CN112300951A - Recombinant pichia pastoris and application thereof in adsorption of heavy metal ions - Google Patents
Recombinant pichia pastoris and application thereof in adsorption of heavy metal ions Download PDFInfo
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Abstract
The invention discloses a recombinant pichia pastoris and application thereof in heavy metal ion adsorption. The invention obtains a recombinant vector pPIC9K-vgb by amplifying a vgb gene shown as SEQ ID NO.1, cutting the vgb gene by EcoRI and NotI, inserting the cut vgb gene into a pPIC9K vector, and then converting Pichia pastoris KM71 after linearization by SalI to obtain recombinant Pichia pastoris; the composite material has better heavy metal ion resistance, the adsorption rate of the composite material to heavy metal ions can reach 69.8%, and the adsorption quantity of the composite material to the heavy metal ions is remarkably improved to 39.87mg/g at most. The recombinant pichia pastoris has the characteristics of high resistance and high adsorptivity to heavy metal ions, and can be well applied to removing the heavy metal ions in water, so that the harm of the heavy metal ions to environmental water pollution is reduced.
Description
Technical Field
The invention belongs to the field of environmental microorganisms, and particularly relates to recombinant pichia pastoris and application thereof in adsorption of heavy metal ions.
Background
Because heavy metal pollution in the environment, particularly in a water body, is increasingly serious and seriously threatens aquatic organisms and human health, a plurality of reports about harm to human health caused by excessive heavy metal exist at present.
The yeasts have strong tolerance to heavy metals, pH values and temperatures and become the preferred material for adsorbing heavy metals, but most of researches select and use screened yeasts for experiments and focus on the first adsorption result of the yeasts as an adsorbent, most of the researches focus on the research of the saccharomyces cerevisiae for adsorbing heavy metal ions, and the researches on the pichia pastoris for adsorbing heavy metal ions are less. The pichia pastoris has high density, simple nutritional requirement and is convenient for large-scale culture, so the pichia pastoris is suitable for the treatment of heavy metal ions in sewage. And at present, few reports on the adsorption of heavy metal ions by recombinant yeasts containing specific genes and how to recycle the yeasts are reported.
The vgb gene codes Vitreoscilla (Vitrosporium angustifoides) hemoglobin, has the functions of improving respiratory chain transmission efficiency and improving the utilization rate of dissolved oxygen, and can promote the growth and metabolism of thalli under the condition of oxygen deficiency. Most of the dissolved oxygen in the sewage is limited, so the recombinant bacteria containing the vgb gene have better application prospect in the field of sewage treatment.
The traditional heavy metal pollution treatment methods have certain limitations, and the adoption of microorganisms for treating heavy metal pollution in the environment has the characteristics of environmental friendliness, wide sources and reproducibility, and is widely concerned by researchers. However, there are few reports on the adsorption of heavy metal ions by recombinant yeasts containing specific genes and how to reuse the yeasts.
Disclosure of Invention
The invention aims to provide efficient recombinant pichia pastoris and application thereof in adsorbing heavy metal ions in water, aiming at the current situation that pichia pastoris is less used for treating heavy metal pollution in water at present.
The recombinant pichia pastoris is prepared by the following method:
amplifying a vgb gene shown as SEQ ID NO.1, carrying out enzyme digestion by EcoRI and NotI, inserting the vgb gene into a pPIC9K vector, constructing to obtain a recombinant vector pPIC9K-vgb, and then carrying out linearization by SalI to transform pichia pastoris KM71, thereby obtaining the recombinant pichia pastoris.
The vgb gene codes vitreoscilla hemoglobin, has the function of improving the respiratory chain transfer rate and improving the utilization rate of dissolved oxygen, and can promote the growth and metabolism of thalli under the condition of oxygen deficiency.
By comparing the adsorption capacities of wild pichia pastoris KM71 and recombinant pichia pastoris KM71 to heavy metal ions Ni2+, Cd2+ and Pb2+, factors influencing the adsorption rate are researched, and the growth curves of the wild saccharomyces and the recombinant saccharomyces under the stress of the heavy metal ions are determined; the recombinant strain with better heavy metal resistance and better adsorption effect is obtained.
The invention also provides application of the recombinant pichia pastoris in adsorption of heavy metal ions.
Preferably, the heavy metal ion is Cd2+、Ni2+And/or Pb2+。
The invention also provides a heavy metal ion adsorbent which contains activated carbon and the recombinant pichia pastoris.
A method for adsorbing heavy metal ions in a water body by using recombinant pichia pastoris is characterized in that the recombinant pichia pastoris and active carbon are mixed to prepare an adsorbent, and then the adsorbent is added into the water body to be treated to adsorb the heavy metal ions.
Preferably, the adsorbent is recovered by centrifugation after use, desorbed by EDTA-2Na, and washed with water to obtain a reusable adsorbent.
According to the invention, by constructing the recombinant yeast KM71 and optimizing the conditions influencing the adsorption rate, the recombinant pichia pastoris which has higher adsorption rate to heavy metals and can be recycled is obtained, and the recycling method is established.
The recombinant pichia pastoris adsorbs Cd in the solution2+、Ni2+And/or Pb2+Has better adsorption capacity, and the maximum adsorption capacity reaches 39.87 mg/g. The recombinant pichia pastoris has good application prospect in the aspect of removing heavy metal ions in wastewater.
The recombinant pichia pastoris has stronger heavy metal resistance and strong adsorption performance, and has good application prospect in the aspect of removing heavy metal ions in sewage. By applying the recombinant pichia pastoris in a heavy metal solution, the passivation and repair capacity of the recombinant pichia pastoris on the effective heavy metal in the solution is found, and the adsorption rate of the recombinant pichia pastoris on the heavy metal ions is influenced by factors such as temperature, pH value and addition amount.
According to the invention, through optimization of the method, an optimal method for removing heavy metal ions in the water body by using the recombinant pichia pastoris and a recycling method for heavy metal ions absorbed by the pichia pastoris are established, so that the efficiency of heavy metal absorption by the pichia pastoris is further improved, and the production cost is reduced. The invention provides a reliable and efficient method for removing heavy metal ions in a water body by using recombinant pichia pastoris, so that the pollution of the heavy metal ions to the environment is reduced.
Drawings
FIG. 1 is a diagram of recombinant vector pPIC9K-vgb introduced into Pichia pastoris KM71: A) pPIC 9K-vgb; B) PCR verification of the pPIC9K-vgb into KM 71.
FIG. 2 shows the results of the comparison of C-KM71(A) and Y-KM71(B) at 5 mg. L-1Ni2+、Cd2+And Pb2+Growth curve under metal ion stress.
FIG. 3 shows the pairs of Ni in C-KM71 and Y-KM712+、Cd2+And Pb2+Adsorption kinetics of (c).
FIG. 4 shows the adsorption of Ni on C-KM71 and Y-KM71 at pH values2+、Cd2+And Pb2+The influence of (c).
FIG. 5 shows the amount of adsorbent added for adsorbing Ni by C-KM71 and Y-KM712+、Cd2+And Pb2+The influence of (c).
FIG. 6 shows the pairs of Ni in C-KM71 and Y-KM712+、Cd2+And Pb2+Adsorption isotherm of (1).
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
Materials and methods
1.1 strains for experiments
Wild Pichia pastoris KM71 was purchased from Wuhan vast Ling Biotech Co., Ltd, and recombinant yeast was KM71 strain (constructed in this laboratory) introduced with vector pPIC 9K-vgb. Wherein the recombinant KM71 strain is cultured for 24h, then 1% methanol is added to induce the expression of vgb gene, and then the wild yeast and the recombinant yeast are cultured for 48h and then thallus is collected. The culture solution was turbid, pH was about 5.0, and OD was measured after shaking600The OD of the two cells was adjusted600The values are consistent. OD of test bacterial solution600The value was 10.0, and the adsorbent was used in subsequent adsorption experiments (wild yeast Y-KM71, recombinant yeast C-KM 71). Precisely transferring 2mL of yeast liquid, centrifuging, washing with water, and drying at 75 ℃ to obtain 12mg of dry weight of 2mL of yeast liquid.
1.2 Experimental reagents and instruments
HCl, NaOH, EDTA-2Na, diatomite, activated carbon and nitric acid; a pH meter, a multifunctional microplate reader for measuring growth curves (Tecan, SPARK), a UV spectrophotometer (T6, general purpose in Beijing Punetron), a centrifuge (ST16R, Thermo).
The concentration of heavy metal ions was determined by inductively coupled plasma mass spectrometer (Agilent7800) with all experimental solutions at 8000 r.min-1Centrifugation was carried out for 3min under the conditions, and the supernatant solution was removed and diluted with a 5% nitric acid solution to measure the concentration, which was repeated 3 times for each sample.
1.3 preparation of heavy Metal solution
Cadmium chloride hemipentahydrate (purity 98%, mclin) was used separately; lead chloride (purity 99.5%, Mecline) and nickel (II) chloride (anhydrous) (purity 98.0%, TCI) are prepared into stock solutions of 3 single elements, and the stock solutions are diluted by deionized water to respectively obtain unitsMetal solutions of elements with concentrations of 29.1, 23.6 mg.L-1As a single element adsorption desorption experimental solution.
1.4 Experimental methods
1.4.1 Effect of heavy metal ions of different concentrations on growth inhibition of adsorbents Y-KM71 and C-KM71 were inoculated into a medium, 1 mg. L was added-1And 5 mg. L-1The growth curve of the yeast under the stress of heavy metals with different concentrations is determined by continuously culturing the single-element heavy metal solution for 48 hours by using a multifunctional microplate reader.
1.4.2 adsorption/desorption equilibration time and desorbent selection 25mL of 3 single-element heavy metal solutions were precisely transferred into 50mL plastic centrifuge tubes, 2mL of Y-KM71 and C-KM71 were added, respectively, at 25 ℃ and 200 r.min-1And (5) shaking, centrifuging and sampling for 15min, 30 min, 60min, 120min, 180 min, 240 min and 300min respectively, and determining the adsorption equilibrium time. Separating the supernatant, selecting 1% HCl, 0.05 mol. L-1NaOH and 0.05 mol. L-1And (3) desorbing the adsorbent by using EDTA-2Na 3 desorbents, and determining desorption equilibrium time and desorption rate.
1.4.3 temperature, pH value and influence of different addition amounts of adsorbents on metal ion adsorbability 25mL of metal solution was precisely transferred into a 50mL plastic centrifuge tube, 2mL of Y-KM71 and C-KM71 were added, and the concentration was tested after standing at 5, 25 and 30 ℃ for 120min, and the influence of different temperatures on the adsorbability was tested. According to the same steps, the method comprises the following steps of 1: 1 hydrochloric acid and 1 mol. L-1NaOH was added to adjust the pH of the solution to 3, 5, 7 and 9, 2mL each of Y-KM71 and C-KM71, at 25 ℃ at 200 r.min-1After oscillating for 120min, the influence of the pH value on the adsorption rate is tested. Adding 0.50 mL, 1.0 mL, 2.0 mL, 5.0 mL and 10.0mL of Y-KM71 and C-KM71 into the metal solution at 25 deg.C and 200r min-1After oscillating for 120min, testing the influence of different addition amounts of the adsorbent on the adsorption rate and the adsorption quantity. 1.4.4 adsorption isotherms of Metal ions of different concentrations 25mL of adsorption isotherm metal solutions of different concentrations were transferred into 50mL plastic centrifuge tubes, 2mL of each of Y-KM71 and C-KM71 were added, and the mixture was heated at 25 ℃ and 200 r.min-1And (5) oscillating for 120min and then sampling and testing.
Influence of 1.4.5C-KM71 composite on heavy metal adsorption according to the results of heavy metal adsorption by two yeasts, 0.01,Compounding 0.02 and 0.03g of diatomaceous earth and activated carbon with 2mL of C-KM71, adding 25mL of heavy metal solution, adjusting pH to 5.0, 6.0, and 7.0, adsorbing, balancing, centrifuging, sampling, testing metal ion concentration, separating supernatant, and purifying with 0.05mol/L-1EDTA-2Na is used for desorbing the adsorbent for 2h, the supernatant is centrifugally separated, 50mL of deionized water is used for washing and centrifuging the adsorbent for 3 times, 25mL of heavy metal solution is added for adsorption for 120min, and then sampling is carried out to test the adsorption rate of the recycled KM71 strain on the heavy metal ions.
1.5 data processing
The adsorption rate of yeast to heavy metal ions is calculated according to formula (1):
A＝(C0×V0-C×V)/C0×V0×100％ (1)
the adsorption amount of yeast to heavy metal ions is calculated according to the formula (2):
B＝(C0×V0-C×V)/m (2)
in the formula, C0For the mass concentration (mg. L) of heavy metal in experiment-1)；V0Volume of heavy metal solution (mL) for experiment; c is the mass concentration (mg. L) of heavy metal in the solution after adsorption equilibrium-1) (ii) a V is the volume sum (mL) of the heavy metal solution and the yeast; m is the weight (mg) of yeast added in the experiment.
The adsorption isotherm data were fitted using the commonly used Freundlich model and Langmuir model, which were given by the equation:
lgqe＝lgKf+1/nlgCe (3)
the Langmuir model equation is:
in the formula, qeAnd CeThe amount of heavy metal ions absorbed by the unit mass of the adsorbent and the concentration of metal ions in the water phase in the adsorption balance are respectively KfFreundlich adsorption coefficient, n is adsorption regression constant, qmaxIs the amount of metal ions when the surface of the adsorbent per unit mass completely forms a monolayer structure,represents the actual maximum adsorption capacity of the adsorbent when the surface of the adsorbent is completely covered with metal ions, KLIs the binding force constant of the adsorption site to the heavy metal.
2 results
1. Construction of recombinant Pichia pastoris KM71 and resistance thereof to heavy metal ions
The recombinant vector pBEP43-vgb (stored in the laboratory) is used as a template to amplify the vgb gene (the sequence of the amplified vgb gene is shown as SEQ ID NO. 1), the adopted upstream primer and downstream primer are 5'-CCGGAATTCATGTTAGACCAGCAAACCAT-3' (namely SEQ ID NO.2), 5'-ATAAGAATGCGGCCGCTTCAACCGCTTGAGCGTAC-3' (namely SEQ ID NO.3), and EcoRI and NotI are inserted into a pPIC9K vector after enzyme digestion. And (3) screening positive clones by PCR of the bacterial liquid, and sequencing and verifying to obtain the correct pPIC9K-vgb vector. The pPIC9K-vgb vector (FIG. 1A) was linearized with SalI and recovered, and then electrotransferred into Pichia pastoris KM71 competence at 1.5 kV. Positive clones were then screened with histidine-deficient MD plates. Each colony is subjected to amplification culture, total DNA is extracted, vgb gene is amplified, and the introduction of the recombinant plasmid pPIC9k-vgb is verified by sequencing (an electrophoretogram of an amplification product is shown in figure 1B). Thus, the recombinant Pichia pastoris KM71 strain containing pPIC9K-vgb is finally obtained.
Inoculating wild and recombinant Pichia yeast KM71 in the culture medium, adding Cd at 1mg/L and 5mg/L2+、Ni2+And Pb2+And (5) continuously observing the growth curve of the pichia pastoris in the heavy metal solution for 48h under different heavy metal stresses. According to the results of the growth curve measurement, the pichia pastoris KM71 enters the logarithmic growth phase at 5h and enters the stationary phase at 40h (fig. 2). 5mg/L heavy metal ion Cd2+、Ni2 +The growth of wild pichia pastoris is inhibited to a certain extent (figure 2B), and heavy metal ions with the same concentration have almost no inhibition effect on the growth of the recombinant pichia pastoris KM71 (figure 2A). The recombinant pichia pastoris KM71 is proved to have higher resistance to heavy metal ions. The reason for this is that the recombinant pichia pastoris KM71 contains vgb gene, which can improve the respiratory chain transmission efficiency of the recombinant pichia pastoris KM71, improve the energy utilization rate, and promote the activity of metal binding related proteins such as metal-sulfur binding protein, so the recombinant pichia pastoris KM71 has good stability and stability in the respiratory chain, and can be used for treating diabetesThe stress of heavy metal ions has better stress resistance. The performance is also more beneficial to the application of the recombinant yeast in the treatment of high-concentration heavy metal pollution in wastewater. Heavy metal ion Cd of 1mg/L2+、Ni2+And Pb2+And 5mg/L Pb2+Has no influence on the growth of wild yeast, and indicates that the wild pichia KM71 has certain heavy metal ion resistance and Pb2+The growth inhibition was less for the wild and recombinant KM71 strain.
2. Adsorption characteristics of pichia pastoris on different heavy metal ions
Heavy metal ions are adsorbed by using wild pichia pastoris KM71 and recombinant pichia pastoris KM71 containing pPIC9 k-vgb. The two kinds of Pichia pastoris have heavy metal adsorbing amount over 60% in 15min, 80% in 60min and 120min, and the heavy metal adsorbing amount is generally divided into two stages, the first stage is cell surface adsorbing stage, i.e., extracellular polymer and functional group in cell wall (carboxyl, phosphoryl, hydroxyl, sulfate and amido) are coordinated and complexed with metal ion, and the second stage is the process of transferring the heavy metal ion from cell surface to cell and participating in cell metabolism under the action of cell surface enzyme. Wild KM71 for Ni in adsorption equilibrium2+、Cd2+And Pb2+The adsorption rates of (1) and (4) were 18.8%, 26.1% and 46.6%, respectively, and the recombinant KM71 was used for Ni2+、Cd2+And Pb2+The adsorption rates of (a) were 22.8%, 29.6% and 52.0%, respectively, as shown in FIG. 3. The reason why the adsorption performance of the recombinant KM71 on heavy metals is higher than that of the wild KM71 is that the vgb gene in the recombinant yeast expresses vitreoscilla hemoglobin under the induction of 1% methanol. The protein located on the periplasm space of the cell can improve the energy transfer efficiency and the oxygen utilization rate of the recombinant KM71, so that the growth rate and the expression level of the metal binding related protein are higher. For Pb2+Has a significantly higher adsorption rate than Ni2+And Cd2+Adsorption of Pb at high or low level2+﹥Cd2+﹥Ni2+Illustrating that the adsorbent is selective for the adsorption of heavy metalsThe ion radius and electronegativity of the metal ions are related, the larger the ion radius of the metal ions with the same valence, the smaller the hydration radius of the metal ions, the easier the metal ions are to be absorbed, and the Pb-adsorbing material is used for adsorbing Pb2+With a marked selectivity, possibly due to Pb2+The radius (0.132) is larger than other ions, and the electronegativity (2.33) is larger than other ions; it is also possible that the KM71 is more resistant to lead ions because the lead ions are less bio-toxic to the KM71 strain.
The desorption balance of heavy metals is achieved by adopting three desorbents of 1 percent HCl, 0.05mol/L NaOH and 0.05mol/L EDTA-2Na for desorption for 60 min. The results are shown in Table 1, 0.05mol/L EDTA-2Na vs Ni2+And Pb2+The desorption result is relatively better, 1% HCl to Cd2+The desorption results are relatively better. In general, these desorbents were unable to completely desorb the metal ions from the adsorbent, and it was also confirmed that pichia KM71 had good adsorption ability to heavy metal ions.
TABLE 1 desorption results of adsorbents for metal ions
Under different temperature conditions, the difference of the adsorption rate of the KM71 strain on heavy metal ions is not obvious, and under the test conditions of 5 ℃, 25 ℃ and 30 ℃, the adsorption rate of KM71 on a plurality of heavy metal ions is less than 5%. The yeast has wide temperature adaptability, thereby being beneficial to the application of the yeast in different environments.
At pH values of 3, 5, 7 and 9, the adsorption rate of the wild and recombinant KM71 strains on heavy metal ions increased and then decreased, with a smaller overall difference (fig. 4). The reason why the adsorption rate of heavy metals is relatively low under low pH conditions is that a large amount of H is contained in the solution+Compete for adsorption sites with heavy metal ions and simultaneously hinder dissociation of active groups; the adsorption rate of the KM71 to metal ions increases with the increase of pH value, and reaches the highest at the pH value of 5-7, which is probably H in the solution+Sharply reduces the competition of metal ions for the adsorption sites of the adsorbent, and reduces the reaction of the negatively charged adsorbent with goldThe electrostatic action of the metal ions is enhanced, so that the metal ions are favorably adsorbed. At the condition of pH9, the adsorption rate of the adsorbent to heavy metal ions is relatively reduced, which may be related to the growth metabolism of yeast itself. The growth pH of the yeast is about 5, when the pH is 9, the over-alkali environment is not favorable for the growth and secondary metabolism of the yeast, the heavy metal ion adsorption capacity of the yeast is reduced, and the metal ions are easy to form insoluble precipitates such as oxides, hydroxides and the like under the alkaline condition, cover the surface of the KM71 yeast strain and are not favorable for the adsorption of the metal ions. In general, the yeast has a wider pH adsorption value, and under the same condition, the adsorption rate of the KM71 strain on heavy metal ions can be improved by adding NaOH to properly adjust the pH value of the solution to be neutral.
When different dosages of KM71 strain were added, the concentration of metal ions in the solution after adsorption equilibrium decreased with the increase of the adsorbent, but the adsorption rate did not increase significantly with the increase of the addition amount of the adsorbent. The amount of metal ions adsorbed by the cells per unit mass was significantly reduced as the amount of the cells was increased (FIG. 5). Since the total amount of the metal ions adsorbed by the KM71 strain was uniform, the total volume of the system increased and the amount of the ions adsorbed per unit volume was small as the amount of the adsorbent added was larger.
3. Adsorption isotherm and condition optimization of recombinant pichia pastoris on metal ions with different concentrations
The recombinant KM71 strain was used to fit the isotherm data of a Freundlich model commonly used for heavy metal adsorption characteristic selection of different ion concentrations in 1.4.4 (FIG. 6), and the unit mass of adsorbent was used for Ni2+、Cd2+And Pb2+The adsorption amount of (A) is increased along with the increase of the ion concentration in the solution, and the equilibrium is reached when the ion concentration reaches a certain concentration. The adsorption capacity of the recombined KM71 strain to metal ions is higher than that of a wild KM71 strain, regression constants n of linear fitting results are all larger than 1, the value of 1/n is between 0.727 and 0.891, the yeast is shown to be easy to adsorb the metal ions, and the isothermal linear regression coefficient is between 0.910 and 0.984. Shows that in the initial stage of isothermal adsorption, yeast has stronger affinity to metal ions, and as adsorption sites on the yeast are gradually occupied, the adsorption sites of the metal ions are less and less,recombinant Pichia pastoris C-KM71 for Cd2+And Pb2+The adsorption strength of the yeast is obviously higher than that of wild yeast; adsorbent per unit mass to Ni2+、Cd2+And Pb2+The adsorption capacity of the strain is increased along with the increase of the ion concentration in the solution, the strain reaches the equilibrium when reaching a certain concentration, and the adsorption capacity of the recombinant C-KM71 strain to metal ions is higher than that of the wild Y-KM71 strain.
4. Optimization of heavy metal adsorption conditions
The adsorption capacity of the recombinant pichia pastoris KM71 on heavy metal ions is superior to that of the wild pichia pastoris KM71, diatomite and activated carbon are selected as a complexing agent of the recombinant KM71, a carrier can be provided for saccharomycetes, groups on the surface of the carrier can be combined with the heavy metal ions to form covalent bonds and ionic bonds, the carrier is blank, the adsorption result of the carrier and the recombinant KM71 in combination with the heavy metal ions is shown in Table 2, the adsorption capacity of the activated carbon is superior to that of the diatomite, and the effect after the activated carbon is combined with the recombinant KM71 is also demonstrated. The selected 0.03g of activated carbon is compounded with the recombined KM71, so that the adsorption force is improved to a certain extent, the complexing agent provides a carrier for the growth of microorganisms, and the complexing agent has a certain enrichment effect on heavy metal ions due to the porous structure and the rich active groups.
Adjusting the pH values to 5, 6 and 7 respectively, centrifugally separating the adsorbent, desorbing by adopting EDTA-2Na, and washing for three times to obtain the recyclable adsorbent. Recombinant KM71 strain pair Ni2+、Cd2+And Pb2+In the second adsorption result, the influence of the pH value on the adsorption rate was small, but the adsorption capacity was greatly improved compared to the untreated adsorbent (table 2). Washing recombinant KM71 strain pair Ni2+、Cd2+And Pb2+The optimal adsorption pH values of the heavy metal ion adsorption material are respectively 6, 6 and 5, the adsorption rates are respectively 35.20%, 50.08% and 69.84%, and compared with untreated C-KM71, the adsorption rates of the heavy metal ion adsorption material are respectively improved by 15.14%, 21.04% and 18.47% through condition-optimized water washing. C-KM71 p Ni2+、Cd2+And Pb2+The adsorption capacity is 21.27mg/g, 25.08mg/g and 39.87mg/g respectively, and the adsorption capacity of heavy metal ions is respectively improved by 11.26mg/g and 13 through condition optimization.43mg/g, 22.90 mg/g. The results show that the water washing has a great influence on the heavy metal adsorption performance of pichia pastoris KM71, mainly because the related functional groups of KM71 can be exposed through the water washing.
Table 2C-KM 71 optimized results of conditions for adsorbing heavy metal ions
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> recombinant pichia pastoris and application thereof in adsorption of heavy metal ions
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 438
<212> DNA
<213> Vitreoscilla (Vitroscilla beggiatides)
<400> 1
atgttagacc agcaaaccat taacatcatc aaagccactg ttcctgtatt gaaggagcat 60
ggcgttacca ttaccacgac tttttataaa aacttgtttg ccaaacaccc tgaagtacgt 120
cctttgtttg atatgggtcg ccaagaatct ttggagcagc ctaaggcttt ggcgatgacg 180
gtattggcgg cagcgcaaaa cattgaaaat ttgccagcta ttttgcctgc ggtcaaaaaa 240
attgcagtca aacattgtca agcaggcgtg gcagcagcgc attatccgat tgtcggtcaa 300
gaattgttgg gtgcgattaa agaagtattg ggcgatgccg caaccgatga cattttggac 360
gcgtggggca aggcttatgg cgtgattgca gatgtgttta ttcaagtgga agcagatttg 420
tacgctcaag cggttgaa 438
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccggaattca tgttagacca gcaaaccat 29
<210> 3
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ataagaatgc ggccgcttca accgcttgag cgtac 35
Claims (6)
1. The recombinant pichia pastoris is characterized by being prepared by the following method:
amplifying a vgb gene shown as SEQ ID NO.1, carrying out enzyme digestion by EcoRI and NotI, inserting the vgb gene into a pPIC9K vector, constructing to obtain a recombinant vector pPIC9K-vgb, and then carrying out linearization by SalI to transform pichia pastoris KM71, thereby obtaining the recombinant pichia pastoris.
2. The application of the recombinant pichia pastoris of claim 1 in adsorption of heavy metal ions.
3. The use of claim 2, wherein the heavy metal ion is Cd2+、Ni2+And/or Pb2+。
4. A heavy metal ion adsorbent comprising activated carbon and the recombinant Pichia pastoris of claim 1.
5. A method for adsorbing heavy metal ions in a water body by using recombinant Pichia pastoris is characterized in that the recombinant Pichia pastoris of claim 1 is mixed with activated carbon to prepare an adsorbent, and then the adsorbent is added into the water body to be treated to adsorb the heavy metal ions.
6. The method of claim 5, wherein the adsorbent is recovered by centrifugation after use, desorbed with EDTA-2Na, and washed with water to obtain a reusable adsorbent.
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Address after: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee after: Institute of Microbiology, Guangdong Academy of Sciences Address before: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) |