CN108004256A - Glucose oxidase gene Glox, albumen, pichia pastoris yeast and its preparation and application - Google Patents

Abstract

The invention belongs to the preparation of new genes and the construction of engineering bacteria, in particular to the preparation, construction and application of a glucose oxidase gene Glox and Pichia pastoris. Using the 5￠end ATGAAGCTCCTTGGCCTCC and the 3￠end CTAATTAACAGTAGCGTTGTAATC as specific primers, using Penicillium elongatus DNA as a template, the glucose oxidase gene Glox was obtained by PCR amplification method, and the engineering strain of Pichia pastoris was successfully constructed. The present invention solves the problem of heterologous expression of recombinant glucose oxidase gene at present, obtains the full-length Glox gene and its constructed shuttle expression vector by using the present invention, further transforms into Pichia pastoris X33 strain, and obtains a strain through screening and identification The strain expressing glucose oxidase had a higher secretion than the original strain. The method of the invention is simple and easy, and the cost is low, which lays a good foundation for the large-scale production of glucose oxidase.

Description

Glucose oxidase gene Glox, protein, Pichia pastoris and its preparation and application

technical field

The invention belongs to the preparation of new genes and the construction of engineering bacteria, in particular to a glucose oxidase gene Glox, protein, Pichia pastoris and its preparation and application.

Background technique

Glucose oxidase is an aerobic dehydrogenase that can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide. The catalytic properties of glucose oxidase enable it to remove glucose, deoxidize, sterilize and other functions, and it is safe, non-toxic and without side effects.

Glucose oxidase is widely distributed in animals, plants and microorganisms. Due to the strong ability of mold to produce enzymes, the currently used research and industrial strains mainly include Aspergillus niger and Penicillium strains. The presence of the purification brings considerable difficulties. Penicillium extensa belongs to a kind of Penicillium, which also has such problems. In recent years, the market demand for glucose oxidase has increased year by year, and the quality requirements have also increased year by year. Therefore, the problems of low yield, low enzyme activity, cumbersome purification, and complicated operation of enzyme activity detection methods in the technical level of glucose oxidase in my country need to be solved urgently.

Heterologous expression of recombinant glucose oxidase gene can effectively solve these problems. In particular, the expression of exogenous proteins by Pichia pastoris has the advantages of high expression, good stability, low culture cost, and easy separation and purification of products. , AOX) promoter and other advantages, can strictly control the expression of foreign genes.

Through the transformation of glucose oxidase production strains by means of genetic engineering, the accurate and efficient production and extraction methods of glucose oxidase can be obtained, and the enzyme production can be increased, which provides a guiding basis for later industrial production and improves its economic benefits.

In the invention patent application with application number 201510893562.8, the applicant disclosed a glucose oxidase gene GOD, a protein encoded by it, and a transformed genetically engineered strain of Pichia pastoris. Glucose oxidase gene GOD was obtained by PCR amplification method using 5' end ATGAAGTCCACTATTATCACCTCCA and 3' end CTAGGCACTTTTT GGCATAGTCTTCA as specific primers and Penicillium pointis DNA as template. The full-length GOD gene and its constructed shuttle expression vector were obtained, and further transformed into Pichia pastoris genetically engineered bacteria. After screening and identification, a strain with higher secretion and expression of glucose oxidase than the original strain was obtained. The obtained transgenic Pichia Yeast engineered bacteria, under the detection condition of 10L fermenter level, induced by methanol for 144 hours, the enzyme activity in the fermentation broth reached 594U/ml, which significantly improved the fermentation enzyme activity; and the glucose oxidase production strain was modified by genetic engineering means, and it can also Accurate and efficient production of glucose oxidase is obtained, and the amount of enzyme production (9g/L) is increased. The problem that the above-mentioned prior art exists is that the large-scale production of GOD adopts Aspergillus niger or Penicillium fermentation widely, but in the process of producing GOD by Aspergillus niger and Penicillium fermentation, the production of a large amount of miscellaneous proteins such as catalase, cellulase and amylase Presence poses considerable difficulties for purification. Therefore, it is a more economical and effective way to develop and produce high-quality GOD preparations through the construction of engineered bacteria to realize the heterologous and efficient expression of glucose oxidase.

The comparative documents searched by the applicant include:

1. In the patent document with application number CN105936910A, the starting strain is the heterologous expression of GOD of Aspergillus niger, but the enzyme activity expression level is only 77U/ml. In addition, it was mutated and the amino acid sequence was changed. We only changed the gene sequence, but the amino acid sequence was consistent with that of the original starting strain of Penicillium expansica. The Pichia pastoris is recombinant Pichia pastoris GS115, and we use X33.

2. The strain used in the patent literature with application number CN106591249A is Aspergillus niger, and the fermentation process of Aspergillus niger has been optimized, but in the process of producing glucose oxidase by mold fermentation, a large amount of catalase, cellulase and amylase, etc. The presence of hybrid enzymes brings considerable difficulties to purification.

3. The improvement of CN105936910A in the patent document with the application number CN105950577A, the above sequence is mutated, which improves the thermal stability of the enzyme. Fermentative enzyme activity is not mentioned.

Contents of the invention

One of the objectives of the present invention is to provide a glucose oxidase gene Glox.

The second object of the present invention is to provide a method for preparing the glucose oxidase gene Glox.

The third object of the present invention is to provide a protein encoded by the glucose oxidase gene Glox.

The fourth object of the present invention is to provide a Pichia pastoris transformed with the glucose oxidase gene Glox.

The fifth object of the present invention is to provide a method for transforming Pichia pastoris genetically engineered bacteria using the glucose oxidase gene Glox.

Overall technical idea of the present invention is:

Glucose oxidase gene Glox, its gene sequence is SEQ ID No.1.

The preparation method of the glucose oxidase gene Glox is obtained by using the 5' end ATGAAGCTCCTTGGCCTCC and the 3' end CTAATTAACAGTAGCGTTGTAATC as specific primers, using the Penicillium sp. DNA as a template, and obtaining it by PCR amplification.

The sequence of the protein encoded by the glucose oxidase gene Glox is SEQ ID No.2.

The Pichia pastoris genetically engineered strain transformed with the glucose oxidase gene Glox has a preservation number of CGMCC No. 13358.

The Pichia genetically engineered bacterium in the present invention has been submitted by the applicant to the General Microorganism Center of China Microbiological Culture Collection Management Committee on November 30, 2016. The deposit number is CGMCC No.13358. The address of the depository institution is located in Chaoyang, Beijing. Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yard 1, Beichen West Road, District. The abbreviation of this depository institution is CGMCC.

Utilize the preparation method of the Pichia pastoris genetic engineering bacterium transformed by glucose oxidase gene Glox, the steps in its preparation method are as follows:

a. Design primers by PCR method to remove the signal peptide coding sequence in the glucose oxidase gene sequence of Penicillium elongatus. The primer sequence is as follows: Glox-F: 5'GGCTGAAGCTTACGTAGAATTCCTTCCACAAGCTGACTTCGACC3'; the underlined part in the sequence is the upstream primer to add restrictions Endonuclease EcoRI recognition site; Glox-R: 5'GAGATGAGTTTTTGTTCTAGAGCGGCCGCCTAATTAA CAGTAGCGTTGTAATC 3'; the underlined part in the sequence is the restriction endonuclease NotI recognition site added downstream;

b. After purification and recovery, the PCR product was connected to the EcoRI and NotI sites of the expression vector pMD-AOX with GIBSON, transformed into E.coli DH5α, verified by restriction enzyme digestion, screened positive clones, and sent for sequencing;

c. The sequencing is correct. The recombinant plasmid pMD-AOX-Glox is linearized after digestion with PmeI, and 80 μl of Pichia pastoris X33 competent cells are transformed by electroporation with 2 μg of the linearized pMD-AOX-Glox plasmid, and then the transformed cells are spread on a medium containing 100 μg /mlG418 on a YPDS plate, cultured at 30°C for 96 hours; Pichia pastorisCGMCC No.13358 was obtained.

Application of Pichia pastoris CGMCC No.13358 in preparing glucose oxidase.

Concrete technical concept of the present invention also has:

The application of Pichia pastoris CGMCC No.13358 in the preparation of glucose oxidase comprises the following process steps:

A. Inoculate Pichia pastoris CGMCC No.13358 in sterile YPD medium for 12-16 hours;

B. Transfer the bacteria cultured in step A to a sterile BMGY medium with a volume of 100ml according to the volume ratio of 3% inoculum, and shake the flask at a temperature of 30°C until OD ₆₀₀ ≈6-7;

C. Inoculate the cultured strains in step B in a fermenter filled with sterile BSM medium at a volume ratio of 10%, under the conditions of 30°C, 550 rpm, and pH=5 Cultivate until the cell OD ₆₀₀ ≈90;

D. Feed 50% glycerol, each liter of glycerin contains 12mL PTM1, the flow rate is 12mL/h/L, and the temperature is 30°C, culture for 4h, OD ₆₀₀ ≈150-170, stop feeding for 0.5-1h, confirm glycerol run out

E. Add the inducer methanol according to the flow rate of 3.0mL-5.0mL/h/L. Each liter of methanol contains 12mL of PTM1, the pH is maintained at 6.0-6.5 with ammonia water, and the enzyme activity is measured every 12 hours. When the enzyme activity occurs Fermentation was stopped at the inflection point of decline.

The substantive characteristics and remarkable technical progress that the present invention obtains are:

1. The present invention obtains a full-length Glox gene sequence through PCR technology, the full-length of the gene is 1815bp, and the GC content is 47.6%. Homology analysis showed that the sequence homology between this gene and the previously reported GOD gene was 94.71%, which proved that this gene was a new glucose oxidase gene.

2. In the process of traditional Penicillium fermentation to produce GOD, there is not only a large amount of mixed enzymes such as catalase, cellulase and amylase, which are difficult to purify. The transgenic Pichia pastoris engineered bacteria obtained by the present invention can realize heterologous high-efficiency expression of glucose oxidase, and the expression of foreign protein by Pichia pastoris has the advantages of good stability, low culture cost, easy separation and purification of products, and the like.

3. It has been confirmed by the applicant's test that the fermented enzyme activity of the transgenic Pichia pastoris obtained in the present invention reaches 305U/mL in a 10L fermenter, and the band is single, and the accurate and efficient production of glucose oxidase is obtained, which greatly reduces the after-effects. The cost of processing provides a guiding basis for later industrial production and greatly improves economic benefits.

Description of drawings

Accompanying drawing of the present invention has:

Fig. 1 is the sequence listing of the glucose oxidase gene Glox and the protein encoded by it in the present invention.

Figure 2 Expands the full-length results of the Penicillium Glox gene.

Using Genomic DNA of Penicillium elongatum as a template, specific primers were designed for PCR amplification, and a specific band less than 2000bp was amplified, and the sequence analysis showed that the fragment was 1815bp long. M is DNA Marker, the molecular weight from large to small is 10000bp, 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1 is the Glox gene amplification product.

Figure 3 is the result of double enzyme digestion detection of the expression plasmid pMD-AOX-GLOX.

The Penicillium elongata Glox gene recovered by PCR amplification and purification with primers Glox-F and Glox-R and the vector pMD-AOX cut with EcoRI and NotI were subjected to Gibson ligation to construct a recombinant plasmid. Transform competent Escherichia coli DH5α with the recombinant plasmid, and screen positive colonies on LB plates containing Amp. After the positive colony plasmid was digested with EcoRI and NotI, two DNA fragments of about 6.5.0kb and 1.8kb were obtained, which were also in line with the expected size. In the figure, M is DNA Marker, the molecular weight from large to small is 10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1000bp, 1-6 is the product of double enzyme digestion of the expression plasmid pMD-AOX-Glox.

Fig. 4 is the SDS-PAGE detection result of the expression of glucose oxidase in YPCS.

The recombinant expression plasmid pMD-AOX-Glox was digested with PmeI and linearized, then electrotransformed Pichia pastoris X33 competent, after growing on the YPDS plate for 3 days, randomly selected positive colonies were transferred to liquid YPCS, and induced by methanol for 72 hours, The glucose oxidase activity in the supernatant of the fermentation broth was measured, and at the same time, the supernatant of the fermentation broth was subjected to protein SDS-PAGE electrophoresis, and a densely stained protein band of about 80kD was seen, with almost no other miscellaneous bands. Marker in the figure is a protein marker, and its molecular weight from large to small is 220kD, 135kD, 90kD, 66kD, 45kD, 35kD, 29kD, 20kD, 14kD, 1-3 is the 10-fold dilution of the fermentation supernatant, 4-6 is the fermentation supernatant The supernatant was diluted 5 times.

Figure 5 is the SDS-PAGE detection result of the expression of glucose oxidase in a 10L fermenter.

In the 10L fermenter, before methanol was not induced, it was in the stage of strain cultivation and carbon source feeding. In these two stages, the bacteria grew in large quantities, and there was no expression of glucose oxidase protein at this time. With the induction of methanol, the activity of glucose oxidase in the fermentation broth gradually increased. After 144 hours of induction, the activity of glucose oxidase in the fermentation broth was 305U/ml, and the protein expression reached 5.2g. SDS-PAGE also showed that the glucose oxidase in the fermentation broth Protein expression is also accumulating. In the figure, M is the protein marker, 1-14 is the expression level of glucose oxidase induced by methanol at 0h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h, 132h, 144h, 156h, and 168h.

Detailed ways

Embodiments of the present invention will be further described below in conjunction with the accompanying drawings, but they are not intended to limit the present invention. The scope of protection of the present invention is based on the contents of the claims, and any replacement of equivalent technical means based on the specification does not depart from the scope of protection of the present invention.

Construction and Identification of Embodiment 1 Recombinant Bacteria

Select the Penicillium expansum CICC 40658 previously preserved in the laboratory to obtain a strain with stable and high enzyme activity, and use the gene of this strain as a template to design primers to obtain the gene Glox. The purified PCR product was connected with the shuttle vector pMD-AOX by the Gibson method. After the connection was completed, the E. coli DH5α competent cells were transformed, and the white positive clones on the transformation plate were picked to obtain the recombinant expression plasmid pMD-AOX containing the Glox gene. -Glox. Validation by double enzyme digestion.

The recombinant plasmid pMD-AOX-Glox was electroporated to transform Pichia pastoris X33 competent cells to obtain genetically engineered bacteria Pichia pastoris X33-Glox.

The transformation of Pichia pastoris adopts the electroporation method: take X33 and culture overnight, pick a single colony on the plate and culture it in 5mLYPD to OD ₆₀₀ ≈1.2, and then transfer it to a 50mL YPD shake flask according to the inoculum size of 10%. Cultivate until OD ₆₀₀ ≈1.5; centrifuge at 2500rpm for 5min, discard the supernatant; resuspend the collected cells with 100mL 0.1M LiAc, 100M DTT, 0.6M sorbitol, 10mM Tris-Hcl (pH=7.5), at 30℃ Incubate at a low speed for 30 minutes; centrifuge again at 2500 rpm for 5 minutes to discard the supernatant, wash with 1M pre-cooled sorbitol repeatedly for 3 times, and resuspend in 1.5mL BEDS. Mix 80 μL protoplasts with 5 μL linearized plasmid DNA (cut with SacI), transfer to an ice-cold electroporation cuvette, and let stand for 5 minutes; electric shock the mixture of cells and DNA (1.5kv, 3.0-4.9ms); add the transformed recombinant bacteria to 1M Sorbitol, stand at 30°C for 2-5h; then centrifuge at 5000rpm for 2min, wash with saline repeatedly for 3 times, and spread on solid YPDS (containing G418) medium. After culturing at 30°C for 72 hours, a single colony was picked to obtain Pichia pastoris CGMCC No.13358.

Enzyme activity assay and protein electrophoresis of embodiment 2 recombinant bacteria

Using the Pichia pastoris CGMCC No.13358 obtained in Example 1 as the production bacteria, after activation, the seed culture solution cultivated to OD ₆₀₀ ≈ 1.2 under the conditions of 30°C and 200rpm was transferred to YPCS with an inoculum of 2%. Culture medium, cultivated at 30°C and 200rpm; when cultured in YPCS medium to OD ₆₀₀ ≈1.2, yeast cells were transferred to YPCS induction medium, and 1% methanol was added every day to continuously induce expression for 72 hours.

culture medium

The seed and slant medium are YPD medium: 2% tryptone, 1% yeast extract, 2% glucose; 2% agar is added to the slant medium.

YPCS medium: tryptone 2%, yeast extract 1%, casein hydrolyzate 2%, sorbitol 0.5%.

A protein band with a molecular weight of about 80 kDa was obtained by protein electrophoresis (SDS-PAGE) as shown in FIG. 4 .

Glucose oxidase enzyme activity assay method: The glucose oxidase activity assay generally adopts the o-one (two) anisidine spectrophotometric method. In a 3mL reaction system containing 0.5mol/L acetate buffer (pH=5.1), 0.17mmol/L o-dianisidine solution, 1.72% glucose solution and 0.1mg/mL horseradish peroxidase, shake at 35°C Mix well, add 0.1mL glucose oxidase solution, and use a spectrophotometer to automatically record the absorbance value changing with time at A=500nm, and calculate the glucose oxidation according to the formula X=(ΔA500×3.1×df)/(7.5×0.1). Enzyme activity unit.

X: sample enzyme activity (U/mL);

ΔA500 is the activity value;

3.1: Reaction volume;

df is the dilution factor;

7.5: Glucolactone A500 extinction coefficient;

0.1: Add the volume of enzyme solution.

The verification of embodiment 3 recombinant bacterial strains enzyme activity on 10L fermenter

A. Inoculate Pichia pastoris CGMCC No.13358 in sterile YPD medium for 12-16 hours;

B. Transfer the bacteria cultured in step A to a sterile BMGY medium with a volume of 100mL at a volume ratio of 3% inoculum, and shake the flask at a temperature of 30°C until OD ₆₀₀ ≈6-7;

C. Inoculate the cultured strains in step B in a fermenter filled with sterile BSM medium at a volume ratio of 10%, under the conditions of 30°C, 550 rpm, and pH=5 Cultivate until the cell OD ₆₀₀ ≈90;

D. Feed 50% glycerol, 12mL PTM1 per liter of glycerin, flow rate of 12mL/h/L, culture at 30°C for 4h, OD ₆₀₀ ≈150-170, stop feeding for 0.5-1h, confirm glycerol run out

E. Add the inducer methanol according to the flow rate of 3.0mL-5.0mL/h/L, each liter of methanol contains PTM112mL, the pH is maintained at 6.0-6.5 with ammonia water, and the enzyme activity is measured every 12 hours. When the enzyme activity occurs Fermentation was stopped at the inflection point of decline. The highest enzyme activity of recombinant bacteria in 10L fermenter can reach 305U/mL.

Media and Reagents

BMGY medium: yeast extract 1%; peptone 2%; potassium phosphate pH6.0 100mM; YNB1.34%; biotin 4×10-5%; glycerol 1% or methanol 0.5%.

PTM1 (trace elements): copper sulfate 6.0g/L; sodium iodide 0.08g/L; manganese sulfate 3.0g/L; sodium molybdate 0.2g/L; boric acid 0.02g/L; cobalt chloride 0.5g/L; Zinc chloride 20g/L; ferrous sulfate 65g/L; sulfuric acid 5.0mL/L; biotin 0.2g/L; V _C 80mg/L; V _B2 0.5M.

BSM medium: 85% H ₃ PO ₄ 26.7mL/L; CaSO ₄ 0.93g/L; K ₂ SO ₄ 18.2g/L; MgSO ₄ ·7H ₂ O 14.9g/L; KOH 4.13g/L; Glycerol 40g /L; PTM14.35mL/L (sterilized by filtration).

sequence listing

<110> Hebei Institute of Microbiology

Institute of Microbiology, Chinese Academy of Sciences

<120> Glucose oxidase gene Glox, protein, Pichia pastoris and its preparation and application

<130> 201510893562.8; CN105936910A; CN106591249A; CN105950577A

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ggtggttcca ccttggttaa cggaggatct tggactcgtc cacacaaggc tcaagttgac 420

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tccttctcac caatgattaa atctttgatg aagaacgcta ataactctgg tattcctgtc 660

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actccaaaag ccgtcggtgt gaattttggt actcacgcca aggtcaactt cgacgtgcac 900

gctagacacg aagttttgct tgcttctggt tccgccgttt ctccacagat tctggagcat 960

tccggagttg gtcttaaggc tgtcctggat aacgtcggtg ttgaacagtt ggttgatctt 1020

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gtttctagag gtggtttcca caacgaaacc gctcttcttg tccagtacga gaactacaga 1260

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ttgaatctgg acctgtggga tcttattcca tttactcgtg gatatgttca catcctggat 1380

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580 585

Claims (7)

1. Glucose oxidase gene Glox, characterized in that its gene sequence is SEQ ID No.1. 2. The preparation method of the glucose oxidase gene Glox, which is characterized in that the 5' end ATGAAGCTCC TTGGCCTCC and the 3' end CTAATTAACAGTAGCGTTGTAATC are used as specific primers, and Penicillium elongatus DNA is used as a template to obtain it by PCR amplification. 3. A protein encoded by the glucose oxidase gene Glox, characterized in that its sequence is SEQ ID No.2. 4. Pichia pastoris transformed with the glucose oxidase gene Glox, characterized in that its preservation number is CGMCC No.13358. 5. the preparation method of Pichia pastoris according to claim 4 is characterized in that adopting following method to prepare: a. Design primers by PCR to remove the signal peptide coding sequence in the glucose oxidase gene sequence of Penicillium expansum CICC 40658. The primer sequences are as follows: Glox-F: 5'GGCTGAAGCTTACGTAGAATTCCTTCCACAAGCTGACTTCGACC3', the underlined part in the sequence is the restriction endonuclease EcoRI recognition site added by the upstream primer; Glox-R: 5'GAGATGAGTTTTTGTTCTAGAGCGGCCGCCTAATTAACAGTAGCGTTGTAATC3', the underlined part in the sequence is the restriction endonuclease NotI recognition added downstream site; b. After purification and recovery, the PCR product was connected to the EcoRI and NotI sites of the expression vector pMD-AOX with GIBSON, transformed into E.coli DH5α, verified by restriction enzyme digestion, screened positive clones, and sent for sequencing; c. Sequence correct recombinant plasmid pMD-AOX-Glox was linearized after digestion with PmeI, and 80 μl Pichia pastoris X33 competent cells were transformed by electroporation with 2 μg linearized pMD-AOX-Glox plasmid, and then the transformed cells were smeared on a medium containing 100 μg /mlG418 on a YPDS plate, cultured at 30°C for 72 hours; Pichia pastoris CGMCCNo.13358 was obtained. 6. the application of Pichia pastoris according to claim 4 in the preparation of glucose oxidase. 7. The application according to claim 6, characterized in that it comprises the following process steps: A. Inoculate Pichia pastoris CGMCC No.13358 in sterile YPD medium for 12-16 hours; B. Transfer the bacteria cultured in step A to a sterile BMGY medium with a volume of 100ml according to the volume ratio of 3% inoculum, and shake the flask at a temperature of 30°C until OD ₆₀₀ ≈6-7; C, according to the volume ratio of 10%, the culture solution in step B is inoculated in the fermenter that fills the culture medium of sterile BSM, under the condition that the temperature is 30 ℃, rotating speed 550 rev/min, pH=5, cultivate to Cell OD ₆₀₀ ≈90; D. Feed 50% glycerol, each liter of glycerin contains 12mL PTM1, the flow rate is 12mL/h/L, and the temperature is 30°C, culture for 4h, OD ₆₀₀ ≈150-170, stop feeding for 0.5-1h, confirm glycerin run out E. Add the inducer methanol according to the flow rate of 3.0mL-5.0mL/h/L, each liter of methanol contains 12mL PTM1, the pH is maintained at 6.0-6.5 with ammonia water, and the enzyme activity is measured every 12 hours. When the enzyme activity occurs Fermentation was stopped at the inflection point of decline.