CN1712518A - Fungus and method for preparing vanillin from isoeugenol converted by microorgan - Google Patents

Abstract

Vanillin bacteria from microbial conversion isobutyl aromatic phenol and its production are disclosed. The process is carried out by fermentation culturing under optimizing condition, converting isobutyl aromatic phenol for 24-96hrs from fermentation liquor or free cell or fixed cell with conversion liquor containing vanillic acid 2-4g/L, conversion reacting for 72hrs in water-organic solvent double-phase system with vanillin concentration 32.5g/L in organic phase, adsorbing vanillin in conversion liquor from resin, extracting from ethyl acetate and obtaining light yellow crystalline vanillin powder. Extraction rate reaches 87%, purity of product reaches 98.1% and recovery rate reaches 93.4%.

Description

The microbial transformation isoeugenol prepares the bacterial classification and the method for Vanillin

Technical field

The present invention relates to a strain and from soil, screen bacillus fusiformis (Bacillus fusiformis) CGMCC1347 (SW-B9) and the cultivation and fermentation thereof that obtains, and be used to transform the method that isoeugenol prepares Vanillin.

Background technology

Vanillin is one of most widely used spices in the industry, is used for foodstuffs industry in a large number, and the main raw material that can be used as fragrance modification and fixation is used for food, toothpaste, perfumed soap, tobacco; In medication chemistry,, can be used for making the common drug of treatment hypertension, heart trouble, tetter and elimination halitosis, diuresis as important material or intermediate; In chemical industry, can be used as chemical assistant, be used for the anti-setting agent and the Ni of plastics, Cr, the brightening agent of metals such as Cd; In agriculture production, Vanillin can be used as crop yield agent and ripener, and preparation weedicide of using and insect attractant etc., is in great demand.Vanillin is at present mainly by chemical process preparation, but is not natural perfume with the Vanillin that chemical synthesis makes.Natural herb aldehyde can extract from vanillic colored pod, but the natural herb aldehyde amount of producing with the method that plant tissue extracts is few and the valency height can not satisfy growing demand.

Along with countries in the world are more and more paid attention to food safety, increasing to the demand of natural herb aldehyde.Natural perfume is meant by the animals and plants material and obtains through physics (comprising distillation, solvent extraction) method, enzyme process or microbial process, can be by being used for human consumption's material after traditional food-processing method (comprise drying, bake, ferment) processing.According to Europe and US-legislation, the material that utilizes animal and plant resource to obtain by physical method, enzyme process or microbial method just can be called crude substance (Muheim Andrea.US6,235,507.2001).Vanillin with biological process is produced belongs to natural product, can biological degradation, meet the consumer psychology that the human consumer pursues natural product.Therefore, utilizing conversion technology to produce biological vanillin food grade,1000.000000ine mesh, is a kind of effective, up-and-coming alternative method.

History with the research of the biological Vanillin of biotransformation method production has had surplus in the of ten year has adopted multiple biomaterial in the research, comprise fungi (Laurence Lesage Meessen et al.US 05866380,1999; US06162637.2000), bacterium (Rabenhorst Jurgen ﹠amp; Hopp Rudolf.US 6133003,2000), actinomycetes (Audrs ﹠amp; More.WO 9634971,1996; Rabenhorst ﹠amp; Hopp.EP0761817,1997; Muller.EP 0885968,1998) genetic engineering bacterium (Overhage J, et al.Journal ofBiotechnology, 2000), vegetable cell (Podstolski A ﹠amp; Havkin-Frenkel D.WO 9903975,1999) and extract the enzyme (Frost JW.WO 0017319,2000) obtain.The investigator has carried out extensive studies on cell, zymetology, gene level, and has obtained a lot of achievements in research.Along with going deep into of research, many investigators begin to be devoted to realize the suitability for industrialized production of biological Vanillin, and existing successful example, as French Laurence Lesage-meessen (Lesage-Meessen, L., et al.J.Biotechnol, 1996) research utilizes the two step method of forulic acid as substrate, earlier forulic acid is converted into vanillic acid with aspergillus niger (Aspergillus niger), use bright red samguineus (Pycnporus cinnabarnus) or Phanerochaete chrysosporium (Phanerochaete chrysosporium) that vanillic acid is reduced into Vanillin again, in fermentation culture, add soybean phospholipid, growth promoters such as vitamin, 6～7 days, the concentration of Vanillin reaches 1,575mg/L.Present inventor's research group has also obtained success (the great .CN 1421523A of Sun Zhi, 2002) in this respect.Prepare in the process of novel method of natural herb aldehyde at the research starting raw material, we find, because the price of forulic acid is higher, therefore the Vanillin price of producing in this way is also than higher, and Eugenol, isoeugenol can extract from Syzygium aromaticum stem oil on technical scale, cheap, obtain easily.And the research (Markus Paul Henry.EP 0542348A2,1993 that also have some to carry out bio-transformation before nearly 10 years successively with microorganism or enzymatic conversion Eugenol or isoeugenol; Mane J ﹠amp; Zucca J.WO 9402621,1994), present inventor's research group has also carried out exploring (the great .CN 03157579.X of Sun Zhi, 2003) to utilizing the thick enzymatic conversion isoeugenol of soybean to generate the Vanillin method.

Having reported many methods with microbial method or Production by Enzymes Vanillin in the past 10 years, all is by microorganism or enzyme suitable precursor to be converted into Vanillin generally.Available precursor has forulic acid, Eugenol, isoeugenol, curcumine etc., and production concentration and transformation efficiency are very low usually.2000, people (ShimoniE such as Shimoni E, et al.Journal of Biotechnology.2000) from soil, is separated to a bacillus subtilis, it can transform isoeugenol with 12.4% molar yield is Vanillin, production concentration is 0.61g/L, and its acellular extraction liquid can produce the 0.9g/L Vanillin.In the microbial metabolism system, be difficult to obtain the Vanillin of high yield, major cause is because Vanillin has cytotoxicity, its concentration surpasses 1g/L will stop microorganism growth, and, Vanillin is a metabolic intermediate, and the multiple pathways metabolism that exists in microflora easily further is converted into VANILLYL ALCOHOL MIN 98 or vanillic acid etc.Therefore, screen a kind of suitable microbial strains, develop suitable transformation system and remove the key that the further conversion of product inhibition and product is a raising Vanillin concentration.

Summary of the invention

The objective of the invention is to filter out and a kind ofly can effectively transform new bacterial strain bacillus fusiformis (Bacillus fusiformis) CGMCC1347 that isoeugenol is a Vanillin, and provide a kind of new bioconversion reaction to generate the method for Vanillin.

The microbial strains SW-B9 that obtains is screened in a strain of the present invention from soil, it is characterized by and to tolerate high concentration of substrate, isoeugenol can be converted into Vanillin, be bacillus fusiformis Bacillus fusiformisSW-B9 (CGMCC 1347) through identifying this bacterial strain.

The cultural method of microbial strains Bacillus fusiformis SW-B9 of the present invention may further comprise the steps:

(1) slant medium is the glucose nutrient agar;

(2) seed culture medium consists of: glucose 0.1%～1%; Corn steep liquor 1%～10%; Urea 0.1%～2%; K ₂HPO ₄3H ₂O 0.01%～0.5%; KH ₂PO ₄0.01%～0.5%, MgSO ₄7H ₂O 0.01%～0.5%; PH 7.0;

(3) fermention medium consists of: corn steep liquor 1～10%; K ₂HPO ₄3H ₂O 0.1%～0.5%; Urea 0.1%～0.5%; MgSO ₄7H ₂O 0.1%～0.5%; PH 7.5;

(4) cultivation with fermentation condition is: 37 ℃ of temperature, shaking speed 180r/min.

A kind of bioconversion method of producing Vanillin of the present invention may further comprise the steps:

(1) microorganism strains is cultivated resulting fermented liquid, wet thallus, freeze-dried vaccine powder or immobilized cell as biocatalyst cell;

(2) isoeugenol and water are mixed with mixed solution as bio-transformation substrate reactions liquid in 0.1%～99.9% ratio;

(3) biocatalyst cell that (1) is obtained, the substrate reactions liquid that adds (2) carries out conversion reaction, conversion reaction conditions is: the concentration of substrate scope is 0.1%～99.9%, the microorganism wet thallus is 10～80g/L to the consumption of substrate solution, initial pH 4.0～8.0, the conversion reaction temperature is 20～42 ℃, and the conversion reaction time is 24～96h;

(4) purify with refining.

Described purification is that the reaction solution organic phase is separated with making with extra care, adding D202, D031, HZ004, HZ801, HD-8 or HD-2 resin adsorbs, re-use organic solvent polymeric adsorbent is carried out wash-out, the eluted product Vanillin is refining with crystallization process, and the unconverted substrate isoeugenol reclaims and is used for transforming once more.

It below is the detailed description of the inventive method.

The screening of microorganism strains and evaluation:

The present invention gathers soil sample from the soil layer that is polluted by different spices, the various soil samples that obtain are suspended in carry out enrichment culture in the 0.9%NaCl solution, and continuously switching does not contain the screening culture medium 3～4 times of glucose, be coated with the isoeugenol is that the selectivity flat board of sole carbon source carries out primary dcreening operation again, separate and obtain and on the selection flat board, to go to reaction solution conversion, the qualitative Vanillin of thin plate chromatography (TLC method) by the growth bacterial strain.The bacterial strain that primary dcreening operation is obtained further carries out multiple sieve, and it is carried out the Vanillin degradation experiment, measures Vanillin concentration with the HPLC standard measure, and finishing screen is selected aimed strain B9.

The B9 bacterial strain that relates among the present invention can be grown on the substratum that with 1% isoeugenol is sole carbon source.In nutrient agar, form white colony, Gram-positive, the VP reaction negative, carry out 16S rDNA sequential analysis by the 8th edition physio-biochemical characteristics evaluation of uncle Jie Shi handbook and fine works molecular biology experiment guide, determine that it belongs to bacillus fusiformis Bacillus fusiformis, intend called after Bacillus fusiformis SW-B9.This bacterial strain has been kept at Zhong Guan-cun, BeiJing, China China Committee for Culture Collection of Microorganisms common micro-organisms preservation center, preservation CGMCC No.1347 on April 8th, 2005.

The cultivation of microorganism strains and fermentation:

Slant medium is conventional glucose nutrient agar (a LB substratum).Seed culture medium consists of: glucose 0.1%～1%; Corn steep liquor 1%～10%; Urea 0.1%～2%; K ₂HPO ₄3H ₂O 0.01%～0.5%; KH ₂PO ₄0.01%～0.5%, MgSO ₄7H ₂O 0.01%～0.5%; PH 7.0; The seed culture condition: with 250mL triangle bottled 25～70mL substratum, sterilize according to a conventional method, cool off, inoculate afterwards in 30～45 ℃, 180r/min shaking table shaking culture 12～24 hours is as seed culture fluid.Fermention medium consists of: corn steep liquor 1～10%; K ₂HPO ₄3H ₂O 0.1%～0.5%; Urea 0.1%～0.5%; MgSO ₄7H ₂O 0.1%～0.5%; PH 7.5; Fermentation condition: adorn 25～70mL substratum in the 250mL triangular flask, sterilization according to a conventional method, cooling, the inoculation back is in 30～45 ℃, 180r/min shaking table shaking culture is added the 1mmol/L Vanillin and is continued to cultivate 8～20 hours as inductor after 12～24 hours, cell culture fluid can be directly used in conversion, or obtain wet thallus after centrifugal, or make lyophilized powder and add reaction solution and transform, also available sodium alginate, carrageenin, gelatin, carrier embeddings such as chitin, perhaps use resin covalent attachment or absorption, method immobilizations such as glutaraldehyde cross-linking make immobilized cell.Carry out bio-transformation with this as biological catalyst, immobilized cell is reuse repeatedly, carries out repeatedly bioconversion reaction.

Bioconversion reaction:

Fermented liquid, wet thallus (free cell), freeze-dried vaccine powder or the immobilized cell etc. that make with aforesaid method are as biological catalyst, with the isoeugenol is substrate, content is 0.1%～99.9%, the humidification biomass is 10～80g/L of substrate solution, initial pH 4.0～8.0, the conversion reaction temperature is 20～42 ℃, and the conversion reaction time is 24～96h.The conversion fluid of gained is measured the product amount with high pressure liquid chromatographic analysis (HPLC) with this understanding.

Immobilized cell repeatedly in batches conversion method be after a collection of conversion finishes, to filter, reclaim immobilized cell, as the catalyzer of conversion reaction next time, the substrate that adds new preparation continues to transform.Immobilized cell conversion test in batches shows and can carry out repeatedly repeatedly continuously.

Can improve the method etc. of substrate solubleness with adding solvent or solubility promoter, improve the bioconversion reaction effect.Add tensio-active agent and can increase the contact of substrate, can strengthen the bioconversion reaction effect with cell.

Carry out water-solvent biphasic reaction with the substrate isoeugenol as the organic phase solvent, the product that generates in time changes organic phase over to, both can avoid the restraining effect of product to microbial cells, it is unstable and cause further transforming and generate the by product vanillic acid in the aqueous solution to reduce product again.

Vanillin and isoeugenol measuring method:

Reference report method (what new Asia etc., assay laboratory, 1999) is surveyed Vanillin with thiobarbituricacid colorimetry (TBA method).Tlc (TLC method) can be measured Vanillin and isoeugenol simultaneously, developping agent: normal hexane-chloroform-anhydrous diethyl ether-Glacial acetic acid 4: 3: 2: 0.1 (v/v/v), developer: iodine steam promptly earlier develops the color Vanillin and different syringic aldehyde with iodine vapor in conjunction with 2,4 dinitrophenyl hydrazine simultaneously, use 2 again, the 4-dinitrophenylhydrazine develops the color specifically to get rid of the interference of other impurity, the scanning of the single wavelength reflection of the condition of scanning: 228nm square tooth, slit 0.4 * 0.4mm Vanillin, SX=3, sensitivity is medium.High performance liquid chromatography (HPLC) also can be measured Vanillin and isoeugenol simultaneously, stationary phase: Lichrospher 100RP-18,5 μ m, 250mm * 4mm, moving phase: with the glacial acetic acid aqueous solution of methyl alcohol and 0.01%, methyl alcohol/0.01% glacial acetic acid aqueous solution 65/35, non-gradient elution: flow velocity 1ml/min ultraviolet 270nm detects.

The separation of Vanillin, extraction:

Separation, extracting method are with the resin absorption Vanillin, then with organic solvent wash-out isoeugenols such as normal hexanes, the unconverted substrate isoeugenol are separated, at last with the eluent ethyl acetate Vanillin with the product Vanillin.

Contain the high density Vanillin in the organic phase isoeugenol of above-mentioned conversion reaction gained conversion fluid, make two phase stratification and tell organic phase (isoeugenol layer) conversion fluid is static, add 1～10 times of volumes of deionized water, add resin absorption such as D202 or D031, HZ004, HZ801, HD-8, HD-2 again and be dissolved in the Vanillin of isoeugenol, also sticked simultaneously the part isoeugenol on the resin, leach resin, with the organic solvent of 1～10 times of resin volume such as wash-outs such as normal hexane, chloroform 1～5 time, extraction unconverted substrate isoeugenol makes it to separate with the product Vanillin.Rotary evaporation reclaims organic solvent; The isoeugenol that recovery obtains can be recycled continuation and be used for the next batch conversion reaction as substrate.

Reclaimed the resin behind the isoeugenol, used the wash-outs such as ethyl acetate, dehydrated alcohol 1～5 time of 1～10 times of resin volume again, filtered and collect filtrate, added method such as anhydrous sodium sulphate and dewater, solvent is reclaimed in vacuum-evaporation.Vacuum concentration near buttery concentrated slurry drying, is promptly got light brown powder Vanillin (crude product).The Vanillin crude product is added 10～100 ℃ of dissolvings of about 1～10 times of amount pure water, be cooled to room temperature, the brown oil of separating out is earlier removed, be cooled to 0～5 ℃ of crystallization again, suction filtration, 30～60 ℃ of dryings promptly obtain refining Vanillin finished product.

The bioconversion method of production Vanillin of the present invention may further comprise the steps:

(1) screens the bacterial strain bacillus fusiformis SW-B9 of acquisition as bacterial classification with the present invention;

(2) the bacillus fusiformis SW-B9 intact cell that cultivate, fermentation obtains is as biological catalyst;

(3) with isoeugenol as bio-transformation substrate and organic phase;

(4) in water-isoeugenol diphasic system, carry out bioconversion reaction, generate Vanillin;

(5) unconverted substrate isoeugenol recyclable being used for transforms once more;

(6) the product Vanillin carries out separation and Extraction with resin absorption, normal hexane and ethyl acetate stepwise elution.

Biotransformation method of the present invention has the following advantages with respect to traditional chemical synthesis or the method extracted from the chinese cymbidium platymiscium: 1. the harmful substance contents of product Vanillin is extremely low, safe without toxic side effect; 2. can carry out scale operation, not be subjected to seasonal effect; 3. production operation is easy, product recovery rate height; 4. compare cost with the vanilla pods extraction method and reduce greatly, even also have substantial degradation than microbial transformation forulic acid method cost; 5. the microorganism cells as biological catalyst is easy to cultivate and safety non-toxic bioconversion reaction mild condition, environmental friendliness.

Characteristics of the present invention are to have isolated the toxic bacterial strain of plant height degree tolerance substrate, this bacterial strain can efficiently transform isoeugenol and generate Vanillin, product purity height after the conversion, metabolic by-prods vanillic acid, methoxyl group quinhydrones, VANILLYL ALCOHOL MIN 98 equal size are very low, unconverted isoeugenol reclaims easily, can be used for transforming once more, to the total molar yield height of substrate.Conversion process of the present invention can directly add isoeugenol with the microorganism cells fermented liquid and transform, and also can add the isoeugenol substrate solution with the lyophilized powder for preparing or its immobilized cell and transform, and immobilized cell can be recycled repeatedly.

Description of drawings

Fig. 1 is a process flow sheet of the present invention.

Embodiment

Below be that bacterial strain bacillus fusiformis SW-B9 transforms the embodiment that isoeugenol prepares Vanillin, be used to illustrate method of the present invention, but the present invention be not limited to listed several examples.

Embodiment 1

Gather the soil sample of being polluted by different spices from Wuxi spices company limited, the various soil samples that obtain are respectively taken by weighing about 10g, be suspended in respectively in the 0.9%NaCl solution, remove bigger impurity particle, be seeded to enrichment medium (isoeugenol 0.2% with 8 layers of filtered through gauze; Glucose 0.5%; Yeast extract paste 0.5%; Peptone 0.5%; K ₂HPO ₄3H ₂O 1.4%; KH ₂PO ₄0.5%; MgSO ₄7H ₂O 0.2%; PH 7.0), behind the cultivation 24h, the screening culture medium of transferring continuously (not containing glucose, other same enrichment mediums) 4 times, coating selectivity flat board (the same screening culture medium of component contains agar 2%).Through primary dcreening operation, be divided into from obtaining 14 strain bacterium and can on the flat board that with the isoeugenol is sole carbon source, grow, be genus bacillus through the microscopy observation, respectively it is gone to reaction solution and transform 3 days, through the legal property of TLC Vanillin, find overwhelming majority energy generating portion Vanillin, choose 14 strains and further carry out multiple sieve, the oxalaldehyde degradation experiment of holding or participate in a prayer service at a temple of going forward side by side, measure Vanillin concentration with TBA and HPLC standard measure, the results are shown in Table 1 and table 2, comprehensively produce Vanillin ability and Vanillin degraded situation, finally selected SW-B9 is as starting strain.

Each bacterial strain of table 1 transforms isoeugenol and generates the Vanillin ability

Bacterial strain	??SW-B1	??SW-B2	??SW-B3	??SW-B4	??SW-B5	??SW-B6	??SW-B7
								Vanillin (g/L)	??0.54	??1.08	??1.01	??0.36	??1.09	??0.36	??0.59
Bacterial strain	??SW-B8	??SW-B9	??SW-B10	??SW-B11	??SW-B12	??SW-B13	??SW-B14
								Vanillin (g/L)	??1.10	??1.17	??0.81	??0.77	??0.78	??0.85	??0.41

Each strains for degrading Vanillin situation of table 2

Bacterial strain	Contrast *	??SW-B1	??SW-B2	??SW-B3	??SW-B4	??SW-B5	??SW-B6	??SW-B7
									Residual Vanillin %	??96.13	??99.71	??90.41	??83.98	??93.75	??95.89	??98.99	??96.61
Bacterial strain	??SW-B8	??SW-B9	??SW-B10	??SW-B11	??SW-B12	??SW-B13	??SW-B14
									Residual Vanillin %	??90.41	??95.26	??100.00	??98.99	??94.70	??98.04	??100.00

^*Contrast: do not connect bacterial classification, other reaction conditionss are identical.

Embodiment 2

SW-B9 bacterial strain to screening carries out the physio-biochemical characteristics evaluation by uncle Jie Shi handbook the 8th edition, the results are shown in Table 3.And carry out 16S rDNA by the method for fine works molecular biology experiment guide and identify, the results are shown in Table 4.Experimental result can determine that it belongs to bacillus fusiformis (Bacillus fusiformis), intends called after Bacillus fusiformis SW-B9 (CGMCC1347).

Table 3 physio-biochemical characteristics qualification result synopsis

Physio-biochemical characteristics	??SW-B9	Subtilis	Bacillus fusiformis	Bacillus sphaericus
					Form: cell Dan Sheng	??-	??+	??-	??+

The circular gemma end of gemma produced amylolysis tyrosine hydrolysis hippuric acid salt hydrolysis gelatin 1 μ g/mL tetracyclin resistance, the 2 μ g/mL tetracyclin resistance D-Glucoses that liquefy produce sour D-sucrose and produce sour D-wood sugar and produce sour D-lactose and produce sour D-Maltose and produce the red test indoles test of 50 ℃ of growths of 37 ℃ of growths of 30 ℃ of growths of 17 ℃ of growths of 5 ℃ of growths of acid pH6.8 growth pH5.7 growth 2%NaCl growth 5%NaCl growth 7%NaCl growth 10%NaCl growth Gram’s staining anaerobism growth glucose aerogenesis VP test methyl catalase urase nitrate reduction

??+ ??+ ??- ??- ??- ??+ ??- ??- ? ? ??- ??- ? ??- ? ??- ? ??- ? ??- ? ??+ ??+ ??+ ??- ??+ ??+ ??+ ??+ ??+ ??- ??+ ??- ??- ??- ??- ??- ? ??+ ??+ ??-

??- ??- ??+ ??- ??- ??+ ??+ ??+ ? ? ??+ ??+ ? ??+ ? ??- ? ??+ ? ??- ? ??+ ??+ ??+ ??+ ??+ ??+ ??+ ??+ ??+ ??+ ??+ ??- ??- ??+ ??- ??- ? ??+ ??- ??+

??+ ??D ??- ??- ??- ??+ ??- ??- ? ? ??- ??D ? ??- ? ??- ? ??- ? ??- ? ??+ ??+ ??+ ??- ??+ ??D ??D ??D ??+ ??- ??+ ??- ??- ??- ??- ??- ? ??D ??D ??-

??+ ??D ??- ??- ??D ??+ ??+ ??D ? ? ??- ??- ? ??- ? ??- ? ??- ? ??- ? ??+ ??+ ??+ ??- ??+ ??D ??+ ??D ??- ??- ??+ ? ??- ??- ??- ??- ? ??D ??- ??-

+, positive more than 90%;-, negative more than 90%; D, 11%～89% positive.

The 16S rDNA sequence (1525bp) of table 4 SW-9 bacterial strain

??1????GACGAACGCT?GGCGGCGTGC?CTAATACATG?CAAGTCGAGC?GAACAGAGAA?GGAGCTTGCT ??61???CCTTCGACGT?TAGCGGCGGA?CGGGTGAGTA?ACACGTGGGC?AACCTACCTT?ATAGTTTGGG ??121??ATAACTCCGG?GAAACCGGGG?CTAATACCGA?ATAATCTGTT?TCACCTCATG?GTGAAACACT ??181??GAAAGACGGT?TTCGGCTGTC?GCTATAGGAT?GGGCCCGCGG?CGCATTAGCT?AGTTGGTGAG ??241??GTAACGGCTC?ACCAAGGCGA?CGATGCGTAG?CCGACCTGAG?AGGGTGATCG?GCCACACTGG ??301??GACTGAGACA?CGGCCCAGAC?TCCTACGGGA?GGCAGCAGTA?GGGAATCTTC?CACAATGGGC ??361??GAAAGCCTGA?TGGAGCAACG?CCGCGTGAGT?GAAGAAGGAT?TTCGGTTCGT?AAAACTCTGT ??421??TGTAAGGGAA?GAACAAGTAC?AGTAGTAACT?GGCTGTACCT?TGACGGTACC?TTATTAGAAA ??481??GCCACGGCTA?ACTACGTGCC?AGCAGCCGCG?GTAATACGTA?GGTGGCAAGC?GTTGTCCGGA ??541??ATTATTGGGC?GTAAAGCGCG?CGCAGGTGGT?TTCTTAAGTC?TGATGTGAAA?GCCCACGGCT

??601???CAACCGTGGA?GGGTCATTGG?AAACTGGGAG?ACTTGAGTGC?AGAAGAGGAT?AGTGGAATTC ??661???CAAGTGTAGC?GGTGAAATGC?GTAGAGATTT?GGAGGAACAC?CAGTGGCGAA?GGCGACTATC ??721???TGGTCTGTAA?CTGACACTGA?GGCGCGAAAG?CGTGGGGAGC?AAACAGGATT?AGATACCCTG ??781???GTAGTCCACG?CCGTAAACGA?TGAGTGCTAA?GTGTTAGGGG?GTTTCCGCCC?CTTAGTGCTG ??841???CAGCTAACGC?ATTAAGCACT?CCGCCTGGGG?AGTACGGTCG?CAAGACTGAA?ACTCAAAGGA ??901???ATTGACGGGG?GCCCGCACAA?GCGGTGGAGC?ATGTGGTTTA?ATTCGAAGCA?ACGCGAAGAA ??961???CCTTACCAGG?TCTTGACATC?CCGTTGACCA?CTGTAGAGAT?ATGGTTTCCC?CTTCGGGGGC ??1021??AACGGTGACA?GGTGGTGCAT?GGTTGTCGTC?AGCTCGTGTC?GTGAGATGTT?GGGTTAAGTC ??1081??CCGCAACGAG?CGCAACCCTT?GATCTTAGTT?GCCATCATTT?AGTTGGGCAC?TCTAAGGTGA ??1141??CTGCCGGTGA?CAAACCGGAG?GAAGGTGGGG?ATGACGTCAA?ATCATCATGC?CCCTTATGAC ??1201??CTGGGCTACA?CACGTGCTAC?AATGGACGAT?ACAAACGGTT?GCCAACTCGC?GAGAGGGAGC ??1261??TAATCCGATA?AAGTCGTTCT?CAGTTCGGAT?TGTAGGCTGC?AACTCGCCTA?CATGAAGCCG ??1321??GAATCGCTAG?TAATCGCGGA?TCAGCATGCC?GCGGTGAATA?CGTTCCCGGG?CCTTGTACAC ??1381??ACCGCCCGTC?ACACCACGAG?AGTTTGTAAC?ACCCGAAGTC?GGTGAGGTAA?CCTTTTGGAG ??1441??CCAGCCGCCG?AAGGTGGGAT?AGATGATTGG?GGTGAAGTCG?TAACAAGGTA?GCCGTATCGG ??1501??AAGGTGCGGC?TGGATCACCT?CCTTA

Wherein, consist of A 25%; C 23%; G 31%; T 21%.

Embodiment 3

Carry out cultivation and fermentation with bacillus fusiformis CGMCC1347 (SW-B9) bacterial strain, slant medium is conventional LB substratum, and seed culture medium consists of: glucose 0.3%; Corn steep liquor 5.5%; Urea 0.3%; K ₂HPO ₄3H ₂O 0.09%; KH ₂PO ₄0.03%, MgSO ₄7H ₂O 0.1%; PH 7.0; With the bottled 25mL substratum of 250mL triangle, in 37 ℃, 180r/min shaking table shaking culture 12 hours is as seed culture fluid.Fermention medium consists of: corn steep liquor 5.5%; K ₂HPO ₄3H ₂O 0.2%; Urea 0.1%; MgSO ₄7H ₂O0.1%; PH 7.5; Dress 50mL substratum in the 250mL triangular flask, in 37 ℃, 180r/min shaking table shaking culture is added the 1mmol/L Vanillin and is continued to cultivate 8 hours as inductor after 12 hours, and final acquisition contains the fermented liquid that dry cell weight is 6.9g/L.

Fermented liquid through the centrifugal 10min of 3000r/min, is obtained wet thallus amount 0.36g, dress isoeugenol 0.4mL in the 250mL triangular flask, add tap water and be settled to 20mL, initial pH 7.0 is in 37 ℃, 180r/min vibration transforms 72 hours, records that Vanillin concentration is 4.11g/L in the conversion fluid.

Embodiment 4

The wet thallus 0.36g that obtains by embodiment 3 methods, dress 12mL isoeugenol in the 250mL triangular flask, the 8mL tap water, 0.02mL tween-80,4.0,37 ℃ of initial pH, the 180r/min conversion of vibrating, every sampling in 12 hours, use the Vanillin in the high effective liquid chromatography for measuring conversion fluid organic phase isoeugenol, result such as table 5 in the conversion process.Reacted 72 hours, Vanillin concentration reaches the highest 32.5g/L in the organic phase.

Conversion results in table 5 water-organic solvent biphasic system

Sample time (h)	??12	??24	??36	??48	??60	??72	??84
								Vanillin concentration (g/L) in the organic phase	??10.2	??15.1	??20.0	??26.4	??30.5	??32.5	??27.2

Embodiment 5

Wet thallus 1g by embodiment 3 methods obtain is modulated into 10% bacteria suspension with 10mL physiological saline.In addition use the 0.9g sodium alginate, 20mL physiological saline is mixed with the sodium alginate soln of 3% concentration, boils dissolving, is cooled to about 45 ℃, with the bacteria suspension mixing and stirring, splashes into the CaCl of 100mL 0.1mol/L with glue head dropper ₂In the solution, after 4 ℃ immersion was hardened 4 hours down, promptly can be used as immobilized cell and be used for bio-transformation.

Transform with 3g immobilized cell and the contrast of 0.36g free cell by example 4 modes, conversion results is as shown in table 6 in batches repeatedly.

Table 6 immobilized cell is conversion results in batches repeatedly

	Vanillin (g/L)
			Number of times	Free cell	Immobilized cell
??1	??17.88	??21.97
			??2	??19.38	??21.21
??3	??17.39	??20.63
			??4	??13.24	??20.33

Embodiment 6

Press the method for embodiment 4, obtain that Vanillin concentration is the conversion fluid of 22.8g/L in the organic phase, get its organic phase 5mL, wherein containing the Vanillin total amount is 114.00mg, adds to add 20g resin HD-8 behind the 50mL water and adsorb centrifugation resin behind the 24h; With the isoeugenol on the 50mL normal hexane wash-out resin, behind the static wash-out 12h again separation resin repeat wash-out once, merge the normal hexane elutriant twice, the solvent normal hexane is reclaimed in rotation vacuum-evaporation, steam the back recovery and obtain isoeugenol 3958mg, the isoeugenol rate of recovery is 93.4%.Then with the Vanillin in the resin behind the 50mL dehydrated alcohol wash-out recovery isoeugenol, with the 30mL dehydrated alcohol wash-out second time, merge ethanol eluate twice once more, etoh solvent is reclaimed in rotation vacuum-evaporation, obtain Vanillin 99.19mg after the steaming, its Vanillin extraction yield is 87%.

With 99.19mg Vanillin crude product, add about 10mL deionized water and be heated to 85 ℃ of dissolvings, be cooled to room temperature, the brown oil of separating out is earlier removed, be cooled to 0～5 ℃ of crystallization again, suction filtration, 35 ℃ of dryings obtain faint yellow powdery crystallization 52mg, and HPLC measures its purity 98.1%.

Claims (4)

1. the microbial strains SW-B9 that obtains is screened in a strain from soil, it is characterized by and can tolerate high concentration of substrate, isoeugenol can be converted into Vanillin, is bacillus fusiformis Bacillus fusiformisSW-B9 (CGMCC1347) through identifying this bacterial strain.

2. the cultural method of Bacillus fusiformis SW-B9 according to claim 1 may further comprise the steps:

(1) slant medium is the glucose nutrient agar;

(2) seed culture medium consists of: glucose 0.1%～1%; Corn steep liquor 1%～10%; Urea 0.1%～2%; K ₂HPO ₄3H ₂O 0.01%～0.5%; KH ₂PO ₄0.01%～0.5%, MgSO ₄7H ₂O 0.01%～0.5%; PH7.0;

(3) fermention medium consists of: corn steep liquor 1～10%; K ₂HPO ₄3H ₂O 0.1%～0.5%; Urea 0.1%～0.5%; MgSO ₄7H ₂O 0.1%～0.5%; PH7.5;

(4) cultivation with fermentation condition is: 37 ℃ of temperature, shaking speed 180r/min.

3. bioconversion method of producing Vanillin may further comprise the steps:

(1) the described microorganism strains of claim 1 is cultivated resulting fermented liquid, wet thallus, freeze-dried vaccine powder or immobilized cell as biocatalyst cell by the described method of claim 2;

(2) isoeugenol and water are mixed with mixed solution as bio-transformation substrate reactions liquid in 0.1%～99.9% ratio;

(3) biocatalyst cell that (1) is obtained, the substrate reactions liquid that adds (2) carries out conversion reaction, conversion reaction conditions is: the concentration of substrate scope is 0.1%～99.9%, the microorganism wet thallus is 10～80g/L to the consumption of substrate solution, initial pH4.0～8.0, the conversion reaction temperature is 20～42 ℃, and the conversion reaction time is 24～96h;

(4) purify with refining.

4. method according to claim 3, it is characterized in that, described purification is that the reaction solution organic phase is separated with making with extra care, adding D202, D031, HZ004, HZ801, HD-8 or HD-2 resin adsorbs, re-use organic solvent polymeric adsorbent is carried out wash-out, the eluted product Vanillin is refining with crystallization process, and the unconverted substrate isoeugenol reclaims and is used for transforming once more.

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