CN109439599B

CN109439599B - Trehalose enzyme production strain and application thereof

Info

Publication number: CN109439599B
Application number: CN201811590020.3A
Authority: CN
Inventors: 董琦; 龚劲松; 张新爽; 许正宏; 史劲松; 董哲卿; 郭鸿飞
Original assignee: Jiangsu Boyang Biological Product Co ltd; Jiangnan University
Current assignee: Jiangsu Boyang Biological Product Co ltd; Jiangnan University
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2020-04-10
Anticipated expiration: 2038-12-25
Also published as: CN109439599A

Abstract

The invention discloses a trehalose enzyme production strain and application thereof, belonging to the technical field of industrial microorganisms. The Erwinia rhapontici C2 is characterized in that the Erwinia rhapontici has been deposited in the China general microbiological culture collection management center in 11.21.2018, and the deposited address is No. 3 Hospital No.1 of North Chen West Lu in the sunward area of Beijing, and the deposited number is CGMCC No. 16760. The invention reports Erwinia rhapontici (Erwinia rhapontici) for producing trehalase for the first time, and researches the enzymatic properties of Erwinia rhapontici. The optimum temperature and pH of trehalase produced by the strain are 40 ℃ and 5 respectively, and the trehalase has high activity and stability under acidic conditions and has wide application prospect in the ethanol industry.

Description

Trehalose enzyme production strain and application thereof

Technical Field

The invention relates to a trehalose enzyme production strain and application thereof, belonging to the technical field of industrial microorganisms.

Background

Trehalose (Trehalose) is a very stable non-reducing disaccharide formed by two molecules of glucopyranose rings connected by α -1, 1-glycosidic bonds, Trehalose was first discovered in 1832, and subsequently isolated in 1858 mushrooms and called Trehalose, which is ubiquitous in nature and widely distributed, including bacteria, fungi, insects, lower plants, vertebrates, particularly in fungi and insects, and is one of the major oligosaccharides recently developed internationally, although Trehalose is widely available, but not many organisms capable of accumulating Trehalose in large amounts in vivo.

The trehalose production methods currently under research and development mainly include microbial extraction, fermentation and enzymatic conversion. However, the cost for extracting trehalose from microorganisms is high, the extraction source is limited, and large-scale industrial production is difficult to realize; the trehalose produced by the fermentation method has the problems of low conversion rate, complex components of the fermentation liquor, difficult extraction and refining of the trehalose and the like, and cannot be widely applied to industrial production. The conversion into trehalose by the action of enzymes involved in trehalose synthesis is currently carried out by a method commonly used in industry using maltose, starch or the like as a substrate. However, when trehalose is produced by an enzyme conversion method, some problems still remain to be solved, for example, mother liquor containing components such as glucose and a small amount of trehalose is still generated after trehalose is prepared and extracted, and resource waste is caused. If the trehalose in the mother liquor can be hydrolyzed into glucose, the effective reuse of the mother liquor can be realized, and the resource waste is reduced. Therefore, the research on the trehalose hydrolase has important research value and application prospect.

Trehalase is a trehalose hydrolase, which can specifically and specifically decompose one molecule of trehalose into two molecules of glucose. In nature, trehalase widely exists in insects, mammals, microorganisms and plants, plays an important role, has a wide application prospect in the ethanol industry, and can improve the utilization rate of residual sugar and the conversion rate of total sugar; has great application prospect in food, agriculture, pesticide and insect treatment. Trehalase was first discovered by Bourquerot in Aspergillus niger 1893, and by Fischer in Saccharomyces cerevisiae in 1895. Subsequently, researchers have continually discovered and identified different trehalases in different organisms, including bacteria, yeasts, fungi, insects, nematodes, plants, and vertebrates. The difference in optimum pH of trehalase in fungi due to the reaction is divided into two categories: neutral trehalase and acid trehalase; wherein the acid trehalase is located in vacuole, is extracellular enzyme, can not be regulated by phosphorylation, and has optimal pH of about 4.5; neutral trehalase is located in cytoplasm and is responsible for the decomposition of main intracellular trehalose, and the optimum pH is about 7.0.

Research has found that the enzyme trehalose decomposes trehalose using an inverse catalytic mechanism: firstly, the nucleophilic group attacks the water molecule, and the water molecule attacks the C atom of the substrate to form a connecting bridge; at the same time, the carboxyl group of the catalytic amino acid residue will provide H⁺，H⁺Will electrophilically attack the O atom in the C-O of the substrate to form an intermediate product; finally, the C-O bond is broken to form the product. The trehalase is applied to production, and the trehalase is required to have the properties of high catalytic activity, good pH stability, good thermal stability and the like, but at present, the strain generated by the trehalase is still relatively few in reports, and the research on the enzymatic characteristics of the trehalase is still not much. The application lays a foundation for international first report that the Erwinia rhapontici (Erwinia rhapontici) produces trehalase, and subsequent researches such as modification of trehalase by using a genetic engineering method, and has good theoretical value and practical significance.

Disclosure of Invention

The invention aims to provide an Erwinia rhapontici C2 strain for producing trehalase, which is classified and named Erwinia rhapontici and is preserved in the China general microbiological culture collection management center in 11.21.2018, the preservation address is No. 3 of West Lu No.1 of the sunward area of Beijing, and the preservation number is CGMCC No. 16760.

The second purpose of the invention is to provide the application of the Erwinia rhapontici C2 in food, agriculture, pesticides or insect treatment.

Further, the application is to apply the Erwinia rhapontici C2 to hydrolysis of trehalose.

Further, the application is to use the Erwinia rhapontici C2 for fermentation production of trehalase.

Further, the Erwinia rhapontici C2 is cultured at 35-38 ℃ and 200-250 rpm for 30-50 h to produce trehalase by fermentation.

Further, the composition of the fermentation medium is (g/L): 10-20 parts of trehalose; KCl 0.5-1.5; KH (Perkin Elmer)₂PO₄0.5～1.5；MgSO₄·7H₂O 0.2～0.8；CaCO₃0.5 to 1.5; the pH value is 6.0-8.0.

The third purpose of the invention is to provide a microbial agent containing the Erwinia rhapontici C2.

Further, the microbial agent is a solid microbial agent.

Further, the microbial agent is a liquid microbial agent.

The invention has the beneficial effects that:

the invention reports Erwinia rhapontici (Erwinia rhapontici) for producing trehalase for the first time, and researches the enzymatic properties of Erwinia rhapontici. The optimal temperature and the pH of the trehalase produced by the strain are 40 ℃ and 5 respectively, the trehalase has high activity and stability under an acidic condition, the trehalase can hydrolyze unused trehalose in an alcohol plant into glucose, the energy consumption can be reduced, the ethanol yield can be increased, and the trehalase has wide application prospects in the ethanol industry.

Drawings

FIG. 1 is the strain morphology of Erwinia rhapontici (Erwinia rhapontici) in plates;

FIG. 2 shows the strain morphology of Erwinia rhapontici (Erwinia rhapontici) under an optical microscope;

FIG. 3 shows the strain morphology of Erwinia rhapontici (Erwinia rhapontici) under electron microscopy;

FIG. 4 is the result of phylogenetic analysis based on the 16S rDNA sequence of Erwinia rhapontici (Erwinia rhapontici);

FIG. 5 shows the optimum temperature and temperature stability of trehalase from Erwinia rhapontici (Erwinia rhapontici);

FIG. 6 shows the pH optima and pH stability of trehalase from Erwinia rhapontici (Erwinia rhapontici).

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 method for detecting the enzyme activity comprises the following steps:

the content of glucose generated by decomposing trehalose by trehalase is measured by a 3, 5-dinitrosalicylic acid (DNS) colorimetric method: a2 mL EP tube was used, and 150. mu.L of diluted enzyme solution and 150. mu.L of 10% soluble trehalose substrate were added to the experimental group. Adding 150 μ L of diluted enzyme solution and 150 μ L of pH 6.5 phosphate buffer solution 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 pH 6.5 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: enrichment screening process of Erwinia rhapontici (Erwinia rhapontici)

Collecting soil samples domesticated by trehalose in a Wuxi Yangxi wetland park and Jiangnan university, shoveling surface soil by using a small shovel when collecting the soil samples, collecting soil from 5-10 cm positions by using a sterilized plastic bag, sampling about 50g, sealing, and recording time, place and environment 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. The number of single colonies is 100-200The left and right plates serve as target plates. During rescreening, the colonies were inoculated into a liquid medium, shake-cultured at 37 ℃ and 220rpm for 36 hours, and then enzyme activity was measured. The obtained strain with higher trehalase activity is C2, and the strain is preserved in China general microbiological culture Collection center (preservation number CGMCC No. 16760).

The composition of the enrichment medium is (g/L): peptone 5; 1.5 of yeast extract; glucose 150; MgSO (MgSO)₄·7H₂O0.5；K₂HPO₄0.5。

The composition of the primary screening culture medium is (g/L): trehalose 15; KCl 1; KH (Perkin Elmer)₂PO₄1；MgSO₄·7H₂O0.5；CaCO₃1; adding agar 20 to the culture medium; the pH was 7.0.

The composition of the liquid culture medium is (g/L): trehalose 15; KCl 1; KH (Perkin Elmer)₂PO₄1；MgSO₄·7H₂O0.5；CaCO₃1；pH 7.0。

Example 2: morphological and physiological characterization of Erwinia rhapontici (Erwinia rhapontici)

The bacterial strain is subjected to streak inoculation on a solid LB culture medium plate, grown bacterial colonies are round, raised and grey white, the center and the periphery of the bacterial colonies are free of other colors and are easy to pick, gram staining is red, cells are mostly in a straight rod shape and are 0.5-1.0 mu m multiplied by 1.0-3.0 mu m long, and the bacterial colonies are singly generated or paired and sometimes in a short chain shape, the bacterial strain is gram negative, oxidase negative and catalase positive, and can generate acid from fructose, galactose, D-glucose, β -methyl glucoside and sucrose, malonate, fumarate, gluconate and malate are used as the only carbon source and energy source, but benzoate, oxalate or propionate cannot be used, and the morphology of the bacterial strain is identified under the plate, the optical microscope and the electron microscope (figures 1-3).

Example 3: molecular biological identification of Erwinia rhapontici (Erwinia rhapontici)

The thalli growing in the liquid fermentation medium was collected, 16S rDNA universal primers 27F (AGAGTTTGATCCTGGCTCAG), 1492R (GGTTACCTTGTTACGACTT) were selected to perform PCR amplification on the bacterial genome, and a 50. mu.L PCR reaction system was prepared in a 200. mu.L PCR tube according to the formulation in 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 performed with 1 XTBE buffer, 10 × Loading buffer, using 5000DL DNA marker as standard marker, and 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 instructions of the kit for purification of PCR products from recovery of small amount of gel from Shanghai Biotech, and the concentration of the extracted genome was determined using Thermo NanoDrop 2000, and sequencing was carried out by Shanghai Ruidi Biotech.

The length of the 16S rDNA sequence of the strain is 1409bp, and the strain is subjected to nucleotide sequence Blast comparison in an NCBI database to determine the species. The final result shows that the 16S rDNA sequence of the strain has more than 99 percent of homology with the related sequence of Erwinia (HM748066.1, MG198678.1 and the like), and has about 98 percent of homology with the related strain of Pantoea (KM019845.1), and finally the strain is classified as the Erwinia strain. The strain is identified as Erwinia rhapontici (Erwinia rhapontici C2) with the number of C2 by combining morphological characteristics and physiological and biochemical characteristics, and the strain is preserved in China general microbiological culture Collection center (the preservation number is CGMCC No.16760) (figure 4).

Example 4: research on enzymatic properties of trehalase of Erwinia rhapontici (Erwinia rhapontici)

(1) The enzyme activity detection method comprises the following steps:

a2 mL EP tube was used, and 150. mu.L of the diluted enzyme solution and 150. mu.L of 0.1g/mL of the soluble trehalose substrate were added to the experimental group. Adding 150 μ L of diluted enzyme solution and 150 μ L of PBS 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.

Enzyme activity (Unit) definition:

under the above experimental conditions, the amount of enzyme required to produce 1. mu. moL of glucose per minute was defined as one unit of enzyme activity.

The enzyme activity of the whole-cell protein of the trehalase is measured and can reach 17.99U/mL.

(2) The optimum reaction temperature and temperature stability study method is as follows:

optimum reaction temperature: the enzyme solution was diluted appropriately with 50mmol/L phosphate buffer (pH 6.5), and the activity of trehalase was measured at 30, 35, 40, 45, 50, 55 ℃ and the like, respectively, with the reaction temperature of the highest activity being the optimum 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 40 ℃, and the relative enzyme activity can reach more than 90% within the range of 35-40 ℃; 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 5).

(3) The optimum reaction pH and pH stability study method is as follows:

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. And detecting the enzyme activity under different buffer solution systems according to a standard enzyme activity detection method, and taking the reaction pH with the highest enzyme activity 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 trehalose enzyme has the optimum pH value of 5, has higher activity under the acidic condition, and particularly has the relative enzyme activity of more than 90 percent within the pH range of 4.5-5.5; the trehalase has good stability in an acidic environment, can keep more than 80% of residual enzyme activity within the pH range of 4-6, is suitable for ethanol industry, and can improve the utilization rate of residual sugar and the conversion rate of total sugar (figure 6).

(4) Effect of Metal ions and chemical Agents 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 adding the metal ion mother liquor into a trehalase enzyme activity detection reaction system at a final concentration of 1mmol/L, and performing standard enzyme activity detection reaction. Reactions without any added metal ions were used as controls.

Influence of chemical reagents: tween, triton, Sodium Dodecyl Sulfate (SDS) and dimethyl sulfoxide (DMSO) are firstly prepared into 10% solution, and then added into an enzyme activity detection reaction system at a final concentration of 1% 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, adding the mother liquor into a reaction system according to the final concentration of 1mmol/L, and carrying out standard enzyme activity detection reaction, wherein the reactions are all controlled by reactions without any chemical reagent.

The study found that Ca compares to a control without any metal ions added²⁺、Zn²⁺、Ni²⁺、Mn²⁺、Cu²⁺、K⁺、Na⁺And Fe²⁺Has promoting effect on enzyme activity, and Sn²⁺And Ba²⁺Has inhibitory effect on enzyme activity, Mg²⁺、Co²⁺And Li⁺Had little effect on enzyme activity (Table 3).

DMSO and DTT promoted the enzyme activity, while TritonX-114, SDS and PMSF inhibited the enzyme activity, and Tween 20, Tween 80, Triton X-100 and EDTA had almost no effect on the enzyme activity, as compared with the control without any chemical reagent (Table 4).

TABLE 3 Effect of Metal ions on trehalase Activity

TABLE 4 Effect of chemical reagents on trehalase Activity

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. The protection scope of the invention is subject to the claims.

Claims

1. An Erwinia rhapontici (Erwinia rhapontici) C2 strain for producing trehalase is characterized in that the Erwinia rhapontici (Erwinia rhapontici) C2 strain has been preserved in the China general microbiological culture collection management center at 11.21.2018.A preservation address is No. 3 of the West Luo No.1 of the Chao-Yang district, Beijing, and a preservation number is CGMCC No. 16760.

2. The erwinia rhapontici C2 as claimed in claim 1 for use in food, agriculture, pesticides or insect treatment.

3. The use according to claim 2, wherein the Erwinia rhapontici C2 is used for hydrolyzing trehalose.

4. The use of claim 2, wherein trehalase is produced by fermentation using Erwinia rhapontici C2.

5. The use of claim 2, wherein the Erwinia rhapontici C2 is cultured at 35-38 deg.C and 200-250 rpm for 30-50 h to produce trehalase by fermentation.

6. The use according to claim 5, wherein the fermentation medium consists of (g/L): 10-20 parts of trehalose; KCl 0.5-1.5; KH (Perkin Elmer)₂PO₄0.5～1.5；MgSO₄·7H₂O 0.2～0.8；CaCO₃0.5 to 1.5; the pH value is 6.0-8.0.

7. A microbial inoculant comprising Erwinia rhapontici C2 according to claim 1.

8. The microbial inoculant according to claim 7, wherein the microbial inoculant is a solid inoculant.

9. The microbial inoculant according to claim 7, wherein the microbial inoculant is a liquid inoculant.