CN107325977B - Method for producing protein glutaminase, purification and application - Google Patents
Method for producing protein glutaminase, purification and application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
- A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
- A21D2/24—Organic nitrogen compounds
- A21D2/26—Proteins
- A21D2/267—Microbial proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01044—Protein-glutamine glutaminase (3.5.1.44)
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The invention relates to a Protein-glutaminase (PG) producing strain, and preparation and application of a fermentation product thereof. The protein glutaminase producing strain is a Chryseobacterium prion protein (YF 810) strain, and the preservation number of the strain is CGMCC NO. 10532. The strain is obtained by enriching and screening from soil, is safe and nontoxic, can produce protein glutaminase by fermentation, and has higher enzyme activity. Through preparation and purification, the protein glutaminase has high purity, is used in the field of food industrial processing, can obviously improve the solubility, foamability, emulsibility and stability of protein, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of food microorganism screening and application, and particularly relates to screening of protein glutaminase producing bacteria and preparation, purification and application of a fermentation product protein glutaminase.
Background
Natural proteins contain a large number of glutamine and asparagine residues, which are cross-linked with other amino acids in the form of hydrogen bonds, resulting in reduced solubility of the protein and further affecting the process characteristics of the protein, such as emulsifiability, foamability, gelling ability, etc. (Fennema OR. food chemistry [ Z ]. Beijing: China light industry Press, 2003316-. This limits the applications of proteins in the fields of food, beverages, health products, medicine, etc. Deamidation is an important approach to solve this problem, and research shows that through deamidation, amide groups in proteins are converted into carboxyl groups, negative charges are increased, isoelectric points of proteins are reduced, and the deamidation also changes the spatial structure of protein molecules, so that hydrophilic groups originally wrapped inside are exposed, and a small degree of deamidation (2% -6%) can significantly improve the functional properties of proteins (Hamada J S, Marshall WE.preparation and functional properties of amino deamidation peptides [ J ]. Journal of Food science 1989,54(3): 598-.
The methods for deamidation reported in the literature include acid methods and enzymatic methods. Although the acid deamidation can obviously improve the solubility of the protein, the hydrolysis of the peptide chain of the protein and the degradation of other amino acid residues are inevitable in the treatment process, which has great influence on the flavor of the protein. Transglutaminase, protease and peptidoglutaminase can all be used for deamidation of proteins. But all have certain disadvantages: protein aggregation can occur when transglutaminase is used in excess; the protease can hydrolyze the whole protein, the substrate specificity is poor, and bitter taste is easy to generate; the peptide glutaminase does not exert an effect on high molecular weight polypeptides and proteins and has a low degree of deamidation.
Protein glutaminase (EC 3.5.1.44), PG for short, is a novel hydrolase which has been studied in recent years and which is capable of removing amino groups from a variety of proteins, polypeptides or short peptides (Yamaguchi S, Jenes D J, Archer D B. Protein-glutamine from microorganism proteins, an enzyme through amino groups in proteins [ J ]. European Journal of Biochemistry,2001,268 (1410) 1421), which is capable of hydrolyzing L- β -glutamine into L-glutamic acid and ammonia and acts only on the glutamine group of proteins or peptides without affecting the glutamine residues and free glutamine, while not causing crosslinking and hydrolysis of proteins [ Marcoa C, soluble C. amino acids ] Protein and Protein derivatives [ 2008. amino acids ] 139, 132), thus improving the solubility, the emulsibility and the like of the protein, and the enzyme can also play a role under an acidic condition, and is an enzyme with a very promising application prospect in the food industry. However, because of the rare production strains, researches on protein glutaminase at home and abroad are mainly focused on the aspects of structure and application, and researches on other strains capable of producing protein glutaminase are very little, which limits the industrial application of protein glutaminase to a certain extent.
Among the bacteria capable of producing protein glutaminase are Chryseobacterium prion (Chryseobacterium proteolyticum), Chryseobacterium mucosum (Chryseobacterium gleum) and Chryseobacterium indogenes (Chryseobacterium indicum) have been reported. Currently, only Chryseobacterium prion is approved for industrial production, but the enzyme production capability is low, and the enzyme activity is about 0.2 IU/mL.
Therefore, it is necessary to select other strains capable of producing the high-enzyme activity protein glutaminase. The invention aims to solve the problems of few protein-producing glutaminase strains and low enzyme activity in the prior art, and provides a novel protein-producing glutaminase strain which can be safely and efficiently applied to food and can improve the solubility, foamability, emulsibility and stability of protein.
Disclosure of Invention
The invention aims to provide a Protein Glutaminase (PG) producing strain, namely a new strain of golden yellow bacillus priolyticus capable of secreting and expressing Protein glutaminase, and preparation and application of a fermentation product Protein glutaminase of the strain in Protein deamidation, namely, the Protein glutaminase prepared by the strain is applied to cake making, and deamidation is carried out on egg white, so that the foamability of the egg white is improved, the softness degree of the cake is increased, and the using amount of the egg white is reduced; the method is applied to the development of the soybean protein functional beverage, and the deamidation effect is carried out on the soybean protein isolate in the soybean protein functional beverage, so that the hydrolysis degree of the soybean protein isolate is improved, the clarity of the soybean protein functional beverage is improved, and the bad flavor is not caused; the protein glutaminase and the soybean polysaccharide are compounded to form a new protein stabilizer, so that the solubility and the stability of the protein in the protein beverage can be improved, and the flavor and the taste are more exquisite and refreshing.
The invention firstly provides a Chryseobacterium prion protein YF810 strain which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 2 month 6 year 2015, the preservation address is No.3 of Xilu No.1 of Beijing Korean area, the preservation number is CGMCC NO.10532, and the 16S rDNA sequence is shown as SeqID No. 3.
The protein glutaminase produces a strain YF810 of Flavobacterium priolyticum (Chryseobacterium proteolyticum), the strain can secrete Protein Glutaminase (PG), the DNA sequence of the enzyme is shown as Seq ID No.10, and the amino acid sequence is shown as Seq ID No. 14.
The protein glutaminase has a DNA sequence of a signal peptide encoding the enzyme as shown in Seq ID No. 11; the amino acid sequence of the signal peptide is shown in Seq ID No. 15.
The protein glutaminase has a DNA sequence encoding a propeptide of the enzyme as shown in Seq ID No. 12; the amino acid sequence of the propeptide is shown in Seq ID No. 16.
In the protein glutaminase, the DNA sequence of a mature enzyme for encoding the enzyme is shown as Seq ID No. 13; the amino acid sequence of the mature enzyme is shown in Seq ID No. 17.
The Chryseobacterium prion protein YF810 grows in a seed culture medium, the growth temperature ranges from 20 ℃ to 40 ℃, and preferably, the growth temperature ranges from 25 ℃ to 30 ℃; the growth pH range is 6.0-9.0, preferably, the optimum growth pH is 6.5-7.5.
The culture medium used for culture is seed culture medium, and its formula is polypeptone 10g/L, yeast extract 2g/L, anhydrous MgSO41g/L, pH 7.0.
In the present invention, PCR amplification is performed on the full-length DNA encoding the protein glutaminase, and the upstream and downstream primers are shown as Seq ID No.4 and Seq ID No.5, respectively.
In the present invention, DNAs encoding the pro peptide of the protein glutaminase and the maturase are PCR-amplified with the upstream and downstream primers shown as Seq ID No.6 and Seq ID No.7, respectively.
In the present invention, the DNA encoding the mature enzyme of the protein glutaminase is PCR-amplified, and the upstream and downstream primers are shown as Seq ID No.8 and Seq ID No.9, respectively.
A protein glutaminase gene of a Chryseobacterium prion strain YF810 was PCR-amplified and sequenced to obtain a DNA sequence (Seq ID No. 10). The protein glutaminase DNA sequence is 963bp (Seq ID No.10), wherein 1-63bp (Seq ID No.11) is the sequence for coding a signal peptide, 64-405bp (Seq ID No.12) is the sequence for coding a propeptide, and 406-963bp (Seq ID No.13) is the sequence for coding a mature enzyme. The protein glutaminase produced by the strain has 320 amino acids (Seq ID No.14), wherein the signal peptide is 21 amino acids (Seq ID No.15), the propeptide comprises 114 amino acids (Seq ID No.16), and the mature enzyme consists of 185 amino acids (Seq ID No. 17).
The invention also provides a sequence of a coding protein glutaminase signal peptide, which is shown as Seq ID No. 11; the protein glutaminase is produced by a Chryseobacterium prion strain YF 810.
The Chryseobacterium prion lyticum YF810 strain can produce protein glutaminase by fermentation, the strain is inoculated in a fermentation culture medium, the temperature is 30 ℃,200 r/min and shake culture is carried out, the strain is fermented for 12 hours, the enzyme yield is the highest and can reach 0.7-2IU/mL, and the enzyme yield is 3.5-10 times of the currently reported enzyme activity of the Chryseobacterium prion lyticum. Wherein the fermentation medium formula comprises lactose 5g/L, soybean peptone 15g/L, and Na2HPO4·12H2O 3.8g/L，KH2PO40.25g/L，MgSO4·7H2O 0.25g/L，FeSO4·7H2O0.05g/L, porous pH stabilizer such as zeolite 10-50g/L, and pH 7.2.
Wherein, 10g/L-50g/L porous pH stabilizer is added into the initial fermentation culture medium, wherein, the porous pH stabilizer comprises zeolite, montmorillonite, active carbon, medical stone, volcanic rock and the like; preferably, 30g/L of porous pH stabilizer zeolite is added to the initial fermentation medium.
Wherein the activity of the protein glutaminase in the fermentation liquor is 0.7-2.0 IU/mL.
The invention also provides a fermentation medium, which comprises the following components in percentage by volume: lactose 5g/L, soybean peptone 15g/L, Na2HPO4·12H2O 3.8g/L，KH2PO40.25g/L，MgSO4·7H2O 0.25g/L，FeSO4·7H20.05g/L of O, 10-50g/L of porous pH stabilizer and 7.2 of pH.
Wherein the porous pH stabilizer comprises zeolite, montmorillonite, active carbon, medical stone, volcanic rock and the like; preferably, the porous pH stabilizer is a zeolite. Preferably, the addition amount of the porous pH stabilizer is 30 g/L.
In the invention, the protein glutaminase produced by the strain is purified, and the process comprises the following steps:
the first step is as follows: concentrating by ultrafiltration
Primarily removing thallus from the fermentation liquor by centrifugation, carrying out filter pressing on the supernatant in a filter press by using a filter membrane of 0.22 mu m to remove insoluble impurities, and concentrating by using an ultrafiltration system;
the second step is that: ethanol precipitation
Precipitating the concentrated solution with anhydrous ethanol, standing, centrifuging to obtain protein precipitate, and adding ddH2Re-dissolving O, and centrifuging again to remove insoluble impurities to obtain a crude protein glutaminase extract;
the third step: desalination
Filtering the crude extract with 0.45 μm filter membrane, and desalting with desalting column; collecting protein fraction at 280nm position with absorption peak; performing ion exchange chromatography with cation exchange chromatography column;
the fourth step: cation exchange
Carrying out cation exchange chromatography on the protein component collected by desalting by using a cation exchange chromatography column; collecting the fraction at a position having an absorption peak at 280 nm;
the fifth step: concentrating
Collecting the components by cation exchange, and dialyzing and concentrating with PEG 20000 to obtain concentrated solution containing enzyme activity;
and a sixth step: gel filtration chromatography
The concentrated solution containing enzyme activity obtained by concentration is filtered by a filter membrane of 0.22 μm, and then is subjected to gel filtration chromatography by using a gel filtration chromatography column, and components are collected at a position with an absorption peak at 280 nm.
In a specific embodiment, the process for purifying the protein glutaminase produced by said strain is: inoculating the seed solution into 25mL fermentation medium with an inoculum size of 2% (V/V), shake culturing at 30 deg.C and 200r/min for 12h to obtain fermentation liquid, centrifuging at 4 deg.C and 10000r/min to remove thallus and obtain supernatant, and filter-pressing with a filter membrane of 0.22 μm in a filter press to remove insoluble impurities. Using QuixstandTMThe ultrafiltration system concentrates 2-6 times, the interception molecular weight is 3000-5000D, the flow rate of the feed liquid is 0.5L/min, and the maximum pressure difference is 0.2 MPa. Precipitating the concentrated solution with 1-4 times volume of anhydrous ethanol pre-cooled at 4 deg.C, centrifuging at 4 deg.C for 20min at 10000r/min, and collecting protein precipitate2And (4) re-dissolving the O, and then centrifuging again to remove insoluble impurities to obtain a crude protein glutaminase extract. The crude extract was filtered through a 0.45 μm filter and then desalted using a HiPrep 26/10 desaling Desalting column. The protein fraction was collected at a position where there was an absorption peak at 280 nm. Performing ion exchange chromatography with cation exchange chromatography column. The collected protein fraction was desalted and subjected to cation exchange chromatography using HiPrep 16/10 SP Sepharose cation exchange chromatography column. After the loading, elution was performed, and the column-bound protein was eluted by a linear gradient of 0-1M NaCl, and fractions having an absorption peak at 280nm were collected in tubes. The product after ion exchange contains enzyme activityAnd (3) dialyzing and concentrating the components by using PEG 20000 to obtain concentrated solution containing enzyme activity. Filtering the concentrated solution with 0.45 μm filter membrane, performing gel filtration chromatography with HiPrep 26/60Sephacryl S-100 HR gel filtration chromatography column, and collecting the fraction at 280nm position with absorption peak to obtain protein glutaminase. Through the purification step, the specific enzyme activity of the protein glutaminase is not lower than 130U/mg, and the total enzyme activity recovery rate can reach 32%.
The invention also proposes the use of said protein glutaminase for deamidation.
The invention also provides application of the protein glutaminase in deamidation of egg white in cake making, so that the solubility, foamability and emulsibility of the protein glutaminase are improved, the softness of a cake is increased, and the using amount of the egg white in cake making can be reduced.
In the process of making the cake, protein glutaminase obtained after purification is added into egg white in different proportions, and the deamidation degree, the emulsibility and the foamability of the egg white are continuously increased along with the increase of the addition amount.
The invention also provides the development of the protein glutaminase for the soybean protein functional beverage, which is used for improving the solubility of the soybean protein functional beverage and improving the clarity of the soybean protein functional beverage, and simultaneously, the protein glutaminase does not bring bad flavor.
In the development of a soybean protein functional beverage, protein glutaminase obtained after purification is added to soybean protein, and the deamidation degree and hydrolysis degree of the soybean protein are increased along with the increase of the addition amount of the protein glutaminase.
The invention also proposes the use of said protein glutaminase in a novel protein stabilizer.
In the formula of the protein stabilizer, the protein glutaminase and the soybean polysaccharide are compounded, so that the solubility and the stability of protein in the protein beverage can be improved, and the flavor and the taste are more exquisite and refreshing.
The invention has the beneficial effects that: firstly, the related Chryseobacterium prion protein (YF 810) strain is safe and nontoxic, and has the advantages of simple nutritional requirement, rapid growth, high protein-producing glutaminase activity and the like; secondly, the strain is applied to ferment protein glutaminase to act on proteins containing glutamine residues such as soybean protein isolate and the like, and the amide groups in the proteins are removed, so that the strain can be better applied to the food production and processing industry, and has the advantages of strong specificity and no bad influence of bad flavor and the like in the action process; thirdly, the protein glutaminase is fermented by applying the strain, and is purified by a purification process, so that the specific enzyme activity and the total enzyme activity recovery rate are high; fourthly, the protein glutaminase can be used as a solubilizer, a stabilizer, a nutrition enhancer, a flavor modifier and the like, and is suitable for the aspects of cake making, the development of soybean protein functional beverages, the development of protein stabilizers and the like. Therefore, the strain has wide economic prospect when being applied to the field of food.
Drawings
FIG. 1 shows the colony morphology of the strain YF810 of Chryseobacterium prion protein (Chryseobacterium proteolyticum) according to the present invention in example 2.
FIG. 2 shows the gram stain results of the YF810 strain of Chryseobacterium proteolyticum according to the present invention in example 2.
FIG. 3 shows the results of scanning electron microscope observation of the strain YF810 of Chryseobacterium proteolyticum according to the present invention in example 2.
FIG. 4 shows the growth curve of the strain YF810 of Chryseobacterium prion protein (Chryseobacterium proteolyticum) according to the present invention in example 3.
FIG. 5 shows an enzyme production curve of the strain YF810 of Chryseobacterium prion protein (Chryseobacterium proteolyticum) according to example 4.
FIG. 6 shows the effect of the amount of the porous pH stabilizer, e.g., zeolite, according to the present invention on the enzyme production of the F810 strain of Chryseobacterium prion protein in example 4.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
A protein glutaminase producing bacterium YF810, taxonomically named: golden yellow bacillus prion, latin literature name: chryseobacterium proteolyticum, deposit unit: china general microbiological culture Collection center, the preservation unit is abbreviated as: CGMCC, storage unit address: west road No.1 hospital No.3, north jing, chaoyang district, preservation date: the preservation number is CGMCC NO.10532 at 2 months and 6 days in 2015.
The preparation and application of the fermentation product protein glutaminase of Chryseobacterium prion protein YF810 strain are specifically implemented by the following mode.
Example 1 screening isolation of Chryseobacterium prion protein YF810 Strain
The YF810 strain is obtained by screening an enrichment medium, wherein the enrichment medium is formed by mixing 54mL of A liquid, 6mL of 1% CBZ, 60 mu L of mother liquid I, 60 mu L of mother liquid II, 60 mu L of mother liquid III and 120 mu L of mother liquid IV.
Wherein, the formula of the solution A is as follows: 5g/L glucose, 0.2g/L KH2PO4，0.2g/L MgSO4·7H2O、0.01％NaCl
The mother liquor I comprises the following components in percentage by weight: 20g/L CaCl2
The formula of the mother solution II is as follows: 2g/L FeSO4·7H2O，5g/L MnSO4·4H2O
The mother liquor III comprises the following components: 5g/L NaWO4·4H2O，5g/L NaMO4·2H2O
The mother solution IV comprises the following components in percentage by weight: 50g/L CuSO4·5H2O
The screening steps are as follows:
firstly, weighing 10g of soil sample, adding 90mL of sterile water, placing a small amount of glass beads into a conical flask, and oscillating for 10min by a shaking table to prepare soil suspension.
And step two, after the soil suspension is kept stand for 10min, 0.6mL of the soil suspension is taken to be inoculated into a conical flask filled with the enrichment medium, and shaking culture is carried out for 6 days at the temperature of 30 ℃ and at the speed of 200 r/min.
And thirdly, taking 0.6mL of the bacterial liquid cultured in the last step, inoculating the bacterial liquid into a conical flask filled with a fresh enrichment medium, arranging 2 parallel soil samples, and performing shaking culture at 30 ℃ and 200r/min for 3 days.
Fourthly, taking 0.1 to 0.9mL of sterile water from the bacterial liquid cultured in the previous step, and diluting the bacterial liquid to 10 percent-5、10-6The plating is carried out by two gradients, and the plating bacteria liquid amount is 100 mu L.
Fifthly, placing the flat plate in an incubator at 30 ℃ for inverted culture for 2-3d and observing.
And sixthly, spraying 3% KOH solution on the surfaces of the yellow or orange colonies, immediately observing, and spraying 3% HCl solution if the colonies turn red. If the colony turns yellow or orange, the suspected bacterial strain is selected and further screened.
Example 2 identification of Chryseobacterium prion protein YF810 Strain
1. Morphological identification
(1) Observing the morphology of the colonies
The suspected bacterial strain is observed after being cultured by an LB plate, the bacterial colony is circular, the diameter is 2mm-4mm, the edge is neat, golden yellow or orange yellow, the bacterial colony is opaque, the surface of the bacterial colony is moist and smooth, and the bacterial colony is glossy, and is shown in figure 1.
(2) Observation by microscope
After gram staining and spore staining, the strain was rod-shaped, immotile, spore-free, gram-negative as shown in FIG. 2, when observed under a normal optical microscope.
The cells were observed under a scanning electron microscope to be smooth without flagellum and without movement, with a width of 0.2 μm-0.4 μm and a length of 0.8 μm-2.2 μm, as shown in FIG. 3.
2. Physiological and biochemical identification
The results of physiological and biochemical identification of the F810 strain of Chryseobacterium prion (Chryseobacterium proteolyticum) using a bacterial micro-biochemical identification tube (purchased from Qingdao Haibo Biotechnology Co., Ltd.) are shown in Table 1.
TABLE 1 physiological and biochemical identification of Chryseobacterium prion protein YF810 strain
Wherein "+" indicates that the strain YF810 of Chryseobacterium prion Ceratoides (Chryseobacterium proteolyticum) reacted positively, and "-" indicates that the strain YF810 of Chryseobacterium Ceratoides (Chryseobacterium proteolyticum) reacted negatively.
According to the physiological and biochemical identification results, the screened YF810 strain can be preliminarily determined to belong to the Chryseobacterium, and the identification result is partially different from the Chryseobacterium prion lyticum 9670T strain screened by Shotaro Yamaguchi, so that the strain is a new Chryseobacterium prion lyticum strain.
3.16S rDNA sequencing
Extracting total genome DNA of YF810 strain, and amplifying 16S rDNA sequence of the strain by using the total genome DNA as a template, wherein the primer sequence is as follows:
Seq ID No.1，F:5'-AGAGTTTGATCCTGGCTCAG-3'
Seq ID No.2，R:5'-CTACGGCTACCTTGTTACGA-3'
the PCR product was detected by 1% agarose gel electrophoresis, recovered by tapping and sent to a commercial company for sequencing. The sequencing work was performed by Shanghai Biotechnology engineering services, Inc. The similarity alignment of the 16S rDNA sequence and the 16S rDNA sequence of the existing bacteria shows that the YF810 strain and the Chryseobacterium prion protein 9670T(AB039830) has the highest homology of 98%, so the strain is Chryseobacterium prion lyticum.
Combining the colony morphology, physiological and biochemical characteristics and the results of 16S rDNA gene sequence analysis, the YF810 strain can be determined to be a new Chryseobacterium prion lyticum, named as YF810(Chryseobacterium proteolyticum YF 810).
EXAMPLE 3 determination of DNA sequence encoding protein glutaminase and prediction of amino acid sequence
1. Determination of full-length DNA sequence of encoded protein glutaminase and prediction of amino acid sequence thereof
Extracting total genome DNA of YF810 strain, using the total genome DNA as a template, and amplifying the full-length DNA sequence of protein glutaminase produced by the strain, wherein the primer sequence is as follows:
Seq ID No.4，F:5'-CCAACCAACTTAACAAAAACTCACCATTAAAC-3'
Seq ID No.5，R:5'-GGAACCCGAACTACCGGAGCAGGATG-3'
the PCR product was detected by 1% agarose gel electrophoresis, recovered by tapping and sent to a commercial company for sequencing. The sequencing work was performed by Shanghai Biotechnology engineering services, Inc.
The DNA sequence determined above, namely Seq ID No. 10; the translation was performed with the software Clone Manager to obtain the corresponding amino acid sequence, Seq ID No. 14.
2. DNA sequencing of the encoded protein glutaminase propeptide and the mature enzyme and amino acid sequence prediction
Extracting total genome DNA of YF810 strain, using the total genome DNA as a template, and amplifying the full-length DNA sequence of protein glutaminase produced by the strain, wherein the primer sequence is as follows:
Seq ID No.6，F:5'-CTGTGCCGATTCCAACGGGAATCAG-3'
Seq ID No.7，R:5'-GAACCCGAACTACCGGAGCAGGATG-3'
the PCR product was detected by 1% agarose gel electrophoresis, recovered by tapping and sent to a commercial company for sequencing. The sequencing work was performed by Shanghai Biotechnology engineering services, Inc.
The DNA sequences thus determined, i.e., Seq ID No.12 and Seq ID No. 13; the translation was performed with the software Clone Manager to obtain the corresponding amino acid sequences, i.e., Seq ID No.16 and Seq ID No. 17.
3. DNA sequence determination of encoded protein glutaminase maturase and amino acid sequence prediction thereof
Extracting total genome DNA of YF810 strain, using the total genome DNA as a template, and amplifying the full-length DNA sequence of protein glutaminase produced by the strain, wherein the primer sequence is as follows:
Seq ID No.8，F:5'-AGTCTGCAAGTCCGGAAGACGTAAG-3'
Seq ID No.9，R:5'-CAGCTGGATACATCCGGTGCAGGTG-3'
the PCR product was detected by 1% agarose gel electrophoresis, recovered by tapping and sent to a commercial company for sequencing. The sequencing work was performed by Shanghai Biotechnology engineering services, Inc.
The DNA sequence determined above, namely Seq ID No. 13; the translation was performed with the software Clone Manager to obtain the corresponding amino acid sequence, Seq ID No. 17.
Therefore, the protein glutaminase produces protein glutaminase secreted by a strain YF810 of Chryseobacterium proteolyticum, the DNA sequence of which is shown in Seq ID No.10, and the amino acid sequence of which is shown in Seq ID No. 14.
Wherein the DNA sequence of the signal peptide of the enzyme is shown as Seq ID No. 11; the amino acid sequence of the signal peptide is shown as Seq ID No. 15; the DNA sequence encoding the propeptide of the enzyme is shown in Seq ID No. 12; the amino acid sequence of the propeptide is shown as Seq ID No. 16; the DNA sequence of the mature enzyme coding the enzyme is shown as Seq ID No. 13; the amino acid sequence of the mature enzyme is shown in Seq ID No. 17.
Example 4 culture of Chryseobacterium prion protein YF810 Strain
Chryseobacterium prion protein (YF) 810 strain was inoculated in 25mL seed medium, with the formulation: 10g/L of polypeptone, 2g/L of yeast extract and anhydrous MgSO41g/L, pH 7.0. Shaking for 12h at 30 deg.C and 200r/min, inoculating to fresh seed culture medium at 2%, shaking for 12h at 30 deg.C and 200 r/min. And obtaining YF810 bacterial liquid.
The growth curve is shown in figure 1, lag phase is 0-6h, logarithmic growth phase is 6-14h, and stationary phase is entered after 14 h. The Chryseobacterium prion protein YF810 strain has simple nutritional requirement and rapid growth, and is suitable for industrial application.
EXAMPLE 5 fermentation culture of protein glutaminase
First step fermentation culture of protein glutaminase
Inoculating YF810 bacterial liquid into 25mL of fermentation medium with the inoculum size of 2%, wherein the formula is as follows: lactose 5g/L, soybean peptone 15g/L, Na2HPO4·12H2O 3.8g/L，KH2PO40.25g/L，MgSO4·7H2O 0.25g/L，FeSO4·7H2O0.05g/L and pH 7.2. Shaking-culturing at 30 deg.C for 12h at 200r/min to obtain fermentation liquid.
Second step enzyme activity determination of protein glutaminase
Centrifuging the fermentation liquor at 4 deg.C for 20min at 10000r/min for removing thallus to obtain supernatant, diluting the supernatant by 5 times, adding 0.1mL of supernatant diluent into a test tube, and bathing at 37 + -0.5 deg.C for 1 min; then, 1mL of a preheated 0.01mol/L Cbz-Gln-Gly solution was added thereto, and the mixture was subjected to a bath reaction at 37. + -. 0.5 ℃ for 60 minutes. Then, 1mL of trichloroacetic acid solution was added thereto, and the mixture was mixed well to terminate the reaction. The content of ammonia in the solution after the reaction was measured by the phenol method. A. the630The absorbance A1 was measured. The control group is prepared by adding trichloroacetic acid solution, reacting for 60min, and adding substrate solution. The rest of the operations were performed in the same experimental group. Measurement A630Is a 2. The amount of enzyme required to produce 1. mu. mol ammonia per minute is defined as one enzyme activity unit (IU). The enzyme activity in the fermentation liquor is calculated by the following formula:
the enzyme activity (U/mL) ═ (A1-A2) x 2.1/0.1 x 1/17.03 x 1/60 x a x 5
A1: absorbance values for the experimental groups;
a2: absorbance of the control;
a: the slope of a standard curve for ammonia as determined by phenol hypochlorite spectrophotometry.
The enzyme activity of the strain is measured by the method, the enzyme production curve is shown in figure 2, the highest enzyme activity occurs in the middle and later logarithmic growth period, the enzyme activity can reach 0.7IU/mL, and the strain is the currently reported golden yellow bacillus prion lyase 9670T3.5 times of enzyme activity.
EXAMPLE 6 protein glutaminase fermentation optimization
Adding 10g/L-50g/L porous pH stabilizer such as zeolite into original fermentation medium, and fermenting at 30 deg.C under 200r/miAnd n, carrying out shake culture for 12h to obtain fermentation liquor. The enzyme activity of each fermentation liquid is measured, the optimization result is shown in figure 3, the enzyme yield of the strain can be improved by adding 1%, 2%, 3%, 4% and 5% (w/v) of zeolite, when the adding amount of the zeolite is 30g/L, the promotion effect is optimal, the enzyme activity can reach 2.0IU/mL which is 3 times of that before optimization, and is 9670 times of that of the currently reported golden yellow bacillus prion T10 times of enzyme activity.
Adding 30g/L zeolite into original fermentation medium, shake culturing at 30 deg.C and 200r/min for 12h to obtain fermentation liquid, and measuring bacteria concentration and ammonia concentration (and pH) of the fermentation liquid. The results are shown in Table 2.
TABLE 2 Chryseobacterium prion protein YF810 strain fermentation medium optimization
The porous pH stabilizer has the advantages that the porosity of the material can lead thalli to be adsorbed in the pores, thereby promoting the growth of the thalli and further improving the unit enzyme activity of fermentation liquor; the pH stabilization of the material allows the pH of the fermentation broth to be maintained in a range suitable for strain growth and enzyme production.
EXAMPLE 7 purification of protein glutaminase
The first step is as follows: concentrating by ultrafiltration
The fermentation liquor is centrifuged to remove the thallus primarily, and the supernatant is filtered by a filter membrane with the size of 0.22 μm in a filter press to remove insoluble impurities. Using QuixstandTMThe ultrafiltration system concentrates 2-6 times, the interception molecular weight is 3000-5000Da, the flow rate of the feed liquid is 0.5L/min, and the maximum pressure difference is 0.2 MPa.
The second step is that: ethanol precipitation
Precipitating the concentrated solution with 1-4 times volume of anhydrous ethanol pre-cooled at 4 deg.C, centrifuging at 4 deg.C for 20min at 10000r/min, and collecting protein precipitate2Re-centrifuging to remove insoluble impurities after O redissolving to obtainCrude extract of protein glutaminase.
The third step: desalination
The crude extract was filtered through a 0.45 μm filter and then desalted using a HiPrep 26/10 desaling Desalting column. The protein fraction was collected at a position where there was an absorption peak at 280 nm.
The fourth step: cation exchange
The collected protein fraction was desalted and subjected to cation exchange chromatography using HiPrep 16/10 SP Sepharose cation exchange chromatography column. Fractions were collected at a position where there was an absorption peak at 280 nm.
The fifth step: concentrating
And (3) performing ion exchange to obtain a component containing enzyme activity, and dialyzing and concentrating by using PEG 20000 to obtain a concentrated solution containing the enzyme activity.
The sixth step of gel filtration chromatography
Filtering the concentrated solution with 0.45 μm filter membrane, performing gel filtration chromatography with HiPrep 26/60Sephacryl S-100 HR gel filtration chromatography column, and collecting the fraction at 280nm position with absorption peak to obtain protein glutaminase.
In the present invention, by this purification step, the specific enzyme activity of the protein glutaminase is not less than 130U/mg, and the total enzyme activity recovery is not less than 32%.
Example 8 use of protein glutaminase in cake making
Thereby need wrap up the air through egg white foaming and can make the cake soft delicious at the in-process of baking in the preparation process of cake, egg white can effectively improve its emulsibility and foamability through the deamidation effect, increases the soft degree of cake. The purified protein glutaminase is applied to the deamidation of egg white to improve the emulsibility and foamability of the egg white.
(1) Weighing 40g of egg white, adding 100mL of 200mM acid buffer solution, placing the egg white into a charging cup, adding protein glutaminase with different proportions, incubating in a water bath kettle at 37 ℃ for 2h, centrifuging, taking supernatant, and determining the ammonia content. Meanwhile, sulfuric acid is added into the same egg white solution to 1M, the mixture reacts for 4 hours at the temperature of 100 ℃, and then the ammonia content in the supernatant is measured to be the total ammonia content. The percentage of ammonia content to total ammonia content in the sample is the degree of deamidation.
(2) Weighing 40g of egg white, adding 100mL of water into a charging cup, adding protein glutaminase in different proportions with 100mL of soybean salad oil, incubating for 2h in a water bath at 37 ℃, beating for 30s at the speed of 1000r/min in a high-speed tissue triturator, centrifuging for 5min at 1500r/min, and recording the volume of an emulsion layer, wherein the volume of the emulsion layer is the emulsification value of the egg white.
(3) Weighing 40g of egg white, adding 100mL of water, placing in a charging cup, adding protein glutaminase with different proportions, stirring for 1min in a high-speed tissue triturator after 30min of action, and quickly pouring into a 500mL measuring cylinder, wherein the foam volume represents the foaming degree value of the egg white.
TABLE 3 Effect of glutaminase addition of different proteins on egg white deamidation, emulsifiability and foaming degree, cake softness and retraction
With the increase of the addition amount of protein glutaminase, the deamidation degree, the emulsification degree and the foaming degree of the egg white are all increased continuously, and the softness and the retraction degree of the cake are also obviously improved.
Example 9 application of protein glutaminase in the development of Soy protein functional beverages
In the development process of the soybean protein functional beverage, the solubility of the soybean protein needs to be improved by a certain method, so that the requirements of high protein content and high clarity of the functional beverage are met, and meanwhile, the bad flavor caused by the increase of the content of the soybean protein can be eliminated. The purified protein glutaminase is applied to the deamidation of the soybean protein to improve the solubility and eliminate the bad flavor.
Weighing 1g and 2g of soy protein isolate respectively, filling into different hydrolysis tubes, and diluting to 100mL with 200mM acid buffer solution to obtain 10g/L and 20g/L soy protein suspension. Adding protein glutaminase into the suspension of different soybean protein isolates respectively to make the ratio of enzyme to substrate reach 20 units/g protein and 50 units/g protein, and then placing the mixed solution in a water bath kettle at 37 ℃ for incubation for 18 h.
(1) Samples with different reaction times are taken, centrifuged, and the supernatant is taken to determine the ammonia content. Meanwhile, sulfuric acid is added into 10g/L and 20g/L soybean protein isolate suspension with the same volume to 1M, reaction is carried out for 4 hours at 100 ℃, and then the ammonia content in supernatant is measured to be the total ammonia content. The percentage of ammonia content to total ammonia content in the sample is the degree of deamidation.
(2) Samples of different reaction times were taken, CCl3COOH crystals were added to reach a CCl3COOH concentration of 12%, and after precipitation of the protein, the protein content in the supernatant was determined. Meanwhile, sulfuric acid is added into the soybean protein isolate suspension liquid with the same volume to 1M, the reaction is carried out for 4 hours at the temperature of 100 ℃, and then the protein content in the supernatant is measured to be the total protein content. The percentage of the protein content in the supernatant to the total protein content after the sample precipitation is the degree of hydrolysis.
TABLE 4 Effect of the amount of glutaminase added to the protein on the clarity and flavor of functional beverages containing soy protein at different concentrations
The protein glutaminase is added into the soybean protein water solution, so that the solubility of the soybean protein functional beverage can be greatly improved, the clarity of the solution is greatly improved, the content of the soybean protein in the beverage is increased, and the appearance requirement of consumers on the soybean protein functional beverage can be met. Meanwhile, the deamidation effect of the protein glutaminase also eliminates the beany flavor to a great extent, and meets the taste requirement of consumers on the soy protein functional beverage.
EXAMPLE 10 use of protein glutaminase in a protein stabilizer
Soybean polysaccharide is a commonly used protein stabilizer and is widely used in the development of various protein drinks and yogurt drinks. The protein glutaminase and the soybean polysaccharide are compounded, so that the solubility and the stability of the protein in the protein beverage can be improved, and the taste is more exquisite and refreshing.
(1) Preparing functional beverage with 0.5 percent and 1 percent of soybean protein isolate: adding protein glutaminase and soybean polysaccharide mixture with different compounding ratios into functional beverage containing 0.5% and 1% of soybean protein isolate respectively, and then placing the mixed solution in a water bath kettle at 37 ℃ for incubation for 2 h. The incubated samples were left for 10d, 20d, 30d, 40d, 60d, respectively.
(2) And (3) determining the precipitation rate: taking samples which are kept stand for different time, shaking up, taking 50mL at 3000r/min, centrifuging for 15min, removing supernate and calculating the precipitation rate. The precipitation rate is (mass of precipitate and tube after centrifugation-mass of empty tube)/(mass of sample and tube-total mass of empty tube).
(3) And randomly enabling consumers to taste the functional drinks containing the 1% of the isolated soy protein and subjected to different treatments in schools, exhibitions and markets, and recording the taste evaluation of the consumers on the drinks subjected to different treatments.
TABLE 5 Effect of the Complex Effect of protein glutaminase and Soybean polysaccharide on protein precipitation Rate
Compared with the existing report that 0.5% of soybean protein is added into functional drinks, the protein glutaminase and soybean polysaccharide are added into the drinks after being compounded, and the addition amount of the soybean protein in the drinks can be increased to 1%, so that a good protein stabilizing effect can be achieved, and therefore, the addition of the protein glutaminase can cooperate with the soybean polysaccharide to stabilize the protein, the use amount of the soybean polysaccharide is reduced, the content of the protein in the drinks is improved, and the nutritional value of the drinks is enhanced.
TABLE 6 evaluation results of consumer taste for differently treated functional beverages
The protein glutaminase and the soybean polysaccharide are compounded to be used as a protein stabilizer to be added into the drink, so that the taste of the drink can be improved, the drink is fresh and fine, and the drink is more popular with consumers.
In the present invention, a higher degree of deamidation can be achieved with a smaller amount of protein glutaminase and a shorter enzyme action time. The higher deamidation degree can greatly improve the solubility, foaming property, emulsifying property and stability of the protein and eliminate bad flavor, which is very important for the application of the protein in the food industry.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
Claims (3)
1. A method of producing protein glutaminase from chrysosporium prion protein (YF 810), said method comprising: inoculating the Chryseobacterium prion lyticum strain into a fermentation medium, and performing shake culture for 12h to obtain a fermentation broth of the protein glutaminase; wherein the temperature of the culture is 30 ℃, and the fermentation medium comprises: lactose 5g/L, soybean peptone 15g/L, Na2HPO4·12H2O 3.8g/L，KH2PO40.25g/L，MgSO4·7H2O 0.25g/L，FeSO4·7H2O0.05g/L, pH 7.2; adding 10g/L-50g/L of porous pH stabilizer into the fermentation medium; the YF810 strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.10532, and the 16S rDNA sequence of the strain is shown in Seq ID No. 3.
2. The method of claim 1, wherein the porous pH stabilizer comprises zeolite, montmorillonite, activated carbon, medical stone, volcanic rock.
3. The method of claim 1 or 2, wherein the porous pH stabilizer is used in an amount of 30 g/L.
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