paenibacillus alginolyticus for producing 2(3H)-Naphthalenone and relates to the paenibacillus alginolyticus. The invention provides a paenibacillus alginolyticus strain for producing the 2(3H)-Naphthalenone, which has a function of killing termites, so as to lay a foundation for synthesizing a termite medicament which is toxic or has low toxins to people and livestock to prevent and treat the termites by carrying out biological fermentation by virtue of microorganisms. The paenibacillus alginolyticus for producing the 2(3H)-Naphthalenone disclosed by the invention is paenibacillus alginolyticus HDZK-BYB36 and is preserved in China Center for Type Culture Collection; the preservation date is April 9th, 2013; the preservation number is CCTCC No: M2013136. The paenibacillus alginolyticus HDZK-BYB36 disclosed by the invention can produce the 2(3H)-Naphthalenone; the compound can kill or inhibit the termites.
Description
Technical field
The present invention relates to a strain solution algin series bacillus.
Background technology
Termite (Termite) belongs to the insect of Arthropoda Insecta Pterygota Isoptera, is comparatively ancient social insect.The financial loss causing because of termite damage buildings and farm crop every year reaches more than hundred billion dollars.Existing cure of termite medicament is chemical agent, has very large toxic side effect, and can cause severe contamination to environment.Utilize endophyte biological fermentation research and development have efficient, nontoxic, the longevity of residure long, to little the killing termite biological agent and will become the main direction of following termite preventing and controlling agent research of environmental influence.
The insects such as termite can digest the lignocellulose in timber, are not they self abilities, but by the effect of the microorganism in its hindgut.If termite has been lost the effect that digests xylogen and cellulosic microorganism in enteron aisle, will be dead because of not digesting timber.Investigator has been found that the Chamaecyparis lawsoniana of Ore. has natural termite-proof, the characteristic such as anticorrosive, and from this tree, has been separated to the kind more than ten such as torreyol, nootkatone and has the biologically active substance of termite-proof effect.The trees gene that the gene of endophyte can be parasitic with it by inference transmits mutually, the endophyte of living in this particular surroundings of plant materials can produce and the same or analogous meta-bolites of host, so infer that the termite-proof compound of this class tree may be produced by endophyte.In addition, control insect pest of the plant is had to potential application to the secondary metabolite that external research has also shown endophyte of plant and exploitation is worth, and can produce that to kill the endophyte of plant of termite compound practical so filter out from natural termite-proof plant.Therefore, utilize the synthetic meta-bolites control termites of endophyte biological fermentation significant.
Summary of the invention
The invention provides strain solution algin series bacillus (Paenibacillus alginolyticus) bacterial strain, can produce nootkatone, have the function of cure of termite, in order to utilize microorganism biological fermentation, synthetic to people and animals, nontoxic or low poisoning termite medicament carrys out control termites and lays a good foundation.
The present invention produces the solution algin series bacillus of nootkatone for separating algin series bacillus (Paenibacillus alginolyticus) HDZK-BYB36, be deposited in Chinese Typical Representative culture collection center (CCTCC), preservation address is Wuhan University of Wuhan City, preservation date is on April 9th, 2013, and deposit number is CCTCC No:M2013136.
Solution algin series bacillus HDZK-BYB36 Gram-positive of the present invention, shaft-like, be catenation, thalline size be (0.7 μ m～0.8 μ m) × (2.0 μ m～3.0 μ m),, without pod membrane, produce gemma, raw in gemma ellipse, sporangiocyst is without obvious expansion, and peritrichous is movable.Solution algin series bacillus HDZK-BYB36 grows on beef-protein medium, and bacterium colony is transparent, projection is smooth, be creamy white, and subcircular, surface drying, tarnish, rapidly, without obvious gauffer shape projection, edge is irregular in growth, and center is outstanding.
Solution algin series bacillus HDZK-BYB36 of the present invention can anaerobic growth, there is mobility, catalase test is positive, Starch Hydrolysis test is positive, V.P test is positive, indole test is negative, nitrate reduction test is positive, gelatin hydrolysis test is positive, casein hydrolysis is positive, can utilize Citrate trianion, can not utilize propionic salt, urea, can not be hydrolyzed tyrosine and arginine, can decomposition glucose, cyclodextrine, wood sugar, pectinose and N.F,USP MANNITOL produces acid.
Solution algin series bacillus HDZK-BYB36 of the present invention can grow on beef-protein medium, can in the beef-protein medium that contains 5g/LNaCl, grow, also can in the beef-protein medium that contains 0.001% (quality) lysozyme, grow, can chromogenesis in late stage of culture.The optimum growth temperature of separating algin series bacillus HDZK-BYB36 is 37 DEG C, and minimum growth temperature is that 15 DEG C, maximum growth temperature are 50～55 DEG C.
Solution algin series bacillus HDZK-BYB36 of the present invention analyzes by 16S rDNA sequence alignment, and has high homology between various in series bacillus genus (Paenibacillus), and homology is more than 99%.By determining that in conjunction with morphological features, growth conditions, Physiology and biochemistry qualification result separating algin series bacillus HDZK-BYB36 belongs to series bacillus genus (Paenibacillus), be to separate algin series bacillus (Paenibacillus alginolyticus).
Solution algin series bacillus HDZK-BYB36 of the present invention can produce nootkatone, and this compound can kill or suppress termite.Solution algin series bacillus HDZK-BYB36 is inoculated into fermentation culture 4d in beef extract-peptone liquid nutrient medium, after fermentation ends, supernatant concentration is made to fermented liquid concentrated solution, fermented liquid concentrated solution can be good at suppressing termite body endosymbiosis bacterium, by the filter paper feeding termite of soaking fermentation liquid, can effectivelyly kill the activity of termite or inhibition termite.
Solution algin series bacillus of the present invention (Paenibacillus alginolyticus) HDZK-BYB36, belong to series bacillus and belong to (Paenibacillus), be deposited in Chinese Typical Representative culture collection center (CCTCC), preservation address is Wuhan University of Wuhan City, preservation date is on April 9th, 2013, and deposit number is CCTCC No:M2013136.
Brief description of the drawings
Fig. 1 is that solution algin series bacillus HDZK-BYB36 of the present invention amplifies the transmission electron microscope photo of 20500 times; Fig. 2 is that the 16S rDNA sequence of the close bacterial strain of including in solution algin series bacillus HDZK-BYB36 of the present invention and GenBank is carried out the constructed phylogenetic tree of sequence analysis; Fig. 3 is the GC-MS collection of illustrative plates of the nootkatone of purifying from separate algin series bacillus HDZK-BYB36 fermented liquid concentrated solution extract; Fig. 4 be from separate algin series bacillus HDZK-BYB36 fermented liquid concentrated solution extract, purify 2, the GC-MS collection of illustrative plates of 6-xylenol.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment is produced the solution algin series bacillus of nootkatone for separating algin series bacillus (Paenibacillus alginolyticus) HDZK-BYB36, be deposited in Chinese Typical Representative culture collection center (CCTCC), preservation address is Wuhan University of Wuhan City, preservation date is on April 9th, 2013, and deposit number is CCTCC No:M2013136.
Present embodiment solution algin series bacillus (Paenibacillus alginolyticus) HDZK-BYB36 Gram-positive, shaft-like, be catenation, thalline size be (0.7 μ m～0.8 μ m) × (2.0 μ m～3.0 μ m),, without pod membrane, produce gemma, raw in gemma ellipse, sporangiocyst is without obvious expansion, and peritrichous is movable.Separate the transmission electron microscope photo of 20500 times of algin series bacillus HDZK-BYB36 amplifications as shown in Figure 1.Solution algin series bacillus HDZK-BYB36 grows on beef-protein medium, and bacterium colony is transparent, projection is smooth, be creamy white, and subcircular, surface drying, tarnish, rapidly, without obvious gauffer shape projection, edge is irregular in growth, and center is outstanding.
Carry out Physiology and biochemistry qualification with reference to " uncle outstanding Bacteria Identification handbook " the 8th edition and " common bacteria system identification handbook " to producing nootkatone solution algin series bacillus HDZK-BYB36 (Paenibacillus alginolyticus): present embodiment solution algin series bacillus HDZK-BYB36 can anaerobic growth, there is mobility, catalase test is positive, Starch Hydrolysis test is positive, V.P test is positive, indole test is negative, nitrate reduction test is positive, gelatin hydrolysis test is positive, casein hydrolysis is positive, can utilize Citrate trianion, can not utilize propionic salt, urea, can not be hydrolyzed tyrosine and arginine, can decomposition glucose, cyclodextrine, wood sugar, pectinose and N.F,USP MANNITOL produce acid.
Present embodiment solution algin series bacillus HDZK-BYB36 can grow on beef-protein medium, can in the beef-protein medium that contains 5g/L NaCl, grow, also can in the beef-protein medium that contains 0.001% (quality) lysozyme, grow, can chromogenesis in late stage of culture.The optimum growth temperature of separating algin series bacillus HDZK-BYB36 is 37 DEG C, and minimum growth temperature is that 15 DEG C, maximum growth temperature are 50～55 DEG C.
Embodiment two: separate and obtain in the bark of present embodiment solution algin series bacillus (Paenibacillus alginolyticus) HDZK-BYB36 by Chamaecyparis lawsoniana (Chamaecyparis lawsoniana).Separation method carries out according to the following steps: one, Chamaecyparis lawsoniana (Chamaecyparis lawsoniana) bark is first used to aseptic water washing (removing the impurity such as the dust on bark surface), be cut into the segment of 2.0cm × 1.0cm × 0.5cm by sterile scissors, process trees segment 1～2min with the mercuric chloride of mass concentration 0.1% again, outwell mercuric chloride, then clean and remove residual mercuric chloride 3 times with distilled water; Two, the segment access water agar of processing is soaked in juice substratum, within 3～5 days, grow bacterium; Three, adopt three zoning collimation methods that the bacterium that grows is inoculated on the beef-protein medium flat board that contains 100 μ g/mL nystatin, then constant temperature culture 2～3 days under 37 DEG C of conditions; Four, adopt existing normal fermentation method and fermentation condition to ferment, by the anti-microbial activity of fermented liquid with kill termite activity analysis, select the bacterium of best results according to result, be the solution algin series bacillus HDZK-BYB36 of present embodiment.
Described in present embodiment, water agar soaks the preparation process of juice substratum and is: 5g timber is cut into small pieces with sterilized scalper, add 100mL water boil 30min, obtain trees and soak juice, get 20mL trees and soak juice, add 15～20g agar, add water to 1000mL, in 121 DEG C of high pressure steam sterilization 30min, point install in culture dish for subsequent use.
The solution algin series bacillus HDZK-BYB36 that separation screening is obtained carries out Molecular Identification, carries out according to the following steps: extract total DNA of bacterial strain, adopt the 16S rDNA universal primer of bacterium, carry out pcr amplification taking genomic dna as template.Then utilize glue to reclaim test kit (purchased from Dalian treasured biotechnology company limited) and reclaim purified pcr product; Afterwards, clone, transform, screening positive clone daughter colony is entrusted the order-checking of Shanghai Sheng Gong Bioisystech Co., Ltd after enlarged culturing.
The 16SrDNA sequence length of separating algin series bacillus (Paenibacillus alginolyticus) HDZK-BYB36 is 1516bp, the sequence number that its sequence is committed to GenBank acquisition is KC874838,16S rDNA sequence in sequencing result and GenBank is carried out sequence analysis, build phylogenetic tree as shown in Figure 2, to determine the race relation of bacterial strain.Homology analysis result shows, this sequence and series bacillus belong between various in (Paenibacillus) high homology, and homology is more than 99%.By determining that in conjunction with morphological features, growth conditions, Physiology and biochemistry qualification result separating algin series bacillus HDZK-BYB36 belongs to series bacillus genus (Paenibacillus), be to separate algin series bacillus (Paenibacillus alginolyticus).
PCR primer by from Shanghai raw work synthetic, other reagent is all purchased from the precious biotechnology company limited in Dalian.
(1) to present embodiment solution algin series bacillus HDZK-BYB36 fermented liquid concentrated solution kill termite activity evaluation, concrete grammar is as follows:
The preparation method of present embodiment solution algin series bacillus HDZK-BYB36 fermented liquid concentrated solution is: by the trees endophyte of present embodiment separation and purification gained---separate algin series bacillus HDZK-BYB36 and be inoculated in beef extract-peptone liquid nutrient medium, under 37 DEG C, 100r/min condition, 4d is cultivated in shaking; Nutrient solution, with 3500r/min centrifugation 10～15min, is got to supernatant liquor, the aseptic Erlenmeyer flask of impouring, under aseptic condition, under 40 DEG C of waters bath with thermostatic control, 150r/min condition, be concentrated into 1/10th of its volume with Rotary Evaporators, put into 4 DEG C of refrigerators and preserve, obtain fermented liquid concentrated solution.
Adopt the anti-microbial activity of agar diffusion method evaluation solution algin series bacillus HDZK-BYB36 fermented liquid concentrated solution to termite parachorium bacterium (indicator), specifically carry out with reference to the method for Okeke et al.2001 report, be recorded in Okeke, M.I., C.U.Iroegbu, E.N.Eze, A.S.Okoli, and C.O.Esimone.2001.Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity.J Ethnopharmacol.78:119-127 (Okeke, M.I., C.U.Iroegbu, E.N.Eze, A.S.Okoli, the evaluation of the anti-microbial activity of and C.O.Esimone.Landolphia owerrience root extract. ethnopharmacology magazine .2001, 78:119-127).Test-results shows to separate algin series bacillus HDZK-BYB36 fermented liquid concentrated solution termite parachorium bacterium is had to good restraining effect.
The termite feeding experiment that kills termite activity evaluation employing of present embodiment solution algin series bacillus HDZK-BYB36 fermented liquid concentrated solution, carry out as follows: solution algin series bacillus HDZK-BYB36 fermented liquid is concentrated by 0,2,10 times, soak respectively filter paper (WhatmanNo.3 with different cycles of concentration fermented liquids,), filter paper weight is that 1g, diameter are 8.5cm; In contrast with the filter paper with distilled water processing meanwhile.Afterwards, the every filter paper of processing is laid in to diameter is 9cm, highly in the culture dish of 1.5cm, on filter paper, puts 300 termites alive, covers culture dish lid.Then, culture dish being placed on to temperature is in 26.5 DEG C, the incubator of relative humidity 80%.Regularly in each culture dish, drip.By calculating termite mortality analysis strain HD ZK-BYB36 fermented liquid termite-proof activity, observe 14d.Test-results (as shown in table 1) shows: strain HD ZK-BYB36 fermented liquid can kill termite, 0,2,10 times of concentrated solution group termite mortality ratio of the strain HD ZK-BYB36 ferment liquid all utmost point is significantly higher than control group termite mortality ratio (p < 0.01), and 2,10 times of concentrated solution groups termite mortality ratio in the time of 14d of strain HD ZK-BYB36 fermented liquid is 100%.Not significantly (p > 0.05) of termite mortality difference when 0,2,10 times of concentrated solution group 14d of strain HD ZK-BYB36 fermented liquid.The explanation of this test-results is separated in algin series bacillus HDZK-BYB36 fermented liquid and is contained the compound that kills termite.
Table 1 is separated algin series bacillus HDZK-BYB36 fermented liquid termite-proof activity
(2) extract separating algin series bacillus HDZK-BYB36 fermented liquid, detailed process is: separate algin series bacillus HDZK-BYB36 fermented liquid first through macroporous resin Image processing, obtain crude extract; Then crude extract is used n-hexane extraction (extracting process is: 25g crude extract adds 500mL normal hexane to be placed on shaking table, 150-180r/min, 24h) again; Reclaim under reduced pressure normal hexane afterwards, obtains enriched material and is the extract of separating the synthetic meta-bolites of algin series bacillus HDZK-BYB36 biological fermentation, 4 DEG C of preservations.Extract is carried out to gas chromatography-mass spectrum (GC-MS), and the GC-MS collection of illustrative plates obtaining as shown in Figure 3.As shown in Figure 3, separate in algin series bacillus HDZK-BYB36 fermented liquid and contain compound nootkatone 2 (3H)-Naphthalenone, molecular weight 218, structural formula is C
15h
22o.Studies have reported that at present and proved that nootkatone can kill termite (McDaniel, C.A.1989.Major termiticidal components of heartwood of Port-Orford-Cedar, the main termite-proof composition of Chamaecyparis lawsoniana (A.Murr.) Parl.Material und Organismen.24:1-15 Chamaecyparis lawsoniana heartwood, material and the .McDaniel of biological tissue, C.A., J.A.Klocke, and M.F.Balandrin.1989.Major antitermitic wood extractive components of Eastern Red Cedar, the main termite-proof tree extractive composition of Juniperus virginiana.Material und Organismen.24:301-313. North America Chinese juniper, material and biological tissue), illustrate that separating algin series bacillus HDZK-BYB36 can produce the compound nootkatone that kills termite.In addition, separate algin series bacillus HDZK-BYB36 and also can produce 2,6-xylenol (Fig. 4), molecular weight 122, structural formula is C
8h
10o.2,6-xylenol is mainly used in the production of polyphenylene oxide resin, agricultural chemicals, polyester and polyether resin, is also the raw material of antiarrhythmic drug mexiletine.2，6-xylenol can make antiarrhythmic drug mexiletine through hydroxypropylation, oxidation, condensation, hydrogenation and salify.
Claims (1)
1. the solution algin series bacillus of nootkatone is produced in a strain, it is characterized in that this bacterial strain is for separating algin series bacillus (Paenibacillus alginolyticus) HDZK-BYB36, be deposited in Chinese Typical Representative culture collection center, preservation address is Wuhan University of Wuhan City, preservation date is on April 9th, 2013, and deposit number is CCTCC No:M2013136.
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| CN105002220B (en) * | 2015-07-27 | 2018-02-13 | 黑龙江大学 | For the preparation method for the endophyte mixed biologic tunning for preventing and treating termite |
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