CN115232760B

CN115232760B - Bacillus alpine and application thereof

Info

Publication number: CN115232760B
Application number: CN202210597863.6A
Authority: CN
Inventors: 王璋倩; 杨锐; 高超; 丛欣; 何毅; 程水源; 董星星; 王沫
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2023-10-20
Anticipated expiration: 2042-05-24
Also published as: CN115232760A

Abstract

The invention discloses bacillus alpine and application thereof, wherein the bacillus alpine is bacillus alpine (Bacillus altitudinis) LH18, and the preservation number is CCTCC NO: m20211529, storage time is 2021, 12, 03. The strain can produce amylase, protease and carboxymethyl cellulose, and can reduce toxic sodium selenite to obtain nano selenium with small particle size, good biological activity and low toxicity; furthermore, in the environment with a certain concentration of selenium, the bacillus alpine LH18 not only converts selenite compounds into nano-selenium with higher safety, but also improves the enzyme activity of amylase produced by the strain, enhances the biological activity of the strain, expands the application range of the strain, can convert selenium when being used for preparing environment restoration preparations, fertilizer additives and the like, plays a role in treating the environment polluted by high selenium, can degrade macromolecular substances in soil, and plays a role in improving soil conditions.

Description

Bacillus alpine and application thereof

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to bacillus alpine and application thereof.

Background

Selenium is an essential trace element for organisms. In recent years, some researches find that nano-form elemental selenium (SeNPs) has strong in vivo activity, high safety and good absorption. The nanometer selenium has antioxidant, immunity regulating, liver protecting, growth promoting, and selenium supplementing effects. As seen from biological efficacy, nano-selenium exhibits higher free radical scavenging ability and anticancer activity.

Currently, the synthesis methods of nano selenium are mainly 3, namely a physical method, a chemical method and a biological method. How to minimize the influence and damage to the ecological environment in the production process is also one of the hot spots of research in various industries at present. By utilizing the microbial synthesis method, not only can the Senps with high safety and stable chemical property be prepared, but also the nano selenium can adsorb heavy metal ions in the environment to form a metal oxide-nano selenium compound, so as to achieve the purpose of removing heavy metal pollution. SeNPs synthesized as reported by Jiang et al Shewanella putrefaciens are capable of combining with HgO in the environment to form HgSe-NPs complex, so as to achieve the biological toxic effect of removing mercury (Hg); losi et al, screened Enterococcus cloacae SLD a-1 has been used for biodegradation of selenate and selenate, a selenoxyanion in agricultural wastewater.

Disclosure of Invention

The invention mainly aims to provide bacillus alpine and application thereof, and aims to provide bacillus alpine, which not only can synthesize nano selenium, but also can produce amylase, protease and carboxymethyl cellulose.

In order to achieve the above object, the present invention utilizes soil collected from the selenium rich region of Enshi of Hubei province, from which a strain of Bacillus alpinus is isolated and purified. The bacillus alpinus obtained by separation and purification of the invention is preserved in China center for type culture collection, and addresses: the university of Wuhan, china, classified and named as bacillus alpine (Bacillus altitudinis) LH18, and the preservation number is CCTCC NO: m20211529, storage time is 2021, 12, 03.

The invention further provides an application of the bacillus alpina in preparing amylase.

Optionally, the preparation of the amylase comprises the steps of:

obtaining seed liquid of bacillus alpinus;

inoculating the seed liquid into a starch liquid culture medium, and fermenting to obtain amylase;

wherein the starch liquid culture medium comprises 20g/L of soluble starch, 3g/L of yeast and 3g/L, mgSO of peptone ₄ ·7H ₂ O 0.2g/L、K ₂ HPO ₄ 1g/L、NaCl 1g/L，pH＝7。

Optionally, the seed solution is inoculated into a starch liquid culture medium for fermentation, and the step of obtaining amylase comprises the following steps:

inoculating the seed liquid into a starch liquid culture medium, adding sodium selenite into the starch liquid culture medium, and fermenting for 24-72 h to obtain amylase;

wherein, in the starch liquid culture medium, the concentration of the sodium selenite is 2.5-10 mmol/L.

Optionally, the concentration of the sodium selenite is 5mmol/L, and the fermentation time is 48h.

In addition, the invention also provides an application of the bacillus alpinus in preparation of protease.

In addition, the invention also provides an application of the bacillus alpinus in preparing carboxymethyl cellulose.

In addition, the invention also provides an application of the bacillus alpinus in preparing fertilizer additives or fertilizers.

In addition, the invention also provides nano-selenium, which is obtained by reducing inorganic selenium by the bacillus alpina.

In addition, the invention also provides an application of the bacillus alpinus in preparing an environment restoration preparation.

According to the technical scheme, the bacillus alpine LH18 is provided, and the strain can produce amylase, protease and carboxymethyl cellulose, and can reduce toxic sodium selenite to obtain nano-selenium with small particle size, good biological activity and low toxicity; furthermore, in the environment with a certain concentration of selenium, the bacillus alpine LH18 not only converts selenite compounds into nano-selenium with higher safety, but also improves the enzyme activity of amylase produced by the strain, enhances the biological activity of the strain, expands the application range of the strain, can convert selenium when being used for preparing environment restoration preparations, fertilizer additives and the like, plays a role in treating the environment polluted by high selenium, can degrade macromolecular substances in soil, and plays a role in improving soil conditions.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a colony morphology of Bacillus alpinus in example 2;

FIG. 2 is a graph of the evolved tree of Bacillus alpina of example 2;

FIG. 3 is a scanning electron microscope analysis chart of the synthesized nano-selenium in example 3 with the proportional size of 50-170 nm;

FIG. 4 is a graph showing the growth kinetics of Bacillus alpinus in various sodium selenite concentrations in example 4;

FIG. 5 is a graph showing the relationship between the reduction rate of inorganic selenium by Bacillus alpinus and the cultivation time in example 4;

FIG. 6 is a graph showing amylase activity assay of Bacillus alpinus of example 4 in different culture times;

FIG. 7 is a graph showing the protease activity of Bacillus alpinus in example 4;

FIG. 8 is a graph showing the activity of carboxymethylcellulase produced by Bacillus alpinus in example 4;

FIG. 9 is a graph showing amylase activity of Bacillus alpinus at various sodium selenite concentrations in example 5.

The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.

The specific conditions were not specified in the examples, and the examples were conducted under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The nano selenium has the activities of resisting cancer, resisting oxidation, reducing blood sugar, inhibiting bacteria and the like, and can be applied to fertilizer, medicine and the like. Currently, the synthesis methods of nano selenium are mainly 3, namely a physical method, a chemical method and a biological method. The physical method adopts high temperature, high pressure or laser catalysis and other conversions, has high cost and large environmental ecological hazard, and is less adopted. The chemical method synthesizes by chemical reaction, which has unsafe factors such as dangerous chemical substances, waste liquid and the like, and the synthesized nano selenium is pure in irregular form, easy to gather, poor in stability and the like. In view of the above, the invention utilizes the soil collected from the Enshi selenium-rich region of Hubei province to separate and purify the soil to obtain the bacillus alpinum LH18, and the strain can reduce the toxic sodium selenite to obtain the nano-selenium with small particle size, good biological activity and low toxicity.

Bacillus is a broad-spectrum antagonistic bacterium, and various Bacillus subgroups such as Bacillus pumilus, bacillus subtilis, bacillus thuringiensis and the like are important in biological control of plant diseases. At present, the research on the bacillus alpine is less at home and abroad, and the functions of the bacillus alpine discovered at present mainly comprise antagonism, phosphate and potassium dissolving functions and the like. The bacillus alpine LH18 provided by the invention can produce amylase, protease and carboxymethyl cellulose, and the enzyme activity of amylase produced by the bacillus alpine LH18 can be further improved in a selenium-containing environment with a certain concentration.

Specifically, the bacillus alpinus obtained by separation and purification of the invention is preserved in China center for type culture collection, and addresses: the university of Wuhan, china, classified and named as bacillus alpine (Bacillus altitudinis) LH18, and the preservation number is CCTCC NO: m20211529, storage time is 2021, 12, 03.

The separation method of the bacillus alpine LH18 comprises the following steps:

weighing a proper amount of soil sample, adding 100mL of sterile water, and putting into a shaking table, and shaking at 180rpm for 30min; then sampling and carrying out plate culture, picking single colony, subculturing and purifying to obtain purified single colony.

And (3) coating the purified single colony on an LB solid culture medium flat plate containing 300mmol/L sodium selenite, culturing, and picking a single colony with better growth vigor, namely the bacillus alpine LH18.

The identification of the 16S rRNA sequence of the bacillus alpine strain LH18 shows that the bacillus alpine strain LH18 and Bacillus altitudinis KF2b are aggregated into a strain with the homology as high as 93%, which indicates that the strain obtained by the separation method is a bacillus alpine strain.

Furthermore, the bacillus alpine LH18 provided by the invention has diversified enzyme producing capability and can produce amylase, protease and carboxymethyl cellulose.

The invention further provides an application of the bacillus alpina in preparing amylase. The amylase can be prepared by fermenting and culturing bacillus alpine LH18, and specifically, the method for preparing the amylase comprises the following steps of:

step S10, obtaining the seed liquid of the bacillus alpina as described above.

Step S20, inoculating the seed solution into a starch liquid culture medium, and fermenting to obtain amylase, wherein the starch liquid culture medium comprises 20g/L of soluble starch, 3g/L of yeast and 3g/L, mgSO of peptone ₄ ·7H ₂ O 0.2g/L、K ₂ HPO ₄ 1g/L、NaCl 1g/L，pH＝7。

When the bacillus alpine LH18 is in a selenium-containing environment, the protease and carboxymethyl cellulose production capacity of the strain is not obviously affected compared with that of the strain cultured in a selenium-free environment, but the enzyme activity of the obtained amylase is obviously improved, and based on the characteristics, an embodiment of a method for improving the amylase production activity of the bacillus alpine is further provided. Specifically, in this embodiment, step S20 may be performed as follows:

step S21, inoculating the seed liquid into a starch liquid culture medium, adding sodium selenite into the starch liquid culture medium, and fermenting for 24-72 hours to obtain amylase; wherein, in the starch liquid culture medium, the concentration of the sodium selenite is 2.5-10 mmol/L.

When the bacillus alpine LH18 is fermented for 24-72 hours in the environment with the concentration of sodium selenite of 2.5-10 mmol/L, the activity of the obtained amylase is obviously improved. Preferably, when the concentration of the sodium selenite is 5mmol/L, the fermentation time is 48 hours, which is helpful for further improving the amylase activity.

Based on the above situation, when the strain is used for treating selenium-polluted soil, the existence of selenium can improve the amylase activity produced by the strain, promote the decomposition of macromolecular substances and improve the soil performance, and meanwhile, the strain can convert selenium into nano-selenium.

The invention further provides an application of the bacillus alpine LH18 in preparation of protease. The protease can be prepared by fermenting and culturing bacillus alpine LH18, and specifically, the method for preparing the protease comprises the following steps of: obtaining seed liquid of bacillus alpine LH18, inoculating the seed liquid into a protease liquid culture medium, and fermenting to obtain protease, wherein the protease liquid culture medium comprises 20g/L of glucose, 12g/L of peptone, 3g/L of beef extract, 5g/L of NaCl and MgSO ₄ 0.5g/L。

The invention further provides an application of the bacillus alpine LH18 in preparing carboxymethyl cellulose. The carboxymethyl cellulose can be prepared by fermenting and culturing bacillus alpine LH18, and specifically, the method for preparing the carboxymethyl cellulose comprises the following steps of: obtaining seed liquid of bacillus alpine LH18, and then inoculating the seed liquid into a carboxymethyl cellulose liquid culture medium for fermentation to obtain carboxymethyl cellulose, wherein the carboxymethyl cellulose liquid culture medium comprises 10g/L of peptone, 10g/L of yeast extract powder, 10g/L of sodium carboxymethyl cellulose, 5g/L of sodium chloride and 1g/L of potassium dihydrogen phosphate.

The nano selenium has the particle size of 50-170 nm, is easy to be absorbed by organisms and plays a role; the nano selenium has low cytotoxicity, and is more suitable for preparing edible products such as food, medicine, fertilizer and the like.

The invention further provides a preparation method of the nano-selenium, which comprises the following steps: inoculating the bacillus alpine LH18 into an LB liquid culture medium containing inorganic selenium salt for culture, and then separating and purifying to obtain the nano selenium.

Wherein the inorganic selenium salt comprises sodium selenite. Further, the concentration of sodium selenite may be 5 mmol/L.

The bacillus alpine LH18 has the function of reducing selenium, so that the bacillus alpine LH18 can be used for treating selenium pollution environment; furthermore, the bacillus alpine LH18 can adapt to the high-selenium environment with the concentration not higher than 250mmol/L and can be suitable for the treatment of the environment polluted by high selenium. In addition, bacillus alpine LH18 exhibits diverse enzyme-producing capabilities, and is capable of producing amylase, protease, and carboxymethyl cellulase. And when the strain is in a selenium-containing environment, compared with the selenium-free environment, the capability of the strain for producing protease and carboxymethyl cellulose is not obviously affected, but the enzyme activity of the obtained amylase is obviously improved, and based on the characteristic, when the strain is used for treating selenium-polluted soil, the existence of selenium can improve the enzyme activity of the amylase produced by the strain, promote the decomposition of macromolecular substances and improve the soil performance.

In specific application, the bacillus alpine LH18 can be prepared into a single microbial inoculum or compounded with other strains to prepare a compound microbial inoculum which is directly used for environmental pollution or mixed with other environmental restoration components to prepare an environmental restoration agent for use.

The bacillus alpine LH18 can reduce toxic sodium selenite to obtain low-toxicity nano selenium. The nano selenium has small particle size, good bioactivity and easy absorption by organism. Meanwhile, the strain can produce amylase, protease and carboxymethyl cellulose, the enzyme activity is not affected by selenium, and the enzyme activity of part of enzymes is improved in a selenium environment. The fertilizer additive or the fertilizer is used for preparing fertilizer additives or fertilizers, which is helpful for improving soil performance, facilitating nutrient absorption of the fertilizer additives or the fertilizer additives by plants and simultaneously helping to cultivate selenium-rich plants.

Taking the functional additive as an example, the fermentation product of the bacillus alpina LH18 or the functional component (such as amylase, protease, nano selenium and the like) extracted from the fermentation product can be used as a main functional component or can be used as one of the functional components to be compounded with other functional components to prepare the additive in specific application.

The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.

EXAMPLE 1 isolation of strains

Soil samples were taken from the soil in the Enshi selenium rich region of Hubei province.

Taking 5g of a soil sample, adding 100mL of sterile water, putting into a shaking table, and shaking for 30min at 180rmp to obtain a mixed solution; the mixture (30. Mu.L) was spread on a plate, and placed in an incubator at 37℃for 24 hours. And selecting colonies, and carrying out plate streaking separation and purification to obtain single colonies. LB solid medium containing 300mmol/L selenite was prepared, and single colony was selected from the above plate and inoculated into the medium for cultivation. The growth condition and the color of the strains are observed, and the strain with better growth vigor and redder color is selected, and identified as bacillus alpine LH18.

Example 2 identification of strains

The strains used in the following experiments were all isolated from example 1.

(1) The strain was cultured on LB plates at 37℃to form colonies, and the characteristics of colony morphology, color, etc. were observed. As a result, as shown in FIG. 1, the colony was milky white, the surface thereof was moist, slightly wrinkled, and the gram stain was purple, and was a gram-positive bacterium.

(2) The physiological and biochemical identification results show that the strain can decompose and utilize glucose, sucrose, mannose, arabinose and xylose for different carbon sources, and the decomposing and utilizing capacity of lactose is negative. The V-P test was negative, and the oxidase test and amylase test were positive. The control "Berger handbook" is essentially identical to Bacillus.

(3) Genome sequencing of strains

Genomic DNA of the strain was extracted and 16S rRNA gene amplification was performed with the general primers 27-F:5 'GAGAGTTTGATCCTGGCTCAG-3' and 1392-R:5 '-ACGGGCGGTGTGTRC-3'. The amplified product is sent to Shanghai biological limited company for sequencing; the resulting sequences were submitted to the ezbiocoud database for alignment and BLAST alignment. The evolutionary tree was constructed using the neighbor-Joining method (MAGA 7.0).

The results are shown in FIG. 2: the bacteria clustered with Bacillus altitudinis 41KF2 b.

Therefore, this bacterium was designated Bacillus altitudinis LH in combination with the genotype and physiological and biochemical identification results.

Example 3 fermentation of Strain and production of nanoselenium

The bacillus alpus LH18 isolated in example 1 was taken. Inoculating the strain into LB liquid culture medium containing sodium selenite at 1% inoculum size, and shake culturing at 180rpm and 37deg.C until the growth log phase to obtain fermentation mixed liquid. Centrifuging the fermentation mixed solution at 12000rpm for 15min, collecting the thallus and nano-selenium mixture at the lower layer, and washing with sterile water for 3 times to obtain the mixture. The mixture was placed in a sterile mortar, and immediately ground with the addition of liquid nitrogen to give a ground slurry. Suspending the grinding slurry with sterile water, performing ultrasound for 6min (the parameter is amplitude transformer 6 and the power is 35%) under ice compress condition, and then adding sterile water to sufficiently suspend the grinding slurry, and ensuring that the final volume is about 1/2 of the original culture solution volume, and the ultrasound time is not more than 10min to obtain selenium suspension. The nano-selenium suspension is sequentially filtered through a filter membrane of 20 mu m, 10 mu m, 5 mu m, 3 mu m, 1.2 mu m and 0.8 mu m, and the filtrate is collected. And adding n-hexane which is 5 times the volume of the filtrate into the filtrate, uniformly mixing, standing, collecting lower liquid, and repeating the operation of washing with n-hexane for 6-7 times until the upper organic phase is transparent and colorless and has no thalli. Collecting water phase, centrifuging at 10000rpm for 10min, collecting precipitate, and cleaning with sterile water for 2-3 times to obtain nanometer selenium.

And (3) storing: the nano selenium is refrigerated and preserved at 0-4 ℃ after being diluted by deionized water.

SEM scanning electron microscope analysis of nano selenium: inoculating seed solution of Bacillus alpinus LH18 into 100mL LB liquid culture medium at 1% inoculum size, shaking culturing at constant temperature of 37 deg.C at 180rpm for growing logarithmic phase, adding 50mmol/L sodium selenite, culturing for 6 hr, centrifuging at 10000rpm for 30min, collecting precipitate, washing with physiological saline for 2 times, and washing with double distilled water for 3 times each for 5min. Centrifuging at 10000rmp for 30min, collecting precipitate, and adding electron microscope fixing solution for SEM scanning. The results of the measurements are shown in figure 4,

as can be seen from FIG. 3, the nano selenium is distributed outside the cells in a spherical shape, the size is uniform, and the particle size is 50-170 and nm.

Example 4 Performance test

(1) Growth kinetics of strains at different sodium selenite concentrations: inoculating bacterial solution of Bacillus alpinus LH18 into a Erlenmeyer flask (OD) containing 100mL LB culture solution ₆₀₀ About 0.02), and sodium selenite at a concentration of 0, 5, 25, 50mmoL/L, respectively. The growth curve of the strain in selenium-containing medium was determined. And culture was continued at 37 ℃. Throughout the culture, cell concentration (OD) was measured every two hours ₆₀₀ ) And a growth curve is drawn. As a result, as shown in FIG. 4, the strain grew well in selenium-containing environments, especially at a concentration of 5mmoL/L, with optimal growth.

(2) And (3) measuring the reduction rate of the nano selenium of the strain: 1-10 mu moL selenite solution is taken in 25 mu moL LHN ₂ After the OH HCl is sufficiently used, the mixture is blown dry by a nitrogen blowing instrument, and 1M Na is added ₂ S, finally, measuring the absorbance value at 500 and nm, and drawing a standard curve by taking the absorbance value as a Y axis and the selenium concentration as an X axis.

Taking the seed liquid of the bacillus alpine LH18 obtained by separating in the activated example 1, respectively inoculating the seed liquid into LB liquid culture medium containing 5mmol/L sodium selenite according to an inoculum size of 1%, taking 6mL of culture liquid every 24 hours, centrifuging for 20min at 12000rmp, and taking a precipitate. Washing the sediment with 1mol/L NaClPrecipitating for 2 times, centrifuging under the same condition, adding 1mol/L Na to the precipitate ₂ S, standing for 1h after uniformly mixing, centrifuging for 20min at 12000rmp, collecting a supernatant, and measuring an absorbance value at 500 nm.

And finally, calculating the concentration of the nano selenium corresponding to the light absorption value according to a quasi-curve formula. Reduction rate (%) = (nano selenium concentration in product/initial selenium treatment concentration) ×100%

Analysis of results: referring to FIG. 5, in a medium with a sodium selenite concentration of 5mmol/L, the reduction rate of Bacillus alpinus LH18 can reach 94.5% at 48 hours.

(3) Determination of Amylase Activity-producing ability

1) Preparation of crude enzyme solution

Starch liquid medium: soluble starch 20g, yeast 3g, peptone 3g and MgSO ₄ ·7H ₂ O 0.2g、K ₂ HPO ₄ 1g of NaCl and 1g of pH 7.

Adding the strain seed liquid into a liquid culture medium according to the inoculation amount of 2%, shake-culturing 50mL (250 mL) conical flasks in shake flasks at 37 ℃ and 180rpm, taking fermentation liquor (0-72 h) in different time periods, centrifuging 8000g, centrifuging for 20min, and taking supernatant as crude enzyme liquid.

2) Determination of alpha-amylase Activity

(1) 4 test tubes were taken and two were noted as measurement tubes.

(2) 1mL of crude enzyme solution was added to each tube, and the mixture was heated in a constant temperature water bath at 70℃for 15min (the water temperature should not vary by.+ -. 0.5 ℃) during which time the beta-amylase was heated to deactivate, and immediately cooled in tap water after removal.

(3) 1mL of pH 5.6 citrate buffer was added to each tube.

(4) To the control tube, 4mL of 0.4N sodium hydroxide was added to inactivate the enzyme activity.

(5) The measurement tubes and the control tube are placed in a constant temperature water bath with the temperature of 40 ℃ (+/-0.5 ℃) for heat preservation for 15min, 2mL of starch solution preheated at the temperature of 40 ℃ is added into each tube respectively, the mixture is shaken uniformly, the mixture is immediately placed in the water bath with the temperature of 40 ℃ for accurate heat preservation for 5min and then taken out, 4mL of 0.4N sodium hydroxide is rapidly added into each measurement tube to terminate the activity of enzyme, and then the next sugar measurement is prepared.

The measurement result of the amylase activity of the bacillus alpine LH18 is shown in figure 6, CK represents no sodium selenite addition, and the amylase activity is 290U/mL.

(4) Protease production enzyme Activity assay

1) Preparation of crude enzyme solution

Liquid fermentation medium: glucose 20g, peptone 12g, beef extract 3g,NaCl 5g,0.5 g MgSO ₄ Water was added to 1L. Adding the strain seed liquid into a liquid fermentation culture medium, shake-culturing 50mL (250 mL) conical flask in a shake flask at 33 ℃ and 180rpm, taking fermentation liquor (0-72 h) in different time periods, centrifuging 13000g for 10min, and taking supernatant as crude enzyme liquid.

2) Preparation of tyrosine standard curve

6 test tubes (numbered 0, 1-5) are taken, 0.00,0.20,0.40,0.60,0.80 and 1.00mL of standard tyrosine solution (50 mug/mL) are respectively added in sequence, water is added to be 1.00mL, 0.55mol/L sodium carbonate is respectively added after shaking, and shaking is carried out. Sequentially adding 1.00mL of Folin-phenol reagent, shaking uniformly, timing, and preserving the temperature in a water bath kettle at 30 ℃ for 15min. The absorbance was then measured at 680nm (as Guan Zuodui light at 0). The tyrosine content (μg) is plotted on the abscissa and the absorbance value is plotted on the ordinate.

3) Enzyme Activity measurement

Enzyme reaction: taking a test tube, adding 2.0mL of 0.5% casein solution, preheating for 5min in a water bath at 30 ℃, adding 1.0mL of preheated Bacillus pumilus protease solution, immediately timing, accurately preserving heat for 10min in the water bath, immediately adding 2.0mL of 10% trichloroacetic acid solution after taking out from the water bath, shaking uniformly, standing for a plurality of minutes, filtering with dry filter paper, and collecting filtrate (sample liquid). Another test tube was taken, 1.0mL of the preheated crude enzyme solution and 2.0mL of 10% trichloroacetic acid solution were added, the mixture was shaken well, the mixture was left for several minutes, then 2.0mL of 0.5% casein solution was added, the mixture was incubated in a water bath at 30℃for 10 minutes, the mixture was filtered with the same dry filter paper, and the filtrate (control solution) was collected.

4) Taking 3 test tubes, respectively adding 1.0mL of water, crude enzyme solution and standard tyrosine solution, then respectively adding 5.0mL of 0.55mol/L sodium carbonate solution and 1mL of Folin-phenol reagent, shaking uniformly, preserving heat according to a standard curve manufacturing method, and measuring absorbance values. And (3) according to the absorbance value, detecting the tyrosine content difference value in the crude enzyme solution and the standard tyrosine solution by using a standard curve, and then calculating the activity unit of the enzyme.

The measurement result of the protease activity of the bacillus alpine LH18 is shown in figure 7, wherein CK represents no sodium selenite is added, and the protease activity is 74U/mL.

(5) Determination of carboxymethyl cellulose production Activity

1) Preparation of crude enzyme solution

Liquid fermentation medium: 10g of peptone, 10g of yeast extract powder, 10g of sodium carboxymethylcellulose, 5g of sodium chloride and 1g of potassium dihydrogen phosphate, and adding water to 1L. Adding the strain seed liquid into a liquid fermentation culture medium, shake-culturing 50mL (250 mL) conical flask in a shake flask at 33 ℃ and 180rpm, taking fermentation liquor (0-72 h) in different time periods, centrifuging 13000g for 10min, and taking supernatant as crude enzyme liquid.

2) And (5) drawing a glucose standard curve. A100 mL volumetric flask is used for constant volume preparation of 1mg/mL standard glucose mother liquor for standby. The standard glucose mother solutions were diluted to glucose solutions having concentrations of 0, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL, respectively, and 2mL were taken. Then adding 1mL of DNS reagent, boiling in boiling water for 8min, cooling, adding 21.5mL of distilled water, after the solution is cooled to be consistent with room temperature, respectively measuring absorbance values with a spectrophotometer with wavelength of 540nm, recording absorbance values, and drawing a glucose standard curve.

3) And (5) measuring the activity of the strain for producing carboxymethyl cellulose (CMC). 6.0mL of 1.0% CMC-Na solution is added into a 10.0mL test tube, 2.0mL of crude enzyme solution (crude enzyme solution: 50mL of fermentation culture medium with 10% inoculum size, 4 ℃ C., 12000 r.m., 15 Min) with different culture times is added, the supernatant is collected, enzymolysis is carried out for 30Min in a water bath pot with 50 ℃ C., 2.0mL of DNS reagent is added, boiling water is carried out for 8Min, the test tube is taken out, and the test tube is put into crushed ice prepared in advance and cooled to room temperature. Distilled water was then added to 10.0mL, the absorbance was measured with a spectrophotometer at a wavelength of 540nm, and the enzyme activity was calculated from the glucose standard curve drawn.

The measurement result of the activity of the carboxymethyl cellulose produced by the bacillus alpine LH18 is shown in figure 8, CK represents that no sodium selenite is added, and the enzyme activity of the carboxymethyl cellulose is 161U/mL.

EXAMPLE 5 increasing amylase enzyme Activity with selenite addition

Starch liquid medium: soluble starch 20g, yeast 3g, peptone 3g and MgSO ₄ ·7H ₂ O 0.2g、K2HPO ₄ 1g of NaCl and 1g of pH 7.

(1) Adding the strain seed liquid into a liquid culture medium according to the inoculation amount of 2%, adding 5mmol/L selenite and culture liquid, shake-culturing 50mL (250 mL) conical flask in a shake flask at 37 ℃ and 180rpm, taking fermentation liquid (0-72 h) in different time periods (24 h, 48h and 72 h), centrifuging 8000g, centrifuging for 20min, and taking supernatant as crude enzyme liquid. The measurement method was as in example 4. The results are shown in FIG. 6, na ₂ SeO ₃ Representing the addition of 5mmol/L sodium selenite. After sodium selenite is added, the activity of alpha-amylase is obviously improved. The optimal culture time is 48h, and the amylase activity is 642U/mL.

(2) Adding the strain seed solution into a liquid culture medium according to the inoculation amount of 2%, adding sodium selenite with different concentrations (the concentration of the sodium selenite in the culture solution after the addition is 2.5mmol/L, 5mmol/L and 10mmol/L respectively) into the culture solution, shake-culturing a 50mL (250 mL) conical flask in a shake flask at 37 ℃ and 180rpm, taking the fermentation broth (0-72 h) for 48h, centrifuging 8000g, centrifuging for 20min, and taking the supernatant as a crude enzyme solution. The measurement method was as in example 4. As a result, as shown in FIG. 9, it was found that the activity of alpha-amylase produced by Bacillus alpicus LH18 was best after adding 5mmol/L sodium selenite to the culture solutions of different sodium selenite concentrations, respectively, and culturing for 48 hours. The addition of selenium increases the amylase activity of the strain compared to CK without selenium in fig. 6.

The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The bacillus alpine is characterized in that the bacillus alpine is bacillus alpine (Bacillus altitudinis) LH18, the preservation number is CCTCC M20211529, and the preservation time is 2021, 12 months and 10 days.

2. The bacillus alpine of claim 1, wherein the bacillus alpine is capable of reducing inorganic selenium to nano-selenium.

3. Use of the bacillus alpinus of claim 1 for preparing amylase.

4. The use of bacillus alpinus according to claim 3 for the preparation of amylase, wherein the preparation of amylase comprises the steps of:

obtaining seed liquid of bacillus alpinus;

5. The use of bacillus alpinus according to claim 4 for preparing amylase, wherein the step of inoculating the seed solution into a starch liquid culture medium for fermentation to obtain amylase comprises the steps of:

6. The use of bacillus alpinus according to claim 5 for preparing amylase, wherein the concentration of sodium selenite is 5mmol/L and the fermentation time is 48h.

7. Use of the bacillus alpinus of claim 1 for preparing protease.

8. Use of the bacillus alpinus according to claim 1 for preparing carboxymethyl cellulose.

9. Use of the bacillus alpinus of claim 1 in the preparation of a fertilizer additive or fertilizer.

10. Use of the bacillus alpinus of claim 1 in the preparation of an environmental remediation formulation.