KR20110112730A - Thermostable xylanase purified from fomitopsis pinicola sp.and method of production thereof - Google Patents

Abstract

The present invention relates to xylanase isolated from pine zanabi mushrooms, and provides xylanase having excellent thermal stability even at high temperatures and having excellent activity even in an acidic environment.

Description

Thermostable xylanase isolated from pine beetle mushrooms and its production method {Thermostable xylanase purified from Fomitopsis pinicola sp.and method of production}

The present invention is a pine grass butterfly ( Fomitopsis) It relates to xylanase having excellent thermal stability isolated from pinicola ) and a method of producing the same.

Xylan is the major hemicellulose in differentiated hardwoods. While saline contains only 15-25% of hemicellulose, hardwoods are made up of 25-32% of hemicellulose, which has attracted attention as a huge reserve of biomass. Xylan is considered to be an important report of fixed carbon in nature, and researches using xylanase, xylanase, are being actively conducted to effectively and economically decompose it.

Most of the currently characterized xylanases exhibit optimal activity at weakly acidic pH levels (pH 5.0-6.0) for most fungal xylanases, while the optimum pH level for bacterial xylanases is generally slightly higher. Most of the characterized xylanases exhibit optimal activity at temperatures between 40 ° C. and 70 ° C.

Xylans are complex and highly variable polysaccharides with β-1,4-binding backbones of xylopyranosyl residues comprising 4-O-methyl-glucoronosyl, 4-O-arabinosyl and acetic acid side chains. Endo-1,4-β-D-xylanase (EC 3.2.1.8) is a commercially noteworthy enzyme because it is by random cleavage of the xylan backbone. A comparison of the amino acid sequences of various endo-1,4-β-D-xylanases indicates that xylanases are primarily classified into two families of GHs: GH10 and GH11. However, other GH families, 5, 7, 8 and 43, were also found to contain distinct catalytic domains with proven endo-1,4-β-D-xylanase activity. Xylanases of family GH11 exhibit low molecular weights with pi 8-9.5 compared to high molecular weight family GH10 with low pi levels.

Currently known xylanase fungi are mostly ascomycetes, approved by enzymes produced by Aspergillus (de Vries and Visser 2001) and Trichoderma (Wong and saddler 1992). Is especially noteworthy. Xylanases and cellulase, together with pectinase, account for about 20% of the world's enzyme market, and explore new microbial and enzymatic systems that are not traditional xylanase and maintain their activity under high temperature and pH conditions. Efforts to develop Lanaze continue.

Currently known domestic xylanase-related patents include a patent for a novel Streptomyces genus VL-2 strain that produces xylanase (Korean Patent No. 10-0411771), a novel cellulose microbiium sp. Patent for the 12 strains and xylanase produced therefrom (Korea Patent Registration No. 10-080561) and the novel Fanibacillus sp. Xylanase (HK-8 2007-0082329) of the HV-8 strain, a novel penivacillus sp. That produces xylanase. HPP-001 strains, and patents for novel xylanase enzymes (Korean Patent Publication No. 2010-0021175), but are not actually used in many fields in Korea, and also the jyl to maintain the activity in an acidic environment or high temperature The development of lanase is inadequate.

The present invention has been made as a result of earnest research in view of the above points, in particular pine Jang Butterfly ( Fomitopsis It is an object of the present invention to provide a xylanase having excellent thermal stability separated from pinicola ) and a method for producing the same.

The above object of the present invention is achieved by separating xylanase from pine beetle mushroom, measuring its specific activity, pH, reducing sugar level, determining molecular weight, measuring activity according to pH or temperature, and evaluating heat inactivation It was.

Xylanase isolated from the pine needles mushroom of the present invention has an excellent effect showing excellent activity even in acidic environment and high temperature.

Figure 1 shows the specific activity, pH and residual reducing sugar values over time when xylanase production by pine straw butterfly KMJ812 in rice straw.
Figures 2a and 2b shows the PAGE and molecular weight determination of xylanase purified from pine needles mushrooms. FIG. 2A is a PAGE of xylanase purified from pine berry butterfly, line 1 is molecular weight marker, line 2 is cell extract, line 3 is DEAE ion-exchange fraction and line 4 is SDS-PAGE of MonoQ ion exchange fraction . Figure 2b shows the determination of the molecular weight of pine needles xylanase by gel filtration chromatography on Sephacryl S-300 high resolution column. The column was adjusted with standard molecular weight proteins such as aldose (168 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease A (13.7 kDa).
Figure 3 shows the effect of pH and temperature on the activity of pine needles xylanase. Figure 3 (A) shows the effect of pH on the activity of xylanase purified from pine needles butterfly KMJ812. Enzyme activity was measured by standard assay methods with varying buffers to achieve the required pH. Citrate (pH 3.0 to 4.5), sodium acetate (pH 4.5 to 6.0) and phosphate (pH 6.0 to 8.0) were used as the buffer. Figure 3 (B) shows the effect of temperature on the activity of xylanase purified from pine needles butterfly KMJ812. Enzyme activity was assayed at various temperatures using standard assay methods. Each number is the average of three measurements and does not change more than 15% from the mean.
Figure 4 shows the heat inactivation of pine needles xylanase. Enzymes were incubated at different times at 40 ° C, 50 ° C, 60 ° C, 65 ° C and 70 ° C. Samples were recovered at each time interval and relative activity was measured.

Purification and characterization of xylanase from several strains has been reported, but the present invention is the first to report on the purification and characterization of xylanase from basidiomycetes, pine needles. Extracellular xylanase purified from pine needles mushrooms is a monomer having a molecular weight of 58 kDa. The molecular weight of pine needles xylanase is the same as many of the extracellular xylanases characterized from other fungi. In general, GH comprising xylanase has been characterized as having a relatively broad substrate specificity, and pineapple butterfly xylanase also exhibits relatively broad substrate specificity.

The xylanase of the present invention comprises the steps of: 1) centrifuging and washing pine mushroom butterfly culture; 2) mixing the washing solution and the supernatant, concentrating and filtration to obtain a crude enzyme solution; And 3) purifying the crude enzyme solution by column chromatography.

Example  One. Xylanase  Screening of Producing Strains

All fungal strains were obtained from the Korean Agricultural Culture Collection (KACC). Initial screening of 112 fungi producing xylanase was performed in agar flat medium containing 0.4% Remazol Brillinat Blue xylan. Based on the observed clearance portion, 15 strains were selected. In addition to fifteen strains, an effective xylanase-producing pine needle butterfly ( Fomitopsis pinicola ) KMJ812 (obtained from KACC) was selected for further study. 15 strains were 8 g / l peptone, 2 g / l yeast extract, 5 g / l KH ₂ PO ₄ , 5 g / l K ₂ HPO ₄ , 3 g / l MgSO ₄ .7H ₂ O, It was inoculated in 3 ml growth medium containing 0.005 g / l thiamine-HCl and haute-spell xylan (Sigma, MO, USA) and incubated with stirring at 200 rpm for 7 days at 28 ° C. The xylanase activity of the culture was analyzed using 1% oatsfeldt xylan as substrate. After the analysis, the strain with the highest xylanase activity was selected.

Example  2. Pine needles  culture

In the flask culture, the mycelium of pine needles butterfly KMJ812 was inoculated into 100 ml of potato dextrose culture. Precultures (5 ml) were inoculated into 200 ml xylan medium in a 1 L fermenter. This culture medium contains 8 g / l peptone, 2 g / l yeast extract, 5 g / l KH ₂ PO ₄ , 5 g / l K ₂ HPO ₄ , 3 g / l MgSO ₄ .7H ₂ O, 0.005 g / l thiamine.HCl and oatsfeld xylan. The effect of carbon or nitrogen sources on xylanase production was investigated after 17 days of incubation in a flask containing 50 g / l of carbon and a medium consisting of various nitrogen sources. The concentration of nitrogen source was adjusted to the same content of nitrogen using the Kjeldahl method. The medium used for xylanase production was 8 g / l peptone, 2 g / l yeast extract, 5 g / l KH ₂ PO ₄ , 5 g / l K ₂ H PO ₄ , 3 g / l MgSO ₄ .7H ₂ O, 0.005 g / l thiamine.HCl and 20 g / l rice straw.

Example  3. Xylanase  For produce Carbon source  Of nitrogen and nitrogen sources

To choose a suitable carbon source for xylanase production, pine grass butterfly KMJ812 was incubated in a medium containing yeast extract (10 g / L) and various carbon sources (cellulose, rice straw, bran, xylan, avice, CMC, cellobiose, glucose, maltose, lactose, sucrose or wood fiber). Among the carbon sources tested, rice straw was found to be the most suitable carbon source for xylanase production, leading to xylanase-specific activity of 5.1 U mg-protein- ¹ in flask culture (see Table 1).

Since the mechanisms governing the formation of extracellular enzymes are affected by the effectiveness of the precursors for protein synthesis, the effects of inorganic and organic nitrogen sources on xylanase synthesis have also been tested (see Table 1). Rice straw (20 g / l) was used as the carbon source. Among various nitrogen sources (peptone, corn leach powder, yeast extract, iodine, ammonium sulfate, potassium nitrate and sodium nitrate), xylanase production is maximized in combination of yeast extract (5 g / l) and peptone (5 g / l) (5.4 U mg-protein- ¹ ) followed by the yeast extract, and other nitrogen sources were not suitable under conditions without pH control. The time course of xylanase production by pine straw butterfly grown on rice straw, yeast extract and peptone was examined (see FIG. 1). Xylanase activity increased up to 17 days and then gradually decreased.

Example  4. Xylanase  Enzyme assay

The xylanase activity of the present invention was assayed using 1% (w / v) oatsfeld xylan as substrate. Xylan was dissolved in 100 mM sodium acetate buffer (pH 5.0). The reaction mixture containing 10 μg of enzyme and 2.5 mg of substrate was incubated at 50 ° C. for 30 minutes. The amount of free reducing sugar was determined according to the 3, 5-dinirosalicylic acid (DNS) method (Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar.Anal Chem 31: 426-428). Xylose was used as a standard. One unit of xylanase activity is expressed in μmol of reducing sugar (xylose equivalent) released for 1 minute under the above conditions. Cellulose activity was assayed as above using low viscosity carboxymethylcellulose (1%) instead of xylan.

Example  5. Xylanase  refine

All procedures were carried out at 4 ° C. and 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol (DTT) was used for purification unless otherwise noted. Proteins measured by Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem 72: 248-254 did. Proteins in the column effluent were found by measuring absorbance at 280 nm. All chromatographic separations were performed using an AKTA FPLC system (AKTA, Sweden).

Step 1: Preparation of Coenzyme. Cells were recovered from the culture by centrifugation at 10,000 xg for 30 minutes. After washing with 20 mM sodium acetate buffer (pH 5.0), the washing solution and the supernatant were mixed, concentrated and desalted by ultrafiltration through a polyester sulfone membrane (30 kDa cutoff) in a stirred cell (Amicon, Beverly, MA, USA). .

Step 2: DEAE Sepharose Chromatography. The dialyzed enzyme solution was charged to a DEAE Sepharose ^™ Fast Flow column (1.6 × 10 cm, Amersham Biosciences) equilibrated with 20 mM sodium acetate buffer at pH 5.0 and then zero in the same buffer at a flow rate of 1.0 ml / min. The protein was eluted with a 180-minute linear gradient of -0.5 M NaCl. 1 mL of each compartment was pooled and assayed for xylanase activity. The active compartments were combined and dialyzed against the same buffer and then concentrated by ultrafiltration for further purification.

Step 3: Sephacryl gel filtration chromatography . The concentrated enzyme solution was charged to a HiPrep 16/60 Sephacryl S-300 HR column (1.0 cm x 120 cm, Amersham Biosciences) equilibrated with 20 mM sodium acetate buffer containing 100 mM NaCl at pH 5.0 and 0.5 ml / min. The protein was eluted with the same buffer at the flow rate of. The active fractions were combined, dialyzed against the same buffer and concentrated by ultrafiltration.

Step 4: MonoQ Ion Exchange Chromatography . The enzyme was further purified by MonoQ ion exchange chromatography column 5/50 GL (1.0 × 10 cm, Amersham Biosciences) previously equilibrated with 20 mM sodium acetate (pH 5.0). The enzyme was eluted with a 180-minute linear gradient of 0-0.5 M NaCl in the same buffer at a flow rate of 0.5 ml / min. Bound active fractions were combined and concentrated, dialyzed against the same buffer and concentrated with a Centrion (Millipore, Bedford, Mass., USA) ultrafiltration apparatus of 30 kDa molecular weight cut off and used as purified enzyme in the next experiment.

After the purification as described above, the results are shown in Table 2.

Fractionation by ultrafiltration resulted in a 13% recovery of xylanase activity, with an approximately 2-fold increase in specific activity. The active fractions were applied to a DEAE Sepharose column and xylase was eluted with approximately 0.1 M NaCl. The subsequent gel filtration step produced three peaks containing protein; The second peak showed xylanase activity. FPLC elution of a MonoQ ion exchange chromatography column with 0.5 M NaCl produced an active xylanase protein peak. This chromatography method yielded 14-fold purified xylanase with a recovery of 0.1%. Analysis of the purified enzyme by gel electrophoresis in the presence of SDS (lanes 2A-4) shows one band with M _r of 57,000. Size exclusion chromatography on Sephacryl S-300 high resolution column eluted the enzyme activity as a symmetric peak corresponding to M _r of approximately 58,000 (FIG. 2B). These results indicate that the enzyme migrates as a monomer upon gel filtration under mild conditions and can therefore also be present and activated as a monomer in solution.

Example  6. Optimal pH  And temperature determination

Optimum pH of the xylanase of the present invention was changed to different buffers (citrate (100 mM, pH 3-4.5), sodium acetate (100 mM, pH 4.5-5.5), phosphate (100 mM, pH 5.5-8) at 50 ° C. It was determined by incubating the purified enzyme within 15 minutes). To determine the optimum temperature, the enzymes were incubated in sodium acetate buffer (100 μmM, pH 5) for 15 minutes at different temperatures of 25-100 ° C. To determine the thermal stability of xylanase, purified enzymes were incubated at different temperatures (40 ° C.-80 ° C.) without substrate. After holding for a period of time (0-25d), residual xylanase activity was determined as above.

The optimal pH of xylanase is 4.5, showing a maximum activity of 98% and 98% at pH 4.0 and 5.0, respectively (FIG. 3A). The isoelectric point (pI) was determined to be 4.7, which is common for extracellular xylanase. The optimum temperature for the hydrolysis action is 70 ° C. with a maximum activity of 94% and 96% at 50 and 80 ° C., respectively (FIG. 3B). At 90 ° C., approximately 50% of the relative activity is maintained.

Example  7. Pine Butterfly Mushroom Xylanase Thermal stability

The thermal stability of the purified xylanase was tested at various temperatures from 40 ° C. to 80 ° C. Purified pine beetle xylanase is very stable and maintained up to 100% when incubated at 40 ° C. for 25 days. After incubation at 60 ° C. for 60 hours, activity of about 90% was maintained. At temperatures above 80 ° C., xylanase activity decreases rapidly with incubation time. Enzymes are each an 410 times, 96 times and 33 times of t _1/2 levels in 50 ℃, 60 ℃ and 70 ℃ (Fig. 4).

Example  8. Xylanase PAGE  And molecular mass determination

In the determination of the subunit molecular weight, SDS- in a 10% gel as described in Laemmli (Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227 (5259): 680-5). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (PAGE) was performed. Protein bands were developed with Coomassie Brilliant Blue R-250 (Sigma). The molecular mass of the purified enzyme was determined by size exclusion chromatography using a Superose ^™ 12 (Amersham Pharmacia Biotech, Uppsala, Sweden) column accompanying the AKTA FPLC system (GE health care). The enzyme was eluted with 20 mM sodium acetate (pH 5.0) at a flow rate of 0.5 ml / min.

Example  9. Xylanase Substrate specificity

Substrate specificity of the purified enzyme was tested in a standard assay system containing the following substrates at the same concentration (1%; w / v): Oatsfeld xylan, Birchwood xylan, Beechwood xylan, Avicel (avicel), carboxymethyl cellulose, p-NP β-d- xylene in pyrano side, p - nitrophenyl -β-d- cell lobby O side, p-NP β- galactosidase-pyrano side, p NP- β-D-lactopyranoside, p -nitrophenyl-α-L-arabinofuranoside.

Table 3 shows the activity of pine needles xylanase with various substrates. The highest activity (105%) was observed in Birchwood xylan, followed by the highest activity (100%) in hautesfeld xylan. The enzyme showed low activity against cellulose substrates such as Avicel (11%) and CMC (7.2%).

Example  10. Xylanase  Determination of Kinetic Parameters and Inhibition Constants

The values of the Michaelis constant (K _m ) and maximum velocity (V _max ) of the xylanase of the present invention were measured at 50 ° C. with hautesfeld xylan in concentrations ranging from 0.5 to 5 mg / ml. It was determined by incubation in 100 mM sodium acetate buffer (pH 5). Inhibition of xylanase by xylose was determined using oatsfeld xylan as substrate. The values of K _m and V _max and K _i were determined from Lineweaver-Burk plots using standard linear regression techniques. As a result, K _m was 3.4 mM, V _max was 80.2 mol min ⁻¹ mg ^−1, and K _i was found to be 13.4 mM.

Example  11.Effects of Metals and Reagents

The effect of various metal ions and reagents of 1 μmM on the xylanase activity of the present invention was determined by 30 minutes pre-incubation of the enzyme with individual reagents in 20 mM sodium acetate buffer (pH 5.0). Activity was then measured at 50 ° C. for 15 minutes in the presence of metal ions or reagents. Assayed activity was reported as 100% without metal ions and reagents. The results of assaying xylanase activity for the presence and absence of metal ions, metal chelating agents (EDTA) and various other compounds are shown in Table 4.

Xylanase activity strongly inhibits the activity of BaCl ₂ , NaCl. Slightly activated by CuCl ₂ , HgCl ₂ and MnCl ₂ , CaCl ₂ , CoCl ₂ , FeCl ₂ , MgCl ₂ and ZnCl ₂ have a much more limited inhibitory effect. Inhibited by EDTA at concentrations ranging from 1-10 mM.

The effect of sulfhydryl compounds on xylanase from pine needles mushrooms was also tested (Table 4). 10 mM 2-mercaptoethanol and cysteine were added to the reaction mixture to increase the enzyme activity by 135.3 and 119.3%, respectively. These results suggest that sulfhydryl compounds retain active enzymes in reduced states. This only indicates that xylanase can easily access disulfide bonds which are readily reduced.

Example  12. Xylanase  Internal amino acid sequence

Purified protein of the present invention was digested by SDS-PAGE and then electroblotted onto polyvinylidene trifluoride membrane (Bio-Rad). 50 μl of 100 μl of the protein was cleaved at 37 ° C. for 4 hours with 100 ng of endoproteinase Asp-N or endoproteinase Lys-C or trypsin (Promega, Madison, WI, USA) for peptide mapping. mM (NH ₄ ) ₂ CO ₃ 20 μg of purified enzyme in pH 8.5 was digested. The resulting peptide fragment was separated by SDS-PAGE (15% polyacrylamide) and the separated peptides were electroblotted and transferred onto polyvinylidene trifluoride membrane. Peptide bands were developed by 0.1% Coomassie Brilliant Blue R-250 staining in 40% methanol. Partial amino acid sequences were determined by Edman degradation using an automatic protein sequencer (model 491A; Applied Biosystems, Division of Perkin-Elmer) at The National Instrumentation Center for Environmental Management (Seoul, Korea). Partial amino acid sequences were used to identify similar proteins through BLAST searches of nonredundant protein databases.

Example  13. Identification of Partial Peptide Fragments

Purified enzyme (1.5-mg) of the present invention was isolated by 10% SDS-PAGE and blotted with polyvinylidene fluoride (PVDF) membrane. Automated Edman degradation of the enzyme protein failed, indicating that the N-terminus of the enzyme was blocked. Xylanase was partially cleaved with trypsin, endoproteinase Asp-N and endoproteinase Lys-C, then separated by 12.5% SDS-PAGE and blotted onto PVDF membranes. Three fragments of Lys-C fragment (LYS), Asp-N fragment (ASP) and trypsin fragment (TRY) were sequenced with automated protein sequencing. LYS fragments include the YKEFFKIGAAVTVK fragment. TRY and ASP Fragments include LSDVEEAIRIVR and NNESLICTNSDGTLD fragments, respectively.

Example  14.Various Xylanase  Property comparison

Table 5 compares the properties of various xylanases from many different microorganisms. Pine needle butterfly xylanase of the present invention has a remarkable specific activity of 74.4 U mg-protein- ¹ against oatsfeld xylan. In comparison, specific activity levels of xylanase purified from other fungi range from 7.4 to 894 U mg-protein ⁻¹ (Table 5). Depending on the optimum temperature, enzymes can be classified into mesophilic (40-60 ° C.), thermophilic (50-80 ° C.) and ultra high temperature (> 80 ° C.). Recently, many efforts have been made to isolate enzymes from thermophilic and even extreme microbes as they produce enzymes with good stability. Talaromyces emersonii ), Thermomyces xylanase from thermophilic strains such as lanuginosus ) and Thermoascus aurantiacus have an optimum temperature of 60-80 ° C. and are very stable in this range. Many endoxylanases from thermophilic microorganisms have the same degree of structural homology as entozylanase from mesophilic microorganisms. Xylanases from the mesophilic microbial pine butterfly show considerable stability at high temperatures. The pine needles xylanase enzyme of the present invention exhibits the highest thermal stability with a half-life of 33 hours at 70 ^° C. (Table 5).

Example  15. Pine Butterfly Mushroom Xylanase  Classification

Family 10 xylanase is known to have a (β / α) ₈ barrel structure and belongs to the so-called 4/7 superfamily of GH. The catalytic domain structure of the natural and complex forms of family 10 xylanase is Pseudomonas fluorescens ) (Pell G, Taylor EJ, GlosterTM, Turkenburg JP, Fontes CMGA, Ferreira LMA, Nagy T, Clark SJ, Davies GJ, Gilbert HJ (2004a) The mechanisms by which family 10 glycoside hydrolases bind decorated substrates.J Biol Chem 279: 9597-9605), Streptomyces olivaceoviridis ) (Fujimoto Z, Kaneko S, Kuno A, Kobayashi H, Kusakabe I, Mizuno H (2004) Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86.J Biol Chem 279: 9606-9614) And Geobacillus stearotermophilus ( Geobacillus) stearothermophilus ) (ZZolotnitsky G, Cogan U, Adir N, Solomon V, Shoham G, Shoham Y (2004). It can be obtained from microorganisms. The internal amino acid sequence of Pinus pinococcus xylanase shows similarity to GH family 10 xylanase. The other fragments of pine needles xylanase, YKEFFKIGAAVTVK, SDVEEAI and ESLICTNSDGTLD, respectively, were shown in Anaerocellum thermophilum ) , Bacteroides fragilis ) And Sphingobacterium spiritivorum . Evidence from enzymatic and bioinformatics experiments suggests that pine beetle xylanase can be classified as a member of GH family 10.

Xylanase of the present invention is characterized by excellent thermal stability and excellent activity in an acidic environment.

Claims (3)

Inoculating pine needles mycelium hyphae in 100 ml of potato dextrose broth; And
In a 1 L fermentor, 5 ml of preculture was added to 8 g / l peptone, 2 g / l yeast extract, 5 g / l KH ₂ PO ₄ , 5 g / l K ₂ HPO ₄ , 3 g / l MgSO ₄ Inoculating xylan medium consisting of 7H ₂ O, 0.005 g / l thiamine.HCl and oatsfeld xylan
Production method of pine myrtle mushroom mycelium comprising a. 1) centrifugation and washing the pine grass butterfly culture medium;
2) mixing the washing solution and the supernatant, concentrating and filtration to obtain a crude enzyme solution; And
3) Purifying the crude enzyme solution by column chromatography
Xylanase production method comprising a. Lys-C (LYS) fragments containing KEFFKIGAAVTVK fragments and trypsin fragments containing Asp-N (ASP) and LSDVEEAIRIVR fragments, showing maximal activity at 50 ° C. to 80 ° C. and pH 4.0 to 5.0. , Xylanase isolated from pine twig butterfly.

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