KR102155046B1 - Novel Trichoderma sp. KMF006 strain producing cellulase with high activity - Google Patents

Abstract

The present invention relates to a strain of KMF006 in the genus Trichoderma that produces cellulose, the strain is highly active endo-beta-1,4-glucanase (endo-β-1,4-glucanase), beta-glucosidase (β-glucosidase) and cellobiohydrolase can be produced.

Description

KMF006 strain of the genus Trichoderma producing highly active cellulose {Novel Trichoderma sp. KMF006 strain producing cellulase with high activity}

The invention in Trichoderma which produces high activity cellulase (Trichoderma sp.) It relates to the strain KMF006.

The renewable fuel standard (RFS) is a system that mandates fuel mixing obligors to mix and supply a certain ratio of renewable fuel with fuel for transportation. The system's goal is to reduce gasoline consumption and expand biofuel use by 2020.

Accordingly, wood-based biomass is emerging as a raw material for new and renewable energy, and interest in manufacturing biofuels using the same is increasing. Since lignocellulosic biomass generates 10 billion to 15 billion tons per year, it has the advantage of being a renewable resource, but it has the disadvantage that it is difficult to decompose cellulose bound to lignin.

In order to process lignocellulosic biomass into biofuel, a process of extracting sugar by decomposing cellulose is required, and the method of extracting sugar is derived from fungi such as fungi that mainly degrade wood and chemical saccharification using a catalyst such as acid alkali. There is an enzymatic saccharification using cellulase. Enzymatic saccharification is an environmentally friendly method because it does not cause problems such as environmental pollution or metal corrosion compared to chemical saccharification. However, since the cost of enzymatic saccharification in the production of biofuels is very high, there is a need for a way to reduce the amount of enzyme used or the unit cost of the enzyme by using a highly active enzyme.

As a result of the present inventors trying to develop a strain producing high activity cellulose, the present invention was completed by separating the strain KMF006 of the genus Trichoderma, which produces cellulose having a higher activity than the existing strain.

1. Korean Patent Registration No. 10-0205247

It is an object of the present invention to provide a strain of KMF006 genus Trichoderma that produces highly active cellulose.

Another object of the present invention is to provide a saccharification composition comprising cellulose obtained from the strain KMF006 of the genus Trichoderma.

In order to achieve the above object, one aspect of the present invention is the genus Trichoderma producing highly active cellulose sp.) KMF006 strain is provided.

In one embodiment of the present invention, the strain KMF006 of the genus Trichoderma may be a strain having a 28S rRNA sequence of SEQ ID NO: 3. The present inventors compared the 28S rRNA sequence of the KMF006 strain with other strains previously reported by NCBI BLAST, and as a result, it was confirmed that it is a novel strain classified into the genus Trichoderma.

As used herein, the term'cellulase' is a generic term for an enzyme that hydrolyzes cellulose, and'cellulose' refers to glucose as β-1,4 glucoside (β-1,4-glucosde) It means a homopolymer connected by a bond.

In one embodiment of the present invention, the cellulose is endo-β-1,4-glucanase (endo-β-1,4-glucanase; EC 3.2.1.4), cellobiohydrolase; EC 3.2. 1.91) and beta-glucosidase (EC 3.2.1.21).

Endo-beta-1,4-glucanase is an enzyme that randomly hydrolyzes the β-1,4-glucoside bonds of cellulose inside, and generally uses soluble carboxymethyl cellulose (CMC) as a substrate. Because it is used, it is also called CMCase.

Cellobiohydrolace hydrolyzes the reducing and non-reducing ends of the product hydrolyzed by endo-beta-1,4-glucanase to produce cellobiose, a glucose disaccharide. The generated cellobiose inhibits the activities of endo-beta-1,4-glucanase and cellobiohydrolace.

Since beta-glucosidase decomposes cellobiose into glucose (glucose), it eliminates the inhibition of the activity of endo-beta-1,4-glucanase and cellobiohydrolace by cellobiose. Therefore, in order to completely decompose cellulose into glucose, it is important to increase the activity of beta-glucosidase.

In one embodiment of the present invention, in order to completely decompose cellulose into glucose, which is a monosaccharide, a complementary action of the three enzymes is required.

In one embodiment of the present invention, the strain KMF006 of the genus Trichoderma may produce cellulose having significantly superior cellulose decomposition activity than Trichoderma reesei , which is an industrial strain for producing cellulose.

Another aspect of the present invention provides a saccharification composition comprising cellulose obtained from the strain KMF006 of the genus Trichoderma.

As used herein, the term'saccharification' refers to a reaction that converts high molecular weight carbohydrates such as starch and fiber into low molecular weight (monosaccharides or disaccharides) saccharides by hydrolysis by the action of enzymes or acids. do.

In one embodiment of the present invention, the strain KMF006 of the genus Trichoderma will produce all of endo-β-1,4-glucanase, cellobiohydrolace, and beta-glucanase required for the process of decomposing cellulose into monosaccharides (glucose). I can. Accordingly, the saccharification composition can produce monosaccharides or disaccharides from cellulose, and thus can be usefully used in biofuel production.

The KMF006 strain of the genus Trichoderma according to an embodiment of the present invention can produce highly active endo-beta-1,4-glucanase, beta-glucosidase, and cellobiohydrolace, so it will be usefully used in biofuel production. I can.

1 shows a schematic diagram of a strain of KMF006 genus Trichoderma prepared with an ITS (internal transcribed spacer) sequence.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example One: Cellulite Isolation of production strain

In order to discover a new strain producing cellulose, the KMF006 strain was isolated from the deteriorated wood and the cellulose activity was measured.

To cellulase activity it is known to measure strain and strain KMF006 commercial cellulase producing strain Trichoderma reseyi (Trichoderma reesei: Korea Culture Center of Microorganisms KCCM11770), Trichoderma corruption to (Trichoderma viride , Blue spot mushroom: National Institute of Forest Science KFRI 21052) and Ganoderma lucidum ( Ganoderma lucidum , Ganoderma lucidum : National Forest Research Institute KFRI 20433) was used.

1-1. Strain culture

Four kinds of cellulose-producing strains were cultured in PDA (potato dextrose agar) for 3 to 4 days, and mycelium was inoculated in 100 ml of PDB (potato dextrose broth) for 3 to 7 days while shaking at 150 rpm at 25°C. During pre-culture.

The main culture was peptone 8 g/L, KH ₂ P0 ₄ 5 g/L, K ₂ HP0 ₄ 5 g/L, MgS0 ₄ .7H ₂ 0 3 g/L, yeast extract 2 g/L and cellulose (~20 micron ) A liquid medium (pH 5.5) containing 20 g/L was used. Pre-culture solution 5% (v/v) was inoculated into 200 ml of the liquid medium, and cultured for 2 to 3 weeks while shaking at 150 rpm at 25°C. During the cultivation period, 500 µl of the strain culture solution was collected every day, centrifuged, and the supernatant was collected and used to measure cellulose activity.

1-2. Endo-beta-1,4- Glucanase ( endo -β-1,4- glucanase , EG) active

An enzyme reaction solution was prepared by dissolving carboxylmethylcellulose sodium salt (CMC-Na) at a concentration of 2% (v/v) in a 0.1M sodium acetate (NaAC) buffer solution (pH 5.0). 5 μl of the strain culture solution was added to 45 μl of the enzyme reaction solution, followed by reaction at 50° C. for 30 minutes, and 50 μl of a copper solution was added, followed by heating at 100° C. for 10 minutes to stop the reaction. The amount of reducing sugar produced was measured by the Somogyi-Nelson method (latest experimental microbiology, p253, 2001). The unit of enzyme activity, 1 unit (U), was defined as the amount of enzyme required to produce 1 μmol of glucose for 30 minutes under certain conditions.

1-3. beta- Glucosidases (β- glucosidase , BGL ) activation

0.1M NaAC buffer solution (pH 5.0) 0.8 ㎖ the p - nitrophenyl -β-D- glycoside pyrano seed (p -nitrophenyl-β-D- glycopyranoside, p NPG) was added to 0.1 ㎖ and strain culture solution 0.1 ㎖ 50 It was reacted at °C for 15 minutes. Since 2M sodium carbonate (Na ₂ CO ₃₎ solution was added to terminate the reaction 0.1 ㎖, the p generated by measuring the absorbance at 405 ㎚ - was confirmed that the amount of the nitrophenol (p -nitrophenol). The enzyme activity unit, 1 unit (U), was defined as the amount of enzyme required to produce 1 μmol of p -nitrophenol for 15 minutes under certain conditions.

1-4. Cello Bio Hydro Race ( cellobiohydrolase , CBH ) activation

P -nitrophenyl-β-D-cellobioside in 0.8 ml of 0.1M NaAC buffer (pH 5.0) 0.1 ml of ( p- nitrophenyl-β-D-cellobioside, p NPC) and 0.1 ml of the strain culture were added and reacted at 50° C. for 15 minutes. Thereafter, 0.1 ml of 2M sodium carbonate solution was added to stop the reaction, and absorbance was measured at 405 nm to confirm the amount of p -nitrophenol produced. The unit of enzyme activity, 1 unit (U), was defined as the amount of enzyme required to produce 1 μmol of p -nitrophenol for 15 minutes under certain conditions.

1-5. Enzyme activity measurement result

The measurement results of cellulase activity of each strain are shown in Table 1 below.

Unit (U/ml) Endo-beta-1,4-glucanase (EG) Beta-glucosidase (BGL) Cello Bio
Hydro Race (CBH) KMF006 33.8 3.50 1.20 T. reesei 13.0 1.70 (No activity measured) T. viride 14.4 1.10 0.30 G. lucidum 6.20 1.30 (No activity measured)

As a result of measuring the enzyme activity, the endo-beta-1,4-glucanase (EG) activity of the KMF006 strain was the highest among the strains to be tested, and Trichoderma Resei, which commercially produces endo-beta-1,4-glucanase. The activity was 2.6 times better than the strain. In addition, the beta-glucosidase (BGL) activity of the KMF006 strain was found to be 2 to 3.2 times more than that of the Trichoderma resei strain, and the cellobiohydrolace (CBH) activity was 4 times superior to that of the Trichoderma viride strain.

Through the measurement result of the enzyme activity, it can be confirmed that the cellulose produced by the strain KMF006 of the present invention has remarkably superior cellulose degradation activity compared to the cellulose produced by the Trichoderma resay strain.

Example 2: KMF006 Strain identification

Genomic DNA was extracted from the KMF006 strain, and PCR was performed with the ITS (internal transcribed spacer) primers shown in Table 2 below.

Name Sequence (5'→ 3') Sequence number ITS 1 TCC GTA GGT GAA CCT GCG G One ITS 2 TCC TCC GCT TAT TGA TAT GC 2

As a result of requesting 28S rRNA sequencing to Macrogen (Seoul, Republic of Korea) using the amplified PCR product, a total of 1,131 bp (SEQ ID NO: 3) was obtained.

As a result of performing an NCBI BLAST search with the nucleotide sequence, the schematic diagram of FIG. 1 was confirmed. Specifically, the KMF006 strain is Trichoderma longibrachiatum (325301884), Trichoderma longibrachiatum (1201281026), Trichoderma resei ( Trichoderma reesei ) (675946435), Trichoderma resei (21239410), Trichoderma resei (1050550597), and the like. However, when comparing the 28S rRNA sequences of the 10 strains disclosed in FIG. 1, the maximum identity was 98%, so the KMF006 strain was determined to be a new strain belonging to the genus Trichoderma. The KMF006 strain of the genus Trichoderma was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center and was given the deposit number KCTC13500BP on March 21, 2018.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Korea Research Institute of Bioscience and Biotechnology Biological Resource Center KCTC13500BP 20180321

<110> Kookmin University <120> Novel Trichoderma sp. KMF006 strain producing cellulase with high activity <130> PN180101 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS 1 primer <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS 2 primer <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 1131 <212> RNA <213> Artificial Sequence <220> <223> Trichoderma sp. KMF006 strain 28S rRNA <400> 3 accccatgtg acgttaccaa tctgttgcct cggcgggatt ctcttgcccg gggggcgttg 60 gcagcccccg gattccccat ggcgcccgcc ggaggaccaa ctccaaactc ttttttctct 120 ccgtcgcggc tcccgtcgcg gctctgtttt atttttgctc tgagcctttc tcggcgaccc 180 tagcgggcgt ctcgaaaaat gaatcaaaac tttcaacaac ggatctcttg gttctggcat 240 cgatgaagaa cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc 300 gaatctttga acgcacattg cgcccgccag tattctggcg ggcatgcctg tccgagcgtc 360 atttcaaccc tcgaacccct ccggggggtc ggcgttgggg atcggcccct caccgggccg 420 cccccgaaat acagtggcgg tctcgccgca gcctctcctg cgcagtagtt tgcacactcg 480 caccgggagc gcggcgcggc cacagccgta aaacacccca aacttctgaa atgttgacct 540 cggatcaggt aggaataccc gctgaactta agcatatcaa taagcggagg aaaagaaacc 600 aacagggatt gccccagtaa cggcgagtga agcggcaaca gctcaaattt gaaatctggc 660 ccttacgggt ccgagttgta atttgtagag gatgcttttg gcaaggcgcc gcccgagttc 720 cctggaacgg gacgccacag agggtgagag ccccgtctgg ctggccgccg agcctctgta 780 aagctccttc gacgagtcga gtagtttggg aatgctgctc aaaatgggag gtatatgtct 840 tctaaagcta aatattggcc agagaccgat agcgcacaag tagagtgatc gaaagatgaa 900 aagcaccttg aaaagagggt taaatagtac gtgaaattgt tgaaagggaa gcgcttgtga 960 ccagacttgg gcgcggcgga tcatccgggg ttctccccgg tgcacttcgc cgcgtccagg 1020 ccagcatcag ttcgtcgcgg gggaaaaagg cttcgggaac gtggctccct cgggagtgtt 1080 atagcccgtt gcgtaatacc ctgcggtgga ctgaggaccg cgcatctgca a 1131

Claims (4)

As a strain of Trichoderma sp. producing highly active cellulose,
The cellulose includes all of endo-beta-1,4-glucanase, beta-glucosidase, and cellobiohydrolase,
The deposit number is KCTC13500BP,
The strain of the genus Trichoderma has the 28S rRNA sequence of SEQ ID NO: 3,
Trichoderma genus strain.

delete delete A saccharification composition comprising cellulose obtained from the strain of claim 1, Trichoderma sp.

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