CN109897805A - A kind of screening technique producing sarcosine oxidase bacterial strain - Google Patents
A kind of screening technique producing sarcosine oxidase bacterial strain Download PDFInfo
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- CN109897805A CN109897805A CN201910270719.XA CN201910270719A CN109897805A CN 109897805 A CN109897805 A CN 109897805A CN 201910270719 A CN201910270719 A CN 201910270719A CN 109897805 A CN109897805 A CN 109897805A
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- bacterial strain
- sarcosine oxidase
- creatine
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- enzyme activity
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- 230000001580 bacterial effect Effects 0.000 title claims abstract description 71
- 108010060059 Sarcosine Oxidase Proteins 0.000 title claims abstract description 48
- 102000008118 Sarcosine oxidase Human genes 0.000 title claims abstract description 48
- 238000012216 screening Methods 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 33
- 102000004190 Enzymes Human genes 0.000 claims abstract description 53
- 108090000790 Enzymes Proteins 0.000 claims abstract description 53
- 230000000694 effects Effects 0.000 claims abstract description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 238000010790 dilution Methods 0.000 claims abstract description 9
- 239000012895 dilution Substances 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 239000008223 sterile water Substances 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 241000233866 Fungi Species 0.000 claims abstract description 3
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 claims description 130
- 229960003624 creatine Drugs 0.000 claims description 65
- 239000006046 creatine Substances 0.000 claims description 65
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 42
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 26
- 239000001963 growth medium Substances 0.000 claims description 25
- 239000002609 medium Substances 0.000 claims description 19
- 239000012153 distilled water Substances 0.000 claims description 15
- 238000011534 incubation Methods 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 14
- 229920002472 Starch Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- 229940041514 candida albicans extract Drugs 0.000 claims description 14
- 230000015556 catabolic process Effects 0.000 claims description 14
- 238000006731 degradation reaction Methods 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 235000019698 starch Nutrition 0.000 claims description 14
- 239000008107 starch Substances 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 14
- 239000012138 yeast extract Substances 0.000 claims description 14
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 13
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 13
- 238000004659 sterilization and disinfection Methods 0.000 claims description 13
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 12
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- 238000005259 measurement Methods 0.000 claims description 8
- 238000000386 microscopy Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 230000008859 change Effects 0.000 claims description 6
- 238000001514 detection method Methods 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 238000000407 epitaxy Methods 0.000 abstract description 2
- 239000000725 suspension Substances 0.000 abstract 1
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 16
- 238000013459 approach Methods 0.000 description 11
- 108010077895 Sarcosine Proteins 0.000 description 8
- 229940043230 sarcosine Drugs 0.000 description 8
- -1 i.e. Chemical compound 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 239000002054 inoculum Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 238000012549 training Methods 0.000 description 6
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 description 2
- 206010029164 Nephrotic syndrome Diseases 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 208000009928 nephrosis Diseases 0.000 description 2
- 231100001027 nephrosis Toxicity 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the technical field of bacteria screening, specifically a kind of screening technique for producing sarcosine oxidase bacterial strain.The method are as follows: the marine source sample suspension that will acquire is diluted with sterile water, a point different gradients are coated on screening and culturing medium after dilution, and being placed on temperature is that culture 4-5 days is carried out under 20-30 DEG C of constant temperature, picking contains the bacterial clump of transparent circle afterwards, is isolated and purified by three rides, and single bacterium colony is obtained, then measure enzyme activity, the highest bacterial strain for producing sarcosine oxidase of one plant of enzyme activity is finally selected, fungi preservation is carried out, that is, is purpose bacterial strain.The method of the present invention can fast and accurately screen the bacterium for producing sarcosine oxidase, bacterial strain obtained has the characteristics that yield is high, efficiency is fast and enzyme activity is high, enzyme activity is up to 120~160U/L, and have the higher advantage of low-temperature epitaxy producing enzyme enzyme activity, be conducive to later stepping practise medicine learning detection application aspect research.
Description
Technical field
The present invention relates to the technical field of bacteria screening, specifically a kind of screening technique for producing sarcosine oxidase bacterial strain.
Background technique
It is reported that only uremic patient just has as many as 1,000,000 in China, timely, accurate renal diagnostic controls nephrotic
It treats extremely important.Currently, the domestic developmental research also ground zero to nephrosis enzyme process detection kit, creatinine detection is nephrosis with enzyme
One of the important enzyme of enzyme process detection.Sarcosine oxidase is widely used in human body blood as a kind of important diagnosis enzyme preparation
The detection of creatinine level in clear, for judging the health degree of renal function.Although many domestic and foreign scholars to its existing research,
In actual production there are still low output, thermal stability is bad, particularly (20-25 DEG C) of the room temperature problem for using enzyme scarcity, and
Studies in China is less at present, and marine low temperature enzyme (5-15 DEG C) medical detection reagent box apply in occupation of highly importantly
Position, thus how quickly to filter out the high enzyme activity of high yield, be adapted to room temperature medicine detection sarcosine oxidase be of great significance.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art, the present invention provides a kind of new screening for producing sarcosine oxidase bacterial strain
Method can more rapidly, more accurately filter out the bacterial strain of the production sarcosine oxidase of high yield, high enzyme activity under such an approach.
For achieving the above object, the invention adopts the following technical scheme:
A kind of screening technique producing sarcosine oxidase bacterial strain,
A kind of screening technique producing sarcosine oxidase bacterial strain, the described method comprises the following steps:
(1) it will be accessed in enriched medium for the marine source sample of examination by 5% inoculum concentration, and be put into constant-temperature table training
Case is supported, shaking flask enrichment culture is carried out at 150rpm, temperature setting is cultivated under 20-30 DEG C of constant temperature, while surveying creatine drop daily
Solution rate is further cultured for 2 days when creatine is degraded to 80% or more, can be carried out in next step；
(2) it produces the separation of sarcosine oxidase bacterial strain: creatine in step (1) is degraded complete culture medium substantially, use
Sterile water dilution, is coated in screening and culturing medium, temperature setting is constant temperature incubation 4-5 days under the conditions of 20-30 DEG C after dilution；
(3) produce the primary dcreening operation of sarcosine oxidase bacterial strain: picking has the bacterial clump of transparent circle, is divided by three rides
From purifying, temperature setting obtains single bacterium colony constant temperature incubation 4-5 days under the conditions of 20-30 DEG C, microscopy is carried out, by observing bacterium
Fall form, Preliminary Identification strain；
(4) produce the secondary screening of sarcosine oxidase bacterial strain: measurement enzyme activity finally selects the highest production sarcosine oxygen of one plant of enzyme activity
The bacterial strain for changing enzyme, is saved, that is, filters out purpose bacterial strain.
Above-mentioned a kind of screening technique for producing sarcosine oxidase bacterial strain, in step (1) in sample access enriched medium
Access amount is the 5% of enriched medium volume.
A kind of above-mentioned screening technique for producing sarcosine oxidase bacterial strain, enriched medium in step (1) are as follows: creatine 5.0-
10.0g, yeast extract 0.5-5.0g, sodium chloride 2.0-5.0g, potassium dihydrogen phosphate 1.0-5.0g, dipotassium hydrogen phosphate 0.5-5.0g, sulphur
Sour magnesium 0.5-5.0g, starch 10.0-15.0g, potassium iodide 1.0-5.0g, agar 20.0-30.0g, distilled water 1.0-1.5L, high temperature
It is saved after sterilizing, temperature is controlled at 100-200 DEG C.
A kind of above-mentioned screening technique for producing sarcosine oxidase bacterial strain, screening flat board culture medium in step (2) are as follows: creatine
5.0-10.0g, yeast extract 0.5-10.0g, sodium chloride 2.0-10.0g, potassium dihydrogen phosphate 1.0-10.0g, dipotassium hydrogen phosphate 0.5-
10.0g, magnesium sulfate 0.5-10.0g, starch 10.0-20.0g, potassium iodide 1.0-10.0g, agar 20.0-40.0g, distilled water
1.0-1.5L is saved after high-temperature sterilization, and temperature is controlled at 100-200 DEG C.
Compared with prior art, the invention has the benefit that the present invention provides a kind of new production sarcosine oxidases
The method of bacterial strain, this method can fast and accurately screen the bacterium for producing sarcosine oxidase, and bacterial strain obtained, which has, to be produced
The feature that amount is high, efficiency is fast and enzyme activity is high, enzyme activity is up to 120~160U/L, and has low-temperature epitaxy producing enzyme enzyme activity higher
Advantage, be conducive to later stepping practise medicine learning detection application aspect research.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further described.
Ooze sample in deep-sea used is from (N:39 ° 7 ', S:121 ° of Liaoning Province, Bohai Sea Gulf, Dalian in following embodiment
41 ') ooze seawater sample.
Embodiment 1
The screening technique of sarcosine oxidase bacterial strain is produced, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training
Case is supported, shaking flask enrichment culture is carried out at 150rpm, temperature setting is cultivated 2.5-3.0 days under 20-30 DEG C of constant temperature, while daily
Creatine degradation rate is surveyed, when creatine is degraded to 80% or more, is further cultured for 2 days, can carry out in next step；Blank group is carried out simultaneously
Culture, blank group is not add the culture medium of creatine, i.e., creatine be zero culture medium, above-mentioned sample sets are added to creatine
Culture medium, and with the variation of incubation time, creatine is constantly degraded, until infinite approach blank group, is defaulted as creatine
Degradation is complete；Wherein, enrichment culture based formulas: creatine 5.0-10.0g, yeast extract 0.5-5.0g, sodium chloride 2.0-5.0g, phosphoric acid
Potassium dihydrogen 1.0-5.0g, dipotassium hydrogen phosphate 0.5-5.0g, magnesium sulfate 0.5-5.0g, starch 10.0-15.0g, potassium iodide 1.0-
5.0g, agar 20.0-30.0g, distilled water 1.0-1.5L are saved after high-temperature sterilization, and temperature is controlled at 100-200 DEG C；
(2) produce sarcosine oxidase bacterial strain separation: by creatine in step (1) degrade substantially completely (i.e. sample values
Infinite approach blank group) culture medium, diluted with sterile water, be coated in screening and culturing medium after dilution, temperature setting is in 20-30
Constant temperature incubation 4-5 days under the conditions of DEG C；Wherein, screening flat board culture medium prescription: creatine 5.0-10.0g, yeast extract 0.5-10.0g,
Sodium chloride 2.0-10.0g, potassium dihydrogen phosphate 1.0-10.0g, dipotassium hydrogen phosphate 0.5-10.0g, magnesium sulfate 0.5-10.0g, starch
10.0-20.0g, potassium iodide 1.0-10.0g, agar 20.0-40.0g, distilled water 1.0-1.5L are saved after high-temperature sterilization, temperature
Control is at 100-200 DEG C；
(3) produce the primary dcreening operation of sarcosine oxidase bacterial strain: picking has the bacterial clump of transparent circle, is divided by three rides
From purifying, temperature setting is cultivated 4-5 days under 20-30 DEG C of constant temperature, obtains single bacterium colony, carries out microscopy, by observing bacterium colony shape
State, Preliminary Identification strain；
(4) produce the secondary screening of sarcosine oxidase bacterial strain: measurement enzyme activity finally selects the highest production sarcosine oxygen of one plant of enzyme activity
Change the bacterial strain of enzyme, carries out fungi preservation；
Above-mentioned creatine degradation rate measurement: the NaOH 1mL of the 1.5mol/L containing l% alpha-Naphthol is added in the sample of 0.5mL,
Add 0.5mL 0.05% biacetyl colour developing 30min, then plus 3mL distilled water, in 530nm survey absorbance；
Enzyme activity determination method: somatic cells to be measured are subjected to broken wall with ultrasonication machine, take supernatant after centrifugation (15min)
Enzyme solution 0.1mL is added to sodium pyrophosphate buffer solution (0.1mol/ of the 0.9mL (pH8.0,25 DEG C) containing 0.1mol/L sarcosine
L in), the acetic acid termination reaction for being added dense DEG C of 0.25mL after 10min as 1.0mol/L is reacted in 37 DEG C, and 1.5mL is added and contains
20% ammonium acetate solution of 0.04% acetylacetone,2,4-pentanedione measures OD value after 37 DEG C of heat preservation 40min at 410nm；Enzyme activity is defined as:
37 DEG C of enzyme amount for decomposing 1 μm of ol sarcosine per minute are an enzyme-activity unit.
Embodiment 2
The screening technique of sarcosine oxidase bacterial strain is produced, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training
Case is supported, shaking flask enrichment culture is carried out at 150rpm, temperature setting is cultivated 2.5 days under 20 DEG C of constant temperature, while surveying creatine daily
Degradation rate is further cultured for 2 days when creatine is degraded to 80% or more, can be carried out in next step；The culture of blank group is carried out simultaneously,
Blank group is not add the culture medium of creatine, i.e., creatine be zero culture medium, above-mentioned sample sets are to be added to the culture of creatine
Base, and with the variation of incubation time, creatine is constantly degraded, until infinite approach blank group, is defaulted as creatine degradation
Completely；Wherein, enrichment culture based formulas: creatine 5.0g, yeast extract 5.0g, sodium chloride 2.0g, potassium dihydrogen phosphate 1.0g, phosphoric acid hydrogen
Dipotassium 0.5g, magnesium sulfate 0.5g, starch 10.0g, potassium iodide 1.0g, agar 20.0g, distilled water 1.0L are saved after high-temperature sterilization,
Temperature is controlled at 121 DEG C；
(2) produce sarcosine oxidase bacterial strain separation: by creatine in step (1) degrade substantially completely (i.e. sample values
Infinite approach blank group) culture medium, diluted with sterile water, be coated in screening and culturing medium after dilution, temperature setting is at 20 DEG C
Under the conditions of constant temperature incubation 4 days；Wherein, screening flat board culture medium prescription: creatine 5.0g, yeast extract 0.5g, sodium chloride 2.0g, phosphoric acid
Potassium dihydrogen 1.0g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, starch 10.0g, potassium iodide 1.0g, agar 20.0g, distilled water
1.0L is saved after high-temperature sterilization, and temperature is controlled at 121 DEG C；
(3) produce the primary dcreening operation of sarcosine oxidase bacterial strain: picking has the bacterial clump of transparent circle, is divided by three rides
From purifying, temperature setting is cultivated 4 days under 20 DEG C of constant temperature, obtains single bacterium colony, carries out microscopy, by observing colonial morphology, just
Step identification strain；
(4) produce the secondary screening of sarcosine oxidase bacterial strain: measurement enzyme activity finally selects the highest production sarcosine oxygen of one plant of enzyme activity
Change the bacterial strain of enzyme, the bacterial strain for choosing enzyme activity 1.82U/ml is saved, as required bacterial strain.
Creatine degradation rate and enzyme activity determination method are the same as embodiment 1.
Embodiment 3
The screening technique of sarcosine oxidase bacterial strain is produced, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training
Case is supported, shaking flask enrichment culture is carried out at 150rpm, temperature setting is cultivated 2.5 days under 25 DEG C of constant temperature, while surveying creatine daily
Degradation rate is further cultured for 2 days when creatine is degraded to 80% or more, can be carried out in next step；The culture of blank group is carried out simultaneously,
Blank group is not add the culture medium of creatine, i.e., creatine be zero culture medium, above-mentioned sample sets are to be added to the culture of creatine
Base, and with the variation of incubation time, creatine is constantly degraded, until infinite approach blank group, is defaulted as creatine degradation
Completely；Wherein, enrichment culture based formulas: creatine 10.0g, yeast extract 5.0g, sodium chloride 2.0g, potassium dihydrogen phosphate 1.0g, phosphoric acid
Hydrogen dipotassium 0.5g, magnesium sulfate 0.5g, starch 10.0g, potassium iodide 1.0g, agar 20.0g, distilled water 1.0L are protected after high-temperature sterilization
It deposits, temperature is controlled at 121 DEG C；
(2) produce sarcosine oxidase bacterial strain separation: by creatine in step (1) degrade substantially completely (i.e. sample values
Infinite approach blank group) culture medium, diluted with sterile water, be coated in screening and culturing medium after dilution, temperature setting is at 25 DEG C
Under the conditions of constant temperature incubation 4 days；Wherein, screening flat board culture medium prescription: creatine 10.0g, yeast extract 0.5g, sodium chloride 2.0g, phosphorus
Acid dihydride potassium 1.0g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, starch 10.0g, potassium iodide 1.0g, agar 20.0g, distilled water
1.0L is saved after high-temperature sterilization, and temperature is controlled at 121 DEG C.
(3) produce the primary dcreening operation of sarcosine oxidase bacterial strain: picking has the bacterial clump of transparent circle, is divided by three rides
From purifying, temperature setting is cultivated 4 days under 25 DEG C of constant temperature, obtains single bacterium colony, carries out microscopy；By observing colonial morphology, just
Step identification strain；
(4) produce the secondary screening of sarcosine oxidase bacterial strain: measurement enzyme activity finally selects the highest production sarcosine oxygen of one plant of enzyme activity
Change the bacterial strain of enzyme, the bacterial strain for choosing enzyme activity 1.61U/ml is saved, as required bacterial strain.
Creatine degradation rate and enzyme activity determination method are the same as embodiment 1.
Embodiment 4:
The screening technique of sarcosine oxidase bacterial strain is produced, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training
Case is supported, shaking flask enrichment culture is carried out at 150rpm, temperature setting is cultivated 2.5 days under 30 DEG C of constant temperature, while surveying creatine daily
Degradation rate is further cultured for 2 days when creatine is degraded to 80% or more, can be in next step；The culture of blank group, blank are carried out simultaneously
Group is not add the culture medium of creatine, i.e., creatine be zero culture medium, above-mentioned sample sets are the culture mediums for being added to creatine, and
And with the variation of incubation time, creatine is constantly degraded, until infinite approach blank group, it is complete to be defaulted as creatine degradation；
Wherein, enrichment culture based formulas: creatine 10.0g, yeast extract 5.0g, sodium chloride 5.0g, potassium dihydrogen phosphate 5.0g, dipotassium hydrogen phosphate
0.5g, magnesium sulfate 5.0g, starch 15.0g, potassium iodide 5.0g, agar 30.0g, distilled water 1.5L are saved after high-temperature sterilization, temperature
Control is at 121 DEG C；
(2) produce sarcosine oxidase bacterial strain separation: by creatine in step (1) degrade substantially completely (i.e. sample values
Infinite approach blank group) culture medium, diluted with sterile water, be coated in screening and culturing medium after dilution, temperature setting is at 30 DEG C
Under the conditions of constant temperature incubation 4 days；Wherein, screening flat board culture medium prescription: creatine 5.0g, yeast extract 5.0g, sodium chloride 3.0g, phosphoric acid
Potassium dihydrogen 1.0g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, starch 10.0g, potassium iodide 1.0g, agar 20.0g, distilled water
1.0L is saved after high-temperature sterilization, and temperature is controlled at 121 DEG C.
(3) produce the primary dcreening operation of sarcosine oxidase bacterial strain: picking has the bacterial clump of transparent circle, is divided by three rides
From purifying, temperature setting is cultivated 4 days under 30 DEG C of constant temperature, obtains single bacterium colony, carries out microscopy；By observing colonial morphology, just
Step identification strain；
(4) produce the secondary screening of sarcosine oxidase bacterial strain: measurement enzyme activity finally selects the highest production sarcosine oxygen of one plant of enzyme activity
Change the bacterial strain of enzyme, the bacterial strain for choosing enzyme activity 1.72U/ml is saved, as required bacterial strain.
Creatine degradation rate and enzyme activity determination method are the same as embodiment 1.
Embodiment 5:
The screening technique of sarcosine oxidase bacterial strain is produced, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training
Case is supported, shaking flask enrichment culture is carried out at 150rpm, temperature setting is cultivated 3 days under 25 DEG C of constant temperature, while surveying creatine drop daily
Solution rate is further cultured for 2 days when creatine is degraded to 80% or more, can be carried out in next step；The culture of blank group is carried out simultaneously, it is empty
White group is the culture medium for not adding creatine, i.e., creatine be zero culture medium, above-mentioned sample sets are the culture mediums for being added to creatine,
And with the variation of incubation time, creatine is constantly degraded, until infinite approach blank group, is defaulted as creatine and has degraded
Entirely；Wherein, enrichment culture based formulas: creatine 10.0g, yeast extract 10.0g, sodium chloride 3.0g, potassium dihydrogen phosphate 5.0g, phosphoric acid hydrogen
Dipotassium 0.5g, magnesium sulfate 5.0g, starch 15.0g, potassium iodide 5.0g, agar 30.0g, distilled water 1.5L are saved after high-temperature sterilization,
Temperature is controlled at 121 DEG C；
(2) produce sarcosine oxidase bacterial strain separation: by creatine in step (1) degrade substantially completely (i.e. sample values
Infinite approach blank group) culture medium, diluted with sterile water, be coated in screening and culturing medium after dilution, temperature setting is at 25 DEG C
Under the conditions of constant temperature incubation 5 days；Wherein, screening flat board culture medium prescription: creatine 10.0g, yeast extract 10.0g, sodium chloride 5.0g, phosphorus
Acid dihydride potassium 3.0g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, starch 10.0g, potassium iodide 1.0g, agar 20.0g, distilled water
1.0L is saved after high-temperature sterilization, and temperature is controlled at 121 DEG C；
(3) produce the primary dcreening operation of sarcosine oxidase bacterial strain: picking has the bacterial clump of transparent circle, is divided by three rides
From purifying, temperature setting is cultivated 5 days under 25 DEG C of constant temperature, obtains single bacterium colony, carries out microscopy；By observing colonial morphology, just
Step identification strain；
(4) produce the secondary screening of sarcosine oxidase bacterial strain: measurement enzyme activity finally selects the highest production sarcosine oxygen of one plant of enzyme activity
Change the bacterial strain of enzyme, the bacterial strain for choosing enzyme activity 1.65U/ml is saved, as required bacterial strain.
Creatine degradation rate and enzyme activity determination method are the same as embodiment 1.
The highest bacterial strain to cultivate under the conditions of 20 DEG C of embodiment of enzyme activity, enzyme activity 1.82U/ in above-mentioned several embodiments
Ml, and the characteristics of belong to marine low temperature enzyme, be the enzyme.
Claims (4)
1. a kind of screening technique for producing sarcosine oxidase bacterial strain, which is characterized in that the described method comprises the following steps:
(1) will marine source sample access enriched medium in cultivate, temperature setting in 20-30 DEG C of constant temperature incubation, while daily
Creatine degradation rate is surveyed, when creatine is degraded to 80% or more, is further cultured for 2 days；
(2) it produces the separation of sarcosine oxidase bacterial strain: creatine in step (1) has been degraded complete culture medium, it is dilute with sterile water
It releases, is coated in screening and culturing medium after dilution, temperature setting is constant temperature incubation 4-5 days under the conditions of 20-30 DEG C；
(3) produce the primary dcreening operation of sarcosine oxidase bacterial strain: picking has the bacterial clump of transparent circle, separate by three rides pure
Change, temperature setting obtains single bacterium colony constant temperature incubation 4-5 days under the conditions of 20-30 DEG C, microscopy is carried out, by observing bacterium colony shape
State, Preliminary Identification strain；
(4) produce the secondary screening of sarcosine oxidase bacterial strain: it is highest finally to select one plant of production sarcosine oxidase enzyme activity for measurement enzyme activity
Bacterial strain, carry out fungi preservation, that is, filter out aimed strain.
2. a kind of screening technique for producing sarcosine oxidase bacterial strain as described in claim 1, which is characterized in that in step (1)
The access amount that sample accesses in enriched medium is the 5% of enriched medium volume.
3. a kind of screening technique for producing sarcosine oxidase bacterial strain as described in claim 1, which is characterized in that in step (1)
Enrichment culture based formulas: creatine 5.0-10.0g, yeast extract 0.5-5.0g, sodium chloride 2.0-5.0g, potassium dihydrogen phosphate 1.0-
5.0g, dipotassium hydrogen phosphate 0.5-5.0g, magnesium sulfate 0.5-5.0g, starch 10.0-15.0g, potassium iodide 1.0-5.0g, agar
20.0-30.0g, distilled water 1.0-1.5L are saved after high-temperature sterilization, and temperature is controlled at 100-200 DEG C.
4. a kind of screening technique for producing sarcosine oxidase bacterial strain as described in claim 1, which is characterized in that in step (2)
Screening flat board culture medium prescription: creatine 5.0-10.0g, yeast extract 0.5-10.0g, sodium chloride 2.0-10.0g, potassium dihydrogen phosphate
1.0-10.0g, dipotassium hydrogen phosphate 0.5-10.0g, magnesium sulfate 0.5-10.0g, starch 10.0-20.0g, potassium iodide 1.0-10.0g,
Agar 20.0-40.0g, distilled water 1.0-1.5L are saved after high-temperature sterilization, and temperature is controlled at 100-200 DEG C.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013941A (en) * | 2012-12-28 | 2013-04-03 | 卫生部北京医院 | Preparation method and application of sarcosine oxidase |
| CN104109658A (en) * | 2014-07-25 | 2014-10-22 | 江南大学 | Creatine hydrolysis enzyme as well as coding gene and application thereof |
| CN108977386A (en) * | 2018-08-08 | 2018-12-11 | 大连大学 | A method of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain |
| CN109022282A (en) * | 2018-08-08 | 2018-12-18 | 大连大学 | A kind of screening technique producing marine low temperature urate oxidase bacterial strain |
-
2019
- 2019-04-04 CN CN201910270719.XA patent/CN109897805A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013941A (en) * | 2012-12-28 | 2013-04-03 | 卫生部北京医院 | Preparation method and application of sarcosine oxidase |
| CN104109658A (en) * | 2014-07-25 | 2014-10-22 | 江南大学 | Creatine hydrolysis enzyme as well as coding gene and application thereof |
| CN108977386A (en) * | 2018-08-08 | 2018-12-11 | 大连大学 | A method of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain |
| CN109022282A (en) * | 2018-08-08 | 2018-12-18 | 大连大学 | A kind of screening technique producing marine low temperature urate oxidase bacterial strain |
Non-Patent Citations (5)
| Title |
|---|
| 刘辉等: "芽胞杆菌BSD-8肌氨酸氧化酶的纯化与性质", 《生物工程学报》 * |
| 孙桂芹: "Bacilus sp.BSD-8肌氨酸氧化酶的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 崔有宏等: "肌酐测定工具酶产生菌―烟草节杆菌02181的筛选及其产酶条件", 《甘肃科学学报》 * |
| 赵更峰等: "产肌氨酸氧化酶菌株的分离及发酵条件研究", 《微生物学报》 * |
| 赵更峰等: "节杆菌(Arthrobacter)42-1菌株的分离、鉴定及产肌氨酸氧化酶条件研究", 《浙江大学学报(农业与生命科学版)》 * |
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