CN110885772A - Pantoea dispersa for producing trehalase and separation, screening and application thereof - Google Patents
Pantoea dispersa for producing trehalase and separation, screening and application thereof Download PDFInfo
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01028—Alpha,alpha-trehalase (3.2.1.28)
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Abstract
The invention discloses a disperse Pantoea for producing trehalase, and separation, screening and application thereof, wherein the trehalase producing strain C3 is identified as the disperse Pantoea dispersa, is preserved in the China general microbiological culture Collection center, has the preservation time of 2019, 3 and 25 days and has the preservation number of CGMCC No. 17445. The strain C3 is obtained by screening a soil sample by using trehalose as a unique carbon-nitrogen source through flat plate primary screening, shaking flask secondary screening, enzyme activity determination and trehalase activity as an index. The Pantoea dispersa C3 can effectively produce trehalase in large quantity, the trehalase has obvious advantages in temperature and pH stability, can be compounded with other enzymes to be applied to the ethanol industry, can improve the utilization rate of raw materials of the ethanol industry, and has wide application prospects in the ethanol industry.
Description
Technical Field
The invention belongs to the technical field of ethanol industry, relates to a disperse Pantoea for producing trehalase and separation, screening and application thereof, and particularly relates to a strain disperse Pantoea (Pantoea dispersa) C3 for producing trehalase, a breeding method thereof and application of the strain in an ethanol industrial process.
Background
Trehalose (trehalase), also called saccharose and mycose, is a storage carbohydrate and an important product of stress metabolism, and is originally discovered by h.a. wiggers in the ergot of rye in 1832, and widely exists in microorganisms, yeasts, algae, low-grade ferns, insects and invertebrates, especially in fungi and mushrooms, and is formed by connecting two molecules of glucopyranose rings by α -1, 1-glycosidic bonds, so that it is a very stable non-reducing disaccharide.
Trehalose hydrolases (Trehalase, EC:3.2.1.28, Trehalase for short), first discovered in aspergillus niger by bourquerot in 1893, are widely present in microorganisms, plants, insects and mammals. Trehalose is the only hydrolase that can specifically hydrolyze trehalose to 2 molecules of glucose monomers. The action mechanisms of glycoside hydrolases can be divided into two classes, the catalytic mechanism and the reverse catalytic mechanism are reserved, and trehalase adopts the latter. In this mechanism, the active residues (aspartic acid or glutamic acid) in the enzyme molecule play an important role, one as a nucleophilic group and the other as a proton donor. The specific reaction mechanism is as follows: (1) nucleophilic groups (amino acid residues) attack the water molecules, which attack the C atoms of the substrate, forming a connecting bridge; (2) the carboxyl group of the catalytic amino acid residue (carboxyl and base) provides an O atom for H + electrophilic attack of the substrate C-O, forming an intermediate; (3) the C-O bond is broken to form the product.
Trehalase has been reported to be derived from many organisms including prokaryotes, plants, and animals. The trehalase is applied to production, and the trehalase is required to have high catalytic activity, good pH stability, good thermal stability and other properties. However, the research on trehalase in China is far from sufficient at present, and particularly, the research on the enzymatic characteristics of trehalase is very insufficient. The trehalase has great application prospect in food, industry, agriculture and insect treatment. Especially in the ethanol industry, trehalase can improve the yield of fermentation product ethanol, especially reduce the total amount of trehalose of 'DP 2 peak' (DP-degree of polymerization-equivalent to disaccharide) at the end of fermentation, and can greatly improve the utilization rate of residual sugar and the conversion rate of total sugar. Therefore, the development of trehalase related research has very important theoretical significance and practical value, and the application prospect is wide.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a strain of trehalose-producing enzyme C3 which is identified as Pantoea dispersa (Pantoea dispersa), the Pantoea dispersa C3 can effectively produce trehalose enzymes in large quantities, the trehalose enzymes can be compounded with other enzymes to be applied to the ethanol industry, the utilization rate of raw materials in the ethanol industry can be improved, and the trehalose-producing enzyme has wide application prospects in the ethanol industry.
The invention also provides separation, screening and application of the trehalose-producing enzyme C3 strain.
The technical scheme is as follows: in order to achieve the purpose, the trehalose-producing enzyme strain C3 is identified as Pantoea dispersa (Pantoea dispersa) and is preserved in the general microbiological center of China Committee for culture Collection of microorganisms, the preservation time is 3 and 25 days in 2019, and the preservation number is CGMCC No. 17445; address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the strain C3 is obtained by selecting fertile soil from river sides and parks in Wuxi countryside of Jiangsu, collecting soil samples, separating and screening, and the strain C3 is streaked and inoculated on a solid LB culture medium plate, so that the periphery of a colony grown is circular, the surface of the colony is raised, the colony is milky white, and the center and the periphery of the colony have no other colors and are easy to pick. The gram stain is red, and the cells are observed under an optical microscope, are mostly in a straight rod shape, are about 0.5-1.0 mu m multiplied by 1.0-3.0 mu m in length, and are single-generation or pair, and sometimes are short-chain.
The screening method of the trehalose-producing enzyme C3 comprises the following steps:
collecting soil samples, and carrying out strain rapid propagation in an enrichment culture medium; primary screening, namely taking the enriched and cultured bacterial liquid, and separating strains capable of utilizing substrates from a screening culture medium with trehalose as a unique carbon-nitrogen source; and (3) re-screening, selecting a single colony in a liquid fermentation culture medium, and finally screening to obtain a pure culture pantoea dispersa (Pantoea dispersa) through re-screening and 16S rDNA identification.
Specifically, the screening method of the trehalase producing strain C3 comprises the following steps: the strain provided by the invention is characterized in that fertile soil is respectively selected from the rural river sides and parks without stannum, and the number of the strain is enlarged through an enrichment culture medium after a soil sample is collected; primary screening, namely taking the enriched and cultured bacterial liquid, and culturing strains capable of utilizing substrates in a screening culture medium containing trehalose as a carbon-nitrogen source; and (3) re-screening, selecting a single colony in a liquid fermentation culture medium, sampling after the culture is finished, carrying out enzyme activity determination, and selecting a strain with high enzyme activity for preservation.
The screening medium comprises the following components: trehalose 5-20g/L, KCl 0.1-5g/L, KH 0.1-5g/L2PO4，0.1-5g/L MgSO4·7H2O，0.1-5g/L CaCO3，pH 6.0-8.0；
The enrichment medium comprises the following components: 1-10g/L peptone; 1-10g/L yeast extract; 10-200g/L glucose; 0.1-5g/L MgSO4·7H2O；0.1-5g/L K2HPO4。
Liquid fermentation medium: peptone 1-10g/L, yeast extract 4-20g/L, glycerin 3-20g/L, CaCO30.1-5g/L、K2HPO40.5-1g/L、KH2PO41-2g/L、pH 5-7。
The enrichment method comprises the following steps:
5g of fresh soil sample is dissolved in 45g of sterile water, and the supernatant is put into a triangular flask containing the enrichment medium and cultured for 24 hours in a shaking flask.
The enrichment medium comprises the following components:
peptone 5 g/L; 1.5g/L of yeast extract; 150g/L of glucose; MgSO (MgSO)4·7H2O 0.5g/L；K2HPO40.5g/L。
The primary screening method comprises the following steps:
taking 1mL of enriched and cultured bacteria liquid, transferring the bacteria liquid into a test tube which is prepared in advance and contains 9mL of sterile physiological saline, and shaking the bacteria liquid on a vortex shaker uniformly to prepare 10-1A sample suspension of concentration; then sucking 1mL of the suspension, adding the suspension into a test tube containing 9mL of sterile physiological saline, shaking uniformly to prepare 10-2The sample suspension with concentration can be made into 10 by sequentially continuing the operation-3、10-4、10-5、10-6、10-7Sample suspension of concentration. And (3) respectively taking 0.1mL of sample suspension liquid and coating the sample suspension liquid on a primary screening culture medium plate, wherein each concentration is 2, and selecting a plate in which bacterial colonies are dispersed and the number of single bacterial colonies is about 100-200 as a target plate.
The screening culture medium comprises the following components:
15g/L trehalose; 1g/L KCl; 1g/L KH2PO4；0.5g/L MgSO4·7H2O；1g/L CaCO3(ii) a Adding 20g/L of agar into a primary screening culture medium plate solid culture medium; the pH was 7.0.
The re-screening method comprises the following steps:
inoculating the colony to a liquid culture medium, performing shake culture at 37 ℃ and 220rpm for 36h, and then performing enzyme activity determination.
The liquid fermentation medium comprises the following components:
15g/L trehalose, 1g/L KCl; 1g/L KH2PO4；0.5g/L MgSO4·7H2O；1g/L CaCO3；pH7.0。
The method for identifying the trehalase producing strain comprises the following steps:
the 16S rDNA sequence is shown as SEQ ID NO.1 by carrying out PCR amplification on the extracted bacterial genome by using 16S rDNA universal primers 27F (5'-AGA GTT TGATCC TGG CTC AG-3') and 1492R (5'-GGTTAC CTT GTT ACG ACT T-3'), the similarity comparison is carried out on the existing sequences in a database by applying BLAST software on an NCBI website, a proper strain and a corresponding sequence are selected from the comparison result as reference, DNAMAN software is adopted to arrange the comparison result, ClustalX software is adopted to carry out complete sequence comparison, a phylogenetic tree is constructed by means of Mega software, and the final result shows that the 16SrDNA sequence of the strain has more than 99 percent of homology with the related sequences of Pantoea (EU931561.1, GQ246183.1 and the like), meanwhile, the sequence homology with related strains of Cronobacter (KY971631.1) and Enterobacteriacea (LC007912.1) is about 98 percent, and finally the related strains are classified as Pantoea strains. Combining morphological characteristics and physiological and biochemical characteristics, it was identified as Pantoea dispersa (Pantoea dispersa).
The invention utilizes the fermentation liquor containing the trehalase produced by the trehalase producing bacterium C3.
The production method of the fermentation liquor containing trehalase comprises the following steps:
inoculating a single colony of the strain C3 into a seed culture medium, culturing at 25-35 ℃ and 100-300rpm for 10-18 hours to obtain a seed solution, inoculating the seed solution into a fermentation culture medium according to the inoculum size of 1-5% of the volume ratio, wherein the fermentation culture temperature is 20-40 ℃, the rotating speed of a shaking table is 100-300rpm, and the culture time is 15-80 hours. Preferably, the rotation speed in the seed culture medium and the fermentation culture medium is 220 rpm.
Specifically, the strain Pantoea dispersa (Enterobacter ludwigii) C3 is firstly cultured in a seed culture medium by seed liquid, and is cultured for 10-18 hours at 25-35 ℃ and 100-300rpm after inoculation; 20-50mL of culture medium is filled in a 250mL triangular flask for shake flask fermentation, the seed liquid in the seed culture medium is inoculated to the fermentation culture medium according to the inoculum size of 1-5% of the volume ratio, the temperature is 20-40 ℃, the rotating speed of a shaking table is 100-300rpm, and the culture time is 15-80 h.
The formula of the seed culture medium is as follows: peptone 4-20g/L, yeast extract 1-10g/L, NaCl 2-20g/L, pH 5-7.5.
The formula of the fermentation medium is as follows: peptone 1-10g/L, yeast extract 4-20g/L, glycerin 3-20g/L, CaCO30.1-5g/L、K2HPO40.5-1g/L、KH2PO41-2g/L、pH 5-7。
The trehalase produced by the trehalase producing bacterium C3 is provided by the invention.
Further, the mycosidase enzymatic properties are as follows:
(1) the optimum temperature is 35 ℃, the optimum pH value is 5.0, the temperature and pH stability have obvious advantages, and the enzyme activity is kept more than 80% after the treatment for 1h in the pH4-5 range;
(2)Sn2+and Fe2+Has effect in promoting enzyme activity, and contains Ca2+、Zn2+、Mn2+、Cu2+、K+、Ba2+、Li+And Co2+Has inhibitory effect on enzyme activity;
(3) tween 20 has promoting effect on enzyme activity; tween 80, Triton X-100, Triton X-114, DMSO, SDS and EDTA have inhibitory effect on enzyme activity.
The trehalose-producing enzyme C3 is applied to the ethanol industry.
The influence of the reaction temperature and the pH on the activity of the trehalase is examined, the optimal temperature and the optimal pH of the trehalase are respectively 35 ℃ and 5, the trehalase has high thermal stability within the temperature range of 30-35 ℃, has high activity and stability under an acidic condition, and is suitable for the ethanol industry. When the influence of metal ions and chemical reagents on the activity of trehalase is studied, Sn is found2+And Fe2+Has effect in promoting enzyme activity, and Ca2+、Zn2+、Mn2+、Cu2+、K+、Ba2+、Li+And Co2+Has inhibitory effect on enzyme activity, Mg2+、Ni2+And Na+Almost has no influence on the enzyme activity; tween 20 has promoting effect on enzyme activity, Tween 80, Triton X-100, Triton X-114, DMSO, SDS and EDTA have inhibiting effect on enzyme activity, and DTT and PMSF have almost no influence on enzyme activity.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention separates and screens a disperse pantoea (Enterobacter ludwigii) C3, and discovers that the disperse pantoea C3 produces trehalase for the first time, and the enzymological properties of the trehalase are as follows: the optimum temperature is 35 ℃, the optimum pH value is 5.0, the temperature and pH stability have obvious advantages, the activity and the stability are high under the acidic condition, the enzyme can be compounded with amylase, protease, glycosidase and other enzymes to be applied to the ethanol industry, the utilization rate of raw materials in the ethanol industry can be improved, and the enzyme has wide application prospects in the ethanol industry.
The Enterobacter lutesceni C3 can efficiently produce trehalase with good activity at lower pH, the corresponding fermentation conditions are optimized, and the activity of the fermentation enzyme reaches 21.806U/mL. Is suitable for the industrial production of ethanol; and the culture condition is simple, the preservation is easy, the industrial production is easy, and the development and application prospect is good.
Drawings
FIG. 1 is a morphological diagram of a strain of Pantoea dispersa (Pantoea dispersa) under an optical microscope;
FIG. 2 is a graph showing the results of phylogenetic analysis based on the 16S rDNA sequence of Pantoea dispersa (Pantoea dispersa);
FIG. 3 is a schematic diagram showing the optimum temperature and temperature stability of a Pantoea dispersa (Pantoea dispersa) trehalase;
FIG. 4 is a schematic diagram showing the optimum pH of a Pantoea dispersa (Pantoea dispersa) trehalase;
FIG. 5 is a graph showing the pH stability of a Enterobacter dispersus (Pantoea dispersa) trehalase.
Detailed Description
The invention will be further described with reference to specific examples, which are intended for illustrative purposes only and are not intended to be limiting. Those skilled in the art can appreciate the features and utilities of the present invention from the description as set forth herein, and that the present invention may be implemented or utilized in various other embodiments.
The enzyme activity of the trehalose-producing disperse Pantoea (Pantoea dispersa) standard detection method is as follows:
the content of glucose generated by decomposing trehalose by trehalase is measured by a 3, 5-dinitrosalicylic acid (DNS) colorimetric method: to the experimental group, 150. mu.L of the enzyme solution and 150. mu.L of a 10% soluble trehalose substrate (aqueous solution) were added in this order in a 2mL EP tube. Sequentially adding 150 μ L of enzyme solution and 150 μ L of phosphate buffer solution with pH of 6.5 into the control group; reacting at 37 ℃ for 15min, and adding 300 mu L of DNS reagent into each of the experimental group and the control group; stopping the reaction in boiling water bath for 5min, taking out and placing in ice-water mixture for ice bath for 2 min; the absorbance of 200. mu.L of the mixture was measured at 540 nm.
The preparation method of the DNS reagent comprises the following steps: 244.4g of potassium sodium tartrate was accurately weighed into 500mL of deionized water and dissolved by heating at 45 ℃. 21g of NaOH and 6.3g of DNS are added into the solution, after the solution is dissolved, 5mL of phenol and 5g of sodium bisulfite are respectively added in sequence, and the volume is determined to be 1L after the solution is cooled. The prepared DNS solution is stored in a brown bottle in a dark place and can be used after being placed for one week.
The preparation method of the 50mmol/L phosphate buffer solution comprises the following steps: respectively preparing 200mmol/L NaH2PO4And Na2HPO4And (3) buffering the two solutions according to a certain proportion to prepare 50mmol/L PBS buffer solution with pH of 6.5.
The preparation method of the 100mg/mL trehalose solution comprises the following steps: and (3) weighing trehalose by taking the 50mmol/L PBS buffer solution as a solution, dissolving to obtain a 10% trehalose solution, and using the trehalose solution as an enzyme activity determination substrate for later use.
The preparation method of the glucose standard curve comprises the following steps: accurately weighing 1.0g of anhydrous glucose dried to constant weight, dissolving in deionized water, and diluting to 100mL to obtain 10.0mg/mL glucose standard solution, preparing glucose solutions with different concentrations according to Table 1, adding 300 muL of DNS reagent, reacting, measuring absorbance value at 540nm, and replacing the glucose standard solution with PBS buffer solution with pH6.5 in a control group.
TABLE 1 preparation of glucose standard koji
The enzyme activity calculation method comprises the following steps:
enzyme activity (1U) definition: under the above experimental conditions, 1mL of the enzyme solution produced 1. mu. mol of reducing sugar per minute and the measurement was repeated 3 times.
Example 1
1. Process for obtaining Pantoea dispersa (Pantoea dispersa) C3
Collecting soil samples in rural river sides and parks without stannum, shoveling surface soil by using a small shovel, collecting soil from 5-10 cm positions by using a sterilized plastic bag, sampling about 50g, sealing well, and recording time, place and environmental conditions. 5g of fresh soil sample is dissolved in 45g of sterile water, and the supernatant is put into a triangular flask containing the enrichment medium and cultured for 24 hours in a shaking flask. Taking 1mL of enriched and cultured bacteria liquid, transferring the bacteria liquid into a test tube which is prepared in advance and contains 9mL of sterile physiological saline, and shaking the bacteria liquid on a vortex shaker uniformly to prepare 10-1A sample suspension of concentration; then sucking 1mL of the suspension, adding the suspension into a test tube containing 9mL of sterile physiological saline, shaking uniformly to prepare 10-2The sample suspension with concentration can be made into 10 by sequentially continuing the operation-3、10-4、10-5、10-6、10-7Sample suspension of concentration. 0.1mL of each sample suspension was spread on a primary screening medium plate at 2 aliquots and colonies were picked to disperse. And taking the plate with the single colony number of about 100-200 as a target plate.
During re-screening, the bacterial colony is inoculated in a liquid fermentation medium, and after shaking culture at 37 ℃ and 220rpm for 36 hours, enzyme activity determination is carried out. The strain separated by the colony is shown to have higher trehalase production potential, the strain is further separated and purified, the number is C3, and the enzyme activity can reach 21.806U/mL.
The composition of the enrichment medium is (g/L): peptone 5; 1.5 of yeast extract; glucose 150; MgSO (MgSO)4·7H2O 0.5；K2HPO40.5。
The composition of the primary screening culture medium is (g/L): trehalose 15; KCl 1; KH (Perkin Elmer)2 PO 41；MgSO4·7H2O 0.5；CaCO 31; adding agar 20 to the culture medium; the pH was 7.0.
The composition of the liquid fermentation medium is (g/L): trehalose 15; KCl 1; KH (Perkin Elmer)2 PO 41；MgSO4·7H2O 0.5；CaCO 31；pH 7.0。
2. Morphological and physiological characterization of Pantoea dispersa (Pantoea dispersa) C3
And (3) streaking the single colony C3 obtained by selective breeding and separation on an LB solid culture medium, and observing the colony morphology, wherein the result shows that the periphery of the colony is circular, the surface is raised and the colony is milky white. The gram stain is red, and the cells are observed under an optical microscope, are mostly in a straight rod shape, are about 0.5-1.0 mu m multiplied by 1.0-3.0 mu m in length, and are single-generation or pair, and sometimes are short-chain. The strain is gram negative, peritrichogenous flagellum movement, oxidase negative and catalase positive, and can produce acid by using D-glucose and other saccharides. Lysine and ornithine decarboxylase and arginine dihydrolase are negative and do not produce H2S, urea is not hydrolyzed (fig. 1).
3. Molecular biological identification of Pantoea dispersa (Pantoea dispersa) C3
Culturing the strain C3 to logarithmic growth phase, collecting thallus, extracting DNA, selecting 16S rDNA universal primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) to perform PCR amplification on the extracted bacterial genome, and configuring a 50 mu L PCR reaction system in a 200 mu L PCR tube according to the formula in the table 2. After mixing the solution, the solution was centrifuged slightly to completely settle at the bottom of the PCR tube, and the PCR tube was placed in a PCR machine.
TABLE 2 PCR reaction System
PCR amplification conditions: pre-denaturation at 94 deg.C for 10min, denaturation at 95 deg.C for 60s, annealing at 58 deg.C for 60s, extension at 72 deg.C for 90s, repeating for 30 times, and final extension at 72 deg.C for 10 min. And detecting the PCR amplification result by using gel electrophoresis after the amplification is finished. Staining was done with 1 XTBE buffer, 10 × Loading buffer, using 5000DL DNA Marker as standard Marker, run at 150V for 30 min. A bright band at 1500bp was observed using a gel imager. Purification of PCR amplification products was carried out according to the kit for purification of PCR products from small gel recovery of Shanghai Biotech, Inc., and sequencing was carried out by Shanghai Ruidi Biotech, Inc.
The length of the 16S rDNA sequence of the strain is 1414bp, similarity comparison is carried out on the existing sequences in a database by applying BLAST software on an NCBI website, a proper strain and a corresponding sequence are selected from comparison results to be used as reference, DNAMAN software is used for sorting the comparison results, ClustalX software is used for carrying out complete sequence comparison, a phylogenetic tree (shown in figure 2) is constructed by virtue of Mega software, the final result shows that the 16S rDNA sequence of the strain has more than 99 percent homology with the related sequences of Pantoea (EU931561.1, GQ246183.1 and the like), and simultaneously has about 98 percent homology with the related sequences of Cronobacter (KY971631.1) and Enterobacteriaceae (LC007912.1), the strain is finally classified into Pantoea strain, and the strain is identified as Pantoea dispersa (Pantoea disperiana) by combining morphological characteristics and physiological and biochemical characteristics, and named Pantoea disperiana (Pantoea disperiana) C3. The strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 17445.
Example 2
Fermentation conditions for Pantoea dispersa (Pantoea dispersa) C3 trehalase
Culturing Pantoea dispersa (Pantoea dispersa) C3 in seed culture medium by seed liquid, inoculating, and culturing at 30 deg.C and 220rpm for 15 hr; 50mL of culture medium is filled in a 250mL triangular flask for shake flask fermentation, the seed culture medium is inoculated to the fermentation culture medium according to the inoculation amount of 5 percent, and the culture is carried out for 40 hours at 30 ℃ and 220 rpm; obtaining the fermentation liquor containing trehalase.
The formula of the seed culture medium is as follows: 10g/L of peptone and 5g/L, NaCl 10g/L, pH 7.2.2 of yeast extract.
The formula of the fermentation medium is as follows: 10g/L of peptone, 20g/L of yeast extract and 8g/L, CaCO of glycerol31g/L、K2HPO41g/L、KH2PO42g/L、pH 7。
Example 3
Properties of Pantoea dispersa (Pantoea dispersa) C3 trehalase
⑴ optimum reaction temperature and temperature stability:
optimum reaction temperature: enzyme solution (namely fermentation liquor in example 2, the same below) is mixed in equal volume with 50mmol/L phosphate buffer solution (pH 6.5), the enzyme activity of the trehalase is measured at the temperature of 30, 35, 40, 45, 50, 55 ℃ and the like under the condition of pH6.5, and the reaction temperature with the highest enzyme activity is taken as the optimal reaction temperature.
Temperature stability: heat-treating the enzyme solution at different temperatures (30, 35, 40, 45, 50, 55 deg.C) for 30min, cooling on ice, measuring residual enzyme activity of trehalase at 37 deg.C according to standard enzyme activity measuring method, and using enzyme activity of untreated enzyme solution as control.
Researches show that the optimum temperature of the trehalase is 35 ℃, and the relative enzyme activity can reach more than 90% within the range of 35-45 ℃; the thermal stability is higher in the range of 30-35 ℃, the residual enzyme activity can be kept above 90%, but the thermal stability is obviously reduced when the temperature is higher than 40 ℃ along with the increase of the temperature (figure 3).
⑵ optimum reaction pH and pH stability:
optimum reaction pH: preparing Britton-Robinson buffer solution, wherein the Britton-Robinson buffer solution is prepared by mixing phosphoric acid, boric acid and acetic acid, and different amounts of sodium hydroxide are added into the mixed solution to form the buffer solution with a wide pH range, wherein the pH value is 1.8-11.9. Preparing BR buffer solution: in 100ml of a mixed solution of phosphoric acid, boric acid and acetic acid (the concentration is 0.04mol/L), sodium hydroxide (the concentration is 0.2mol/L) with different volumes is added to form a buffer solution with wide pH range. The 10% trehalose substrate solution is prepared from the buffer solution, and the enzyme solution is diluted by the buffer solution appropriately. Enzyme activity detection is carried out under different buffer solution systems (pH4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 and 9) at 37 ℃ according to a standard enzyme activity detection method, and the reaction pH with the highest enzyme activity is taken as the optimal reaction pH.
pH stability: and placing the enzyme solution in buffer solution environments with different pH values and on ice for 1h, and then carrying out enzyme activity detection under standard conditions, wherein the enzyme activity of untreated enzyme solution is used as a control.
Researches show that the optimum pH value of the trehalase is 5, the trehalase has high activity under an acidic condition, and the relative enzyme activity can reach more than 90 percent especially within the pH range of 4.5-5.5 (figure 4); the trehalase has good stability in an acidic environment, can keep more than 80% of residual enzyme activity within the pH range of 4-5, is suitable for ethanol industry, and can improve the utilization rate of residual sugar and the conversion rate of total sugar (figure 5).
⑶ Effect of addition of Metal ions and chemical reagents on trehalase Activity
Influence of Metal ions: first, 200mmol/L Ca is prepared2+、Zn2+、Ni2+、Mn2+、Cu2+、K+、Na+、Fe2+、Sn2 +、Ba2+、Mg2+、Co2+And Li+And (3) waiting for each metal ion mother liquor, then adding the mother liquor into the enzyme solution for treatment for 30min at the final concentration of 1mmol/L, and then carrying out standard enzyme activity detection reaction (the temperature is 37 ℃, and the pH value is 6.5). Reactions without any added metal ions were used as controls.
Influence of chemical reagents: tween, triton, Sodium Dodecyl Sulfate (SDS), dimethyl sulfoxide (DMSO), and the like are firstly prepared into a solution with the mass fraction of 10%, and then the solution is added into an enzyme solution according to the final concentration of 1% of the mass fraction for treatment for 30min to carry out standard enzyme activity detection reaction. Preparing 200mmol/L mother liquor with ethylenediamine tetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), Dithiothreitol (DTT) and the like, then respectively adding the mother liquor into enzyme liquid according to the final concentration of 1mmol/L, and treating for 30min to carry out standard enzyme activity detection reaction, wherein the reactions are all controlled by reactions without any chemical reagent.
Studies have found that Sn is compared to a control without any added metal ions2+And Fe2+Has effect in promoting enzyme activity, and Ca2+、Zn2+、Mn2+、Cu2+、K+、Ba2+、Li+And Co2+Has inhibitory effect on enzyme activity, Mg2+、Ni2+And Na+Had little effect on enzyme activity (Table 3).
TABLE 3 Effect of Metal ions on trehalase Activity
TABLE 4 Effect of chemical reagents on trehalase Activity
The above experiments prove that Pantoea dispersa (Pantoea dispersa) C3 of the present invention can efficiently produce trehalase in large quantities, the optimum temperature of the trehalase is 35 ℃, and the optimum pH is 5; the trehalase has obvious advantages in temperature and pH stability, and the residual enzyme activity can be kept above 90% within the range of 30-35 ℃; the trehalase can keep more than 80% of residual enzyme activity within the pH range of 4-5; can be compounded with other enzymes to be applied to the ethanol industry, can improve the utilization rate of raw materials of the ethanol industry, and has wide application prospect in the ethanol industry.
Example 4
Fermentation conditions for Pantoea dispersa (Pantoea dispersa) C3 trehalase
Culturing Pantoea dispersa (Pantoea dispersa) C3 in seed culture medium by seed liquid, inoculating, and culturing at 25 deg.C and 100rpm for 18 hr; 50mL of culture medium is filled in a 250mL triangular flask for shake flask fermentation, the seed culture medium is inoculated to the fermentation culture medium according to the inoculation amount of 3 percent, and the culture is carried out for 80 hours at the temperature of 20 ℃ and the rpm of 100; obtaining the fermentation liquor containing trehalase.
Example 5
Fermentation conditions for Pantoea dispersa (Pantoea dispersa) C3 trehalase
Culturing Pantoea dispersa (Pantoea dispersa) C3 in seed culture medium by seed liquid, inoculating, and culturing at 35 deg.C and 300rpm for 10 hr; 50mL of culture medium is filled in a 250mL triangular flask for shake flask fermentation, the seed culture medium is inoculated to the fermentation culture medium according to the inoculation amount of 1 percent, and the culture is carried out for 15 hours at 40 ℃ and 300 rpm; obtaining the fermentation liquor containing trehalase.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention.
Sequence listing
<110> Jiangsu Boyang biological products Co., Ltd
University of south of the Yangtze river
<120> a strain of Pantoea dispersa for producing trehalase and separation, screening and application thereof
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<213> Pantoea dispersa (Pantoea dispersa)
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tgcaagtcga acggcagcac agaagagctt gctctttggg tggcgagtgg cggacgggtg 60
agtaatgtct gggaaactgc ccgatggagg gggataacta ctggaaacgg tagctaatac 120
cgcataacgt cgcaagacca aagtggggga ccttcgggcc tcacaccatc ggatgtgccc 180
agatgggatt agctagtagg tggggtaatg gctcacctag gcgacgatcc ctagctggtc 240
tgagaggatg accagccaca ctggaactga gacacggtcc agactcctac gggaggcagc 300
agtggggaat attgcacaat gggcgcaagc ctgatgcagc catgccgcgt gtatgaagaa 360
ggccttcggg ttgtaaagta ctttcagcgg ggaggaaggc ggtgaggtta ataacctcgc 420
cgattgacgt tacccgcaga agaagcaccg gctaactccg tgccagcagc cgcggtaata 480
cggagggtgc aagcgttaat cggaattact gggcgtaaag cgcacgcagg cggtctgtta 540
agtcagatgt gaaatccccg ggcttaacct gggaactgca tttgaaactg gcaggcttga 600
gtctcgtaga ggggggtaga attccaggtg tagcggtgaa atgcgtagag atctggagga 660
ataccggtgg cgaaggcggc cccctggacg aagactgacg ctcaggtgcg aaagcgtggg 720
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcga cttggaggtt 780
gtgcccttga ggcgtggctt ccggagctaa cgcgttaagt cgaccgcctg gggagtacgg 840
ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt 900
ttaattcgat gcaacgcgaa gaaccttacc tggccttgac atccagagaa cttagcagag 960
atgctttggt gccttcggga actctgagac aggtgctgca tggctgtcgt cagctcgtgt 1020
tgtgaaatgt tgggttaagt cccgcaacga gcgcaaccct tatcctttgt tgccagcggt 1080
tcggccggga actcaaagga gactgccggt gataaaccgg aggaaggtgg ggatgacgtc 1140
aagtcatcat ggcccttacg gccagggcta cacacgtgct acaatggcgc atacaaagag 1200
aagcgacctc gcgagagcaa gcggacctca taaagtgcgt cgtagtccgg attggagtct 1260
gcaactcgac tccatgaagt cggaatcgct agtaatcgta gatcagaatg ctacggtgaa 1320
tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtgggtt gcaaaagaag 1380
taggtagctt aaccttcggg agggcgctta ccac 1414
Claims (7)
1. A strain of trehalase producing bacterium C3, identified as Pantoea dispersa (Pantoea dispersa), has been preserved in China general microbiological culture Collection center (CGMCC), with the preservation time of 3 and 25 days in 2019 and the preservation number of CGMCC No. 17445.
2. The method for screening of trehalose-producing enzyme C3 according to claim 1, comprising the steps of:
collecting soil samples, and carrying out strain rapid propagation in an enrichment culture medium; primary screening, namely taking the enriched and cultured bacterial liquid, and separating strains capable of utilizing substrates from a screening culture medium with trehalose as a unique carbon-nitrogen source; and (3) re-screening, selecting a single colony in a liquid fermentation culture medium, and finally screening to obtain a pure culture pantoea dispersa (Pantoea dispersa) C3 through re-screening and 16S rDNA molecular identification.
3. A fermentation broth containing trehalase produced by the trehalase-producing bacterium C3 of claim 1.
4. A method for producing the trehalase-containing fermentation broth of claim 3, comprising the steps of:
inoculating a single colony of the strain C3 into a seed culture medium, culturing at 25-35 ℃ and 100-300rpm for 10-18 hours to obtain a seed solution, preferably inoculating the seed solution into a fermentation culture medium according to the inoculum size of 1-5% of the volume ratio, wherein the fermentation culture temperature is 20-40 ℃, the rotating speed of a shaking table is 100-300rpm, and the culture time is 15-80 hours.
5. A trehalase produced by using the trehalase-producing bacterium C3 of claim 1.
6. The trehalase according to claim 5, characterized in that the trehalase enzymatic properties are as follows:
(1) the optimum temperature is 35 ℃, the optimum pH value is 5.0, the temperature and pH stability have obvious advantages, and the enzyme activity is kept more than 80% after the treatment for 1h in the pH4-5 range;
(2)Sn2+and Fe2+Has effect in promoting enzyme activity, and contains Ca2+、Zn2+、Mn2+、Cu2+、K+、Ba2+、Li+And Co2+Has inhibitory effect on enzyme activity;
(3) tween 20 has promoting effect on enzyme activity; tween 80, Triton X-100, Triton X-114, DMSO, SDS and EDTA have inhibitory effect on enzyme activity.
7. Use of the trehalose-producing enzyme C3 of claim 1 in the ethanol industry.
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| CN112080448A (en) * | 2020-09-14 | 2020-12-15 | 江苏省奥谷生物科技有限公司 | Culture medium and method for producing trehalase through bacterial fermentation |
| CN114164140A (en) * | 2021-10-28 | 2022-03-11 | 中国林业科学研究院华北林业实验中心 | Efficient phosphorus-solubilizing bacterium MQR6 and fermentation product and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112080448A (en) * | 2020-09-14 | 2020-12-15 | 江苏省奥谷生物科技有限公司 | Culture medium and method for producing trehalase through bacterial fermentation |
| CN112080448B (en) * | 2020-09-14 | 2022-08-12 | 江苏省奥谷生物科技有限公司 | Culture medium and method for producing trehalase through bacterial fermentation |
| CN114164140A (en) * | 2021-10-28 | 2022-03-11 | 中国林业科学研究院华北林业实验中心 | Efficient phosphorus-solubilizing bacterium MQR6 and fermentation product and application thereof |
| CN114164140B (en) * | 2021-10-28 | 2023-12-12 | 中国林业科学研究院华北林业实验中心 | Efficient phosphorus-dissolving bacteria MQR6 and fermentation product and application thereof |
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