RU2542486C1 - STRAIN OF Esherichia coli EX pQE30 BACTERIUM, PRODUCER OF ENDOXYLANASE OF Geobacillus stearothermophillus 22 BACTERIUM - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain Esherichia coli EX pQE30 has been obtained by transformation of cells of E.coli XL1-blue bacterium with pQE30-endo-xylanase-lx1 plasmid developed on the basis of vector pQE30. The invention allows obtaining a strain that will produce endoxylanase with stability at the temperature of above 55°C.

EFFECT: possibility of using a strain as a part of complex ferment preparations for hydrolysis of plant biomass.

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Description

The invention relates to the field of biotechnology and can be used to obtain endoxylanase (endo-β-1,4-xylanase), applicable in technological processes of enzymatic hydrolysis of plant biomass.

Endoxylanases (EC 3.2.1.8) belong to a wide class of glycoside hydrolases - a large class of enzymes that carry out a wide range of reactions, including the breakdown of cellulose and hemicellulose to monosaccharides. In particular, xylanase class enzymes catalyze the breakdown of xylan to xylo-oligosaccharides. The biological function of endoxylanase is the cleavage of xylans at 1-4 beta-xylosidase bonds.

The industry mainly uses cellulase of fungal origin. There are also promising thermostable exoglucanases isolated from the genomes of bacteria of the genus Clostridium. Clostridium exoglucanases are most active at temperatures of 60-75 ° C and pH 5.0-6.5. Crystalline cellulose in plant biomass is largely coated with hemicellulose and lignin, and therefore, for the hydrolysis of plant biomass it is important to use not only a complex of cellulolytic enzymes, but also enzymes capable of degrading hemicellulose. Moraïs showed a significant increase in the enzymatic activity of the endoglucanase and exoglucanase complex by the addition of endoxylanase and exoxylanase compared to both free enzymes and separately assembled cellulase and xylanase complexes [1].

Commercially available preparations containing a complex of cellulases are known: "CelloluxA" manufactured by JSC Sibbiopharm based on the fungus Trichoderma reesei and Viscozyme L manufactured by Novozyme based on the fungus Aspergillus sp. The disadvantage of both drugs is the low efficiency of hydrolysis of the hemicellulose component of plant biomass, which leads to the loss of part of the target product.

Known recombinant strain of Aspergillus aculeatus, producing enzymes having xylanase activity and used in baking [2].

Known strain Penicillium verruculosum VKM F-3972D, a producer of a complex of cellulases, xylanase and xyloglucanase, for the hydrolysis of cellulose and hemicellulose [3]. The strain P.verruculosum PV2007 VKM F-3972D is obtained by multistage mutagenesis and selection from the original culture of P.verruculosum 28K VKM F-3764D. The spore suspension of the original strain is irradiated with ultraviolet light. Irradiated spores are plated on Petri dishes with selective media, cultured at 28 ° C for 2 days, and Congo Red is stained. Mutants with improved production are selected visually for the increased areas of enlightenment around the colonies. The most active mutants selected on the plates are tested for the productivity of the synthesis of cellulases, cellobiases (beta-glucosidases), xylanases and xyloglucanases when cultured in a liquid medium in flasks. The most active variants selected during cultivation in flasks are again (repeatedly) subjected to irradiation and selection on dishes and flasks, as described above.

Closest to the claimed strain, the prototype, is a strain of the fungus Penicillium canescens, a producer of secreted endo (1-4) beta-xylanase, constructed by transformation and genetic engineering [4]. This strain for 120-144 hours of fermentation in media with beet pulp allows you to accumulate up to 800 units of activity of secreted endo (1-4) beta-xylanase and not more than 0.13 units of cellulase activity in 1 ml of culture fluid.

A disadvantage of the known strain is the low thermal stability of the resulting endoxylanase and the presence of a large number of side activities.

The objective of the invention is to obtain a bacterial strain producing endoxylanase with high thermal stability, producing pure endoxylanase without cellulase impurities for use as part of complex biocatalysts in the hydrolysis of plant biomass.

The problem is solved by the proposed strain of Esherichia coli EX pQE30, containing the recombinant plasmid pQE30-endo-xylanase-lxl producing bacterial endoxylanase Geobacillus stearothermophilus 22 isolated from a hot spring in the Northern Baikal region.

Endoxylanase has high thermal stability (above 55 ° C), which allows its use in biocatalysts for hydrolysis of plant biomass. The strain is obtained by transforming the cells of the bacterium E. coli XL1-blue DNA of the plasmid pQE30-endo-xylanase-lxl using standard procedures [5]. The selection of transformants is carried out on LB medium with ampicillin (100 mg / l). The plasmid pQE30-endo-xylanase-lxl is assembled on the basis of the pQE30 vector and contains the origin of replication pMB1, the bla ampicillin resistance gene (encoding β-lactamase), which provides for the selection of cells with a plasmid, the T5 phage promoter under the control of the lactose operon operator, and the constructed gene encoding a hybrid protein containing a sequence of 6 histidines and an endoxylanase sequence of Geobacillus stearothermophilus 22.

A genetic and physical map of the pQE30-endo-xylanase-lxl expression construct carrying the gene encoding the endoxylanase enzyme is shown in FIG. 1, where: endo-xylanase is the sequence encoding the endoxylanase of the Geobacillus stearothermophillus 22 strain; AmpR is the ampicillin (beta-lactamase) resistance gene, T5 is the T5 phage promoter, ORI is the origin of replication.

Figure 2 shows the amino acid sequence of the endoxylanase strain of Geobacillus stearothermophillus 22 and the nucleotide sequence of the gene encoding it.

The strain Esherichia coli EX pQE30 has the following characteristics.

Cultural and morphological characters.

The strain is an optional aerobic. When growing on LB agar, the colonies are smooth, round, shiny, grayish, cloudy, the edge is even. When grown in liquid media, LBs form intense turbidity. Cells are straight, rod-shaped, motile, gram-negative. The strain is resistant to ampicillin.

Genetic signs, antibiotic resistance.

The strain is resistant to ampicillin due to the presence in the cell of the plasmid pQE30-endo-xylanase-lxl, carrying the gene for resistance to ampicillin.

Storage conditions of the strain.

The strain Escherichia coli EX pQE30 is stored in Luria Bertani medium with glycerol (t = -70 ° C) for at least a year or on Luria Bertani agar medium (t = + 8 ° C) with 100 μg / ml ampicillin in Petri dishes or in glass tubes with slanted agar at 4 ° C. Reseeding on fresh Wednesday do once a month.

The invention is illustrated by the following examples.

Example 1. Obtaining plasmids pQE30-endo-xylanase-lxl and strain Esherichia coli EX pQE30.

The cloning of endoxylanase enzymes found in the genome of a strain of 22 bacteria of the genus Geobacillus from the laboratory collection was performed in E. coli. Cloning of the genes in the expression construct was performed using primers containing uracil. Amplification of the insert fragments and the vector construct was carried out using the pfr turbo ^cx enzyme (Stratagene). Gene fragments and vector constructs were processed using the USER ^™ enzyme complex and MalI methyl-dependent restrictase (SibEnzyme Novosibirsk). The enzymatic reaction was carried out in a taqSE polymerase enzyme buffer (SibEnzyme Novosibirsk) at 37 ° C for 30 minutes. For ligation, the processed fragments of the insert and the fragment of the vector construct were mixed, heated to 43 ° C. To the mixture was added heated equal volume of a single ligase buffer containing ligase and dATP. Ligation was carried out in a thermocycler using the following temperature program: 43 ° С - 2 minutes, 30 cycles (43 ° С - 10 seconds, 37 ° С - 5 seconds, 32 ° С - 5 seconds, 27 ° С - 10 seconds, 22 ° C - 15 seconds), 25 ° C storage.

The resulting ligation products were precipitated with an equal volume of isopropanol and dissolved in 8 μl of deionized water.

The recombinant strain of Esherichia coli EX pQE30 carrying the plasmid pQE30-endo-xylanase-lxl was obtained by electroporation.

Example 2. Identification of recombinant endoxylanase

Protein expression was studied by recombinant strain of Esherichia coli EX pQE30. For the study, biomass was generated and the expression of the cloned gene was induced.

The accumulation and induction of biomass was carried out as follows. A colony of the recombinant strain was seeded in 20 ml of LB medium (yeast extract 5 g / l, tryptone 10 g / l, NaCl 10 g / l) containing ampicillin 100 μg / ml in a 100 ml conical flask, cultured overnight on an orbital neck BioSan ES- 20/60 at 37 ° C and 200 rpm.

1/20 of the overnight culture was added to 20 ml of LB medium in a conical flask. Cultivated on the orbital neck of the BioSan ES-20/60 at 37 ° C and 250 rpm to achieve an optical density of OD ₆₀₀ = 0-6. Upon reaching the required optical density, the following components were added to the culture: ampicillin 200 μg / ml, IPTP (Isopropyl-β-D-1-thiogalactopyranoside) 0.5 mmol / liter. Further cultivation was performed on the orbital neck of BioSan ES-20/60 at 37 ° C and 250 rpm for 5 hours.

The protein extract was separated using SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis). For this, 150 μl of the cell culture of E. coli clones carrying pQE30 expression vectors with the integrated enzyme endoxylanase were precipitated by centrifugation at 16100 g for 2 min. To the pellet was added 30 μl of lysis buffer containing 0.05 M Tris-HCl pH, 6.8; 2% SDS (sodium dodecyl sulfate), 0.002% bromophenol blue, 10% glycerol, 5% mercaptoethanol, the mixture was resuspended and sonicated in an ultrasonic bath for 15 minutes. The resulting lysate was centrifuged for 2 min at 16100 g, the supernatant was boiled for 5 min at 95 ° C and applied onto SDS polyacrylamide gel for electrophoresis according to the Laemmli method [6] in a mini-gel (8 × 10 cm, 0.75 mm thick). For electrophoresis of proteins after chromatographic purification, the samples were mixed in a 1: 1 ratio with application buffer (0.05 M Tris-HCl pH 6.8; 2% SDS); 0.002% bromphenol blue; 10% glycerin; 5% mercaptoethanol), then boiled for 5 minutes. 20 μl of each sample was applied to the lane, proteins were concentrated in 4% SDS page-SDS (acrylamide / bisacrylamide in the ratio of 37.5: 1, 0.1% SDS, 0.125 mm Tris-HCl pH 6.8, 0.1 % TEMED, 0.05% PSA) at a current of 10 mA per gel and was separated in 12% SDS page-SDS (acrylamide / bisacrylamide in the ratio of 37.5: 1, 0.1% SDS, 0.375 mm Tris-HCl pH 8.8, 0.05% TEMED, 0.05% PSA) at a current of 15 mA per gel in the electrophoretic chamber Mini-PROTEAN® Tetra Cell (Bio-Rad, USA). The gels were stained with Coomassie Brilliant Blue G-250 and photographed using a VersaDoc MP4000 gel documentation system (Bio-Rad, USA).

In FIG. Figure 3 shows the electrophoregram of the proteins of E. coli clones carrying the pQE30 expression vector with the integrated enzyme endoxylanase (LX1). 12% SDS-PAGE, Coomassie Brilliant Blue G-250 dye. 1-3, 5 - no expression; 4 - LX1; 6 is a broad spectrum molecular weight marker SDS-PAGE Molecular Weight Standards, Broad Range (Bio-Rad, USA). The arrow marks the fraction cut for mass spectrometric analysis.

The protein was identified by a tryptic mass map using the Mascot search algorithm (http://matrixscience.com/home.html). The search parameters were set as follows: mass error ± 0.05 Yes, the number of trypsin underexposure sites is 1, possible modifications are methionine oxidation and urea methylation of cysteines, the database is NCBI. The spectrum of tryptic hydrolyzate of endoxylanase is presented in figure 4.

Example 3. Determination of optimum pH and temperature for the manifestation of xylosidase activity of endoxylanase

The optimum pH and temperature for the xylosidase activity of endoxylanase were determined using a synthetic substrate 4-nitrophenyl-β-D-xylopyranoside, one of the enzymatic cleavage products of which is p-nitrophenol.

To determine the activity of the enzyme at various pH and temperature, reaction mixtures were prepared containing 8 μl of 10 mM aqueous solution of 4-nitrophenyl-β-D-xylopyranoside and 72 μl of 0.02 M phosphate buffer with pH values from 4 to 8 in increments at 0.5.

All reaction mixtures were prepared in human-cooled cells of a 96-well PCR plate. For each pH value, 6 repetitions of each point from a selected range of pH values were prepared. Then, 10 μl of the enzyme solution in 0.01 M phosphate-buffered saline was added to each well. The plate was incubated for 15 min in an iCycler thermocycler (BioRad) in a mode of stable maintenance of a temperature gradient of 45-70 ° С, then the reaction was stopped by adding 200 μl of a 1 M aqueous solution of sodium carbonate and 10 μl of a 0.5 M aqueous solution of sodium citrate in each well. The amount of p-nitrophenol formed as a result of the reaction was determined on an Epoch spectrophotometer (BioTek) at a wavelength of 420 nm. Based on the amount of product formed and the reaction time, relative enzyme activity values were calculated. The data are presented in figure 4, which shows that in the pH range from 4.5 to 5.5 and at a temperature of 55 to 65 ° C, a stable plateau is observed, in this range the enzyme is stable, although it exhibits a low level of activity. When the pH values shift to the neutral and slightly alkaline ranges (from 6.0 to 7.5), a marked increase in the enzyme activity is observed at relatively low temperatures (from 45 to 55 ° C). The optimal temperature and pH for endoxylanase when working with 4-nitrophenyl-β-D-xylopyranoside were pH = 5.5-6.0 at 55 ° C.

The proposed strain has the ability to produce thermostable pure endoxylanase without admixture of cellulases.

Information sources

1. Moraïs S., Barak Y., Caspi J., Hadar Y., Lamed R., Shoham Y., Wilson D.B., Bayer E.A. Cellulase-xylanase synergy in designer cellulosomes for enhanced degradation of a complex cellulosic substrate // MBio. 2010.1 (5). doi: pii: e0028510.10.1128 / mBio.00285-10.

2. Patent US 6197564 B1, publ. March 6, 2001

3. Patent RU 2361918 C1, publ. July 20, 2009

4. Patent RU 2293115 C1, publ. 02/10/2007

5. Miller E.M., Nickoloff J.A. Escherichia coli Electrotransformation // Methods in Molecular Biology. 1995. V. 47. P.105-113.

6. Laemmli, U.K., Nature 1970, 227, 680-685.

<110> Federal State Budgetary Institution of Science Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (ICG SB RAS)

<120> The strain of the bacterium Esherichia coli EX PQE30, producer of endoxylanase bacteria Geobacillus stearothermophillus 22

<160> SEQ ID NO

<210> 1

<211> 1026

<212> DNA

<213> Geobacillus stearothermophillus 22

<400> 1

Claims (1)

The strain of the bacterium Esherichia coli EX PQE30 obtained by transformation of the E. coli strain XL1-blue plasmid pQE30-endo-xylanase-lxl producing thermostable endoxylanase of the bacterium Geobacillus stearothermophillus 22.

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