KR101062978B1 - Production Optimization and Gene Cloning of Endoglucanases Derived from the New Bacterium Penicillium Pinophilum ＫＭＪ601 - Google Patents

Abstract

The present invention provides a method for producing endoglucanase (endo-1,4-glucanase, EG), one of cellulose degrading enzymes from a novel strain, Penicillium pinophilum , establishing an optimal condition, an EG enzyme, and an enzyme thereof. The present invention relates to a gene encoding, and more particularly, to a method for producing high activity EG from penicillium pinofilum KMJ601, establishing optimum conditions, and an EG enzyme and a gene encoding the enzyme.

Expression Production, Cellulase, Endoglucanase Gene, Penicillium Pinophyllum

Description

Optimization of endo-β-1,4-glucanase production by a novel strain Penicillium pinophilum KMJ601 and its gene cloning}

The present invention relates to a method for mass-producing an endo-β-1,4-glucanase enzyme from the new strain penicillium pinofilum KMJ601, the endoglucanase enzyme, and a gene encoding the enzyme.

Cellulose is one of the most abundant organic and renewable resources on earth, but most of the cellulose is now a major source of environmental pollution, inundated by agricultural and forestry waste. Therefore, the development of an efficient saccharification process can be a great help in solving food, fuel and environmental problems. The world's agricultural and forestry waste is more than 3 billion tonnes annually, with more than 800 million tonnes produced in Asia alone. It consists mainly of cellulose and hemicellulose, so the production of monosaccharides, including glucose by their saccharification, is an important raw material production technology of bioenergy resources.

There have been many studies for glycosylating cellulose with high efficiency for a long time, and a lot of research has been focused on the development of glycase. As a result, various uses of these glycosylases have been developed and commercialized in various fields, and new applied researches are being actively conducted. Cellulase is widely used in the textile industry, the paper industry, the detergent industry, the feed industry, etc. In addition to the food industry, it is applied to various uses such as the production of low-calorie foods and the fermentation of food waste.

On the other hand, the cell wall of the plant is composed of polymers such as cellulose (insoluble β-1,4-glucan fiber), hemicellulose (hemicellulose, non-cellulose polysaccharide), and lignin (lignin, polysaccharide of complex polyphenol structure). Cellulose, the most abundant of its components, is a homopolymer with glucose units linked by β-1,4 bonds to break it down into monosaccharides, endo-β-1,4-glucanase (endo-β-1,4-glucanase). ) [EC 3. 2. 1. 4], exo-β-1,4-glucanase [EC 3. 2. 1. 91], beta-glucosidase ( Three enzymes are required: β-glucosidase (EC 3. 2. 1. 21). Endo-glucanase randomly cleaves β-1,4 glucose bonds from the inside and exo-glucanase cleaves into cellobiose, a glucose disaccharide at the non-reducing sugar end. Cellobiose is finally degraded into glucose by beta-glucosidase.

Conventionally, the production of these enzymes mainly used fungi (fungi), in particular in the industrial aspect the production of enzymes (Aspergillus) and Trichoderma (Trichoderma). As a cellulase-producing strain, Trichoderma reesei ATCC 56764 has been intensively studied as a representative strain, but it has a disadvantage in that the production concentration and activity of the enzyme are not high enough. The process of producing ethanol from biomass has many advantages such as regeneration of raw materials and environmental friendliness of the produced fuel, but the production cost is very high, which impedes practical use. The largest part of the cost of this ethanol production process is about 60% of the total cost of glycosylase production. Therefore, there is an urgent need for the development of highly active cellulase and highly active strains producing the same.

The present invention solves the above problems and the object of the present invention is to provide a method for selecting a strain producing high activity cellulase and using the strain to produce an optimal cellulase.

Still another object of the present invention is to provide a method for cloning a cellulase enzyme gene from the strain.

In order to achieve the above object, the present invention provides a penicillium pinophilum KMJ601 (Accession No. 93063P), which produces a highly active cellulase endo-β-1,4-glucanase.

The present invention also provides a method for producing endo-β-1,4-glucanase comprising culturing the strain of the present invention.

In one embodiment of the present invention, the culture temperature in the culture step is preferably 23 ~ 32 ℃, the stirring conditions are preferably 100 to 300 rpm, but is not limited thereto.

The present invention also provides an endo-β-1,4-glucanase having the amino acid sequence set forth in SEQ ID NO: 4. Endo-β-1,4-glucanase of the present invention is preferably exemplified in SEQ ID NO: 4, but one or more substitutions, deletions, additions, etc. of the amino acid sequence of SEQ ID NO: 4 All mutant endo-β-1,4-glucanases with activity of -1,4-glucanase are also included in the scope of protection of the present invention.

In a preferred embodiment of the present invention, the enzyme having endo-β-1,4-glucanase activity has a sequence identity of at least 80%, at least 85%, at least 90%, to the amino acid sequence set forth in SEQ ID NO: 4, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%.

In the present invention, the polypeptide has a certain ratio (eg, 80%, 85%, 90%, 95%, or 99%) of sequence identity to another sequence, when aligning the two sequences, By comparison it is meant that the amino acid residues in this ratio are identical. The alignment and percent homology or identity may be determined by any suitable software program known in the art, such as those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al. (Eds) 1987 Supplement 30 section 7.7.18). Can be determined using Preferred programs include the GCG Pileup program, FASTA (Pearson et al. 1988 Proc. Natl Acad. Sci USA 85: 2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, MD, and Altschul et al. 1997 NAR25: 3389-3402. Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, PA), which preferably uses basic parameters. Another sequence software program that can be used is the TFASTA Data Searching Program available from Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).

The present invention also provides a gene encoding the endo-β-1,4-glucanase. In a preferred embodiment of the present invention, the gene preferably has the nucleotide sequence set forth in SEQ ID NO: 5, but the gene encoding the mutated present invention protein is also included in the protection scope of the present invention, in consideration of the genetic code Also included in the genes of the present invention are sequences having at least 85%, preferably 90%, more preferably at least 95% homology with the sequence set forth in SEQ ID NO: 5.

Endo -β-1,4- glucanase of the invention penny silryum Pinot pilreom separated from the water or supernatant of the culture supernatant (Penicillium pinophilum), and can be purified, and Penny using a genetically engineered recombinant techniques silryum Pinot pilreom (Penicillium pinophilum) In addition, it can be produced and separated by strain or artificial chemical synthesis.

When using recombinant technology, it is possible to use factors used for ease of expression of conventional recombinant proteins, such as antibiotic resistance genes, reporter proteins or peptides that can be used for affinity column chromatography, which techniques belong to the present invention. Those skilled in the art fall within a easily implemented range.

The endo-β-1,4-glucanase gene of the present invention is isolated from penicillium pinocylum cells. First, chromosomal DNA is obtained from a strain having an endo-β-1,4-glucanase gene. The separation method of chromosomal DNA can use a well-known method.

For example, after incubating the Penicillium Pinophyllum strain with an appropriate carbon source in LB medium or M9 medium, for example, the method of Marmer et al. (Journal of Molecular Biology, 3, p. 208) , 1961) and the like to prepare chromosomal DNA. The chromosomal DNA obtained by this method was digested with a suitable restriction enzyme (eg Sau3AI, etc.), and the digested product having a suitable fragment length was cleaved with a linkage restriction enzyme (eg BamHI, etc.). Link to and construct a genetic library. The suitable fragment length here is about 4000-25000 base pairs when using a normal plasmid vector, and about 15000-30000 base pairs when using a cosmid or phage vector. As a method of separating and taking a DNA fragment of an appropriate length, a known method such as a method using a sucrose density gradient or agarose gel (Molecular Cloning, Cold Spring Harbor Laboratory Publication (1982)) can be used.

As the vector, a phage vector or plasmid vector capable of autonomous propagation in host microorganisms is used. As a phage vector or cosmid vector, pWE15, M13, (lambda) EMBL3, (lambda) EMBL4, (lambda) FIXII, (lambda) DASHII, (lambda) ZAPII, (lambda) gt10, (lambda) gt11, Charon4A,

Charon21A etc. are mentioned, As a plasmid vector, pBR system, pUC system, pBluescriptII system, pGEM system, pTZ system, pET system, etc. are mentioned, for example. In addition, various vectors such as vectors capable of autonomous propagation in a plurality of host microorganisms such as E. coli and Pseudomonas genus

You can also use a shuttle vector. Also for such a vector, desired fragments can be obtained by cleaving with a suitable restriction enzyme as described above.

The linkage between the chromosomal DNA fragment and the vector fragment may be performed by using a DNA ligase, for example, by using a ligation kit (manufactured by Takara Co., Ltd.). In this way, the chromosomal DNA fragment and the vector fragment are linked to prepare a mixture of recombinant plasmids containing various DNA fragments (hereinafter referred to as "gene library"). Herein, in the production of gene libraries, in addition to using chromosomal DNA fragments of appropriate length, extracting and purifying mRNA from Bacillus licheniformis strain, and synthesizing cDNA fragments using reverse transcriptase (Molecular Cloning, Cold Spring Harbor Laboratory) , 1982). In addition, the gene library can be transformed or transduced into E. coli once, and then a large amount of the gene library can be amplified (Molecular Cloning, Cold Spring Harbor Laboratory, 1982).

Introduction of the recombinant vector into a host microorganism is performed by a well-known method. For example, if the host microbe is Escherichia coli, the Calcium Chloride Method (Vol. 53, p. 159, 1970), the Method of Enzymology (Vol. 68, p. 253)

1979) or the Electroporation Act (Current Protocols in Molecular Biology, Vol. 1, p. 184, 1994). In addition, introductory packaging method (Current Protocols in

Molecular Biology, Vol. 1, p. 571, 1994). In addition, a method by junction transfer is also available.

Next, a probe for obtaining a DNA fragment containing the endo-β-1,4-glucanase gene of the penicillium pinofyllum strain is prepared. The nucleotide sequence of the endo-β-1,4-glucanase gene designs an oligonucleotide from the nucleotide sequence as described in SEQ ID NO: 5. These oligonucleotides can be synthesized using, for example, a custom synthesis service of Amarsham-Pharmacia Biotech.

Next, a polymerase chain reaction (hereinafter, referred to as "PCR") is performed by using the designed oligonucleotide as a primer, and using the chromosomal DNA of the penicillium pinofilum strain as a template, and then endo-β-1,4-glucan Partially amplifies the aze gene. The PCR amplification fragment thus obtained was a fragment having a homology close to 100% to the endo-β-1,4-glucanase gene of the penicillium pinophyllum strain, and a high S as a probe for colony hybridization. The / N ratio can be expected, and the stringency control of the hybridization can be made easy. The PCR amplification fragment is labeled with a suitable reagent, and colony hybridization is performed on the chromosomal DNA library to select an endo-β-1,4-glucanase gene (Current Protocols in Molecular Biology, 1). Vol., P. 603, 1994).

Endo-β-1,4-glucanase gene by recovering the plasmid from the E. coli selected by any of the above methods using the alkaline method (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994) DNA fragments containing can be obtained. The base sequence of the DNA fragment can be determined by, for example, a Sanger's sequencing method (Molecular Cloning, Vol. 2, p. 133, 1989) and the like. The sequencer 377A (Perkin Elmer) etc. can be used by the diprimer method or the diterminator method.

After determining the entire nucleotide sequence by the above method, the DNA fragment prepared by any method, such as chemical synthesis, PCR using chromosomal DNA as a template, or digestion by restriction enzymes of the DNA fragment having the nucleotide sequence. By hybridizing with a probe, it is possible to obtain the gene of the present invention.

SEQ ID NO: 4 indicates the amino acid sequence encoded by the gene, but as described above, as long as the polypeptide having the amino acid sequence has endo-β-1,4-glucanase activity, for example, There may be variations such as deletion, substitution, addition, etc. with respect to one or several amino acids. In addition to having the nucleotide sequence represented by SEQ ID NO: 5, the gene of the present invention also includes decondensates encoding the same polypeptides which differ only in degenerate codons. Herein, mutations such as deletions, substitutions, and additions can be introduced by site mutation introduction methods (Current Protocols in Molecular Biology, Vol. 1, p. 811, 1994) and the like.

The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when producing the recombinant vector. As a host, the genus Esherichia, Pseudomonas, Ralstonia

Genus Ralstonia, genus Alcaligenes, genus Comamonas, genus Burkholderia, genus Agrobacterium, genus Flabobacterium, genus Vibrio , In Enterobacter, Rizzo

Genus Rhizobium, Genus Gluconobacter, Genus Acinetobacter, Genus Moraxella, Genus Nitrosomonas, Genus Aeromonas, Genus Paracoccus Genus, Bacillus genus, Clostridium genus, Lactobacillus genus, Corynebacterium genus, Arthrobacter genus, Achromobacter genus, Micro Mycobacterium genus Micrococcus

Genus, Genus Streptococcus, Genus Streptomyces, Genus Actinomyces, Genus Nocardia, Methylbacterium

Various bacteria, such as the genus (Methylobacterium), are mentioned. In addition to the above bacteria, yeasts such as Saccharomyces genus and Candida genus, as well as various molds, may be mentioned as hosts.

For example, when a bacterium such as E. coli is used as a host, the recombinant vector according to the present invention can autonomously replicate in the host and contain a promoter, an endo-β-1,4-glucanase gene. It is preferable to have a configuration necessary for the expression of DNA, transcription termination sequence and the like.

Any promoter can be used as long as it can be expressed in a host. For example, a trp promoter, a trc promoter, a tac promoter, a lac promoter, a PL promoter, a PR promoter, a T7 promoter, a T3 promoter, or the like can be used. Derived promoters can be used. As a method of introducing recombinant DNA into bacteria, the calcium chloride method, electroporation method and the like described above can be used.

When yeast is used as a host, for example, YEp13, YCp50, pRS-based, pYEX-based vectors and the like can be used as expression vectors. As a promoter, a GAL promoter, an AOD promoter, etc. can be used, for example. As a method of introducing recombinant DNA into yeast

For example, the electroporation method (Method Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), acetic acid Lithium method (J. Bacteriol., 153, 163-168 (1983)) and the like.

Recombinant vectors also contain fragments for expression inhibition with various functions for inhibiting or amplifying or inducing expression, markers for selection of transformants, resistance genes for antibiotics, or secretion out of cells. It is also possible to have a gene encoding the desired signal again.

Production of the endo-β-1,4-glucanase enzyme according to the present invention is carried out by culturing a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same, Or endo-β-1,4-glucanase, which is a gene product, in the culture supernatant), and is obtained by obtaining an endo-β-1,4-glucanase enzyme from the culture.

As a method for culturing the transformant of the present invention, any conventional method used for culturing a host may be used.

As the culture method, any method used for culturing ordinary microorganisms, such as batch type, flow batch type, continuous culture, and reactor type, can be used. Transformants obtained by using bacteria such as Escherichia coli as a host As a medium for culturing, medium or synthetic medium such as LB medium, M9 medium and the like can be given. Incidentally, the culture temperature is cultured in the above-mentioned temperature range to accumulate the endo-β-1,4-glucanase enzyme in the cells and recover.

The carbon source is required for the growth of microorganisms, and for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol and butanol; Polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid and gluconic acid; Fatty acids such as propionic acid, butanoic acid, pentanic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid can be used.

Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, as well as those derived from natural products such as peptone, meat juice, yeast extract, malt extract, caseinate and corn steep liquor. Moreover, as an inorganic substance, 1 potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, etc. are mentioned, for example. You may add antibiotics, such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin, to a culture liquid.

When culturing a microorganism transformed using an inducible expression vector, a promoter may be added to the medium suitable for the type of promoter. For example, isopropyl-β-D-thiogalactopyranoside (IPTG), tetracycline, indole acrylic acid

(IAA) etc. can be mentioned as an inducer.

Acquisition and purification of arabinose isomerase is carried out by centrifugation of the cells or supernatants from the cultures obtained, by cell disruption, extraction, affinity chromatography, cation or anion exchange chromatography, gel filtration, etc. I can do it.

Confirmation that the obtained purified substance is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis, western blotting or the like.

In addition, the culture of transformants using microorganisms as a host, the production of endo-β-1,4-glucanase enzymes by the transformants and accumulation in cells, and endo-β-1 from cells, Recovery of the 4-glucanase enzyme is not limited to the above method.

Hereinafter, the present invention will be described.

In the production method of the cellulase of the present invention, cellulose 10-30g / L, corn steep liquor 8-10g / L, yeast extract 2-5g / L, potassium monophosphate 3-5g / L, potassium diphosphate 3-5g / It is preferable to use a production medium for cellulase containing L and thiamine hydrochloride 0.005 ~ 0.01 g / L, but is not limited thereto.

In addition, the present invention is a cellulase production method, the stirring speed is 100 to 500 rpm, preferably 100 to 300 rpm, the culture temperature is 23 to 33 ℃ preferably 23 to 30 ℃, but preferably incubated at this time No.

In another aspect, the present invention is characterized by cloning the cellulose dehydrogenase EG gene from penicillium pinopylum through Southern hybridization and colony hybridization.

The high activity cellulase produced by the new strain penicillium pinofilum KMJ601 isolated from mushrooms through the present invention exhibits a higher degradation rate than conventional cellulose degrading enzymes, thus producing bioenergy, textile, paper, detergent, feed, and food. It can be applied to various applications in the industry, such as the production of low calorie foods and the fermentation of food waste. In addition, by identifying the entire sequence of the highly active endo-β-1,4-glucanase gene of penicillium pinofilum, it can be applied to mass production of highly active endo-β-1,4-glucanase.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

Example 1 Screening of Cellulase Producing Bacteria

In order to isolate cellulase-producing strains, 10ul of various cultures of mushrooms were suspended in 10 ml of physiological saline, and 10ul (1x10 ⁴ cfu ml ^-1 ) of the suspension was taken. agar) and incubated at 27 ^o C for 3 days. After colonies were formed in solid agar medium, stained with 0.1% Congo red reagent, and decolorized with 1 M sodium chloride to select the bacteria that produced fibrinolytic ring around the colonies. It was.

Solid agar to which carboxymethyl cellulose was added as described above using Tricoderma Reese ZU-02, which was used as a conventional cellulase producing strain as the control (C), the first selected strain (S1 to S7) through the above search process. After confirming the fibrinolytic ability in the medium, the best S3 strain was selected.

Example 2: Identification of Strains

In order to identify the S3 strain isolated in Example 1, the ITS-5.8S rDNA sequence was analyzed at the Korea Microorganism Conservation Center. The ITS-5.8S rDNA sequence of the S3 strain is shown in SEQ ID NO: 6.

As a result of analyzing the soft relationship with the similar species of the ITS-5.8S rDNA sequence of the S3 strain, it was identified as penicillium pinopylum (FIG. 2).

The S3 strain was named Penicillium pinophilum KMJ601, and was deposited with KACC 93063P on November 7, 2008 at the National Institute of Agricultural Science.

Example 3: Basic Production Experiment of Cellulase by the Strain in an Erlenmeyer Flask

Basic production experiment of the cellulase by the penicillum pinopylum KMJ601 strain isolated in Example 1 was carried out under the following conditions. The isolated strain was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of production medium (Table 1) and incubated at 200 rpm and 28 ° C. for 5 days. The culture medium from which the cells were removed was analyzed to determine the activity of endo-β-1,4-glucanase. As a result, it was identified as endo-β-1,4-glucanase 7.3 U / mg-protein.

ingredient Concentration (g / L) Corn Soak 8 Yeast extract 2 Potassium monophosphate (KH ₂ PO ₄ ) 5 Potassium Diphosphate (K ₂ HPO ₄ ) 5 Straw 20 Thiamine Hydrochloride 0.005

Table 1 is a table showing the composition of the cellulase base production medium.

Example 4: Optimal Media Component Tests for Cellulase Production of Strains

(1) Endo-β-1,4-glucanase activity according to the concentration of carbon source

Cellulase production experiments of the inventive strain penicillum pinofilum KMJ601 according to cellulose concentration in a 7 L fermenter were performed.

Species cultivation: A single colony of penicillium pinopylum was inoculated into a 500 mL flask containing 50 mL of pre-culture medium (PDB: potato dextrose broth) and incubated at 200 rpm and 30 ° C. in a shaker for 3 days.

ingredient Concentration (g / L) Potato starch 4 Dexrose 20

Table 2 is a table showing the composition of the composition of the seed medium (PDB).

Main culture: 200 mL of the seed culture solution was inoculated into a 7 L fermenter containing 4000 mL of production medium, and the main culture was performed at 200 rpm, 28 ° C., and pH 4 for 5 days. The volume of the production medium was 4L and the stirring speed during the fermentation was 200rpm, the culture temperature was adjusted to 28 ℃. Experimental results of varying the initial cellulose concentration of 10 ~ 40g / L as shown in Table 3 and showed the maximum cellulase activity at a cellulose concentration of 20g / L.

g / L Endo-β-1,4-glucanase (u / mg)

Cellulose 10 1.53 20 2.15 30 1.00 40 0.99

Table 3 shows the production of endo-β-1,4-glucanase at different rice straw concentrations.

(2) Endo-β-1,4-glucanase activity test according to the concentration of nitrogen source (corn immersion liquid)

Experiments were performed for each concentration of nitrogen source (corn immersion liquid) using a cellulose concentration of 20 g / L in a 7 L fermenter. The culture method and the fermentation medium were the same as in Example 3, and the results of the cultivation by varying the nitrogen source concentration from 6 to 12 g / L were as shown in Table 4, and showed the maximum activity in 8 g / L corn steep liquor.

g / L Endo-β-1,4-glucanase (U / mg-protein) Corn Soak


6 2.15 8 2.78 10 2.01 12 0.94

Table 4 shows the activity of endo-β-1,4-glucanase at concentrations of various corn steep liquors.

(3) Endo-β-1,4-glucanase activity test according to yeast extract concentration

Rice straw and corn steep liquor concentration in the 7L fermentation tank was 20g / L, 8g / L, respectively, the concentration of the yeast extract was tested. The culture method and the fermentation medium were the same as in Example 3, and the results of the cultivation with different concentrations of yeast extract at 1-5 g / L were as shown in Table 5, showing the maximum activity in 2 g / L yeast extract.

g / L Endo-β-1,4-glucanase (U / mg-protein) Yeast Extract



One 2.78 2 3.14 3 2.56 4 1.77 5 1.58

Table 5 shows endo-β-1,4-glucanase enzymatic activity at various yeast extract concentrations.

(4) Endo-β-1,4-glucanase activity test according to potassium monophosphate concentration

The concentration of cellulose, corn steep liquor and yeast extract in the 4 L fermentation tank were 20g / L, 8g / L and 2g / L, respectively. The culture method and the fermentation medium were the same as in Example 3, and the results of the cultivation at different concentrations of potassium monophosphate at 3-6 g / L were as shown in Table 6, showing the maximum activity at 5 g / L potassium monophosphate.

g / L Endo-β-1,4-glucanase (U / mg-protein) Potassium monophosphate


3 3.14 4 3.32 5 4.34 6 2.76

Table 6 shows the various potassium monophosphate concentrations. Endo-β-1,4-glucanase enzyme activity is shown.

(5) Endo-β-1,4-glucanase activity test according to potassium diphosphate concentration

In the 7L fermenter, the concentrations of potassium phosphate were measured using cellulose, corn steep liquor, yeast extract and potassium monophosphate concentrations of 20g / L, 8g / L, 2g / L and 5g / L, respectively. The culture method and the fermentation medium were the same as in Example 3, and the results of the incubation at different concentrations of potassium diphosphate at 3 to 5 g / L were as shown in Table 7, and showed maximum activity at 5 g / L potassium diphosphate.

g / L Endo-β-1,4-glucanase (U / mg-protein)

Potassium Diphosphate

3 4.34 4 4.66 5 5.48 6 3.92

Table 7 shows endo-β-1,4-glucanase enzymatic activity at various potassium diphosphate concentrations.

(6) Endo-β-1,4-glucanase activity test according to thiamine hydrochloride concentration

The cellulose, corn steep liquor, yeast extract, potassium monophosphate, and potassium diphosphate in the 7 L fermentation tank were tested at concentrations of 20 g / L, 8 g / L, 2 g / L, 5 g / L, and 5 g / L, respectively. Was performed. The culture method and the fermentation broth were the same as in Example 3, and the cultivation of the thiamin hydrochloride at a concentration of 0.003 to 0.008 g / L was as shown in Table 8, showing the maximum activity at 0.005 g / L thiamin hydrochloride.

g / L Endo-β-1,4-glucanase (U / mg-protein) Thiamine Hydrochloride




0.003 5.48 0.004 5.49 0.005 6.31 0.006 5.00 0.007 4.67 0.008 4.43

Table 8 shows endo-β-1,4-glucanase enzymatic activity at various potassium diphosphate concentrations.

Example 5 Experimental Optimal Culture Conditions for Highly Active Endo-β-1,4-Glucanase Production

The concentrations of cellulose, corn steep liquor, yeast extract, potassium monophosphate, potassium diphosphate and thiamin hydrochloride in 7 L fermenters were 20 g / L, 8 g / L, 2 g / L, 5 g / L, 5 g / L, and 0.005 g / L, respectively. The optimization experiment of the culture environmental conditions was performed. As a result of comparing the endo-β-1,4-glucanase activity by varying the agitation speed from 100 to 400 rpm and the incubation temperature from 23 to 32 ° C., as shown in Table 9, the maximum endo- agitation was performed at 200 rpm and the incubation temperature of 28 ° C. β-1,4-glucanase activity was shown.

Endo-β-1,4-glucanase (U / mg-protein) Incubation temperature (℃) 23 6.31 25 6.43 28 6.99 30 6.39 32 5.21 Stirring Condition (rpm) 100 6.93 200 7.30 300 6.89 400 5.21

Table 9 shows a table of conditions for the optimization of enzyme activity.

Example 6: Optimal Enzyme Activity Determination Experiment

Culture medium and culture conditions are shown in Table 1 of Example 3 and Table 9 of Example 5, respectively, and the activity was measured for 5 days in a 7L fermenter, the supernatant was used. The activity of endo-β-1,4-glucanase was measured using the somogyi-nelson method using reducing sugars. Tricodermalase ZU-02 was used as an active control bacterium and the culture conditions were consistently applied. As a result, it was confirmed that the endo-β-1,4-glucanase enzyme activity produced by the penicillium pinofilum KMJ601 strain was 7.3 U / mg-protein, which is significantly higher than the control group (Table 10) (FIG. 3).

Strain Endo-β-1,4-glucanase (U / mg-protein) Tricoderma RJ ZU-02 4.7 Penicillium Pinophilum KMJ601 7.3

Table 10 compares the activity of endo-β-1,4-glucanase with controls.

Example 7: Cloning of the Highly Active Endo-β-1,4-Glucanase Gene from Penicillium Pinofilum KMJ601

To obtain the nucleotide sequence of the endo-β-1,4-glucanase gene, which is a cellulase degrading enzyme, the fungus Penicillium Pinophyllum KMJ601 was used. In general, genes with similar functions are known to be somewhat similar in size to each nucleotide sequence. Therefore, the gene of the endo-β-1,4-glucanase enzyme of penicillium pinofilum KMJ601 was estimated to have a size of about 1.2 kb, and the known endo-β-1,4-glucanase of other mushrooms was estimated. Based on the enzyme sequence, the entire gene of endo-β-1,4-glucanase enzyme of penicillium pinopylum KMJ601 was cloned.

E. coli XL1-Blue and pUC18 vectors were used for cloning. As a culture medium for E. coli, LB medium having a general composition was used, and the medium of Table 1 was used for culturing penicillium pinofilum KMJ601. As a plate medium of E. coli, agar plates containing LB agar, 3-5% sugar, 0.3-0.5% beef extract, 0.9-1.1% bactopeptone, and 1.3-1.7% agar composition were used. 50 μg / ml ampicillin was added as needed.

The culture method was inoculated in a 250 ml Erlenmeyer flask containing 50 ml of Penicillium Pinophilum KMJ601 and incubated at 28 ° C. and 200 rpm for 5 days, and for 16 hours at 37 ° C. and 200 rpm for E. coli. It was.

Most DNA was identified by agarose gel (TAE buffer, 0.5%) electrophoresis. The purification of DNA bands on the gel was carried out using QiaXII gel extractor (QIAGEN, USA), and the ligation reaction between DNAs was T4 DNA. Ligase (NEB) was used. In addition, RNA extraction of penicillium pinopylum was performed using a Qiagen plant total RNA kit (QIAGEN), and a reverse transcriptase for cDNA synthesis was used with Oligo-dT RT-mix (intron).

Penicillium pinopylum chromosomes were isolated for cloning the endo-β-1,4-glucanase synthase gene. In order to amplify a portion of the penicillium pinofilum endo-β-1,4-glucanase gene, a nonspecific primer was prepared based on an EG sequence already known from other mushrooms. Using this, a portion of the endo-β-1,4-glucanase enzyme gene corresponding to the size of 1.1 kb was amplified in the penicillium pinopylum chromosome by the chain polymerization reaction.

Forward Primer 5'CAGCARACKGYYTGGGGMCARTG -3 '(SEQ ID NO: 1)

Reverse Primer 5'ACGTTRCCRCCACCRGTYTCKG-3 '(SEQ ID NO: 2)

The genomic DNA of penicillium pinopylum was completely cleaved using restriction enzymes BamHI, EcoRI, HindIII, SalI, and XbaI, which do not have a cleavage site, in the partial base sequence amplified. And a radiolabeled probe was made using a DNA fragment obtained through the polymerase chain reaction. Using this, the DNA fragment containing the gene to be searched by Southern hybridization was searched (FIG. 4). When chromosomes were cut with BamHI, HindIII, and XbaI, the size of the DNA containing the gene for the endo-β-1,4-glucanase enzyme as a result of Southern hybridization was about 20 to 23 kb, which was too large. The genes were searched for using the fragments cut with EcoRI of 2.5 kb and the fragments cut with SalI of about 5.5 kb. The penicillium pinopylum chromosome was digested with EcoRI, separated from the 2.5 kb DNA fragment and the 5.5 kb DNA fragment isolated from SalI, cloned into pUC18 and named pUC-EG (FIG. 5).

Colony hybridization was performed using the 1.1 kb sized probe prepared in the pUC-EG library to determine clones with genes of the desired endo-β-1,4-glucanase enzyme. And the base sequence was analyzed using the determined clone, and the total gene sequence of endo-β-1,4-glucanase was found to be 1,426 bp (FIG. 6). As expected, this was similar in size to the endo-β-1,4-glucanase enzyme genes found in several other fungi. It was also confirmed that the penicillium pinofilum endo-β-1,4-glucanase enzyme has a nucleotide sequence common to the glycohydrolase family 5.

Conserved sequence

FGVMNEPH (SEQ ID NO: 3)

In addition, the gene scan ( http://genes.mit.edu/GENSCAN.html ) site and NCBI blast site showed that the intron portion of the penicillium pinofilum endo-β-1,4-glucanase It was confirmed that it was not included.

1 is a photograph comparing fibrin degradability in a solid agar medium containing carboxymethyl cellulose using Trichoderma Reese ZU-02 as the first selected strain (S1 to S7) and the control (C).

Figure 2 shows the results of analyzing the soft relationship with the similar species of the ITS-5.8S rDNA sequence of the strain of the present invention.

Figure 3 is the result of comparing the activity of endo-β-1,4-glucanase in the optimal expression conditions of the strain of the present invention with the activity of the control Tricoderase.

FIG. 4 shows the exploration of Southern hybridization of fragments with cellulose EG synthetase in the penicillium pinophyllum KMJ601 chromosome, where lane 1 is the penicillium pinocylum KMJ601 chromosome. No restriction enzyme was treated on genomic DNA, lanes 2, 3, 4, 5, and 6 were each treated with restriction enzymes EcoRI, SalI, BamHI, HindIII, and XbaI.

Figure 5 is a vector pUC-EGdml structure containing the endo-β-1,4-glucanase synthase gene on the chromosome of the penicillium pinopylum and cloned into a vector used in E. coli.

Figure 6 is a nucleic acid sequence of the entire gene of cellulose endo-β-1,4-glucanase production enzyme.

Figure 7 is a protein sequence of the entire gene of cellulose endo-β-1,4-glucanase production enzyme.

FIG. 8 compares the protein sequence of penicillium pinopylum endo-β-1,4-glucanase to the protein sequence of endo-β-1,4-glucanase of known penicillium decompens.

<110> Konkuk University Industrial Cooperation Corp. <120> Optimization of endo-beta-1,4-glucanase production by a novel          strain Penicillium pinophilum KMJ601 and its gene cloning <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cagcarackg yytggggmca rtg 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acgttrccrc caccrgtytc kg 22 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> conserved sequence <400> 3 Phe Gly Val Met Asn Glu Pro His   1 5 <210> 4 <211> 411 <212> PRT <213> Penicillium pinophilum <400> 4 Met Thr Ile Ile Ser Lys Phe Gly Ile Gly Val Leu Ile Ala Met Val   1 5 10 15 Ser Glu Ala Thr Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Ile Gly              20 25 30 Trp Thr Gly Pro Val Ser Cys Val Ser Gly Ser Tyr Cys Ala Pro Gly          35 40 45 Asn Ala Tyr Tyr Ser Gln Cys Leu Pro Gly Ala His Leu Asn Gly Pro      50 55 60 Ser Thr Thr Thr Ala Arg Thr Thr Ile Thr Thr Ser Ser Ser Phe Thr  65 70 75 80 Thr Ser Val Ser Thr Thr Ala Ile Pro Thr Ser Thr Gly Lys Val Gln                  85 90 95 Phe Ala Gly Val Asn Ile Ala Gly Phe Asp Phe Gly Met Val Thr Ser             100 105 110 Gly Thr Gln Asp Leu Thr Gln Ile Val Asp Glu Ser Val Asp Gly Val         115 120 125 Thr Gln Ile Lys His Phe Val Asn Asp Asp Thr Phe Asn Met Phe Arg     130 135 140 Leu Pro Thr Gly Trp Gln Tyr Leu Val Asn Asn Leu Gly Gly Gln 145 150 155 160 Leu Asp Ala Thr Asn Phe Gly Gln Tyr Asp Lys Leu Val Gln Gly Cys                 165 170 175 Leu Ser Thr Gly Ala His Cys Ile Val Asp Ile His Asn Tyr Ala Arg             180 185 190 Trp Asn Gly Ala Ile Ile Gly Gln Gly Gly Pro Ser Asp Ala Gln Phe         195 200 205 Val Asp Leu Trp Thr Gln Leu Ala Thr Lys Tyr Lys Ala Asp Ser Lys     210 215 220 Val Val Phe Gly Val Met Asn Glu Pro His Asp Leu Thr Ile Ser Thr 225 230 235 240 Trp Ala Ala Thr Val Gln Lys Val Val Thr Ala Ile Arg Asn Ala Gly                 245 250 255 Ala Thr Ser Gln Met Ile Leu Leu Pro Gly Thr Asp Tyr Thr Ser Ala             260 265 270 Ala Asn Phe Val Glu Asn Gly Ser Gly Ala Ala Leu Ala Ala Val Val         275 280 285 Asn Pro Asp Gly Ser Thr His Asn Leu Ile Phe Asp Val His Lys Tyr     290 295 300 Leu Asp Ser Asp Asn Ser Gly Thr His Ala Glu Cys Val Thr Asn Asn 305 310 315 320 Val Asp Ala Phe Ser Ser Leu Ala Thr Trp Leu Arg Ser Val Gly Arg                 325 330 335 Gln Ala Leu Leu Ser Glu Thr Gly Gly Gly Asn Val Gln Ser Cys Ala             340 345 350 Thr Tyr Met Cys Gln Gln Leu Asp Phe Leu Ser Ala Asn Ser Asp Val         355 360 365 Tyr Leu Gly Trp Thr Ser Trp Ser Ala Gly Gly Phe Gln Ala Ser Trp     370 375 380 Asn Tyr Ile Leu Thr Glu Val Pro Asn Gly Asn Thr Asp Gln Tyr Leu 385 390 395 400 Val Gln Gln Cys Phe Val Pro Lys Trp Lys Asn                 405 410 <210> 5 <211> 1426 <212> DNA <213> Penicillium pinophilum <400> 5 atgacaatca tctcaaaatt cggtattggc gtgttgatcg caatggtcag tgaggccact 60 gcgcaacaga ctgtttgggg acaatgtcag cttacaacat atttctgaac caaatgaatt 120 ggcttctaat ctttattttc taaaggtggt ggtatcggct ggactggacc agttagttgt 180 gtttctggct cctactgcgc gcctggaaat gcctactact cgcagtgtct tccaggatct 240 ggtaggtgtt ttactctcaa taatgtccga atatctcaat cgctcacttg aactaaggac 300 cgtcaacgac cacggcaaga acgacaataa ccacgtcgag cagctttacg acaagtgtca 360 gcacgacggc aattcccacg agtaccggta aggtacagtt cgccggagtg aacattgccg 420 gcttcgactt tggcatggtt accagtggca cacaggatct aactcagatt gtcgatgagt 480 ccgtcgacgg cgtcacacaa atcaagcatt tcgttaatga tgataccttc aacatgttcc 540 gcttgcctac tgggtggcag tatctcgtca acaataacct aggtggccag cttgacgcga 600 caaatttcgg ccagtacgat aagctcgttc agggttgcct ttctacgggt gcgcactgca 660 tcgttgacat ccacaactat gcccgctgga atggagccat cattggccaa ggtggtccgt 720 cggatgcaca attcgtggat ttgtggactc agcttgcgac caaatataag gctgatagca 780 aggtagtctt tggcgtcatg aatgagcccc atgatctaac tatcagcaca tgggctgcca 840 ctgtacaaaa ggtcgttaca gcaattcgta atgctggcgc aacttcacag atgatcttgc 900 tccctggtac agactacaca agtgccgcaa acttcgtgga aaatggatcc ggtgcagccc 960 tggcggcagt ggtaaaccca gatggatcta ctcataactt gatcttcgat gttcataagt 1020 acctggattc tgacaatagt ggcacccatg ccgagtgtgt caccaacaat gtcgacgctt 1080 tctcaagtct cgcaacctgg ctgcgatctg ttggtcgcca ggctctgctc tctgaaaccg 1140 gcggcggtaa cgttcagagt tgtgcaacgt acatgtgcca acagcttgat tttctcaagt 1200 aagtatacat ctacttctct gaaaatttgc attgaaaaat gcgtcatgcc atatctgata 1260 tgctgatatt gcctgtagtg cgaactctga tgtctatctc ggatggactt cctggtcagc 1320 tggtggcttt caggcatcat ggaactatat tttgacggaa gtgccaaatg gcaataccga 1380 tcagtactta gttcagcagt gcttcgtccc caagtggaag aactaa 1426 <210> 6 <211> 526 <212> DNA 5.8 s rDNA sequence <400> 6 cctgcggaag gatcattacc gagtgcgggc ctcgcggccc aacctcccac ccttgtctct 60 ctacccctgt tgctttggcg ggcccaccgg ggccacctgg tcgccggggg acgcacgtcc 120 ccggacccgc gcccgccgaa gcgctctgtg aaccctgatg aagatgggct gtctgagtac 180 gatgaaaatt gtcaaaactt tcaacaatgg atctcttggt tccggcatcg atgaagaacg 240 cagcgaaatg cgataagtaa tgtgaattgc agaattccgt gaatcatcga atctttgaac 300 gcacattgcg ccccctggca ttccgggggg catgcctgtc cgagcgtcat ttctgccctc 360 aagcacggct tgtgtgttgg gtgtggtccc cccggggacc tgcccgaaag gcagcggcga 420 cgtccgtctg gtcctcgagc gtatggggct ctgtcactcg ctcgggaagg acctgcgggg 480 gttggtcacc accatatttt accacggttg acctcggatc aggtag 526  

Claims (8)

delete delete delete delete delete An endo-β-1,4-glucanase having the amino acid sequence set forth in SEQ ID NO: 4. A gene having the nucleotide sequence of SEQ ID NO: 5 encoding endo-β-1,4-glucanase of claim 6. delete

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