CN103773700A - Penicillium oxalicum strain for producing high-temperature polygalacturonase and application of penicillium oxalicum strain - Google Patents
Penicillium oxalicum strain for producing high-temperature polygalacturonase and application of penicillium oxalicum strain Download PDFInfo
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Abstract
The invention relates to a penicillium oxalicum strain for producing high-temperature polygalacturonase and an application of the penicillium oxalicum strain. The penicillium oxalicum strain for producing the high-temperature polygalacturonase is preserved with the serial number of CCTCC No.M2013627 in China center for type culture collection located in the Wuhan university of Wuhan city of China. The strain can be applied to the fermentation production of high-temperature polygalacturonase. A complex enzyme (crude enzyme) of the polygalacturonase produced by the strain protected by the invention shows the maximum reaction activity at the temperature of 60 DEG C and still keeps about 85% of enzyme activity at the reaction temperature of 65 DEG C, and no relevant reports are found so far; therefore, the strain is a penicillium oxalicum strain for producing the high-temperature polygalacturonase.
Description
Technical field
The invention belongs to biotechnology and engineering field, particularly from occurring in nature be rich in screening the environment of pectic substance, separation, purifying obtain a strain can produce the penicillium oxalicum bacteria strain of high temperature polygalacturonase (
penicillium oxalicum).
Technical background
pectin be one mainly by D-galactosonic acid through the polysaccharose substance that α-(Isosorbide-5-Nitrae)-glycosidic link is polymerized, be extensively present in higher plant tissue, especially at fruit, stem piece, in berry, content is the abundantest, is the important component part of cell interbed and primary cell wall.All the time, the degraded of pectic substance is attracting Chinese scholars extensive concern and research, particularly in fruit and vegetable juice processing industry, the existence of pectic substance can make the viscosity of liquid increase, to the clarity of garden spgarden stuff, strainability, crushing juice rates etc. all can cause and have a strong impact on.
Polygalacturonase is the general name of the prozyme that a class can depolymerized pectin class material.It is extensively present in bacterium, fungi, actinomycetes and animals and plants.According to the difference of effect substrate and reactive mode, polygalacturonase roughly can be divided into protopectinase, Rohapect MPE, polygalacturonase and pectin lyase.Protopectinase Main Function, in the protopectin-of capacitive not, makes it become the soluble pectin of high-polymerization degree; Rohapect MPE mainly makes pectin degreasing by the methoxyl group in hydrolysis of pectin; Polygalacturonase and pectin lyase make on pectin main chain α-(Isosorbide-5-Nitrae)-glycosidic link between D-galacturonic acid rupture by hydrolytic action and trans-elimination respectively, make the pectin generation depolymerization of high-polymerization degree.
Polygalacturonase is that a class has significant application value prozyme, it is one of industrial enzyme preparation of global volume of production and marketing maximum, the particularly processing of garden spgarden stuff, fruit wine of main application food-processing industry, can significantly improve the crushing juice rate of squeezing garden spgarden stuff, reduce the viscosity of fruit juice fruit wine, increase clarifying effect, thereby guarantee the quality of fruit juice fruit wine.Polygalacturonase is the important component part of polygalacturonase, is also a kind of polygalacturonase of people's most study all the time.It is hydrolyzed the glycosidic link of polygalacturonic acid (pectic acid) by circumscribed and mode inscribe, be under the jurisdiction of glycoside hydrolase 28 families (GH28).Endopolygalacturonase (endo-PG) glycosidic link adjacent with carboxyl that can only split, is hydrolyzed polygalacturonic in random mode, generates a series of oligogalacturonans, and the viscosity of substrate is reduced rapidly; Polygalacturonic acid excision enzyme (exo-PG) cuts 1-2 galacturonic acid residue from the non-reduced end of polygalacturonic acid, generates digalactosyl aldehydic acid or galacturonic acid mono.
The polygalacturonase that derives from microorganism is most important a kind of polygalacturonase in industrial application, and the industries such as in fruit juice manufacture, brewing fruit wine, feed processing, crudefiber crop are come unstuck, papermaking all have a large amount of reports.Some kinds during the microorganism that can produce polygalacturonase mainly has that aspergillus, mould, head mold, reaping hook are mould etc. and belongs to.Although the enzymatic property of the polygalacturonase that domestic and foreign literature all produces these kinds has been reported a lot, but, along with the increase day by day of polygalacturonase demand and importance, screen the new polygalacturonase production bacterial strain with potential using value still very necessary.
The polygalacturonase with pyroreaction characteristic is even more important in industrial application (particularly fruit juice processing).Because high temperature not only can reduce the pollution of harmful microorganism, but also can reduce the viscosity for the treatment of solution, thereby the shearing force of making and pumping, centrifugal, filtration expense reduce.At present, the microorganism kind report that can produce the polygalacturonase with pyroreaction characteristic is relatively less, and the penicillium oxalicum bacteria strain that can directly produce such prozyme more rarely has pertinent literature report.
Summary of the invention
The object of this invention is to provide and a kind ofly can produce the microorganism strains of the polygalacturonase with pyroreaction characteristic and the application aspect polygalacturonase fermentative production thereof.
The solution of the present invention is: a kind of penicillium oxalicum bacteria strain that produces high temperature polygalacturonase, this bacterial strain deposit number is CCTCC No.M2013627.This bacterial strain is that contriver obtains with microbiology means isolation and selection from citrus orchard soil, the Chinese Typical Representative culture collection center being preserved on December 03rd, 2013, this is centered close to No. 16, Luo Jia Shan road, wuchang, wuhan district of Hubei China province Wuhan University, and deposit number is CCTCC No.M2013627.This bacterial strain, after biolog microorganism automatic identifying system is identified, determines that it is penicillium oxalicum (Penicill
ium oxalicum).Its ITSDNA sequence and Genbank nucleic acid database are listed
penicillium oxalicum strain a1s2_d38the ITSDNA sequence (KC344971.1) of bacterial strain,
penicillium oxalicum strain 114-2the ITSDNA sequence (KF152942.1) of bacterial strain has 99% sequence homology.
As further restriction of the present invention, described penicillium oxalicum strain fermentation produces to such an extent that the catalyzed reaction temperature of high temperature polygalacturonase is 55-65 ℃, under 55-65 ℃ of condition, keeps more than 85% enzyme activity.
As further restriction of the present invention, it is 36-72 hour after fermentation culture starts that described penicillium oxalicum strain fermentation produces high temperature polygalacturonase stationary phase.
Produce an application for the penicillium oxalicum bacteria strain of high temperature polygalacturonase, deposit number is the application of penicillium oxalicum bacteria strain aspect high temperature polygalacturonase fermentative production that CCTCC No.M2013627 produces high temperature polygalacturonase.
As further restriction of the present invention, described penicillium oxalicum strain fermentation produces to such an extent that the catalyzed reaction temperature of high temperature polygalacturonase is 55-65 ℃, the polygalacturonase optimal reactive temperature that this bacterial strain produces is 60 ℃, all can keep more than 85% enzyme activity 55-65 ℃ of scope.
As further restriction of the present invention, it is 36-72 hour after fermentation culture starts that described penicillium oxalicum strain fermentation produces high temperature polygalacturonase stationary phase.
As further restriction of the present invention, described penicillium oxalicum strain fermentation used medium is: 1-2% pectin, 0.1-0.2% ammonium sulfate, 0.01-0.05% magnesium sulfate, 0.1-0.3% potassium primary phosphate, 0.01-0.05% calcium chloride, 0.5-1.0% SODIUMNITRATE, 0.1-0.5% tween 80, initial pH value is 5.0-6.0.As medium optimization scheme, this strain fermentation culture medium is: 1% pectin, and 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80, initial pH value is 5.5.
As further restriction of the present invention, described penicillium oxalicum strain fermentation condition is: under 30-37 ℃ of condition, shaking speed 200-300rpm cultivates 36-72 hour.As culture condition preferred version, optimal culture condition is: 30 ℃, 200rpm shaking table is cultivated 60 hours, and this bacterium is in the time cultivating 60h, and in fermented liquid, polygalacturonase vigor reaches maximum value, is about 3445.3u/ml.
(1) for making the present invention openly abundant, the strains separation that the present invention protects and Breeding Process comprise that three steps carry out: sampling, primary dcreening operation, multiple sieve and purifying.
Sampling: the soil gathering in the citrus orchard of suburb, Yangshuo County, hazard prevention, acquisition time in October, 2013, picker Lu Bo.
Primary dcreening operation: prepare primary dcreening operation substratum (pectin 30g, NaNO
35g, KCl0.5g, MgSO
40.3g, (NH4)
2sO
41.4g, CaCl
20.3g, KH
2pO
43.8g, agar powder 20g, tetrabromophenol sulfonphthalein 0.2g, water 1000ml), 5.5,121 ℃ of sterilizing 20min of Initial pH.Step: the random about 0.5g sample of picking from the soil gathering, be suspended in 10ml sterilized water, fully shake up, after standing for some time, get supernatant liquor and dilute suitable multiple, evenly coat in primary dcreening operation substratum, cultivate 2-3 days for 30 ℃.The larger bacterium colony of the yellow hydrolysis circle of picking does further screening.
Multiple sieve and purifying: prepare liquid fermentation medium (1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80), initial ph value, 5.5,121 ℃ of sterilizing 20min.The colony inoculation that primary dcreening operation is obtained is in liquid fermentation medium, 30 ℃, 200rpm shaking table is cultivated 48 hours, getting fermented liquid measures polygalacturonase vigor and can ferment and produce polygalacturonase (enzymatic reaction condition is as 0.5% polygalacturonic acid take the bacterium colony determining primary dcreening operation and obtain, pH4.8 acetic acid-sodium acetate buffer, 50 ℃ of reaction 15min, produce 1ug galacturonic acid residue with 1min hydrolysis substrate and are defined as an enzyme activity unit).Then bacterium colony high enzyme activity is done after further separation and purification, it is stable that screening obtains a strain growth, the bacterial strain that enzymatic productivity is high, and its polygalacturonase vigor reaches maximum value and is about 3445.3u/ml, called after PC1.
(2), for making the present invention openly abundant, the classification of the bacterial strain that the present invention protects is identified and is comprised Molecular Identification and the evaluation of biolog microorganism automatic identifying system.
Molecular Identification process: PC1 inoculation, in PDA substratum, is cultivated after 5-6 days for 30 ℃ and collected spore, by 10
6the inoculum size of individual spore/ml is inoculated in liquid fermentation medium, and 30 ℃, 200rpm shaking table is cultivated after 48 hours and collected somatic cells, and liquid nitrogen freezing grinds after fragmentation, extracts the genomic dna of thalline with phenol-chloroform method.Take this DNA as template, adopt universal primer ITS1(5-TCCGTAGGTGAACCTGCGG-3 ') and ITS4(5 '-TCCTCCGCTTATTGATATGC-3 ') carry out polymerase chain reaction (PCR), the ITSDNA sequence of this thalline that increases.The reaction system of 50ul is containing PCR damping fluid 5ul, ITS1 and the each 2ul of ITS4 primer, each dNTP2ul, DNA profiling 1ul, TaqDNA polysaccharase 0.3ul, ddH
2o37.7ul.Reaction conditions: 94 ℃ of 5 min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min.Gained reaction product is carried out determined dna sequence analysis, obtain the ITSDNA sequence of this bacterial strain 596pb, this sequence is carried out to the search of homologous sequence compare of analysis at the Genbank of NCBI website nucleic acid database, result shows, the ITSDNA sequence (KC344971.1) of the listed Penicillium oxalicum strain a1s2_d38 bacterial strain of its ITSDNA sequence and Genbank nucleic acid database, the ITSDNA sequence (KF152942.1) of Penicillium oxalicum strain 114-2 bacterial strain has 99% sequence homology.
Biolog microorganism automatic identifying system is identified: this inoculation, in 2% malt extract nutrient agar (20g wort dehydrated medium, 18g bacterium level agar, 1L sterilized water), is placed in to 26 ℃ of incubators and is cultured to product spore (about 5-6 days).Get appropriate spore make spore suspension in the FF-IF inoculation liquid of producer's configuration with aseptic inoculation rod is sticky, by the bacterial suspension inoculation preparing to FF plate (every hole 100ul), after inoculation, cover lid being positioned in 26 ℃ of incubators is cultivated 3 days, cultivates to finish rear use MicrologTM 3 softwares and carry out interpretation of result.
The above analysis result, the bacterial strain PC1 that identifies the product polygalacturonase that institute's seed selection obtains be penicillium oxalicum (
penicillium oxalicum).
(3), for making the present invention openly abundant, the product enzyme time of bacterial strain and the characteristic of the polygalacturonase that produces thereof are specific as follows:
Determining of the product enzyme time of bacterial strain: in liquid fermentation medium, inoculum size is 10 by PC1 inoculation
6individual spore/ml, 200rpm, in 30 ℃ of shaking tables, cultivate, in the vigor of different incubation time section sampling and measuring polygalacturonases, (enzymatic reaction condition is: 0.5% polygalacturonic acid, pH4.8 acetic acid-sodium acetate buffer, 50 ℃ of reaction 15min, produce 1ug galacturonic acid residue with 1min hydrolysis substrate and are defined as an enzyme activity unit).Result shows: this bacterium is in the time cultivating 60h, and in fermented liquid, polygalacturonase vigor reaches maximum value, is about 3445.3u/ml.36-72h is product enzyme stationary phase, and after 72h, enzymatic productivity significantly reduces.At 40-72h, the relatively traditional bacterial strain of strain enzyme-producing efficiency is high in advance stationary phase for strain enzyme-producing of the present invention with traditional strain enzyme-producing stationary phase.
Determining of optimal reactive temperature: by PC1 bacterial strain by 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium, and 200rpm cultivates 48h for 30 ℃, gets the centrifugal 15min of fermented liquid 8500rpm, gets after clear enzyme solution dilution certain multiple respectively at 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, measures enzyme activities for 70 ℃.Result shows: the optimal reactive temperature of this enzyme is 60 ℃, and temperature can keep more than 85% reactive behavior in the time of 55-65 ℃ of scope, and when temperature exceedes 65 ℃, enzyme activity sharply declines, and when temperature of reaction is 70 ℃, enzyme activity only has 22.3% of the highest enzyme work.And traditional enzyme can keep more than 85% enzyme activity so far there are no report 55-65 ℃ of scope.
Determining of optimal reaction pH value: by PC1 bacterial strain by 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium, 200rpm, cultivate after 48h for 30 ℃, the centrifugal 15min of fermented liquid 8500rpm, get that clear enzyme solution dilution suitable multiple is placed on that pH value is respectively 4.4,4.8,5.2,5.4,5.8,6.4, carry out enzymatic reaction in 0.5% polygalacturonic acid reaction system of 6.6 acetic acid-sodium acetate buffer solution preparation, measure enzyme work.Result shows: the polygalacturonase optimal reaction pH that penicillium oxalicum PC1 produces is 5.2, optimal reaction pH scope is 4.8-5.4, and pH value exceedes at 5.4 o'clock, and enzyme activity sharply declines, show that this enzyme is acid pectase, can be widely used in enzymatic reaction under acidic conditions (4.8-5.4).
The research of temperature stability: PC1 bacterial strain is pressed to 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium, 200rpm, cultivate after 48h for 30 ℃, get the centrifugal 15min of fermented liquid 8500rpm, get 0.5% polygalacturonic acid solution dilution of supernatant liquor pH5.2 acetic acid-sodium acetate buffer preparation to suitable multiple, respectively at 50 ℃, in 55 ℃ of water-baths, be incubated, with the enzyme liquid without insulation processing in contrast, at interval of regular hour section sampling cooling rapidly, then measure pectinase activity.Result shows that this polygalacturonase keeps 15min, 30mim at 50 ℃, and 60min, when 90min, its residue enzyme activity is respectively: 87.6%, 79.0%, 59.8%, 43.3%, when temperature rise to 55 ℃, residue enzyme activity is respectively 64.1%, 34.5%, 25.4%, 27.6%, as can be seen here along with the rising of temperature and the prolongation of soaking time, the thermostability of enzyme declines.
The present invention possesses following good result: the penicillium oxalicum bacteria strain PC1(Penicillium oxalicum that provides a strain can produce high temperature polygalacturonase), the polygalacturonase optimal reactive temperature that this bacterial strain produces is 60 ℃, all can keep more than 85% enzyme activity 55-65 ℃ of scope, this so far there are no relevant report.
Accompanying drawing explanation
Fig. 1: the product enzyme time of CCTCC No.M2013627 penicillium oxalicum bacteria strain PC1.
Fig. 2: CCTCC No.M2013627 penicillium oxalicum bacteria strain PC1 produces polygalacturonase optimal reactive temperature.
Fig. 3: CCTCC No.M2013627 penicillium oxalicum bacteria strain PC1 produces polygalacturonase optimal reaction pH value.
Fig. 4: CCTCC No.M2013627 penicillium oxalicum bacteria strain PC1 produces polygalacturonase temperature stability.
Embodiment
Below in conjunction with embodiment and description the present invention, these descriptions are not that content of the present invention is further limited.
embodiment 1: the isolation and selection of bacterial strain
Strains separation and Breeding Process comprise that three steps carry out: sampling, primary dcreening operation, multiple sieve and purifying.
Sampling: gather suitable soil in the citrus orchard of suburb, Yangshuo County, hazard prevention.
Primary dcreening operation: prepare primary dcreening operation substratum (pectin 30g, NaNO
35g, KCl0.5g, MgSO
40.3g, (NH4)
2sO
41.4g, CaCl
20.3g, KH
2pO
43.8g, agar powder 20g, tetrabromophenol sulfonphthalein 0.2g, water 1000ml), 5.5,121 ℃ of sterilizing 20min of Initial pH.Step: the random about 0.5g sample of picking from the soil gathering, be suspended in 10ml sterilized water, fully shake up, after standing for some time, get supernatant liquor and dilute suitable multiple, evenly coat in primary dcreening operation substratum, cultivate 2-3 days for 30 ℃.The larger bacterium colony of the yellow hydrolysis circle of picking does further screening.
Multiple sieve and purifying: prepare liquid fermentation medium (1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80), initial ph value, 5.5,121 ℃ of sterilizing 20min.The colony inoculation that primary dcreening operation is obtained is in liquid fermentation medium, 30 ℃, 200rpm shaking table is cultivated 48 hours, get fermented liquid and measure polygalacturonase vigor, bacterium colony high enzyme activity is done after further separation and purification, it is stable that screening obtains a strain growth, the bacterial strain that enzymatic productivity is high, called after PC1.
embodiment 2: identification of strains
The classification evaluation of embodiment 1 being screened to the bacterial strain PC1 bacterial strain obtaining comprises Molecular Identification and the evaluation of biolog microorganism automatic identifying system.
Molecular Identification process: PC1 inoculation, in PDA substratum, is cultivated after 5-6 days for 30 ℃ and collected spore, by 10
6the inoculum size of individual spore/ml is inoculated in liquid fermentation medium, and 30 ℃, 200rpm shaking table is cultivated after 48 hours and collected somatic cells, and liquid nitrogen freezing grinds after fragmentation, extracts the genomic dna of thalline with phenol-chloroform method.Take this DNA as template, adopt universal primer ITS1(5-TCCGTAGGTGAACCTGCGG-3 ') and ITS4(5 '-TCCTCCGCTTATTGATATGC-3 ') carry out polymerase chain reaction (PCR), the ITSDNA sequence of this thalline that increases.The reaction system of 50ul is containing PCR damping fluid 5ul, ITS1 and the each 2ul of ITS4 primer, each dNTP2ul, DNA profiling 1ul, TaqDNA polysaccharase 0.3ul, ddH
2o37.7ul.Reaction conditions: 94 ℃ of 5 min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min.Gained reaction product is carried out determined dna sequence analysis, obtain the ITSDNA sequence of this bacterial strain 596pb, as shown in SEQ ID NO:1 sequence in sequence table, this sequence is carried out to the search of homologous sequence compare of analysis at the Genbank of NCBI website nucleic acid database, result shows, the ITSDNA sequence (KC344971.1) of the listed Penicillium oxalicum strain a1s2_d38 bacterial strain of its ITSDNA sequence and Genbank nucleic acid database, the ITSDNA sequence (KF152942.1) of Penicillium oxalicum strain 114-2 bacterial strain has 99% sequence homology.
Biolog microorganism automatic identifying system is identified: this inoculation, in 2% malt extract nutrient agar (20g wort dehydrated medium, 18g bacterium level agar, 1L sterilized water), is placed in to 26 ℃ of incubators and is cultured to product spore (about 5-6 days).Get appropriate spore make spore suspension in the FF-IF inoculation liquid of producer's configuration with aseptic inoculation rod is sticky, by the bacterial suspension inoculation preparing to FF plate (every hole 100ul), after inoculation, cover lid being positioned in 26 ℃ of incubators is cultivated 3 days, cultivates to finish rear use MicrologTM 3 softwares and carry out interpretation of result.
The above analysis result, the bacterial strain PC1 that identifies the product polygalacturonase that institute's seed selection obtains be penicillium oxalicum (
penicillium oxalicum).
embodiment 3: strain characteristic experiment
Bacterial strain is by 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium and (contains 1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80,5.5,121 ℃ of sterilizing 20min of initial pH value.), 30 ℃, 200rpm shake-flask culture.Enzymatic reaction condition is: 0.5% polygalacturonic acid substrate, and pH4.8 acetic acid-sodium acetate buffer, 50 ℃ of reaction 15min, produce 1ug galacturonic acid residue with 1min hydrolysis substrate and are defined as an enzyme activity unit.Result shows: this bacterium is in the time cultivating 60h, and in fermented liquid, polygalacturonase vigor reaches maximum value, is about 3445.3u/ml.36-72h is product enzyme stationary phase, and after 72h, enzymatic productivity significantly reduces, as shown in Figure 1.
Bacterial strain is by 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium (containing 1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80, initial pH value 5.5, 121 ℃ of sterilizing 20min), 30 ℃, after 200rpm shake-flask culture 48 hours, the centrifugal 15min of fermented liquid 8500rpm, supernatant liquor is with carrying out enzymatic reaction (reaction conditions: 0.5% polygalacturonic acid substrate after the suitable multiple of distilled water diluting, pH4.8 acetic acid-sodium acetate buffer, reaction times 15min), take the highest reaction enzymes vigor as reference, measure its enzyme activity.Result shows: the optimal reactive temperature of this enzyme is 60 ℃, and temperature can keep more than 85% reactive behavior in the time of 55-65 ℃ of scope, and when temperature exceedes 65 ℃, enzyme activity sharply declines, and when temperature of reaction is 70 ℃, enzyme activity only has 22.3% of the highest enzyme work., as shown in Figure 2.
Bacterial strain is by 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium (containing 1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80, Initial pH 5.5, 121 ℃ of sterilizing 20min), 30 ℃, after 200rpm shake-flask culture 48 hours, the centrifugal 15min of fermented liquid 8500rpm, getting supernatant liquor dilution suitable multiple is placed in the 0.5% polygalacturonic acid reaction system that different pH value acetic acid-sodium acetate buffer solution prepares and carries out enzymatic reaction, 60 ℃ of temperature of reaction, reaction times 15min, using the highest reaction enzymes work as reference, measure its enzyme activity.Result shows: the polygalacturonase optimal reaction pH that penicillium oxalicum PC1 produces is 5.2, and optimal reaction pH scope is 4.8-5.4, and pH value exceedes at 5.4 o'clock, and enzyme activity sharply declines, and shows that this enzyme is acid pectase, as shown in Figure 3.
Bacterial strain is by 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium (containing 1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80, initial pH value 5.5, 121 ℃ of sterilizing 20min), 30 ℃, after 200rpm shake-flask culture 48 hours, the centrifugal 15min of fermented liquid 8500rpm, get after 0.5% polygalacturonic acid solution dilution suitable multiple of supernatant liquor pH5.2 acetic acid-sodium acetate buffer preparation in differing temps and soaking time processing, then carry out enzymatic reaction, to process as reference without insulation, measure its enzyme activity.Result shows that this polygalacturonase keeps 15min, 30mim at 50 ℃, and 60min, when 90min, its residue enzyme activity is respectively: 87.6%, 79.0%, 59.8%, 43.3%, when temperature rise to 55 ℃, residue enzyme activity is respectively 64.1%, 34.5%, 25.4%, 27.6%, as can be seen here along with the rising of temperature and the prolongation of soaking time, the thermostability of enzyme declines, as shown in Figure 4.
embodiment 4: the application of bacterial strain
Utilize the bacterial strain that embodiment 1 isolation and selection obtains to produce high temperature polygalacturonase for the preparation of fermenting, step is as follows:
1) actication of culture
Bacterial classification is kept at 4 ℃ of refrigerators or is kept at-80 ℃ of refrigerators with dried frozen aquatic products with inclined-plane.Utilize CCTCC No:M2013627 grass
When acid Penicillium notatum bacterial strain PC1 pectinase by fermentation, first the bacterial classification of preservation is taken out to refrigerator and recover normal temperature, inoculation PDA nutrient agar (potato 200g, glucose 20g, agar 20g, water 1000ml, pH nature, 121 ℃ of sterilizing 20min), in 30 ℃ of incubators, cultivate 5-6 days to producing spore, collect spore and prepare spore suspension, cultivating for producing enzymic fermentation.
2) producing enzymic fermentation cultivates
Spore suspension is by 10
6individual spore/ml inoculum size is inoculated in liquid fermentation medium (containing 1% pectin, 0.14% ammonium sulfate, 0.03% magnesium sulfate, 0.2% potassium primary phosphate, 0.03% calcium chloride, 0.5% SODIUMNITRATE, 0.1% tween 80, Initial pH 5.5,121 ℃ of sterilizing 20min) the middle 48-60h that cultivates, collect nutrient solution, the centrifugal 15min of 8500rpm, gained supernatant is the polygalacturonase crude enzyme liquid that bacterial strain PC1 produces.
The above embodiment of the present invention scheme is only can not limit the present invention to explanation of the present invention; in claim, point out that the present invention protects theme; therefore; any change in implication and the scope suitable with claims of the present invention, is all considered to be in the scope that is included in claims.
Sequence table
<110> Guangxi Academy Of Sciences
<120> penicillium oxalicum bacteria strain and application thereof of producing high temperature polygalacturonase
<130> 2014
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 596
<212> DNA
<213> CCTCC No.M2013627 penicillium oxalicum bacteria strain PC1ITS
<400> 1
CCTTCCGTAG GGTGAACCTG CGGAAGGATC ATTACCGAGT GAGGGCCCTC TGGGTCCAAC 60
CTCCCACCCG TGTTTATCGT ACCTTGTTGC TTCGGCGGGC CCGCCTCACG GCCGCCGGGG 120
GGCATCCGCC CCCGGGCCCG CGCCCGCCGA AGACACACAA ACGAACTCTT GTCTGAAGAT 180
TGCAGTCTGA GTACTTGACT AAATCAGTTA AAACTTTCAA CAACGGATCT CTTGGTTCCG 240
GCATCGATGA AGAACGCAGC GAAATGCGAT AAGTAATGTG AATTGCAGAA TTCAGTGAAT 300
CATCGAGTCT TTGAACGCAC ATTGCGCCCC CTGGTATTCC GGGGGGCATG CCTGTCCGAG 360
CGTCATTGCT GCCCTCAAGC ACGGCTTGTG TGTTGGGCTC TCGCCCCCCG CTTCCGGGGG 420
GCGGGCCCGA AAGGCAGCGG CGGCACCGCG TCCGGTCCTC GAGCGTATGG GGCTTCGTCA 480
CCCGCTCTGT AGGCCCGGCC GGCGCCCGCC GGCGAACACC ATCAATCTTA ACCAGGTTGA 540
CCTCGGATCA GGTAGGGATA CCCGCTGAAC TTAAGCATAT CAATAAGCGG AGGAGA 596
Claims (8)
1. a penicillium oxalicum bacteria strain that produces high temperature polygalacturonase, is characterized in that, this penicillium oxalicum bacteria strain deposit number is CCTCC No.M2013627.
2. the penicillium oxalicum bacteria strain of product high temperature polygalacturonase according to claim 1, it is characterized in that, described penicillium oxalicum strain fermentation produces to such an extent that the catalyzed reaction temperature of high temperature polygalacturonase is 55-65 ℃, under 55-65 ℃ of condition, keeps more than 85% enzyme activity.
3. the penicillium oxalicum bacteria strain of product high temperature polygalacturonase according to claim 1, is characterized in that, it is 36-72 hour after fermentation culture starts that described penicillium oxalicum strain fermentation produces high temperature polygalacturonase stationary phase.
4. one kind is produced the application of the penicillium oxalicum bacteria strain of high temperature polygalacturonase, it is characterized in that, deposit number is the application of penicillium oxalicum bacteria strain aspect high temperature polygalacturonase fermentative production that CCTCC No.M2013627 produces high temperature polygalacturonase.
5. the application of the penicillium oxalicum bacteria strain of product high temperature polygalacturonase according to claim 4, it is characterized in that, described penicillium oxalicum strain fermentation produces to such an extent that the catalyzed reaction temperature of high temperature polygalacturonase is 55-65 ℃, under 55-65 ℃ of condition, keeps more than 85% enzyme activity.
6. the application of the penicillium oxalicum bacteria strain of product high temperature polygalacturonase according to claim 5, it is characterized in that, described penicillium oxalicum strain fermentation used medium is: 1-2% pectin, 0.1-0.2% ammonium sulfate, 0.01-0.05% magnesium sulfate, 0.1-0.3% potassium primary phosphate, 0.01-0.05% calcium chloride, 0.5-1.0% SODIUMNITRATE, 0.1-0.5% tween 80, initial pH value is 5.0-6.0.
7. the application of the penicillium oxalicum bacteria strain of product high temperature polygalacturonase according to claim 5, is characterized in that, described penicillium oxalicum strain fermentation condition is: under 30-37 ℃ of condition, shaking speed 200-300rpm cultivates.
8. the application of the penicillium oxalicum bacteria strain of product high temperature polygalacturonase according to claim 5, is characterized in that, it is 36-72 hour after fermentation culture starts that described penicillium oxalicum strain fermentation produces high temperature polygalacturonase stationary phase.
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| CN105779419A (en) * | 2016-04-25 | 2016-07-20 | 广西科学院 | Cloning, definition and application of internally tangent polygalacturonase gene with temperature resistance and acid stability |
| CN105925549A (en) * | 2015-12-30 | 2016-09-07 | 广西科学院 | Cloning, expression and application of endo-polygalacturonase gene |
| CN113604522A (en) * | 2021-08-02 | 2021-11-05 | 广西大学 | Penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binder |
| CN113755483A (en) * | 2021-03-12 | 2021-12-07 | 江苏悠恒生物技术有限公司 | Mutagenesis method of high-yield pectinase enzyme activity strain and optimization of solid state fermentation conditions thereof |
| CN114292832A (en) * | 2022-01-27 | 2022-04-08 | 大连海洋大学 | Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925549A (en) * | 2015-12-30 | 2016-09-07 | 广西科学院 | Cloning, expression and application of endo-polygalacturonase gene |
| CN105779419A (en) * | 2016-04-25 | 2016-07-20 | 广西科学院 | Cloning, definition and application of internally tangent polygalacturonase gene with temperature resistance and acid stability |
| CN113755483A (en) * | 2021-03-12 | 2021-12-07 | 江苏悠恒生物技术有限公司 | Mutagenesis method of high-yield pectinase enzyme activity strain and optimization of solid state fermentation conditions thereof |
| CN113604522A (en) * | 2021-08-02 | 2021-11-05 | 广西大学 | Penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binder |
| CN114292832A (en) * | 2022-01-27 | 2022-04-08 | 大连海洋大学 | Thermophilic high-temperature-resistant polygalacturonaseMlPG28BEncoding gene and preparation method thereof |
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