CN108192848A - A kind of Psychrobacter bacterial strain of galactopoiesis carbohydrase and the method that low temperature lactase is prepared using the bacterial strain - Google Patents
A kind of Psychrobacter bacterial strain of galactopoiesis carbohydrase and the method that low temperature lactase is prepared using the bacterial strain Download PDFInfo
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Abstract
Method the invention discloses a kind of Psychrobacter new strains (Cryobacterium sp.LW097) and using this bacterial strain production low temperature lactase, while provide the low temperature lactase.By using Psychrobacter new strains Cryobacterium sp.LW097 (preserving number is CCTCC M 2017015), establish strain improvement, preservation and the method for rejuvenation, by culture medium prescription optimal screening and optimization of fermentation condition, establish stable fermentation process, the lactase of production has high enzyme activity at low temperature, has application value in Low lactose milk Dairy Processing.
Description
Technical field
The present invention relates to a kind of low temperature lactase, the producing strains and preparation method thereof of the enzyme.Specifically, the present invention relates to
It is that one kind is generated by Psychrobacter (Cryobacterium sp.) new strains LW097, at a lower temperature have greater activity
Lactase and its production method of low temperature lactase is prepared using the microbial strains, belong to microorganisms technical field.
Background technology
All contain lactose in most of mammal breast milks such as cow's milk.After the mankind eat the lactose in dairy products, mainly by dividing
The lactase Phlorizin hydrolase being distributed on intestinal brush border cell membrane is hydrolyzed, but asian population is due to genetic background and diet
The reasons such as difference, more than 90% people's alactasia, lactose cannot be digested and absorb after eating dairy products, generation abdominal pain,
The symptom (Husain 2010) of the lactose intolerances such as abdominal distension, nausea and vomiting.Beta galactosidase (β-galactosidase,
EC3.2.1.23), be commonly called as lactase, lactose that can be in extracorporeal hydrolysis dairy products, so as to effectively avoid lactose intolerant patient by
Lead to the adverse reaction of edible dairy products generation in itself alactasia.In addition, hydrolyze dairy products using beta galactosidase
In lactose, can overcome the problems, such as simultaneously the dairy products such as condensed milk, milk powder and ice cream lactose be precipitated, Shelf-life, improve
The sugariness of dairy products.
Beta galactosidase currently used for decomposing lactose in dairy products is mainly derived from the Crewe dimension of generally recognized as safe (GRAS)
Bacterial strain (the Oliveira et of saccharomyces (Kluyveromyces sp.) and aspergillus (Aspergillus sp.)
Al.2011), medium temperature lactase is belonged to.The beta galactosidase of trade name Lactozym is as tieed up from Kluyveromyces Lactis
The medium temperature beta galactosidase of yeast (Kluyveromyces lactis).The enzyme optimal reactive temperature is 50 DEG C, less than 20
DEG C cryogenic conditions under hydrolysis lactose efficiency be only under room temperature 10% or so, enzyme activity is also vulnerable to the suppression of Ca2+ and Na+
It makes (Wierzbicka-Wos et al.2011).Actual production increases the putrid and deteriorated wind of cow's milk according to ordinary-temp hydrolysis
Danger；And low temperature hydrolysis is selected then to increase production cycle extension, cost, it seriously limits the production capacity of low lactose milk and raises
Production cost.Therefore, it is obtained for exploitation and is more suitable for the beta galactosidase that low lactose milk produces under cryogenic conditions, various countries try
Figure is detached from the microorganism in various low temperature habitats to be obtained enzymatic property and meets low temperature beta galactose needed for dairy products low temperature process
Glycosides enzyme.
Since each Institute of Micro-biology production enzyme viability usually has differences, action pH range and peak enzymolysis-ability pH such as enzyme,
The operative temperature range of enzyme and the thermal stability of optimum temperature and enzyme etc., in the world to the research of production cold-adapted enzyme microorganism
Focus primarily on south poles glacier and abyssal environment.
It is below the main bacteria seed of current low temperature Lactase Research and its lactose enzyme viability of generation.
The D2 bacterial strains of Arthrobacter (Arthrobacter sp.), the lactase optimum temperature generated are 25 DEG C,
40% enzyme activity can only be retained at 10 DEG C, 50 DEG C of processing lose activity (Loveland et al.1994) for 10 minutes substantially.
Meat Bacillus (Carnobacterium piscicola) BA bacterial strains, the best use temperature of the lactase generated
It it is 30 DEG C, stability is poor, need to be preserved in the solution for adding 10% glycerine, and 40 DEG C of processing 10min lose vigor completely
(Coombs and Brenchley 1999)。
Planococcus (Planococcus) bacterial strain, the lactase optimal pH generated are 6.5, and the best use temperature is
42 DEG C, 55 DEG C of processing 10min can lose vigor (Sheridan and Brenchley 2000) completely.
The Pseudoalteromonas Pseudoalteromonas sp.22b bacterial strains detached out of euphausiid shrimp body generate breast
Carbohydrase optimum temperature is 6 DEG C, and the best use temperature of the lactase generated is 40 DEG C (pH 6.0-8.0), K+, Na+, Mn2
+, Mg2+ has the lactase certain activation, and Cu2+ has the lactase inhibiting effect, and Ca2+ is to the lactase without bright
Aobvious inhibition or activation (Turkiewicz et al.2003).
Arthrobacter (Arthrobacter) bacterial strain detached from South Pole gulch (Antarctic Dry Valley) soil,
A kind of its lactase the best use temperature generated is 18 DEG C, 0 DEG C of enzymatic activity for retaining 50%, to thermo-responsive, 37 DEG C of processing
10min enzyme activity disappears.
Flavobacterium Flavobacterium sp.4214 bacterial strains, it is 25 DEG C to generate lactase optimum temperature, is generated
Lactase the best use temperature for 42 DEG C, (Sorensen et al.2006) more sensitive to 25 DEG C of temperatures above.
From the Arthrobacter psychrolactophilus F2 bacterial strains of Arthrobacter, a kind of lactase generated
RBglAp the best uses temperature loses activity for 10 DEG C, pH8.0,50 DEG C processing 5min.
Rahnella aquatilis (Rahnella aquatilis) the bacterial strain 14-1 detached from Xinjiang Tianshan No.1 glacier, production
The optimal reactive temperature of raw lactase is 35 DEG C, and optimal reaction pH value is 6.5-7.0 (Liu Wen jade 2008).
Propiram great yeast (Guehomyces pullulans) 17-1 bacterial strains long, the best use of the lactase generated
Temperature is 50 DEG C, and the best use pH is 4.0 (Song et al.2010).
Paracoccus Paracoccus sp.32d bacterial strains, the best use temperature of the lactase generated is 40 DEG C, most preferably
Action pH is 7.5 (Wierzbicka-Wos et al.2011).
The Thalassospira sp.3SC-21 of sea rotation Pseudomonas, the best use temperature of the lactase generated is 20 DEG C,
6.5,45 DEG C of processing of the best use pH lose enzyme activity.
The drawing engler's Pseudomonas Rahnella sp.R3 bacterial strains detached from Xinjiang Tianshan No.1 glacier generate the energy of lactase
Power is 3.01U/ml, and the optimum temperature of the enzyme is 25 DEG C, and enzyme activity during 7.0,15 DEG C of optimal pH is highest enzyme activity
92%, it is 31% (Shen Lian lotuses 2013) at 4 DEG C.
Invention content
For the bacterial strain performance of production low temperature lactase cannot still meet low temperature lactase industrialized production skill both at home and abroad at present
The requirement of art.The purpose of the present invention is to provide a kind of low temperature lactases, generate the microorganism fungus kind of the enzyme and produce the low temperature
The method of lactase.The low temperature lactase can effectively hydrolyze the lactose in dairy products under cryogenic, through pasteurization
Can loss its enzymatic activity after heat treatment.
The present invention carries out separation, culture and the sieve of microorganism fungus kind by the soil sample to Xinjiang Tianshan glacial deposit layer
Choosing obtains the low temperature resistant microbial strains of a batch, and therefrom separates low temperature lactase producing bacterial strain, number LW097,
So as to provide one plant of low temperature lactase producing strains, there is the good characteristic of production low temperature lactase.
Meanwhile the present invention also provides a kind of low temperature lactase, pass through by using the bacterial strain of the present invention present invention determine that
Specific zymotechnique and obtain.
The present invention provides a kind of low temperature lactase bacterial strains, are named as LW097, can generate low temperature lactase.The bacterium
Strain was preserved in budapest treaty microorganism International Depository Authority before the applying date：China typical culture collection center
(CCTCC).Address：Wuhan City, Hubei Province Wuchang District Bayi Road Luo Jia Shan, postcode：430072, preservation date is January 9 in 2017
Day, preserving number：CCTCC NO: M 2017015.5~20 DEG C of the strain culturing temperature, most suitable 16 DEG C of cultivation temperature；Preferred growth
In 1/4TSB media surfaces (soybean broth hydrolysate 1.25g, casein hydrolysate 4.25g, sodium chloride 0.5g, glucose
0.75g, disodium hydrogen phosphate 0.425g, distilled water 1000ml), it is cultivated 72 hours through 16 DEG C, the white transparence of bacterium colony, bacterium colony
Diameter 1.2mm identifies method according to microbiology, carries out phylogenetic analysis to the 16S rDNA sequences of the bacterial strain, really
Determine LW097 bacterial strains and belong to Psychrobacter category (Cryobacterium sp.).Psychrobacter new strains Cryobacterium
Sp.LW097 can produce low temperature lactase, have effect in the range of 4.0~10.0,5~50 DEG C of pH, peak enzymolysis-ability condition is
25~30 DEG C of temperature, pH 6.0.The present invention further established the system process technology of culture presevation, selection and breeding.
The present invention low temperature lactase producing strains Cryobacterium sp.LW097,16S rDNA sequences be<210>
1。
Homologous sequence in the 16S rDNA sequences of LW097 and Genebank is carried out to when evolutionary analysis, LW097
The homology of 16S rDNA sequences and Cryobacterium arcticum PAMC 27867 reach 99%, with more plants
The homology that Cryobacterium belongs to bacterial strain all reaches more than 99%, and carries out evolutionary analysis to its homologous sequence, LW097 with
The Cryobacterium such as Cryobacterium psychrotolerans Z2_S_TSA32 (KC213912.1) belong to bacterial strain with
65% supporting rate is polymerized to cluster, and Phylogenetic is as shown in Figure 1, it is Psychrobacter to fix tentatively the bacterial strain
Cryobacterium sp.LW097。
May be either bacterial strain or sieve naturally that the present invention is protected as the generation strain of low temperature lactase of the present invention
The original strain of choosing or the dissociant to be made a variation for protection bacterial strain by natural variation or artificial induction, by of the present invention
Method, can be achieved technique effect set forth in the present invention.
As the method for production of above-mentioned dissociant, including physical mutagenesis, at treatment with UV radiation, 60Coradiation
The various Irradiations such as reason, ion implanting processing, laser radiation treatment；Mutagenesis, such as nitrosoguanidine (NTG), sulfuric acid diethyl
The processing of the chemical mutagens such as ester, it is excellent to go out production performance with conventional lactase producing strains separation screening culture medium and method optimal screening
Different bacterial strain.
It as the generation strain of low temperature lactase of the present invention, can also be through Protocols in Molecular Biology, from original strain
Or the low temperature lactase gene is obtained in induction variation strain, acquired low temperature lactase gene sequence is<210>2；<210
>3；<210>4；<210>One kind in 5；With prokaryotic micro-organisms, such as Escherichia coli, bacillus subtilis, lactobacillus plantarum bacterium, breast
Yogurt coccus etc., with eukaryotic microorganisms, such as saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis, as genetic recipient bacterium,
Genetic engineering production bacterial strain is built, as long as the low temperature lactose enzyme amino acid sequence of its expression is<210>6；<210>7；<210>8；<
210>One kind in 9 also can generate strain as the low temperature lactase of the present invention.
Low temperature lactase as the present invention generates strain Cryobacterium sp.LW097, and following method pair can be used
It is preserved, activated and is screened and its shake flask fermentation obtains the low temperature lactase of the present invention.
The low temperature lactase producing strains Cryobacterium sp.LW097 of the present invention are preserved using conventional inclined-plane passage
Method, as long as the method is that strain of the present invention is inoculated in the inclined-plane culture primary surface for being suitable for bacterial growth, 15~20 DEG C of cultures 3
~4 days, vacuum freeze-drying method can be stored 3 months or so or be utilized to the low-temperature preservation at 4 DEG C by strain system of the present invention
Into dry powder strain, low temperature or preservation under room temperature were up to 1 year or more.
The strain of long term storage is activated, is screened as follows when in use.By the strain of the present invention of long term storage
It is inoculated in 1/4TSB culture mediums or other is suitable for the media surface of the bacterial growth, 15~20 DEG C are cultivated 3~4 days；This hair
Bright preferential selection 1/4TSB culture medium (soybean broth hydrolysate 1.25g, casein hydrolysate 4.25g, sodium chloride 0.5g, grape
Sugared 0.75g, disodium hydrogen phosphate 0.425g, distilled water 1000ml).The strain after culture is inoculated in lactase screening and culturing medium again
On, screening and culturing medium is as follows：Soybean broth hydrolysate 1.25g, casein hydrolysate 4.25g, sodium chloride 0.5g, glucose
0.75g, disodium hydrogen phosphate 0.425g, agar 15g, distilled water 1000ml, X-Gal 0.01%, ITPG 0.1mM, through 16 DEG C of trainings
It supports 72 hours, generates blue colonies.Generating blue colonies means that bacterial strain can generate lactase.
Fermented and cultured experiment is carried out using the experimental strain of the present invention through filtering out, obtains the low temperature lactose of the present invention
Enzyme.During fermented and cultured, slant strains can be directly inoculated in fermentation medium or bacterial strain is first subjected to liquid increasing
Culture is grown, then is inoculated in fermentation medium with 5%~15% inoculum concentration and carries out fermented and cultured.
As the nutrient source of culture medium, the nutrient source commonly used in culture can be widely used.As long as carbon source can be used as same
For the carbon compound of change either containing the carbon compound, microorganism fungus kind is available, is suitable for fermented and cultured generates invention
The carbon source of the low temperature lactase of bacterium, for example, can be used starch, corn flour, glucose, brewer's wort, sucrose, hydrolysis sugar, can
Polysaccharide etc..Its dosage is different according to the selection difference of other nutrient sources and the difference of condition of culture, in the present invention
It is preferred that lactose is optimal carbon source, dosage 4%.
As nitrogen source, as long as the nitrogen compound that can assimilate as nitrogen source either contains the nitrogen compound, example
Such as, ammonium salt, nitrate, soy meal, meat extract, corn impregnation liquid, corn pulp, yeast extract etc. can be used.The selection of its nitrogen source
And dosage, it can be different according to the ingredient of other nutrient sources and the difference of dosage, as long as choosing is suitable for the low temperature lactase of the present invention
The culture of producing strains and the generation of enzyme.Preferred tryptone+yeast extract is best nitrogen source in the present invention, and dosage is divided
It Wei 1.5% and 0.5%.
As inorganic salts, phosphate, magnesium salts, calcium salt, manganese salt of phosphoric acid hydrogen ammonia, potassium dihydrogen phosphate etc. etc. can be properly added
Salt.Condition of culture is how many and different according to the component of culture medium, is generated in the present invention but as long as being suitable for thalline culture
Lactase condition.
It is cultivated in general, following condition may be selected.That is, cultivation temperature is 15~20 DEG C, preferably 16 DEG C；During culture
Between about 3~4 days, as long as terminating culture when the output of low temperature lactase of the present invention reaches highest；The pH of culture medium
Can 6~8 production range, particularly 7 or so more suitable for lactase of the present invention production.Through cultivating as described above,
Purpose product low temperature lactase is mainly generated in somatic cells.
From culture solution as obtained above acquire low temperature lactase, can according to commonly used in acquire desmoenzyme method,
Such as the processing of multigelation, ethyl acetate, ultrasonic disruption, high-speed homogenization crush, further by centrifugation, ammonium sulfate precipitation, thoroughly
Analysis, ultrafiltration concentration, freeze-drying, chromatographic isolation etc., separation and purification and carry out.
That is, the low temperature lactase of the present invention can be obtained by following methods：By filtration method or centrifugal separation from zymotic fluid
Isolated thalline after the thalline of collection is rinsed and is resuspended using phosphate buffer, passes through multigelation method, ultrasound
Crush method or high-speed homogenization crush method make enzyme add solubility salt, the salting out method for precipitating enzyme from releasing into the cell；
Hydrophilic organic solvent is added, enzyme activity is made to be mingled with the organic solvent precipitation method of precipitation；Use the absorption of ion exchange resin etc.
Spin off method；Gel filtration；Addition stablizes adjuvant or does not add the spray drying process of stable adjuvant；Independent or multiple combinations
Separation or process for purification using freeze-drying etc..
By the elaboration of the present invention as above, obtain Examples hereinafter and further verify, learn low temperature lactase of the present invention
Enzymatic property.The low temperature lactase peak enzymolysis-ability pH that bacterial strain LW097 of the present invention is generated is 6.0 or so, in the range of pH4~9
It can keep higher enzymatic activity；There is enzymatic activity in the range of 5~50 DEG C, optimum temperature is at 30 DEG C or so；The low temperature
The stability of lactase at low temperature is preferable, more sensitive to heat, and 120min is kept the temperature at 20 DEG C, and enzyme activity can still keep 80% left side
It is right；Metal ion has the activity of low temperature lactase certain influence, and wherein K+, which has its enzyme activity, certain to be swashed
Effect living, Cu2+, Fe2+ have significant inhibiting effect, Ca2+, Zn2+, Mn2+, Mg2+, Na+ and Fe3+ couple to its enzyme activity
Its enzyme activity influences smaller.
By implementing the specific technical indicator of the present invention, realize the content of present invention, following advantageous effect can be reached.
The present invention Psychrobacter new strains Cryobacterium sp.LW097 and on microbial taxonomy for
Its equal bacterial strain and its dissociant is effectively used for generating the low temperature lactase of the present invention.Further more, advantages of the present invention
It is, the low temperature lactose can be effectively generated using the method for low temperature lactase of the bacterial strain manufacture with above-mentioned property
Enzyme.
A kind of Psychrobacter bacterial strain, which is characterized in that the Psychrobacter bacterial strain Cryobacterium sp.LW097 are
With preserving number CCTCC NO:M 2017015 is preserved in the bacterial strain of China typical culture collection center.
A kind of production method of low temperature lactase, which is characterized in that include the following steps：
A, actication of culture：To the Psychrobacter new strains Cryobacterium sp.LW097 described in claim 1 into
Row strain activation and culture；
B, producing enzyme is fermented：Producing enzyme fermented and cultured is carried out using the activated spawn that step A is obtained, you can obtains low temperature lactose
Enzyme.
Matched in step A using culture medium A, the culture medium A according to mass volume ratio, lactose 1%~2%, yeast leaching
Cream 1%~2%, NaCl 0.5%~1%, tryptone 0.5%~1%, distillation water dissolution adjustment pH to 6.8~7.2；It is living
The temperature for changing culture is 15~20 DEG C, is cultivated 4~5 days, and the seed culture fluid of activation is made.
Seed culture fluid made from step A is inoculated according to 5~15% inoculum concentrations in following fermentation medium B, is cultivated
Base B matches according to mass volume ratio, lactose 1%~4%, tryptone 4%~5%, yeast extract 1%~2%, magnesium sulfate
0.03%~0.3%, potassium dihydrogen phosphate 0.003%~0.007%, calcium chloride 0.009%~0.013%, manganese sulfate
0.0001%~0.0002%, ferrous sulfate 0.002%~0.005%, the water of surplus, pH is 6.5~7.5, after sterilizing, 16
DEG C, 150~300 revs/min of lower shaken cultivation 56h~60h after culture, are centrifuged and are collected thalline, and~70 DEG C and 50 DEG C are anti-
After multiple freeze thawing 3~5 times, centrifuge and collect supernatant and obtain low temperature lactase crude enzyme liquid, the crude enzyme liquid yield of enzyme is up to 5~20U/
ml。
As an improvement, in order to further obtain enzyme after purification, can be added in into the low temperature lactase crude enzyme liquid of gained
Ammonium sulfate precipitation, the precipitation that ammonium sulfate saturation degree is taken to be stood between 50-60% add in phosphoric acid to obtained precipitation
In salt buffer, after phosphate buffer dissolving precipitation, then with bag filter dialysis desalting, the enzyme activity of gained low temperature lactase can
Up to 5~20U/ml.
The phosphate solution pH is 7.2~7.4.
Description of the drawings
Fig. 1 show the 16S rDNA of the Psychrobacter new strains Cryobacterium sp.LW097 based on the present invention
The evolutionary analysis topology diagram of sequence.
Fig. 2 show the low temperature lactose generated with the Psychrobacter new strains Cryobacterium sp.LW097 of the present invention
The figure of enzyme reaction pH and relative activity relationship.
Fig. 3 show the low temperature lactose generated with the Psychrobacter new strains Cryobacterium sp.LW097 of the present invention
The figure of enzyme reaction temperature and relative activity relationship.
Fig. 4 show the low temperature lactose generated with the Psychrobacter new strains Cryobacterium sp.LW097 of the present invention
Enzyme handles the figure of residual enzymic activities at each temperature.
Fig. 5 show the low temperature lactose generated with the Psychrobacter new strains Cryobacterium sp.LW097 of the present invention
Enzyme handles the figure of residual enzymic activities under each reaction pH.
Fig. 6 show each metal ion species and the Psychrobacter new strains Cryobacterium sp.LW097 of the present invention is produced
The influence of raw low temperature lactase enzymatic activity.
Fig. 7 is shown with ortho-nitrophenyl (ONP) as standard items, the relationship between OD values and ortho-nitrophenyl (ONP) content.
In the following, illustrating the present invention for embodiment, still, the present invention is not limited to following embodiments.
Specific embodiment
Embodiment 1：Produce the culture of low temperature lactase Psychrobacter (Cryobacterium sp.) bacterial strain LW097
The low temperature lactase producing strains LW097 of the present invention is inoculated in and adds 0.01%X~Gal, the 1/ of 0.1mM IPTG
In 4TSB plating mediums, wherein soybean broth hydrolysate 1.25g, casein hydrolysate 4.25g, sodium chloride 0.5g, glucose
0.75g, disodium hydrogen phosphate 0.425g, distilled water 1000ml, 1.0kg/cm2 steam sterilizing 15 minutes.It is small through 16 DEG C of cultures 72
When, then the bacterium colony of production low temperature lactase bacterium of the invention is in blue.
Embodiment 2：Produce the fermented and cultured of low temperature lactase Psychrobacter (Cryobacterium sp.) bacterial strain LW097
By production low temperature lactase Psychrobacter (Cryobacterium sp.) bacterial strain LW097 access seed trainings of the present invention
Support base：7.0,16 DEG C of cultures 4~5 of lactose 10g, yeast extract 10g, NaCl 4g, tryptone 5g, distilled water 1000ml, pH
My god；Then, it is inoculated in following fermentation medium with 5% inoculum concentration.Lactose 40g, potassium dihydrogen phosphate 0.05g, tryptone
15g, calcium chloride 0.11g, yeast extract 5g, manganese sulfate 0.001g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, pH 7.0,250ml
In triangular flask, per bottled 50ml, 1.0kg/cm2 steam sterilizings 15 minutes.16 DEG C, 200 revs/min of lower shaken cultivation 56h.Culture
Afterwards, it centrifuges and collects thalline, after~70 DEG C and 50 DEG C of multigelations, centrifuge and collect the supernatant acquisition thick enzyme of low temperature lactase
Liquid, yield of enzyme is up to 5U/ml.Ammonium sulfate precipitation is added in into the low temperature lactose enzyme solution of gained, takes saturation degree in 50-
It stands and is precipitated between 60%, with bag filter dialysis desalting, the enzyme activity of gained low temperature lactase is up to 5U/ml.
Embodiment 3：The assay method of low temperature lactase enzymatic activity
The measure of low temperature lactose enzyme activity is carried out using colorimetric method.With ortho-nitrophenyl~β~D~galactoside
(oNPG) for substrate specificity, with the unit enzyme solution of unit volume within the unit interval its enzymolysis product o-nitrophenol (oNP)
Production quantity carries out the calculating of enzyme activity.
Enzyme activity defines：Under certain conditions, 1min hydrolyzes 1 μm of ol oNP of oNPG generations and is defined as 1 enzyme-activity unit.Root
ONP contents are calculated according to standard curve and 420nm the OD values measured.Under these conditions, 1min hydrolyzes oNPG and generates 1 μm of ol
ONP is defined as 1 enzyme-activity unit.Specific method is as follows：
A：The drafting of oNP standard curves：ONP139.0mg (ortho-nitrophenol) standard items is taken to be put into 100ml volumetric flasks, are added
95% ethyl alcohol 10ml is allowed to dissolve, then is settled to 1000ml with the phosphate buffer of pH7.0, is configured to 1 mmolL~1oNP
Mother liquor is drawn the solution 2ml, 4ml, 6ml, 8ml, 10ml, 12ml, 14ml, is added separately in the volumetric flask of 100ml respectively,
With mixing after the phosphate buffer constant volume of pH 7.0, above-mentioned every milliliter of dilution respectively containing 0.02 μm of ol of oNP, 0.04 μm of ol,
0.06 μm of ol, 0.08 μm of ol, 0.10 μm of ol, 0.12 μm of ol, 0.14 μm of ol, using the phosphate buffer of pH 7.0 as blank, in wave
Absorbance is surveyed at long 420nm, using absorbance value as ordinate, standard curve is drawn with a concentration of abscissas of oNP, sees Fig. 7.
B：The measure of lactase activity：In microwell plate, it is (molten to add in the 50.0 μ L substrates oNPG containing 4mg/ml to every hole
In 0.1M phosphate buffers, pH 7.0) solution A and 50.0 μ L crude enzyme liquids, at 15 DEG C after heat preservation 15min, add in 200.0 μ L
0.5M precooling sodium carbonate terminates reaction, and product o-nitrophenol (o~nitrophenyl, oNP) shows Huang under alkaline environment
Color measures light absorption value at 420nm wavelength.When measuring enzyme activity, crude enzyme liquid is diluted in right amount with 0.1M phosphate buffers, makes OD420
Value is in 0~0.8 range of linearity)
C：The calculating of enzyme activity is as follows：
E：Enzyme activity force value (U/ml)；OD420：The light absorption value measured at 420nm wavelength；N：Crude enzyme liquid extension rate；t：Thick enzyme
The time of liquid and substrate reactions.
Embodiment 4：Influence of the action pH to low temperature lactase enzyme activity
Action pH and Optimal pH measure, and using oNPG as substrate, are surveyed by aforementioned low temperature lactase activity test method
It is fixed.The pH of measure is respectively the enzyme activity E under the different pH condition in the range of 3~10.To measure under the highest pH of enzyme activity
Enzyme activity value is control, sets its enzyme activity 100%, then the relationship of action pH and enzymatic activity is as shown in Figure 2.Bacterial strain of the present invention
The low temperature lactase that LW097 is generated its peak enzymolysis-ability pH at 30 DEG C is 6.0, and higher enzyme can be kept in the range of pH 6~8
Activity.
Embodiment 5：Influence of the operative temperature to low temperature lactase enzyme activity
Action pH and Optimal pH measure, and using oNPG as substrate, are surveyed by aforementioned low temperature lactase activity test method
It is fixed.The different temperatures that ranging from 5 DEG C~60 DEG C of measuring temperature.To measure the enzyme activity value at the highest temperature of enzyme activity as control,
Its enzyme activity 100% is set, then the relationship of operative temperature and enzymatic activity is as shown in Figure 3.In 5 DEG C~60 DEG C of temperature range
Interior, enzymatic activity is presented in the low temperature lactase that bacterial strain LW097 of the present invention is generated, and best hydrolysis temperature is 30 DEG C or so.10
In the range of~30 DEG C of measuring temperature have good enzymatic property, can be kept at 20 DEG C 70% or so enzyme activity,
More than 60% enzyme activity can be still kept at 10 DEG C.
Embodiment 6：The thermal stability of low temperature lactase
The low temperature lactose enzyme solution that embodiment 2 obtains is kept the temperature under the conditions of 30 DEG C, pH6.0, then the enzyme as described in embodiment 3
Activity determination method was measured its residual enzyme activity every 20 minutes.It is not right not carry out the enzyme activity force value of isothermal holding
According to setting its enzyme activity as 100%, then treatment temperature and the relationship of residual activity at this time is as shown in Figure 4.Bacterium of the present invention
Low temperature lactase caused by strain LW097 has preferable thermal stability below 30 DEG C, and 30 DEG C of heat preservations can still be kept for 120 minutes
60% or so enzyme activity has higher thermal stability.
Embodiment 7：The pH stability of low temperature lactase
The low temperature lactase crude enzyme liquid of 2 gained of embodiment is pressed 1：1 ratio mixes respectively with the buffer solution of pH2~11,
12h is kept the temperature at 4 DEG C, enzyme solution is then adjusted back into Optimun pH (pH6.0), remnant enzyme activity is measured at 30 DEG C.With
Enzyme activity determination value under pH6.0 is control, sets its enzyme activity as 100%, at this time handle pH and residual activity
Relationship is as shown in Figure 5.Low temperature lactase caused by bacterial strain LW097 of the present invention keeps steady in the range of 6.0~pH of pH 8.0
It is fixed.
Embodiment 8：Metal ion is to the influence of low temperature lactase enzyme activity
Metal ion presses above-mentioned enzyme assay method to the influence of low temperature lactase, in the phosphate-buffered of pH 6.0
Ca2+, Zn2+, Cu2+, Mn2+, Mg2+, Na+, K+, Fe2+ and Fe3+ are added in liquid, its final concentration is made to reach 10mM.By implementation
Activity determination method described in example 3 is measured.With do not add in metal ion be control, set its enzyme activity as
100%, then metal ion is as shown in Figure 6 to the influence of enzyme activity.Wherein K+ has its enzyme activity certain activation to make
With Cu2+, Fe2+ have its enzyme activity significant inhibiting effect, and Ca2+, Zn2+, Mn2+, Mg2+, Na+ and Fe3+ are to its enzyme
It is smaller to learn activity influence.
Embodiment 9：A kind of production method of low temperature lactase, which is characterized in that include the following steps：
A, actication of culture：To the Psychrobacter new strains Cryobacterium sp.LW097 described in claim 1 into
Row strain activation and culture, obtains seed culture fluid；
B, producing enzyme is fermented：Producing enzyme fermented and cultured is carried out using the activated spawn that step A is obtained, you can obtains low temperature lactose
Enzyme.
Matched in step A using culture medium A, the culture medium A according to mass volume ratio, lactose 1%~2%, yeast leaching
Cream 1%~2%, NaCl 0.5%~1%, tryptone 0.5%~1%, distillation water dissolution adjustment pH to 6.8~7.2；It is living
The temperature for changing culture is 15~20 DEG C, is cultivated 4~5 days, and the seed culture fluid of activation is made.
Seed culture fluid made from step A is inoculated according to 5~15% inoculum concentrations in following fermentation medium B, is cultivated
Base B matches according to mass volume ratio, lactose 1%~4%, tryptone 4%~5%, yeast extract 1%~2%, magnesium sulfate
0.03%~0.3%, potassium dihydrogen phosphate 0.003%~0.007%, calcium chloride 0.009%~0.013%, manganese sulfate
0.0001%~0.0002%, ferrous sulfate 0.002%~0.005%, the water of surplus, pH is 6.5~7.5, after sterilizing, 16
DEG C, 150~300 revs/min of lower shaken cultivation 56h~60h after culture, are centrifuged and are collected thalline, and~70 DEG C and 50 DEG C are anti-
After multiple freeze thawing 3~5 times, centrifuge and collect supernatant and obtain low temperature lactase crude enzyme liquid, the crude enzyme liquid yield of enzyme is up to 20U/ml.
Ammonium sulfate is added in into the low temperature lactase crude enzyme liquid of gained, its saturation degree is made to reach 55%, standing is precipitated
4 DEG C afterwards, 5000 revs/min of centrifugation 10min collect supernatant, and it is 60% to continue slowly to add in ammonium sulfate to saturation degree into supernatant,
Standing precipitated after 4 DEG C, 5000rpm centrifugation 10min collect precipitation, will obtain precipitation add in pH7.2~7.4 PBS phosphorus
In phthalate buffer, after phosphate buffer dissolving precipitation, dialyse then at 4 DEG C of bag filters using interception as 10000Da de-
Salt, the enzyme activity of gained low temperature lactase is up to 15U/ml.
<110>Shihezi Univ
<120>A kind of Psychrobacter bacterial strain of galactopoiesis carbohydrase and the method that low temperature lactase is prepared using the bacterial strain
<141>
<160> 5
<210> 1
<211> 1487
<212>Nucleotide sequence
<213>Psychrobacter（Cryobacterium sp.LW097）
<220>
<221> misc_feature
<223>16S rRNA gene nucleotide series
<400> 1
GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTGAAGAGGAG
CTTGCTCCTTGGATCAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTCTGGGATAAGCACTGGAA
ACGGTGTCTAATACCGGATACGAACTTCAGCCGCATGGCTAGGAGTTGGAAAGAATTTCGGTCAAGGATGGACTCGC
GGCCTATCAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCAC
ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATG
CAGCAACGCCGCGTGAGGGACGACGGCCTTCGGGTTGTAAACCTCTTTTAGTAGGGAAGAAGCGAAAGTGACGGTAC
CTGCAGAAAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTATT
GGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAAACCCGAGGCTCAACCTCGGGCCTGCAGTGGGTA
CGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACAC
CAATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACC
CTGGTAGTCCATGCCGTAAACGTTGGGAACTAGATGTGGGGACCATTCCACGGTCTCCGTGTCGCAGCTAACGCATT
AAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGC
ATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATATAGAGGAAACGGCTGGAAACAGTCGCC
CCGCAAGGTCTCTATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA
GCGCAACCCTCGTCCTATGTTGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAG
GTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTG
CAATACCGCAAGGTGGAGCGAATCCCAAAAAGCCGGTCTCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAGT
CGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGT
CATGAAAGTCGGTAACACCCGAAGCCAGTGGCCAAACCCGCAAGGGATGGAGCTGTCGAAGGTGGGATCGGTGATTA
GGACTAAGTCGTAACAAGGTAGCCGTAC
<210> 2
<211> 2031
<212>Nucleotide sequence
<213>B-gal322 lactase genes
<220>
<221> misc_feature
<223>B-gal322 lactase gene nucleotide sequences
<400> 1
ATGTCGATCCACACCCCATTCCCCGCCAGAGCGCCCGCGACAGACCGGTGGTTGAAGGGCACGACCGGGCTTC
GTTACGGTGGCGACTACAACCCGGAGCAGTGGCCCCGCGAGACCTGGCTGGAAGACATCAAGCTGATGAAGCAGGCC
GGGATCAACCTGGTCAGCATCGCGATCTTCGCCTGGGGCATCCTGGAGCCACGCGAGGGCGAATACGACTTCAGCAA
GCTCGACGACATCTTCGGCCTGCTGCACGAGGCCGGCATCGACATCGACCTCGCCACGCCCACCGCGGCCCCGCCGG
CCTGGTTCTGGAAGAAATACCCGGACTCCCGGCCGGTCACCCGGGACGGCATCACCCTGGGCAACGGTTCCCGCGGC
ATGGTCAGCCCGTCGAGCCCCGACTACCGCCGGGCCGCCGCGGCCATCACCGAGCAGCTCGCCCGCCGGTATGCGAA
CCACCCCGCCCTTGTGCTCTGGCACGTGCACAACGAGTACGGTGCGCCCATCAGCGACTCCTACGACGACCACTCCG
TCGCCGCCTTCAGGGTCTGGCTGCAGAAGCGTTACGGCACCCTTGACACCCTCAATGAGAAGTGGGGCACCACCTTC
TGGGGACAGGTGTACGGCGAGTGGGACGAGATCGACGCGCCCCGCCAGTCGGCCTCGGTGAGCAACCAGGCGCAGCG
CCTGGACTTCCACCGGTTCACCTCCGACGCGTTGCTCGAGTGCTACATCAACGAGCGCGATGTCATCCGCCGGTTCA
CCCCGGACATCCCCGTGACCACCAACTTCATGGCCACCAATTGCCTCTCCCCCGACTACTGGAAGTGGGCCAGGGAG
GTGGACATCGTCGCCAACGACCACTACCTGGTGGCCGAACGCGCCGACAACCATGTGTTGCTGGCCATGGATGCCGA
CCTGTCCCGGTCGCTGGCCGGCGGCGCCCCGTGGATCCTGATGGAGCACTCCACCGGCGCCGTCAACTGGCAGCCCC
GCAACATCGCCAAGCGTGCCGGCGAGATGGCCCGCAACTCACTCTCCCACCTGGCCCGCGGCGCCGACGGCATCCTG
TTCTTCCAGTTCCGGGCCTCCAGGTTCGGCGTTGAGAAGTTCCACTCCGCGATGCTGCCGCACGCCGGAGCCGAGAC
CCGGGTCTGGCGCGAGGTCGTCGCCCTGGGCGAAACCCTCGGGTCCCTCGAGGCCGTGCGCGGCTCCCGCGTGCAGG
CTCGCGTCGCGCTGCTCTGGGACACCGAGTCGTTCTGGGCACAGGACTTCGAATGGCGCCCGAGCGCCGAACTGGGC
CACCGCGAACGCCTCGAGGCCTTCTACACGGCGCTGTGGAACCGCAACGTGTCGGTGGACTTCGCGCATCCGCGCGA
CGACCTCACCGGCTACGACCTCGTTATCGCGCCCAGCCTCTACCTCGCCGGTCCGGCGGCCTCGGAGAACCTCACCG
GCTACGTGCGGGACGGCGGCCACCTGGTCGTCTCCTACTTCTCCGGCATCGTCGACGAGAACGACACCGTCTACCCG
GGAGCCGCGCCCGGCGCGCTCCGGGAGGTGCTCGGCCTGACCATCCCGGAGTTCCTGCCCCTGCACGAGAACGAGAC
CGTCACCCTCAGCGATTCCCGCACCGGAACCGCCTGGACCGACGACATCGAGGTCTCCACCGCTGAGGTGCTGGCCC
GGTACGTGGACGGCCCGGCCGCCGGCGGACCGGCCCTCACCCGCAACGCGTTCGGTGCGGGGTCGGCCTGGTACATC
TCGACCAAGCTCACCGGAACCGACCTGGACTCCCTGCTGCTGGACGTCCTCAACACCGCCGGCATCGAGGCGGCCCG
CGGCATCGACGGCGTCGAGACCATCATCCGGTCCACCGACACCGACGAGTTCCTGTTCGTGATCAACCACACGGATG
CCGACCAGTCCTGGCCGGCCTCCGGCACCGAGTTACTCACCCAGGCCACGGTCGACGGCACCGTCCTGGTACCGGCC
GGACTCACCCGCGTGGTGCACAGCGTCCGATAG
<210> 3
<211> 2154
<212>Nucleotide sequence
<213>B-gal435 lactase genes
<220>
<221> misc_feature
<223>B-gal435 lactase gene nucleotide sequences
<400> 1
ATGAGCGCCGTGAGCACGGTCTCGACCAACGAGCATCGCTGGGTGCGCTGGCCCGAGGCGCACACCCTCGCCG
ACGGCACCGAGAGCCTGCCCGCCGACGCCGCCCGCATCGGCTACGGCGCCGACTACAACCCCGAACAGTGGCCCGAA
GAGGTCTGGGCCGAAGACATGCGCCTGATGCGCGAGGCCGGTGTCAACATCGTCAGCGTCGGCATCTTCTCCTGGGC
GCTGCTGCAGCCCACCCCGGACAGCTGGGATTTCGGTTGGCTCGACCGCGCCATCGATCTGCTGCACGCCAACGGCA
TCGCCGTCGACCTGGCCACCGCCACCGCCAGCCCGCCGCCGTGGCTCACCGAGCTGCACCCGGAGGTGCTGCCGGTG
AACCGGGTCGGCGAAACCATCTGGCCCGGCGGCCGCCAGCAGTGGCGCCCCACCTCACCGGTGTTCCGCCGCTACGC
GCTGGCCCTGGTCGAGAAAATGGCCGAGCGCTACGCCAACCACCCGGCCCTGTCGATGTGGCACATCAGCAACGAGC
TCGGCTGCCACAACGTCTACGACTTCTCGGACGACGCGGCAGCGGCGTTCCGCGCCTGGCTCGAGGCCCGCTACGGC
ACTCTCACCGAACTCAACCGGGCCTGGGGCACCGCCTTCTGGTCGCAGAACTACACCTCCTGGAACCAGATCCTGCC
GCCGCGGCTGGCCGCGACGCATCCGAACCCCACCCAGCAGCTCGACTTCGCCCGGTTCTCGAGCGACGCCCTCAAGG
GCTACCTCGTCGCCGAGCGGGAGATCCTGCGCCAGATCACGCCCGACGTGCCGATCACCACCAACTTCATGGTGATG
GGCGAGTCCCGCGGCATGGACTACGCCGACTGGGCCGGCGAGATCGACCTGGTCTCCAACGACCACTACCTGCTCGT
GGCCGACCCCGCCGCGTTCGAGGAGCTGTCGTTCTCGGCCAACCTCACCGGCCAGATATCCGGCCGGGCGCCGTGGT
TCCTGATGGAGCACTCCACCAGCGCCGTCAACTGGCAGCCGGTGAACCAGGCCAAAACCCCCGGCCAGCTGCGCCGG
GACAGCCTCACCCACCTGGCCCACGGCGCCGACGCGATCTGCTTCTTCCAGTGGCGCCAGTCCTTGGCCGGCGCCGA
GAAGTTCCACTCCGCGATGCTCCCGCACGCGGGGGAGAACAGCTCGGTGTTCCGCAGCGTCACCCGCTTCGGCGGCG
AACTCGCTTCCCTGGCAGAGGTCGCCGGCAGCCGCACCAAGCAGGCCCGCATCGGAATCCTCTTCGACTGGGACTCC
TGGTGGGCCTCCGAACTCGACTCGCACCCCACCGAGCTGCTGCGCTACAAGGCCGAGGCCATGGCCTGGTACACCGC
CGCGCTCACCATTGGCCTGCAGGCCGACATTGTGCCGGCCCGGTCGGTGCTGAACGGTCACGGCCTCGCCGGGTACG
ACCTCGTCATCGCGCCGATGCTCTACATAGTGCCGCAGCCGCTGGCGGATGCCCTGGCCGACTACGCCCGCACCGGC
GGCCACCTGGTGGTCGGCGTGTTCTCCGGCATTGCCGACGCCGACGACCACATCCACCCCGGCGGTTACCCCGGCGC
GTTCCGCGAGCTGCTCGGCGTGCGCGCCGAGGAGTTCGGCGGCCTGCAGCCGGGCCAGACCGTCGGCCTGTCCGGCG
ACCTGCCGGCCGGGTCAACCGGGTCGCTGCTCACCCACGACGTCACCGTGGCCTCCGATGTCCAGGTGCTCGCCCGC
TTCCAGGACGGCCCGTTCCCCGGTGTGCCGGCGGTGACCAGCCGCGTGGTCTCGACCGAGTCCGGCGGCCGGGCCAG
CTTCGTCGCCACGGTCCTCGACCCCGACGCGTTGGTGGCTTTGGTCGGCCGGTTCGCCGCCGCCGCCCACATCGTCT
CGCCGCTGCCGAGAGACGCGCACGGCATCGTGCAGCTCGCCGTGCGCCAGTCCGACGACATGGACTATCTCTTCTAC
ATCAACCACTCCGACAAGGCCGTGACCGTGCCGCTGGGCAAGGCCGATGGCACGCTCGTGGCCGTCGACCTCGGCGG
CAGCACCCTCGCCGCGGACTCGCTCACCCTCCCGGCCACCGGCGTCGCCGTGCTCGGCCGCGCCCGAACGGCGGCCT
GA
<210> 4
<211> 2064
<212>Nucleotide sequence
<213>B-gal2567 lactase genes
<220>
<221> misc_feature
<223>B-gal2567 lactase gene nucleotide sequences
<400> 1
ATGACCACGCCAGTAGCCACCCCGGCCGTGACCCGTCAGGTCACCCCGACGACACCGTTCGTGCAGAACGGCA
TCGCCTACGGCTGTGACTACAACCCCGAGCAATGGGGTCCGGAAGTCTGGGCCGAGGACGTGCTCCTCATGCAGCAG
GCCGGCGTCGACCTCGTCGCGATCAACATCTTCGGCTGGTCCGCCCTGGAGCCGGCCCCGGGGGAGTACGACTTCAC
CCTGCTCGACACCATCGTGGACCTGCTGCACGCTCACGGCATCCGGGTGAACCTGGGTACCGGCACCTCGTCGCCGC
CGCCGTGGTTGACCACCCTGCACCCCGAGATCCTGCCGCAGACCGAAGACGGCACCACCCGCTACCCCGGCGGTCGC
CAGGCCTGGTGCCCCAGCTCGCCGGTGTTCCGCGAGCACGCGCTGCGCCTGGTCGAGCAGGTCGCCAAGCGCTTCGG
CGACCATCCCGCCGTGGCCCTCTGGCACGTCTCCAACGAACTCGGCTGCCACAACGCGCTCTGCTACGACGACGACA
CCGCCGCCGCGTTCCGGCTCTGGCTCGAGGCCAAGTACGGCACCATCGCCGCCCTCAACGCCGCCTGGGGCACCAGC
TTCTGGAGCCAGCGTTACACCGACTGGGCCGAGGTGCACACCCCCAGGCTCACCCTCTCCAGCCGCAACCCCGGACA
GGTGCTCGACTTCCAGCGGTTCAGCTCCGACGAGCTGCTGGGCTACTACAAGGCCGAAGAAGCCGTCATCCGCCGGC
ACAGCAGCCTGCCCGTCACGACGAACTTCATGGTCGCCGCGCACATTCGCAACCTCGATTACTGGTCCTGGGCATCC
GCCGTTGACGTGATCGCCAACGACCACTACCTCGATCACCGCCTCGCCGACCCCACCACCGAGCTGGCCTTCGCCGC
CGACCTCACCCGCGGCCTGGCCGGCGGCGGCGCGTGGCTGCTGATGGAGCAGGCCACCAGCGCCGTCAACTGGCAGC
CGCTGAACCTGGCCAAGACGCCGGGGGAGCTGACCCGCAACTCGCTGGCCCACGTGGCCCGCGGCGCCGAGGCGATC
TGCTTCTTCCAGTGGCGCGCCTCCCAGCAGGGCTCGGAGAAGTTCCACTCCGCACTGGTGCCGCACGCCGGCACCGA
CACCGTGCTCTGGCGTGAAGTCGTTGAGCTGGGCGCCACCCTCGACAGGCTCGACGAGATCATCGGCACGCGCGTCG
TCGCGGATGCCGCGATCCTGTTCAGCTGGGAGGCCTGGTGGGCCGGCGACGGCGAATCCCGGCCGTCGCAGTCGGTG
ACCTACCTCGAGCAGGTGCACGCCGCGTACACCGCGCTGCACAGGCTCGGCGTCACCGTCGACATCGTCTCCCCGGA
TGCCGACCTGGCCGGCTACAACCTCGTCGTGGTGCCCGCTCTCTACCTGGTCACCGACGCCCAGGCCGACAACCTGT
CCGGTTACGTCGCCGCCGGTGGCCACACCCTGGTCACGTTCTACAGCGGCATCGTCGACGAAGACGACAGGGTGCGC
CTCGGCGGCTACCCCGGCGCGTTCCGCGACCTGCTCGGCGTGCGCGTCGAAGAGTTCGTGCCGCTCCGCCCCGGCAC
CACCGTGGGCCTGGGCGCCCCGATCGGGCTGGCGGCCGGTGCCGCCGGGACCCTGTGGACCGAACGCCTGCAGCTGA
CCGGCGCGGAGGCCGTGGCGCACTACACCGACGGCGCCCTGCCCGGCGTGCCCGCGCTCACCCGCAACCGGCACGGC
GCCGGCACCGCCTGGTACCTGGCCACCGCGCCAGACGCCGACACCTACCGCGACGTGCTCCGCACCATCGCCGGGCA
CGCTCACGTCACAGCGGTCGGTCCGGAGGGCGACGGCCTCATCGAGGTCATCCGCCGCGCCGCGCCCGGCCGCTCCT
ACCGCTTCATCATCAACCACGGCGACACCGACCTGGAGGTGAGCGCGGCGGGGGTCGAGCTCATCACCGGCGCCGAC
ATCGCCCGCACCCTGCGCGTACCGGCCGGAGCCGTACGCGTTCTCAGAGAGGACACCGCATCATGA
<210> 5
<211> 1761
<212>Nucleotide sequence
<213>B-gal436 lactase genes
<220>
<221> misc_feature
<223>B-gal436 lactase gene nucleotide sequences
<400> 1
ATGGGCGCGTTCGAGATCGGCGACGAGCACTTCCTCCTCAACGGTGAGCCGTTCCGGGTGCTCGCCGGGGCCA
TCCACTATTTCAGGGTGCACCCGGATCACTGGGCCGACCGCATCCACAAGGCCCGCCTGATGGGCCTGAACACCATC
GAGACCTACGTGGCCTGGAACGCGCACTCGCCGGCTCGTGGCGGTTTCGACACCGAGGGCCAGCTCGACCTGGCCCG
TTTTCTCGACCTGATCGCCGCCGAGGGCATGTACGCCATCGTGCGGCCCGGCCCGTTCATCTGCGCCGAGTGGGACA
ACGGCGGCCTGCCCGGCTGGCTGTTCACCGACCCGGCCGTGGGGGTGCGGCGGAACGAGCCGCTCTATATGGCGGCC
GTGGCCGAGTACTTCGAGCAGCTGCTGCCGATCGTGGCGTCCCGGCAGATCGACCGGGGCGGCCCGGTCATCCTGGT
GCAGATCGAGAACGAGTACGGCGCCTACGGCGACGACAAGGATTACCTGCGCGCGCTCGTCGAGCTCAACCGGGCCG
GCGGCATCACGGTGCCGCTGACTACCATCGACCAGCCCACCGACCAGATGCTCTCCGACGGCAGCCTGCCCGAGTTG
CACAAAACCGGCTCGTTCGGCTCCCGCGCTACCGAGCGCCTGGCCGTGCTCCGCCGCCACCAGCCGACCGGACCGCT
GATGTGCGCCGAGTTCTGGAACGGCTGGTTCGACCACTGGGGTGCGCACCACCACACCACTTCCGCGGAGGACTCCG
CCCGCGAGCTCGACGACCTGCTCGCCACCGGCGCCAGTGTGTCGCTGTACATGTTCACCGGCGGCACCAACTTCGGC
TTCACCAACGGCGCCAACGACAAGGGTGTCTTCCAGCCCACGGTCACTTCTTACGACTACGACGCGCCCCTCTCGGA
GAGCGGTGAGGTCACAGCCAAGTACCTCGCCTTCCGCGAGGTGCTCGCCAAGTACGCGCCGGTGACCGGGTCGTTGC
CGGCCCCGGCCGCGCCCGCGCCGGCCTTCGAGGTCGCCCTCGACGAGTTCGTTTCGCTGTGGGACGCGTTACCCGAG
CTGGCCGACATAAGCGCCGCTCCGACCGCCGGGCTGCCCAGCATGGATGCCCTCGGCCAGTACACCGGGTTCGCCCT
GTACCGCAGCCGGTTGACTCCGGGTGCCCGCGTGCTGTCCTTCGGCGAGGTGCGCGACCGCGCCCAGGTTTTCGTCG
ACGGCAACCCCGTCGGTGTGCTGCAGCGCGACCACCATGACCGCTCGATCGGCCTACCGCCGGGGGAGCGCCTCGAC
CTGCTCGTGGAGGACCAGGGCAGGGTCAACTACGGCCCGCGCATCGGCGAGGACAAGGGCCTGATCGGCCCGGCCAC
CCTCGACGGTGTCACCCTGGCCGACTGGCAGGTGCTGCCGCTCGACGTCGACGGGTTCGTCGCATCCGGCAGCGCCC
GCTTCGCCCTCGACCTCGCCGGTTCCGGCTCGCTCAGCGGTCCCGCGTTCGTGCGCGGCCGGTTCACCGCCGAGCCC
GGCAAGGACCTGTTCCTCAGCACCGCCGGCTGGGGCAAGGGCCAGGTCTGGATCAACGGGTTCAACCTCGGCCGGTT
CTGGGACCGTGGACCGCAGACCACCCTCTACGTGCCCGGTCCGGTGCTGAGGGCCGACAACGAGCTGGTCATCCTCT
GCCTGCACGGCACCGAGAGCACCCTCGCCCACTTCGTCCCCCGCCCCGACCTAGGCCACACCGACTTCTAG
<210> 6
<211> 676
<212>Amino acid sequence
<213>B-gal322 lactases
<220>
<221> misc_feature
<223>B-gal322 lactose enzyme amino acid sequences
<400> 1
MSIHTPFPARAPATDRWLKGTTGLRYGGDYNPEQWPRETWLEDIKLMKQAGINLVSIAIFAWGILEPREGEYD
FSKLDDIFGLLHEAGIDIDLATPTAAPPAWFWKKYPDSRPVTRDGITLGNGSRGMVSPSSPDYRRAAAAITEQLARR
YANHPALVLWHVHNEYGAPISDSYDDHSVAAFRVWLQKRYGTLDTLNEKWGTTFWGQVYGEWDEIDAPRQSASVSNQ
AQRLDFHRFTSDALLECYINERDVIRRFTPDIPVTTNFMATNCLSPDYWKWAREVDIVANDHYLVAERADNHVLLAM
DADLSRSLAGGAPWILMEHSTGAVNWQPRNIAKRAGEMARNSLSHLARGADGILFFQFRASRFGVEKFHSAMLPHAG
AETRVWREVVALGETLGSLEAVRGSRVQARVALLWDTESFWAQDFEWRPSAELGHRERLEAFYTALWNRNVSVDFAH
PRDDLTGYDLVIAPSLYLAGPAASENLTGYVRDGGHLVVSYFSGIVDENDTVYPGAAPGALREVLGLTIPEFLPLHE
NETVTLSDSRTGTAWTDDIEVSTAEVLARYVDGPAAGGPALTRNAFGAGSAWYISTKLTGTDLDSLLLDVLNTAGIE
AARGIDGVETIIRSTDTDEFLFVINHTDADQSWPASGTELLTQATVDGTVLVPAGLTRVVHSVR*
<210> 7
<211> 717
<212>Amino acid sequence
<213>B-gal435 lactases
<220>
<221> misc_feature
<223>B-gal435 lactose enzyme amino acid sequences
<400> 1
MSAVSTVSTNEHRWVRWPEAHTLADGTESLPADAARIGYGADYNPEQWPEEVWAEDMRLMREAGVNIVSVGIF
SWALLQPTPDSWDFGWLDRAIDLLHANGIAVDLATATASPPPWLTELHPEVLPVNRVGETIWPGGRQQWRPTSPVFR
RYALALVEKMAERYANHPALSMWHISNELGCHNVYDFSDDAAAAFRAWLEARYGTLTELNRAWGTAFWSQNYTSWNQ
ILPPRLAATHPNPTQQLDFARFSSDALKGYLVAEREILRQITPDVPITTNFMVMGESRGMDYADWAGEIDLVSNDHY
LLVADPAAFEELSFSANLTGQISGRAPWFLMEHSTSAVNWQPVNQAKTPGQLRRDSLTHLAHGADAICFFQWRQSLA
GAEKFHSAMLPHAGENSSVFRSVTRFGGELASLAEVAGSRTKQARIGILFDWDSWWASELDSHPTELLRYKAEAMAW
YTAALTIGLQADIVPARSVLNGHGLAGYDLVIAPMLYIVPQPLADALADYARTGGHLVVGVFSGIADADDHIHPGGY
PGAFRELLGVRAEEFGGLQPGQTVGLSGDLPAGSTGSLLTHDVTVASDVQVLARFQDGPFPGVPAVTSRVVSTESGG
RASFVATVLDPDALVALVGRFAAAAHIVSPLPRDAHGIVQLAVRQSDDMDYLFYINHSDKAVTVPLGKADGTLVAVD
LGGSTLAADSLTLPATGVAVLGRARTAA*
<210> 8
<211> 687
<212>Amino acid sequence
<213>B-gal2567 lactases
<220>
<221> misc_feature
<223>B-gal2567 lactose enzyme amino acid sequences
<400> 1
MTTPVATPAVTRQVTPTTPFVQNGIAYGCDYNPEQWGPEVWAEDVLLMQQAGVDLVAINIFGWSALEPAPGEY
DFTLLDTIVDLLHAHGIRVNLGTGTSSPPPWLTTLHPEILPQTEDGTTRYPGGRQAWCPSSPVFREHALRLVEQVAK
RFGDHPAVALWHVSNELGCHNALCYDDDTAAAFRLWLEAKYGTIAALNAAWGTSFWSQRYTDWAEVHTPRLTLSSRN
PGQVLDFQRFSSDELLGYYKAEEAVIRRHSSLPVTTNFMVAAHIRNLDYWSWASAVDVIANDHYLDHRLADPTTELA
FAADLTRGLAGGGAWLLMEQATSAVNWQPLNLAKTPGELTRNSLAHVARGAEAICFFQWRASQQGSEKFHSALVPHA
GTDTVLWREVVELGATLDRLDEIIGTRVVADAAILFSWEAWWAGDGESRPSQSVTYLEQVHAAYTALHRLGVTVDIV
SPDADLAGYNLVVVPALYLVTDAQADNLSGYVAAGGHTLVTFYSGIVDEDDRVRLGGYPGAFRDLLGVRVEEFVPLR
PGTTVGLGAPIGLAAGAAGTLWTERLQLTGAEAVAHYTDGALPGVPALTRNRHGAGTAWYLATAP
DADTYRDVLRTIAGHAHVTAVGPEGDGLIEVIRRAAPGRSYRFIINHGDTDLEVSAAGVELITGADIARTLRV
PAGAVRVLREDTAS*
<210> 9
<211> 586
<212>Amino acid sequence
<213>B-gal436 lactases
<220>
<221> misc_feature
<223>B-gal436 lactose enzyme amino acid sequences
<400> 1
MGAFEIGDEHFLLNGEPFRVLAGAIHYFRVHPDHWADRIHKARLMGLNTIETYVAWNAHSPARGGFDTEGQLD
LARFLDLIAAEGMYAIVRPGPFICAEWDNGGLPGWLFTDPAVGVRRNEPLYMAAVAEYFEQLLPIVASRQIDRGGPV
ILVQIENEYGAYGDDKDYLRALVELNRAGGITVPLTTIDQPTDQMLSDGSLPELHKTGSFGSRATERLAVLRRHQPT
GPLMCAEFWNGWFDHWGAHHHTTSAEDSARELDDLLATGASVSLYMFTGGTNFGFTNGANDKGVFQPTVTSYDYDAP
LSESGEVTAKYLAFREVLAKYAPVTGSLPAPAAPAPAFEVALDEFVSLWDALPELADISAAPTAGLPSMDALGQYTG
FALYRSRLTPGARVLSFGEVRDRAQVFVDGNPVGVLQRDHHDRSIGLPPGERLDLLVEDQGRVNYGPRIGEDKGLIG
PATLDGVTLADWQVLPLDVDGFVASGSARFALDLAGSGSLSGPAFVRGRFTAEPGKDLFLSTAGWGKGQVWINGFNL
GRFWDRGPQTTLYVPGPVLRADNELVILCLHGTESTLAHFVPRPDLGHTDF*
Claims (8)
1. a kind of Psychrobacter bacterial strain, which is characterized in that the Psychrobacter bacterial strain Cryobacterium sp.LW097 be with
Preserving number CCTCC NO:M 2017015 is preserved in the bacterial strain of China typical culture collection center.
2. a kind of production method of low temperature lactase, which is characterized in that include the following steps：
A, actication of culture：Bacterium is carried out to the Psychrobacter new strains Cryobacterium sp.LW097 described in claim 1
Kind activation culture, obtains seed culture fluid；
B, producing enzyme is fermented：Producing enzyme fermented and cultured is carried out using the activated spawn that step A is obtained, you can obtains low temperature lactase.
3. the production method of low temperature lactase as claimed in claim 2, it is characterised in that：Culture medium A, institute are used in step A
It states culture medium A according to mass volume ratio to match, lactose 1%~2%, yeast extract 1%~2%, NaCl0.5%~1%, pancreas egg
White peptone 0.5%~1%, distillation water dissolution adjustment pH to 6.8~7.2；The temperature of activation culture is 15~20 DEG C, culture 4~5
My god, the seed culture fluid activated is made.
4. the production method of low temperature lactase as claimed in claim 2 or claim 3, it is characterised in that：Seed made from step A is trained
Nutrient solution is inoculated according to 5~15% inoculum concentrations in following fermentation medium B, and culture medium B matches according to mass volume ratio, lactose
1%~4%, tryptone 4%~5%, yeast extract 1%~2%, magnesium sulfate 0.03%~0.3%, potassium dihydrogen phosphate
0.003%~0.007%, calcium chloride 0.009%~0.013%, manganese sulfate 0.0001%~0.0002%, ferrous sulfate
0.002%~0.005%, the water of surplus, pH is 6.5~7.5, and after sterilizing, training is vibrated under 16 DEG C, 150~300 revs/min
56h~60h is supported, after culture, centrifuges and collects thalline, after -70 DEG C and 50 DEG C of multigelations 3~5 times, is centrifuged in collection
Clear to obtain low temperature lactase crude enzyme liquid, the crude enzyme liquid yield of enzyme is up to 5~20U/ml.
5. the production method of low temperature lactase as claimed in claim 2 or claim 3, it is characterised in that：To the low temperature lactase of gained
Ammonium sulfate is added in crude enzyme liquid, its saturation degree is made to reach more than 60%, standing is precipitated, and obtained precipitation is added in pH7.2
In~7.4 PBS phosphate buffers, after phosphate buffer dissolving precipitation, then with bag filter dialysis desalting, gained low temperature breast
The enzyme activity of carbohydrase is up to 5~20U/ml.
6. the production method of low temperature lactase as claimed in claim 4, it is characterised in that：To the thick enzyme of low temperature lactase of gained
Ammonium sulfate precipitation is added in liquid, the precipitation that ammonium sulfate saturation degree is taken to be stood between 50-60%, to obtained precipitation
It adds in the PBS phosphate buffers of pH7.2~7.4, after phosphate buffer dissolving precipitation, then with bag filter dialysis desalting,
The enzyme activity of gained low temperature lactase is up to 5~20U/ml.
7. the low temperature lactase gene extracted in a kind of sp.LW097 from Psychrobacter bacterial strain Cryobacterium, feature exist
It is in its nucleic acid sequence<210>2；<210>3；<210>4；<210>One kind in 5.
8. the low temperature lactase extracted from Psychrobacter bacterial strain Cryobacterium sp.LW097 as claimed in claim 7
Gene, it is characterised in that：Its amino acid sequence is<210>6；<210>7；<210>8；<210>One kind in 9.
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Cited By (2)
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| CN107151640A (en) * | 2017-06-16 | 2017-09-12 | 石河子大学 | A kind of Psychrobacter bacterial strain of galactopoiesis carbohydrase and the method that low temperature lactase is prepared using the bacterial strain |
| CN116121225A (en) * | 2022-09-17 | 2023-05-16 | 石河子大学 | Low-temperature trehalase, coding sequence thereof, high-yield recombinant strain and application |
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Cited By (4)
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| CN107151640A (en) * | 2017-06-16 | 2017-09-12 | 石河子大学 | A kind of Psychrobacter bacterial strain of galactopoiesis carbohydrase and the method that low temperature lactase is prepared using the bacterial strain |
| CN107151640B (en) * | 2017-06-16 | 2020-01-10 | 石河子大学 | Lactobacillus acidophilus strain for producing lactase and method for preparing low-temperature lactase by using same |
| CN116121225A (en) * | 2022-09-17 | 2023-05-16 | 石河子大学 | Low-temperature trehalase, coding sequence thereof, high-yield recombinant strain and application |
| CN116121225B (en) * | 2022-09-17 | 2024-04-26 | 石河子大学 | Low-temperature trehalase, coding sequence, recombinant strain and application thereof |
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