CN100347290C - Producing microorganism for trans-glycosylation beta-galactosidase - Google Patents
Producing microorganism for trans-glycosylation beta-galactosidase Download PDFInfo
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Abstract
The present invention discloses a bacterium for producing trans-glycosylation beta-galactosidase, which is named as enterobacter cloacae B5, and the preserving number of the bacterium for producing trans-glycosylation beta-galactosidase is CGMCC NO. 1401. The bacterium is a Gram negative bacillus; the shape of a bacterium body changes along with the extension of the culturing time on an LB culture medium. The bacterium body changes from long-rod shape to a short-rod shape, an ellipsoidal shape and finally shrinks into a spherical shape, the size of each of chromosomes is 0.5 to 1.5*0.7 to 9.0 micrometres, and the chromosomes are arranged singly or in pairs or in a short-chain shape. The bacterium body moves through peritrichous flagellum without forming gemmae; the bacterium can use citrate or acetate as a unique carbon source, and glucose is fermented at 37 DEG C to generate acid and gas. Glycerine is fermented without generating gas, hydrolyzing aesculin or generating indole; V. P. is used for measuring positive, and methyl red is used for measuring negative; nitrate is recovered; lysine has ornithine decarboxylase, double-hydrolase arginine and urease without carrying out decarboxylase. The 16S rDNA nucleotide sequence of the bacterium is shown in SEQ ID No. 1. Beta-galactosidase produced by the bacterium can use lactin as substrate to catalyze and produce oligomerization galactoside.
Description
Technical field
The present invention relates to a kind of enterobacteria, relate in particular to a kind of enterobacter cloacae that can produce glycosyl transferred beta-galactosidase.
Background technology
Oligomeric galactose is one of natural constituents in the breast milk, and its most basic most important physiological function comes from its proliferation function to bifidus bacillus.Because oligomeric galactose has good thermostability and acid resistance, its health-care effect has caused people's very big concern.At present, the production of oligomeric galactose is mainly synthetic by microbial enzyme method, is that substrate commentaries on classics glycosyl is synthetic with the lactose by beta-galactosidase enzymes or Sumylact L promptly.The development research of China's oligomeric galactose also is in initial period, and autonomous microbial enzyme method industrial production still belongs to blank, the whole dependence on import of the Sumylact L that uses in the dairy industry.Therefore, seek to produce the microorganism that changes the active beta-galactosidase enzymes of glycosyl more by force, significance is arranged for filling up the blank that domestic autonomous microbial enzyme method produces oligomeric galactose.
By retrieval, not having the enterobacter cloacae beta-galactosidase enzymes in the world at present is the report of substrate catalytic production oligomeric galactose with the lactose.
Summary of the invention
In existing microbial enzyme method industrial production, the microbe species of glycosyl transferred beta-galactosidase is limited, particularly can produce the deficiency of the microorganism that changes the active beta-galactosidase enzymes of glycosyl more by force, the purpose of this invention is to provide the new isolating enterobacter cloacae that changes the active beta-galactosidase enzymes of glycosyl more by force that produces of a strain, the beta-galactosidase enzymes that this bacterium produces is that substrate can the catalytic production oligomeric galactose with the lactose.
The bacterial strain that changes the active beta-galactosidase enzymes of glycosyl more by force that produces provided by the invention, enterobacter cloacae (Enterobacter cloacae) B5 by name, (be called for short: CGMCC), deposit number is CGMCC No.1401 to be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 30th, 2005.
The glycosyl transferred beta-galactosidase bacteria that the present invention relates to---enterobacter cloacae is to separate to obtain from soil.The pedotheque microorganism obtains by twice screening active ingredients after the lactose enrichment is selected to cultivate, promptly elder generation carries out the flat board selection after sole carbon source is cultivated the sample microbial enrichment to separate, and then screening beta-galactosidase enzymes hydrolytic activity obtains with changeing the galactosyl activity.
Producing of the present invention relates to changeed the enterobacter cloacae of the active beta-galactosidase enzymes of glycosyl more by force, has following biological property:
Morphological feature is: gram negative bacillus; On the LB substratum, thalli morphology changes with the prolongation of incubation time, and from the elongated rod shape to the rod-short, elliposoidal, contract to sphere at last, painted size is 0.5-1.5 * 0.7-9.0 micron; Single, paired or short chain shape is arranged; Move with peritrichous; Do not form gemma.
Physiological and biochemical property is: can utilize Citrate trianion or acetate to be sole carbon source; 37 ℃ of glucose fermentations produce sour aerogenesis; Glycerol fermentation is aerogenesis not; Not hydrolysis polychrom; Edwardsiella hoshinae; V.P. measure positive; Methyl red is measured negative; Reduction nitrate; Not decarboxylation of Methionin has ornithine decarboxylase, arginine dihydrolase, urase; Further feature sees table 1 for details.
Table 1 enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 physiological and biochemical property
| Feature | Enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 |
| The oxidizing ferment catalase produces the red V.P. citrate of indole methyl (western Meng Shi) nitrate reduction hydrolysis aesculin urase lysine decarboxylase ornithine decarboxylase arginine dihydrolase KCN growth D-Glucose and produces sour aerogenesis sugar/alcohol product yogurt sugarcane sugar cellobiose gossypose maltose trehalose D-MANNOSE L-rhamnose PEARLITOL 25C Arabinose D-glucitol glycerine ONPG | - + - - + + + - + - + + + + + + + + + + + + + + + + + |
Annotate :+. the positive;-. feminine gender
The enterobacter cloacae that the present invention relates to (Enterobacter cloacae) B5 CGMCC No.1401 goes down to posterity stable in the lactose slant medium; Well-grown in the LB liquid nutrient medium; Well-grown in producing the enzymic fermentation substratum, and produce glycosyl transferred beta-galactosidase; Culture temperature is 35～40 ℃; Incubation time is 15～24 hours, and shaking speed is 180 rev/mins.
Above-mentioned LB culture medium prescription: peptone 10 ± 1g/L, yeast powder 5 ± 1g/L, sodium-chlor 7 ± 1g/L, pH 7.0～75, sterilize 20 minutes for 121 ℃;
Screening used selection substratum of above-mentioned enterobacter cloacae or slant culture based formulas is: lactose 10 ± 2g/L, and peptone 5 ± 2g/L, yeast powder 10 ± 2g/L, sodium-chlor 3 ± 2g/L, pH 7.0～7.5, and solid medium adds agar 20 ± 2g/L; Sterilized 30 minutes for 112 ℃.
Above-mentioned product enzymic fermentation culture medium prescription is: lactose 10 ± 2g/L, and peptone 5 ± 2g/L, yeast powder 10 ± 2g/L, sodium-chlor 3 ± 2g/L, pH 7.0～7.5, sterilize 30 minutes for 112 ℃.
Wherein, above-mentioned culture temperature is preferably 37 ℃.
Wherein, above-mentioned incubation time is preferably 16～18 hours.
Wherein, screen used selection substratum of above-mentioned enterobacter cloacae or slant medium formula optimization and be: lactose 10g/L, peptone 5g/L, yeast powder 10g/L, sodium-chlor 3g/L, pH 7.5, and solid medium adds agar 20g/L.
Wherein, above-mentioned product enzymic fermentation culture medium prescription preferably: lactose 10g/L, peptone 5g/L, yeast powder 10g/L, sodium-chlor 3g/L, pH 7.5.
The 16SrDNA nucleotide sequence length of the bacterial strain enterobacter cloacae that the present invention relates to (Enterobacter cloacae) B5 CGMCC No.1401 is 1534 bases, shown in SEQ ID No.1 in the sequence table.
By at (the National Center for Biotechnology Information of U.S. biotechnology information center, NCBI) online analysis, listed 16S rDNA sequence among the 16S rDNA sequence of bacterial strain enterobacter cloacae of the present invention (Enterobacter cloacae) B5 and the GenBank is carried out homology relatively, the result shows, the 16S rDNA sequence similarity of the 16SrDNA gene order of bacterial strain B5 and a few strain bacterium of enterobacter is very high, but incomplete same, illustrate that bacterial strain of the present invention is an isolation identification first.In conjunction with bacterial strain B5 morphology and physiological and biochemical property,, be enterobacter cloacae (Enterobacter cloacae) with the bacterial strain B5 of the present invention dientification of bacteria according to uncle's outstanding Bacteria Identification handbook (the 9th edition).And (be called for short: CGMCC), deposit number is CGMCC No.1401 to be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 30th, 2005.
The application of glycosyl transferred beta-galactosidase bacteria provided by the invention in the production of preparation glycosyl transferred beta-galactosidase: glycosyl transferred beta-galactosidase bacteria of the present invention can be cultivated in producing the enzymic fermentation nutrient solution and produce glycosyl transferred beta-galactosidase, in the process of producing oligomeric galactose, multiparity enzymic fermentation nutrient solution is cultivated and is obtained mycetocyte, after freeze thawing, be the beta-galactosidase enzymes crude enzyme liquid.
The application of glycosyl transferred beta-galactosidase bacteria provided by the invention in the production of preparation oligomeric galactose: in the production of preparation oligomeric galactose, the present invention is after earlier enterobacter cloacae (Enterobacter cloacae) B5 CGMCCNo.1401 multiparity enzymic fermentation nutrient solution being cultivated, make the beta-galactosidase enzymes crude enzyme liquid, described beta-galactosidase enzymes is substrate catalytic production oligomeric galactose with the lactose.
By retrieval, not having the enterobacter cloacae beta-galactosidase enzymes both at home and abroad at present is the report of substrate catalytic production oligomeric galactose with the lactose.And the test that utilizes bacterial strain enterobacter cloacae of the present invention (Enterobacter cloacae) B5 CGMCC No.1401 to carry out shows: the enterobacter cloacae beta-galactosidase enzymes that bacterial strain of the present invention relates to can be that substrate reactions generates oligomeric galactose with the lactose, and yield is higher, is about 54.5%.This bacteria growing is fast, cultivates simply, and stable hereditary property is changeed glycosyl efficient height, has very strong suitability for industrialized production advantage, can be used for the industrial production oligomeric galactose, to fill up the blank that domestic autonomous microbial enzyme method is produced oligomeric galactose.
Embodiment
Embodiment 1: bacterial screening
1. can utilize the separation and purification of lactose for sole carbon source growth bacterial strain
The 10g soil sample is added enrichment culture in the nutrient solution that 100mL is sole carbon source with the lactose, nutrient solution is diluted to 10 with sterilized water
-1, 10
-3, 10
-5, 10
-7Gradient is respectively got 200 μ L and is evenly coated on the lactose selection flat board.The bacteria screening flat board was in 37 ℃ of cultivations 24 hours, and the fungi screening flat board was cultivated 48～60 hours in 28 ℃.10
-5The bacterium colony kind that gradient plate grows is many, and is convenient to purifying.The different bacterium colony of picking form carries out separation and purification by plate streaking respectively, repeats 3 times.Isolated strains is carried out microscopy, determine purity: bacterium compares with streptococcus aureus (Staphylococcusaureus) and intestinal bacteria (Escherichia coli), carry out gramstaining, microscopy can be distinguished gram-positive microorganism and Gram-negative bacteria; Fungi is made the water logging sheet, and microscopy can be observed mycelia feature and spore producing method, and preliminary evaluation is to belonging to.The purifying bacterial strain that at last microscopy is obtained is saved in respectively on the test tube slant, 4 ℃ of storages.
It can be the sole carbon source growth with the lactose that above-mentioned separation and purification process obtains 15 strain bacterium altogether, wherein bacterium 9 strains, fungi 6 strains.15 strain bacterium are numbered respectively, bacterium be B1 ..., B9; Fungi be F1 ..., F6.
Above-mentioned bacteria screening is selected substratum (also as slant medium) with the bacterium enrichment, and its prescription is: lactose 10g/L, and peptone 5g/L, yeast powder 10g/L, sodium-chlor 3g/L, pH 7.0～7.5, and solid medium adds agar 20g/L.
Above-mentioned fungi screening is selected substratum (also as slant medium) with the fungi enrichment, and its prescription is: murphy juice (potato 200g/L), lactose 10g/L, agar 20g/L, natural pH.
2. produce the screening of beta-galactosidase enzymes bacterial strain
The 15 strain bacterium that above-mentioned separation is obtained are seeded to respectively in the 30mL product enzymic fermentation nutrient solution, and bacterium was cultivated 18 hours in 37 ℃ of shaking tables, and fungi was cultivated 40 hours in 28 ℃ of shaking tables, and shaking speed is 180 rev/mins.Bacterium and yeast culture liquid are in 12,000 rev/mins of centrifugal 5 minutes acquisition mycetocytes; Mould obtains mycetocyte with filter paper (two circle board 101 fast qualitative filter paper) suction filtration.Measure mycetocyte beta-galactosidase enzymes hydrolytic activity then: with mycetocyte and beta-galactosidase enzymes hydrolysis substrate---o-NP-β-D-galactoside (o-nitrophenyl-β-D-galactopyranoside, ONPG) mixing is reacted, if ONPG is hydrolyzed, think that promptly this bacterium produces beta-galactosidase enzymes.
Get mycetocyte 50mg, add ONPG (colourless) the solution 450 μ L of 2mmol/L, 40 ℃ were reacted 10 minutes, added the Na of 1mL0.5mol/L
2CO
3The solution termination reaction, 12,000 rev/mins centrifugal 2 minutes.If it is yellow that supernatant liquor is, illustrate that former colourless ONPG has been hydrolyzed into the xanchromatic o-NP by beta-galactosidase enzymes, mycetocyte has the existence of beta-galactosidase enzymes.If supernatant liquor is surveyed OD
400, can live quantitatively to lytic enzyme.The unit of activity of enzyme regulation: the enzyme amount that discharges 1 μ mol o-NP with 1 minute hydrolysis ONPG is an enzyme activity unit.
Above-mentioned screening process obtains 9 strain beta-galactosidase bacterias altogether, and wherein 5 strain bacteriums, 4 fungal strains are respectively B1, B2, B4, B5, B9, F1, F2, F3, F4.
Above-mentioned product enzymic fermentation culture medium prescription is: lactose 10g/L, peptone 5g/L, yeast powder 10g/L, CaCl
20.11g/L, MnSO
40.001g/L, MgSO
47H
2O 0.3g/L, KH
2PO
40.05g/L, FeSO
47H
2O 0.03g/L, pH 7.0～7.5 (bacterium) or pH 6.0～6.5 (fungi).
3. produce the screening of glycosyl transferred beta-galactosidase bacterial strain
The mycetocyte of above-mentioned 9 kinds of beta-galactosidase bacterias is made crude enzyme liquid, change the active screening of galactosyl.
Get the 50mg mycetocyte, be suspended in the potassium phosphate buffer of 50 μ L pH7.0,50mmol/L, after-20 ℃ of following temperature are freezing fully, put room temperature and thaw, repeat freeze thawing again 2 times, the gained suspension is the beta-galactosidase enzymes crude enzyme liquid.Get 100 μ L crude enzyme liquids, 30% (w/v) lactose solution that adds the preparation of 300 μ L pH7.0 potassium phosphate buffers, being divided into 2 equal portions reacts respectively at 40 ℃, 50 ℃, bacterial reaction 4 hours, fungi reaction 8 hours, 12,000 rev/mins centrifugal 5 minutes, supernatant liquor promptly contains reaction product---oligomeric galactose.
Reaction product is carried out thin-layer chromatography, and (Thin-Layer Chromatography TLC) analyzes, and determines to change the glycosyl activity.TLC thin plate (Silica gel60, No.553, Merck) behind the point sample, at developing agent (propyl carbinol: ethanol: exhibition layer water=5: 3: 2), spray painting developer (3 of 20% sulphuric acid soln+0.5%, the 5-orcin), in 120 ℃ the baking 10 minutes, the colour developing of sugared spot according to its kind from pale brown look to intense violet color.If generated new oligosaccharides spot near the lactose spot, then the beta-galactosidase enzymes of Chan Shenging has the glycosyl of commentaries on classics activity; Do not change the glycosyl activity otherwise then do not exist.The oligosaccharides spot is big more, many more, illustrates that the commentaries on classics glycosyl activity of enzyme is strong more.
The result determines that 2 strain bacteriums (B1, B5) and 1 fungal strain (F3) have is changeed the glycosyl activity preferably.
The commentaries on classics glycosyl product of bacterial strain B1, B5 and F3 is carried out high performance liquid chromatography, and (High Performance LiquidChromatography HPLC) analyzes and the thin layer scanning analysis, and the yield of oligosaccharide of B5 is the highest as a result, is about 54.5%.
The B5 bacterium is accredited as enterobacter cloacae by 16S rDNA homologous sequence comparison and physio-biochemical characteristics, and strain identification sees embodiment 2 for details.By retrieval, not having enterobacter cloacae beta-galactosidase enzymes involved in the present invention in the world at present is the report of substrate catalytic production oligomeric galactose with the lactose.
Above-mentioned HPLC analyzes equipment used and condition: Tianjin, island (SHIMADZU) high performance liquid chromatograph; Tianjin, island RID-10A differential detector; BIO-RAD Aminex HPX-42C post (300mm * 7.8mm); Moving phase is tri-distilled water, and flow velocity is 0.2mL/min, 80 ℃ of column temperatures; Interpretation of result software is Class-VP6.0.Change the glycosyl product with 0.2 μ m membrane filtration after, be diluted to the sugar soln of 5% (w/v), sample introduction analysis.
Above-mentioned thin layer scanning is analyzed equipment used and condition: Tianjin, island (SHIMADZU) CS-9301 dual wavelength flying spot thin layer chromatography scanner, the detection wavelength is 550nm.Above-mentioned thin layer chromatography board is carried out scanning analysis.
Embodiment 2: strain identification
1. morphology and physiological and biochemical property
The bacterial strain enterobacter cloacae that the present invention separates (Enterobacter cloacae) B5 CGMCC No.1401, morphological feature is: gram negative bacillus; On the LB substratum, thalli morphology changes with the prolongation of incubation time, and from the elongated rod shape to the rod-short, elliposoidal, contract to sphere at last, painted size is 0.5-1.5 * 0.7-9.0 micron; Single, paired or short chain shape is arranged; Move with peritrichous; Do not form gemma; Physiological and biochemical property is: can utilize Citrate trianion or acetate to be sole carbon source; 37 ℃ of glucose fermentations produce sour aerogenesis; Glycerol fermentation is aerogenesis not; Not hydrolysis polychrom; Edwardsiella hoshinae; V.P. measure positive; Methyl red is measured negative; Reduction nitrate; Not decarboxylation of Methionin has ornithine decarboxylase, arginine dihydrolase, urase; Further feature sees table 1 for details.
2.16S rDNA pcr amplification and homology analysis
Enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 with the present invention separates is inoculated in the 5mLLB substratum, and 37 ℃ of shaking tables were cultivated 16 hours, get the 1.5ml culture, 12,000 rev/mins centrifugal 2 minutes, gained precipitation is suspended from the TE damping fluid of 565 μ L; Add 30 μ L mass volume ratios and be 10% sodium laurylsulfonate (SDS) and the Proteinase K of 5 μ L20mg/mL, mixing was in 37 ℃ of incubations 1 hour; Add the abundant mixing of 100 μ L5mol/L NaCl, add 80 μ L CTAB/NaCl solution again, mixing was in 65 ℃ of incubations 20 minutes; Ice bath is 30 minutes then; Add isopyknic phenol/chloroform/primary isoamyl alcohol, mixing; 12,000 rev/mins centrifugal 5 minutes, supernatant liquor is changed in the new pipe, add isopyknic phenol/chloroform/primary isoamyl alcohol again, mixing; 12,000 rev/mins centrifugal 5 minutes, supernatant is changed in the new pipe, add 2 times of volume dehydrated alcohols, mixing precipitates up to DNA gently; 12,000 rev/mins centrifugal 5 minutes, DNA precipitation is with washing 2 times in 70% ethanol; Vacuum-drying 10 minutes heavily is dissolved in the TE damping fluid of 50 μ L.
The TE buffer formulation is as follows: the Tutofusin tris of 10mmol/L (Tris), and the disodium ethylene diamine tetraacetate of 1mmol/L (EDTA), transferring pH is 8.0.
The CTAB/NaCl solution formula is as follows: the cetyltriethylammonium bromide of 10% (w/v) (CTAB) is dissolved among the NaCl of 0.7mol/L.
Enterobacter cloacae genomic dna with extraction is a template, employing bacterial 16 S rDNA sequence universal primer F27 (5 '-AGAGTTTCMTGGCTCAG-3 ') and R1541 (5 '-AAGGAGGTGATCCAGCC-3 '), carry out pcr amplification.Pcr amplification system cumulative volume is 50 μ L, contains ultrapure water 35 μ L, 10 * PCR buffer, 5 μ L, dNTP2.4mmol/L, each 1 μ mol/L of primer, MgCl
21.5mmol/L, template DNA 100ng, Taq enzyme 2.5U.Pcr amplification condition: 95 ℃ of pre-sex change 5 minutes; React 28 circulations (72 ℃ were extended 2 minutes for 95 ℃ of sex change 1 minute, 52 ℃ of annealing 1 minute); 72 ℃ were extended 10 minutes after 28 loop ends.The PCR product is about about 1.5kb, through agarose gel electrophoresis, and ethidium bromide staining, uv analyzer detects, reclaim then, be connected on the plasmid pUCm-T carrier Transformed E .coli Top10 competent cell, coating penbritin flat board extracts plasmid DNA and detects, and Pst I enzyme is cut checking.The correct plasmid that will have 16S rDNA checks order.
Enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 bacterial strain 16S rDNA sequence length is 1534 bases, and nucleotide sequence is as follows:
agagtttgat catggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgaac 60
ggtagcacag agagcttgct ctcgggtgac gagtggcgga cgggtgagta atgtctggga 120
aactgcctga tggaggggga taactactgg aagcggtagc taataccgca taacgtcgca 180
agaccaaaga gggggacctt cgggcctctt gccatcagat gtgcccagat gggattagct 240
agtaggtggg gtaacggctc acctaggcga cgatccctag ctggtctgag aggatgacca 300
gccacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg 360
cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaagaaggcc ttcgggttgt 420
aaagtacttt cagcggggag gaaggtgttg aggttaataa cctcagcaat tgacgttacc 480
cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga agggtgcaag 540
cgttaatcgg aattactggg gcgtaaagcg cacgcaggcg gtctgtcaag tcggatgtga 600
aatccccggg ctcaacctgg gaactgcatt cgaaactggc aggctagagt cttgtagagg 660
ggggtagaat tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accggtggcg 720
aaggcggccc cctggacaaa gactgacgct caggtgcgaa agcgtgggga gcaaacagga 780
ttagataccc tggtagtcca cgccgtaaac gatgtcgact tggaggttgt gcccttgagg 840
cgtggcttcc ggagctaacg cgttaagtcg accgcctggg gagtacggcc gcaaggttaa 900
aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc 960
aacgcgaaga accttaccta ctcttgacat ccagagaact tagcagagat gctttggtgc 1020
cttcgggaac tctgagacag gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctta tcctttgttg ccagcggtcc ggccgggaac 1140
tcaaaggaga ctgccagtga taaactggag gaaggtgggg atgacgtcaa gtcatcatgg 1200
cccttacgag tagggctaca cacgtgctac aatggcgcat acaaagagaa gcgacctcgc 1260
gagagcaagc ggacctcata aagtgcgtcg tagtccggat tggagtctgc aactcgactc 1320
catgaagtcg gaatcgctag taatcgtgga tcagaatgcc acggtgaata cgttcccggg 1380
ccttgtacac accgcccgtc acaccatggg agtgggttgc aaaagaagta ggtagcttaa 1440
ccttcgggag ggcgcttacc actttgtgat tcatgactgg ggtgaagtcg taacaaggta 1500
accgtagggg aacctgcggc tggatcacct cctt 1534
Listed 16S rDNA sequence among the 16S rDNA sequence of bacterial strain enterobacter cloacae of the present invention (Enterobacter cloacae) B5 CGMCC No.1401 and the GenBank is carried out homology relatively, the 16S rDNA sequence similarity of the 16S rDNA sequence of B5 and a few strain bacterium of enterobacter is very high, but incomplete same, illustrate that bacterial strain of the present invention is an isolation identification first.In conjunction with B5 bacterium morphology and physiological and biochemical property,, be enterobacter cloacae (Enterobacter cloacae) with the B5 dientification of bacteria according to uncle's outstanding Bacteria Identification handbook (the 9th edition).This bacterium is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 30th, 2005, and (be called for short: CGMCC), deposit number is CGMCCNo.1401.
Embodiment 3: utilize enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 to produce oligomeric galactose
1. spawn culture
Bacterial strain enterobacter cloacae (Enterobacter cloacae) the B5 CGMCCNo.1401 that selects the present invention to separate activates, transfers and go into to produce the cultivation of enzymic fermentation nutrient solution with ordinary method.
Above-mentioned product enzymic fermentation culture medium prescription is: lactose 10g/L, peptone 5g/L, yeast powder 10g/L, CaCl
20.11g/L, MnSO
40.001g/L, MgSO
47H
2O 0.3g/L, KH
2PO
40.05g/L, FeSO
47H
2O 0.03g/L, pH 7.0～7.5 (bacterium) or pH 6.0～6.5 (fungi).
Grope to optimize product enzymic fermentation nutrient solution through serial experiment, the product enzymic fermentation culture medium prescription of final enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 is reduced to: lactose 10g/L, peptone 5g/L, yeast powder 10g/L, sodium-chlor 3g/L, pH 7.5.
After enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 is seeded to the activation of fresh slant medium, connect one and encircle the above-mentioned product enzymic fermentation of 50mL nutrient solution, cultivated 16 hours in 37 ℃ of shaking tables, shaking speed is 180 rev/mins, or after cultivating again switching go into the identical product enzymic fermentation of 500mL nutrient solution enlarged culturing, enterobacter cloacae (Enterobacter cloacae) the B5 CGMCC No.1401 product enzyme of grow in above-mentioned substratum is good; After cultivate finishing, 12,000 rev/mins centrifugal 5 minutes, results enterobacter cloacae cell.
Stability experiment shows that this bacterial strain has very high stability, and number generation back enzymatic productivity does not reduce.
Above-mentioned slant culture based formulas is: lactose 10g/L, and peptone 5g/L, yeast powder 10g/L, sodium-chlor 3g/L, agar 20g/L, pH 7.5.
2. change the glycosyl reaction
Get the enterobacter cloacae cell of above-mentioned 50mg preparation, the 50mmol/L potassium phosphate buffer that adds 250 μ L, pH 7.0 washs once, the enterobacter cloacae cell is resuspended to the identical damping fluid of 50 μ L again, below-20 ℃ fully freezing after, putting room temperature thaws, repeat freeze thawing again 2 times, the gained suspension is the beta-galactosidase enzymes crude enzyme liquid.
Get the above-mentioned crude enzyme liquid of 100 μ L, add 30% (w/v) lactose solution of 300 μ L, pH 7.0 potassium phosphate buffers preparation, in 50 ℃ of shaking baths reactions 4 hours, 12,000 rev/mins centrifugal 5 minutes, supernatant is changes the glycosyl product.
Above-mentioned commentaries on classics glycosyl product is carried out efficient liquid phase chromatographic analysis and thin layer scanning analysis, and the oligomeric galactose yield is about 54.5%.
Above-mentioned HPLC analyzes equipment used and condition: Tianjin, island (SHIMADZU) high performance liquid chromatograph; Tianjin, island RID-10A differential detector; BIO-RAD Aminex HPX-42C post (300mm * 7.8mm); Moving phase is tri-distilled water, and flow velocity is 0.2mL/min, 80 ℃ of column temperatures; Interpretation of result software is Class-VP6.0.Change the glycosyl product with 0.2 μ m membrane filtration after, be diluted to the sugar soln of 5% (w/v), sample introduction analysis.
Above-mentioned thin layer scanning is analyzed equipment used and condition: Tianjin, island (SHIMADZU) CS-9301 dual wavelength flying spot thin layer chromatography scanner; The detection wavelength is 550nm.Getting 0.5uL changes the glycosyl product behind point sample on the TLC thin plate, at developing agent (propyl carbinol: ethanol: launch spray painting developer (3 of 20% sulphuric acid soln+0.5% water=5: 3: 2), the 5-orcin), in 120 ℃ of bakings 10 minutes, after the sugared spot colour developing, carry out scanning analysis.
Sequence table
SEQ ID No.1
＜110〉Shandong University
＜120〉a kind of glycosyl transferred beta-galactosidase bacteria
<141>2005-6-16
<211>1534
<212>DNA
＜213〉enterobacter cloacae (Enterobacter cloacae)
＜221〉enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 16S rDNA
<222>(1)...(1534)
<400>
agagtttgat catggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgaac 60
ggtagcacag agagcttgct ctcgggtgac gagtggcgga cgggtgagta atgtctggga 120
aactgcctga tggaggggga taactactgg aagcggtagc taataccgca taacgtcgca 180
agaccaaaga gggggacctt cgggcctctt gccatcagat gtgcccagat gggattagct 240
agtaggtggg gtaacggctc acctaggcga cgatccctag ctggtctgag aggatgacca 300
gccacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg 360
cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaagaaggcc ttcgggttgt 420
aaagtacttt cagcggggag gaaggtgttg aggttaataa cctcagcaat tgacgttacc 480
cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga agggtgcaag 540
cgttaatcgg aattactggg gcgtaaagcg cacgcaggcg gtctgtcaag tcggatgtga 600
aatccccggg ctcaacctgg gaactgcatt cgaaactggc aggctagagt cttgtagagg 660
ggggtagaat tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accggtggcg 720
aaggcggccc cctggacaaa gactgacgct caggtgcgaa agcgtgggga gcaaacagga 780
ttagataccc tggtagtcca cgccgtaaac gatgtcgact tggaggttgt gcccttgagg 840
cgtggcttcc ggagctaacg cgttaagtcg accgcctggg gagtacggcc gcaaggttaa 900
aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc 960
aacgcgaaga accttaccta ctcttgacat ccagagaact tagcagagat gctttggtgc 1020
cttcgggaac tctgagacag gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctta tcctttgttg ccagcggtcc ggccgggaac 1140
tcaaaggaga ctgccagtga taaactggag gaaggtgggg atgacgtcaa gtcatcatgg 1200
cccttacgag tagggctaca cacgtgctac aatggcgcat acaaagagaa gcgacctcgc 1260
gagagcaagc ggacctcata aagtgcgtcg tagtccggat tggagtctgc aactcgactc 1320
catgaagtcg gaatcgctag taatcgtgga tcagaatgcc acggtgaata cgttcccggg 1380
ccttgtacac accgcccgtc acaccatggg agtgggttgc aaaagaagta ggtagcttaa 1440
ccttcgggag ggcgcttacc actttgtgat tcatgactgg ggtgaagtcg taacaaggta 1500
accgtagggg aacctgcggc tggatcacct cctt 1534
Claims (4)
1. a strain glycosyl transferred beta-galactosidase bacteria, it is characterized in that: this bacterium is called enterobacter cloacae (Enterobactercloacae) B5, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 30th, 2005, deposit number is CGMCC No.1401.
2. the application of the described glycosyl transferred beta-galactosidase bacteria of claim 1 in the production of preparation glycosyl transferred beta-galactosidase.
3. the application of the described glycosyl transferred beta-galactosidase bacteria of claim 1 in the production of preparation oligomeric galactose.
4. the application of glycosyl transferred beta-galactosidase bacteria as claimed in claim 3 in the production of preparation oligomeric galactose, it is characterized in that the glycosyl transferred beta-galactosidase that described enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 produces is substrate catalytic production oligomeric galactose with the lactose.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101144067B (en) * | 2007-08-23 | 2011-08-10 | 广东省昆虫研究所 | Transgene enterobacter cloacae CEI-a and its construction method and use |
| CN102154239B (en) * | 2011-01-06 | 2012-12-12 | 中山大学 | New gene of beta-galactosidase with high glycosyl transfer efficiency and application thereof |
| CN102286419B (en) * | 2011-09-21 | 2012-10-03 | 甘肃农业大学 | Enterobacter SYA2 and method for preparing lactase with same |
| CN102690767B (en) * | 2012-05-31 | 2013-08-28 | 黑龙江省科学院微生物研究所 | Klebsiella oxytoca efficient in phosphorus solubilizing and nitrogen fixation and capable of inhibiting growth of pathogenic fungi |
| CN115141768B (en) * | 2022-06-17 | 2024-03-26 | 浙江工业大学 | Enterobacter cloacae Zjut HJ2001 and application thereof in fermentation production of galactooligosaccharides |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2001321164A (en) * | 2000-05-12 | 2001-11-20 | Japan National Oil Corp | New bacterial cellulose-producing microorganism and promoted recovering method of petroleum by utilizing the same |
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| JP2001321164A (en) * | 2000-05-12 | 2001-11-20 | Japan National Oil Corp | New bacterial cellulose-producing microorganism and promoted recovering method of petroleum by utilizing the same |
Non-Patent Citations (1)
| Title |
|---|
| 一株曲霉( Aspergillus sp.AF)β-半乳糖苷酶的转糖基活性研究 袁绍鹏，肖敏，孙正，牵镎? 新民，钱新民.食品与发酵工业，第29卷第11期 2003 * |
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