KR101541787B1 - Alkaline resistance xylanase produced from Streptomyces sp. CS628 and its use - Google Patents

Abstract

The present invention relates to an alkali-resistant novel xylanase from Streptomyces sp. And its use. The xylan degrading enzyme Xyn628 according to the present invention was stable in a wide range of pH 5 to 13, indicating that it was an alkaline resistant xylanase. Xyn628 produced xylobiose and xylotriose as major hydrolysis products from xylan, indicating endotypic xylanase. Xyn628 has been shown to be deficient in cellulase activity, and due to its low molecular weight and various biochemical properties, the enzyme can be more suitably used in bio-bleaching and other bioindustries.

Description

The present invention relates to a novel xylanase degrading enzyme derived from Streptomyces sp. CS628 strain and its use. CS628 and its use}

The present invention relates to a novel alkaline resistant xylanase from Streptomyces sp. CS628 strain and its use.

Plant biomass, mainly lignocellulosic biomass, is the most abundant and widespread renewable raw material for the continuous production of a variety of expensive biological products [Ref. 1]. Recently, considerable research attention has been focused on the production of microbial xylenolytic enzymes using agricultural waste products because they are inexpensive and readily available, and the lignocellulose, especially hemicellulose, is highly abundant.

Nonetheless, agricultural waste is unnecessarily occupying space and causing environmental pollution. The use of these inexpensive and easy-to-obtain industrial wastes not only solves the problem of improper disposal but also adds value.

Xyloglycosides are a key component of the hemicellulose fraction as xylans and are inherently present in most of the agricultural biomass [Ref. 2].

Thus, large scale production of xyloglucoside was performed from lignocellulosic biomass, especially from hemicellulose rich. The importance of xylo-oligosaccharides as an important ingredient is that this is increasingly of interest in that it has diverse health benefits in foods, medicines, etc. [3].

Xylen is the second most abundant polysaccharide on the planet and is the main hemicellulose of plant cell walls that make up 20-40% of the total plant biomass. Xylen is a heteropolysaccharide composed of a linear skeleton of 1,4-linked D-xylose units, which forms a physical bond between lignin and other polysaccharides and forms a linkage between beta-1, 4-endo xylanase (EC 3.2 .1.8), β-xylosidase (EC 3.2.1.37), and several other side chain cleavage enzymes [4-5].

In addition, many microorganisms, including bacteria, fungi and yeast, produce different types of endo-xylanase, and microbial xylenolytic enzymes are widely used in animal feed, paper, pulp, waste disposal, baking, brewing, [6]. When xylenolytic enzyme is used for the pretreatment of kraft-pulped pulp, the low molecular weight xylenolytic enzyme forms small holes continuously and enters the inside for hydrolysis [Reference: 7-8]. In addition, xylenolytic enzymes that are active at low molecular weight, alkaline pH and free of cellulose are very easy to use for biological bleaching in the paper and pulp industries [9]. The paper and pulp industries require low molecular weight xylenolytic enzymes because the reprecipitated and repositioned xylenes on the fiber require higher amounts of oxidants. But the problems facing these industries are cost and availability of enzymes. Better microbial strains, efficient fermentation and recovery systems as well as production from agricultural waste will always be a welcome approach in these directions.

To date, the xylanase enzymatic activity has been reported to be in the order of Streptomyces rameus L2001 [Reference: 10], Streptomyces cyaneus SN32 [Reference: 11], Streptomyces catreousis Streptomyces chartreusis L1105 [Reference: 12], Streptomyces sp. SWU10 [Reference: 13], Streptomyces sp. CS802 [Reference: 14], Streptomyces mattensis Streptomyces sp. S38 [Reference: 16], among others, as well as Streptomyces spp. , Streptomyces matensis [Reference: 15] and Streptomyces sp.

In the present invention, the strong xylenolytic enzyme, named Xyn628, produced in a bran medium and isolated from Streptomyces strain by purification, and biochemical characteristics of the enzyme will be described.

A new thermolabile alkaline phospholipase D from Streptomyces sp. Was used to isolate the cells. CS 628. Biotechnol Bioproc E. 2010; 15: 595-602.

The inventors of the present invention have made intensive studies in order to find a method for efficiently utilizing agricultural wastes. As a result, it has been found that Streptomyces species We found that Xyn628 xylenolytic enzyme can be economically produced by culturing in a culture medium supplemented with bran, which is readily available from the environment and is cheap in terms of agricultural by-product, and which effectively treats agricultural wastes, It has been found that it is possible to efficiently produce a functional ingredient from agricultural wastes and have completed the present invention.

Accordingly, an object of the present invention is, in one aspect, a method for preparing a high alkaline solution having a molecular weight of ~ 18.1 kDa, which is separated from a Streptomyces strain and measured by SDS-PAGE and xylan zymography, at a temperature of 60 ° C and a pH of 11.0 And exhibits stable activity over a wide range of pH 5 to 13, and is an endotypic xylenolytic enzyme that produces xylobiose and xylotriose as main hydrolysis products, and has cellulase activity The present invention provides a xylan-degrading enzyme which is characterized in that it is deficient.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. .

The xylan degrading enzyme Xyn628 according to the present invention was stable in a wide range of pH 5 to 13, indicating that it was an alkaline resistant xylanase. Xyn628 produced xylobiose and xylotriose as major hydrolysis products from xylan, indicating endotypic xylanase. Xyn628 has been shown to be deficient in cellulase activity, and due to its low molecular weight and various biochemical properties, the enzyme can be more suitably used in bio-bleaching and other bioindustries.

Figure 1 shows the elution profile of Xyn628 following gel permeation chromatography using a Sepharose CL-6B column (1.7 cm ~ 77.5 cm).
Figure 2 shows SDS-PAGE and the results of the digestion of the purified xylanase from Xyn628.
Fig. 3 is a diagram showing the hydrolysis of Xanthan beech xylan with time lapse. Fig.
Figure 4 is a scanning electron micrograph showing the bran and corncob meal decomposition by Xyn 628;

In one aspect, the present invention provides, in one aspect, a method for the production of a polypeptide having a molecular weight of ~ 18.1 kDa as determined by SDS-PAGE and xylan zymography, isolated from a Streptomyces strain, under conditions of 60 ° C and high alkali at pH 11.0 Activity and exhibits stable activity over a wide range of pH 5 to 13, and is an endotypic xylenolytic enzyme that produces xylobiose and xylotriose as major hydrolysis products, and is an endotypic xylenolytic enzyme that is deficient in cellulase activity Wherein the xylan-degrading enzyme is selected from the group consisting of:

In another aspect the present invention provides a xylan degrading enzyme wherein the strain is Streptomyces sp. CS628 (strain Accession No. KCTC 18252P).

The invention provides, in yet another alternative aspect, a xylenolytic enzyme wherein the first 12 amino acids of the N-terminus are those of SEQ ID NO: 1.

In a still further aspect, the present invention provides a method characterized by the use of xylenolytic enzymes in the production of biotin or xyloligosaccharides.

In a still further aspect, the present invention provides a method for producing Streptomyces sp. Strain CS628;

Wherein the selected strain of 2% wheat bran, 0.5% yeast extract, 1% tryptone, 0.75% of potassium phosphate _{(KH 2 PO 4), 0.15} % J. potassium phosphate (K ₂ HPO ₄₎ and 0.05% of magnesium sulfate (MgSO ₄₎ Lt; RTI ID = 0.0 > 30 C < / RTI > and 120 rpm for 4 days in a shaking incubator;

Centrifuging the culture obtained in the above step at 6,000 x g for 45 minutes at 4 DEG C to obtain a crude supernatant;

Precipitating the crude supernatant with 30 to 75% ammonium sulfate;

The precipitated proteins are recovered by centrifugation at 6,000 x g for 1 hour, dialyzed against 10 mM Tris / HCl (pH 7.5), and concentrated with an ultrafiltration membrane;

The enzyme solution was added to a Sepharose CL-6B column (1.7 cm x 77.5 cm), the column was equilibrated with 10 mM Tris / HCl, pH 7.5 and eluted with the same buffer at a flow rate of 5.29 cm / h (2.5 mL in each fraction) And a step of washing the xylanase.

Hereinafter, the present invention will be described more specifically.

The present inventors isolated a strong low molecular weight xylenolytic enzyme from Streptomyces sp. Into Korean soils and cultured in a medium containing wheat bran. Extracellular xylenolytic enzyme, Xyn628, was purified by gel permeation chromatography (Sepharose CL-6B column) and biochemically characterized. The purified xylenolytic enzyme had a molecular weight of ~ 18.1 kDa as measured by SDS-PAGE and xylan zymography, which is the smallest of the purified xylanase enzymes reported so far from Streptomyces. The N-terminal amino acid sequence of Xyn628 is different from the xylenolytic enzyme reported as AYIKEVVSRAYM (SEQ ID NO: 1). Its enzyme activity was maximal at pH 11.0 and 60 ° C and stable at a wide range of (5.0-13.0) and 60 ° C. Xyn628 activity was increased by various detergents.

This xylenolytic enzyme produced xylobiose and xylotriose as major hydrolysis products from xylans, which means that it is endo xylanase in nature. Importantly, the scanning microscope analysis showed that Xyn628 was able to stably break down agricultural wastes such as corncobs and wheat bran by a small unit of enzyme (20 U / g).

In short, in view of the simplicity of the purification, very low molecular weight, alkali resistance, thermal stability and the ability to produce xyloglucosides, Xyn628 can be used as a bio-bleaching agent, The potential applicability in

<Examples>

Hereinafter, the present invention will be described more specifically by the following representative examples, but the present invention is not limited in any way by these embodiments.

The xylose, beech and birch xylan used in the following examples were purchased from Sigma-Aldrich (St Louis, MO, USA). Sepharose CL-6B was purchased from Amersham Bio-Sciences AB (Uppsala, Sweden). Thin layer chromatography silica gel plates were purchased from Merck (Darmstadt, Germany). Xylobiose, xylotriose and xylotetraose were purchased from Megazyme (Ireland). All other chemicals were of analytical grade.

Example 1: Microbial strain, enzyme production and purification

Streptomyces strain CS628 was isolated from soil in Chonnam province, Korea and was identified as a producer of strong xylanase in solid medium containing xylan [Reference: 17]. This strain was identified as previously reported by the inventors [Ref. 18].

The 16S rDNA sequence of this strain was obtained from Streptomyces cyanecfuscatus (Accession no. AY999770) and S. cavourensis subsp. Washingtonis NRRLB-8030T (Accession no. DQ026671 ), And this strain was named Streptomyces sp. CS628.

Screening the strains, and the xylan-rich as agricultural waste of bran and sole carbon source, 2% wheat bran, 0.5% yeast extract, 1% tryptone, 0.75% of potassium phosphate _{(KH 2 PO 4), 0.15} % J. potassium phosphate (K ₂ HPO ₄ ) and 0.05% magnesium sulfate (MgSO ₄ ). The cultivation was carried out in a 1 liter flask containing 250 mL medium for 4 days with shaking incubator at 30 캜 and 120 rpm. After 4 days, the culture was centrifuged at 6,000 x g for 45 minutes at 4 ° C. The crude supernatant was precipitated with 30-75% ammonium sulfate.

Proteins were recovered by centrifugation at 6,000 x g for 1 hour, dialyzed against 10 mM Tris / HCl (pH 7.5), and concentrated using an ultrafiltration membrane (YM 5 kDa, Millipore Corp., Amicon Danvers, MA, USA). The dialyzed enzyme solution was then added to a Sepharose CL-6B column (1.7 cm x 77.5 cm). The column was pre-equilibrated with 10 mM Tris / HCl, pH 7.5 and washed with the same buffer at a flow rate of 5.29 cm / h (2.5 mL in each fraction). One purification of the active xylanase fragment (Figure 1) was obtained by this purification procedure, and its homogeneity was checked by SDS-polyacrylamide gel electrophoresis. Figure 1 shows the elution profile of Xyn 628 after gel permeation chromatography using a Sepharose CL-6B column (1.7 cm x 77.5 cm).

Xylenolytic enzymes were produced and harvested 4 days later, at which time they showed maximal activity (data not shown). Production of xylenolytic enzyme (6757 U / mL) using wheat bran as substrate was 9.38 times higher than that of xylenolytic enzyme from S. cyaneus SN3210 using bran. However, the maximal xylenolytic activity produced in S. olivaceoviridis E-86 23 and S. rameus L200110 was 1385 U / mL and 1810.9 U / mL, respectively, when using corncobs. Wheat bran, cornstarch, raspberry, cornstarch, and tobacco stalks are abundant in hemicellulose and are considered potential feedstocks for industrial use. The use of these materials for industrial purposes not only addresses the proper disposal of waste but also provides additional income to the farm and creates employment. Thus, strain CS628 has been observed to be a potent xylenolytic enzyme producer using wheat bran as a substrate, and is inexpensive, readily available and applicable to a variety of bio industries.

Example 2: Enzyme activity and protein measurement

The activity of xylanase was analyzed by Miller's method [Ref. 19]. The reaction mixture contained 0.1 mL of enzyme (10 mM, KCl / NaOH, pH 11.0) diluted in 0.1 mL of 0.5% (w / v) beech xylan and reacted at 60 ° C for 5 minutes. Controls were started simultaneously with the reactants in ice water (0 ° C). The amount of reducing sugar was determined by the dinitrosalicylic acid (DNS) method using xylose as a standard. One unit of xylanase was defined as the amount of enzyme catalyzing the release of 1 μmol xylose equivalent per minute under standard analytical conditions. Cellulase activity was assessed using artificial substrates such as sigma cell cellulose, carboxymethylcellulose, avicel and para-nitrophenyl D-cellrobioxide (pNPC) and para-nitrophenyl-beta-D glycopalanoside (pNPG) Respectively. Protein concentration was also determined according to the Bradford method [Ref. 20] using bovine serum albumin as standard.

A summary of the Xyn628 purification steps from the culture supernatant CS628 is shown in Table 1.

Purification step Total protein
(mg) Total active (U) Specific activity (U / mg) yield(%) water
Drainage (NH _{4) 2} SO ₄ Fractionation 2.29 459836 200802 100.0 1.0 Sepharose CL-6B 0.15 155324 1035493 33.78 5.16

Upon completion of the chromatography, 5.16-fold purity and 33.78% activity were recovered. The purity multiplier is S. cyaneus SN32 [Reference: 11] and S. Sp. CS802 [Reference: 14], while the activity recovery was almost similar to that of S. chartreusis L1105 [Reference: 12].

Example 3: Gel electrophoresis

The purified enzyme was subjected to SDS-PAGE on a 12% (w / v) polyacrylamide gel according to a known method [reference: 21]. As a reference protein, a protein size marker (MBI, Fermentas) was used. Proteins were stained with Coomassie Brilliant Blue R-250 and discolored and observed with a solution containing methanol: glacial acetic acid: distilled water = (1: 1: 8 by volume). Molecular weight was determined by comparison with the relative mobility of the reference protein. In addition, xylan zymography was carried out as described in the reference [22].

Analysis of the purified xylenolytic enzyme on SDS-PAGE and analysis of the activity (self-immunogram) stained by gel electrophoresis are shown in FIG. Figure 2 is a diagram showing SDS-PAGE and zymography results of purified xylenolytic enzyme from Xyn628. In lanes, Mr is the protein molecular weight marker, lane 1 is the purified xylenolytic enzyme after Sepharose CL-6B on SDS-PAGE, and lane 2 is the purified band on the zymography.

The enzyme migrated as a single band with a molecular weight of about 18.1 kDa, and its purity was homogeneous.

The xylenolytic enzyme exhibited a relatively clear band on the zymogram gel detected by Congo red staining, indicating that it is active. According to SDS-PAGE and zymography, this xylenolytic enzyme exhibited the smallest size among the purified straptomies reported so far. However, the xylenolytic enzymes produced by S. cyaneus SN32 [Reference 11], S. rameus L2001 [Reference: 10] and S. matensis DW6715 were similar to the molecular weights of Xyn628.

Example 4: Effect of pH and temperature

The optimal pH was adjusted to 10 mM pH buffer (pH 3.0 to 7.5), Tris / HCl (pH 7.0 to 9.5), and CAPS (pH 9.0 to 11.0) at various pH values (3.0 to 13.6) And potassium chloride-sodium hydroxide (KCl / NaOH (pH 11.0-13.6)). The optimal temperature for xylenolytic enzymes was performed by reacting the enzyme at different temperatures (40-80 ° C) in 10 mM KCl / NaOH (pH 11.0). The pH stability was determined by reacting the enzyme samples with various pH buffers (10 mM) at 0-4 ° C for 12 hours and measuring the residual enzyme activity. To determine enzyme temperature stability, enzyme samples were reacted at various temperatures (below 80 ° C) for 60 minutes and residual activity was determined under standard active assay conditions.

Enzyme activity and stability were significantly influenced by pH and temperature. The effect of pH on the xylenolytic enzyme was determined within a pH range of 2.0 to 13.6. Xyn628 showed maximal activity at pH 11.0 and activity at pH 5.0 to 13.0. At pH 5.0 and 13.0, the relative activity was 86% and almost 80% of the maximum activity, respectively. In addition, Xyn628 was very stable at wide pH over the range of pH 5.0-12.5 and showed maximum stability at pH 11.0. The stability of this enzyme showed about 50% residual activity at pH 12.5. This unique feature indicates that Xyn628 is an extremely alkaline xylanase. The reason for being stable at a wide range of pHs may be due to the reversible denaturation of the protein and may have no effect on the enzyme activity upon reaction at different pH. Another report mentions that the amino acid (s) near the catalytic center of the enzyme are involved in such highly alkaline properties [Ref. 24]. However, Xyn628 has been shown to be effective against S. cyaneus SN32 [11], S. rameus L2001 [10], S. matensis DW67, [15], and S. olivaceoviridis E-86 [ Lt; RTI ID = 0.0 &gt; alkaline &lt; / RTI &gt; medium. In addition, Xyn628 showed activity at 50 ㅀ C to 70 ㅀ C and maximum activity at 60 ㅀ C. At a temperature of 50 to 75 캜, the enzyme activity was 70% or more of the maximum activity. The enzyme was completely stable even after reaction at 50 ° C for 1 hour. This maximum activity and stability are comparable to many other Streptomyces xylansolases [15-16, 23, 25]. Nevertheless, it has been found that almost 50% of the maximal activity remains after 1 hour of reaction at 65 ° C. Although partially stable at 65 ° C, the frequency of maximum activity at high temperatures appears to be due to the protective effect of the substrate on Xyn 628 for the optimal time. Since many industrial processes operate at extreme pH (acidic and / or alkaline) and elevated temperatures, the enzymes must meet such process requirements and, for extended periods of time, or at least during processing times that we can find in Xyn 628 It must be able to withstand such harsh conditions. Tolerance to a wide range of pH values, high optimal temperatures, and its substantial thermal stability make Xyn628 applicable to a variety of biotechnologies, including bio-bleaching, xylan hydrolysis, and bio-ethanol production.

Example 5: Influence of various additives

Xyn628 activity was studied in the presence of various additives such as detergents, metal ions, reducing and oxidizing agents. (Na ⁺ and K ⁺ ) and divalent (Ca ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Cu ²⁺ , Mg ²⁺ and Fe ²⁺ ) Metal ions were added to the reaction mixture at a concentration of 1 mM. Conversely, the effects of oxidation and reducing agents on Xyn628 activity were tested by adding hydrogen peroxide, sodium borate, sodium perborate,? -Mercaptoethanol and dithiothreitol at a concentration of 5 mM. On the other hand, the effect of the chelating agent on Xyn628 activity was evaluated by EDTA and ethylene glycol tetraacetic acid (EGTA). In addition, the effect of various types of detergents on Xyn628 activity was performed at a concentration of 0.25%. Xyn628 activity of the detergent was investigated by the addition of Triton X-100, Tween-20, Tween-80, polyoxyethylene-4-lauryl ether, deoxycholic acid (DCA) and SDS. In all cases, the enzyme-free enzyme activity was considered 100% under standard activity assay conditions. The effects of various additives on Xyn628 activity are shown in Table 2.

density Types of ions Relative activity ^e (%) Detergent ^a Triton X-100 0.25% Non-ionic 109 ± 3.3 ^a Tween-20 0.25% Non-ionic 130 ± 2.3 ^a Tween-80 0.25% Non-ionic 118 ± 3.0 ^a Polyoxylethylene-4-laurylether 0.25% Non-ionic 97 ± 1.5 ^a Deoxycholic Acid 0.25% Anionic 16 ± 3.1 ^a Sodium Dodecyl Sulphate 0.25% Anionic 19 ± 3.9 Modulators ^b hydrogen peroxide 5 mM 18 ± 1.8 ^b Sodiumperborate 5 mM 43 ± 2.9 ^c β-mercaptoethanol 5 mM 97 ± 4.5 ^c 1,4-dithiothreitol 5 mM 128 ± 4.7 ^d EDTA 1 mM 18 ± 1.5 ^d EGTA 1 mM 26 ± 2.4 None - 100 ± 2.1

^a detergent; ^b oxidizing agent; ^c reducing agent; ^d chelating agent,

^e Results are expressed as mean ± standard deviation (n = 3)

Xyn628 activity was highly influenced by various detergents, metal ions, metal chelating agents (EDTA and EGTA), reducing and oxidizing agents. Among the detergents, Tween-20 (130%) affected the activity, followed by Tween-80 (118%) and Triton X-100 (109%), followed by polyoxyethylene- (97%) had little effect. In addition, the effects of? -Mercaptoethanol and DTT were almost insignificant, which means that the cysteine residues do not participate in the activity of the enzyme, and similar results have been found in the prior literature of the present inventors ]. An increase in Xyn628 activity by detergents has been reported in Streptomyces [Refs. 24, 26]. On the other hand, enzyme activity was significantly inhibited by SDS (19%). In contrast, SDS [Refs. 27-28] was obtained from Streptomyces sp. B-12-2, and Bacillus sp. But did not affect xylenolytic enzymes from other sources such as 41M-1.

delete

The overall inactivation due to SDS has also been reported in different sources [ref. 29], where the inertity was due to the superposition loosening of the enzyme under such conditions. However, Xyn628 showed excellent activity in the presence of all types of detergents except SDS, which means that Xyn628 is unique and more applicable in the biotechnology industry.

In addition, Xyn628 activity was highly influenced by various metal ions, metal chelating agents (EDTA and EGTA), which means its metal type, and is similar to our previous report [Ref. 14]. However, the relative activities of Ca ²⁺ , Mg ²⁺ , Cu ²⁺ , Co ²⁺ , Zn ²⁺ , Mn ²⁺ , Fe ²⁺ , K ⁺ and Na ⁺ 111 ± 3.5, 62 ± 3.2, 0 ± 3.0, 95 ± 3.7, 52 ± 4.3, 1 ± 2.7, 47 ± 3.7, 102 ± 3.0, and 97 ± 4.8%. Xyn628 was a bivalent metal ion other than Ca ^{2 +} . The results are as follows. Induced xylanase activity by metal ions suggests that several metal ions, particularly Ca ^2+, are required to maintain structural stability. However, Cu ²⁺ ions are known to catalyze the autoxidation of cysteine, which leads to intermolecular and intramolecular disulfide bond formation or formation of sulfene acid [Ref. 30]. It has been found that most of the xylenolytic enzymes, particularly those derived from streptomyces, are inhibited by Mn ²⁺ ions [Reference: 10, 12-13, 15]. In addition, Xyn628 activity was completely inhibited by the oxidizing agents H ₂ O ₂ and sodium-perborate, which means that they are sensitive to oxidizing agents.

Example 7: N-terminal amino acid sequence analysis

The N-terminal amino acid sequence of Xyn628 was determined by Edman degradation using a Procise Model 492 protein sequencer (Applied Biosystems, CA, USA). The first 12 amino acid sequence of the N-terminus of Xyn628 was AYIKEVVSRAYM (SEQ ID NO: 1). These sequences did not show any significant homology with reported enzymes of a similar type. This uniqueness suggests novelty of this enzyme.

Example 8: Substrate specificity and kinetic parameters

To evaluate the substrate specificity of the purified enzyme, the enzyme was reacted with 0.5% (w / v) of each substrate and the activity was measured under standard active assay conditions. In addition, the kinetic parameters were determined using hyperbolic computer software program. The Michaelis-Menten constant ( K _m ) and maximum velocity ( V _max ) were determined by the Hanes plot. In the experiment, five different concentrations (2.5-40 mg / mL) of beech xylan and a constant enzyme concentration (0.18 x g) were prepared and the experiment was performed under standard conditions under standard conditions.

The purified Xyn628 enzyme was analyzed on a variety of commercial substrates and the highest activity of xylanase activity was observed in beech xylan compared to birch xylan (53%). The enzyme did not show any cellulose activity when using avicel, carboxymethylcellulose, p-nitrophenylglucopyranoside (pNPG) and p-nitrophenyl-β-D-cellobiose (pNPC) ). These results demonstrate that Xyn628 is cellulase free, similar to the xylenolytic enzyme reported in Streptomyces [10, 28], which is one of the important features as a bio bleach agent in the paper and pulp industries.

The kinetic constants ( K _m ) and ( V _max ) of the enzyme calculated by the Hanes plot using beech xylan were 6.4 ± 0.8 mg / ml and 437 ± 21 mmol / min mg, respectively. K _{m of} Xyn628 The value is S. rameus L2001 [Reference: 12], but its high V _max value was slightly lower than that of S. sp. CS802 [Reference: 14].

Example 9: Availability of enzymes in hydrolysis and xylooligosaccharide production

To determine the mode of action of Xyn628 and its availability in the production of xylooligosaccharides, a simple and practical evaluation method called thin layer chromatography (TLC) was performed according to known methods [Ref. 22]. The reaction was terminated by adding 5 x L of 10% (v / v) trichloroacetic acid to the enzyme reaction solution (50 x L) at different time intervals (up to 120 min) and the samples were loaded onto a silica gel plate 60F 254 (E. Merck, Germany).

 Plates (6 cm x 6.5 cm) were developed with chloroform: acetic acid: water (6: 10: 2, v / v) and then the plates were sprayed with a methanol: sulfuric acid mixture (95: 5, v / v) Heat was applied at 130 캜 for several minutes on a hot plate. A mixture of xyloglucosides (10 mg / mL) consisting of xylose (X1), xylobiose (X2), xylotriose (X3) and xylotetraose (X4) was used as standard. In addition, to evaluate the degradation of bran and corncob meal by Xyn 628, they were analyzed by scanning electron microscopy according to the method described in [10]. For the experiment, untreated corncobs and bran were washed with distilled water to neutral pH. Bran and cornstarch were then treated with Xyn628 at 20 U / g and 2000 U / g at a stable temperature (50 ° C) for 3 hours at an optimal pH (11.0), while the enzyme samples replaced with buffer were used as control samples.

Figure 3 shows the time course hydrolysis of beech xylan by Xyn628. Figure 3 is a diagram showing the time course hydrolysis of beech xylenes by Xyn 628; S is a mixture of xylose (X1), xylobiose (X2), xylotriose (X3) and xylotetraose (X4).

Xyn628 produced a series of xylo-oligosaccharides predominantly following xylobiose (X2) and xylotriose (X3), indicating that it is an endo-xylanase in nature. Generally, in the case of exo-acting enzymes, only monomers or dimers are accumulated through an enzymatic reaction. In this example, Zarobiose and some oxy oligosaccharides were produced by the action of enzymes, indicating that Xyn628 is an endotypic xylenolytic enzyme, and a similar type of result is also found in the case of xylan degradation from Streptomyces . [Reference: 14-15] Enzymes.

In recent trends, xylooligosaccharides are of great interest not only in the food industry but also in modern medical applications due to their prebiotic potential. In particular, xylobiose and xylotriose can be used directly as food ingredients because they are known to actively regulate intestinal microflora and suppress the growth of pathogenic microorganisms [31]. However, high xylobiose and low xylose are preferred during the xyloligosaccharide production process, because hydrolysates containing xylose greater than 12% are less effective for industrial purposes [Ref. 32]. Figure 4 is a scanning electron micrograph (magnification 1,000 x) showing the bran and corncob meal breakdown by Xyn 628;

In addition, as shown in Fig. 4, Xyn628 effectively decomposed bran and corncob meal even at very low capacity (20 U / g), as analyzed by scanning electron microscopy (SEM). These results indicate that Xyn628 is suitable for decomposition of bran, corncobs, and many other xylen-rich agricultural by-products, and thus can be used in the production of value-added products such as xylooligosaccharides, bioethanol, and the like.

Basically, successive decomposition of agricultural by-products by Xyn 628 is significantly reduced by using xyloglucoside production costs lower, reproducible, readily available and unnecessarily occupying space-consuming xylan-rich agricultural byproducts .

More importantly, low molecular weight xylenolytic enzymes as bleaching agents in the pulp and paper industry are desirable because they readily permeate into the reprecipitated xylan on the surface of the pulp. This alleviates the problem of xylan barriers on the surface of lignin containing pulp during subsequent chemical bleaching steps [Ref. 33].

However, the lack of cellulosic activity is more important for the production of xyloglucosides [Ref. 15]. In addition, a number of reports of ideal xylenolytic enzymes useful for bio-bleaching have already been published, which must be active and stable in high temperature and alkaline kieselguhr, and should also have cellulase activity [15,34]. Therefore, Xyn628 has these characteristics and is highly likely to apply to a variety of bioindustries.

In conclusion, we purified and biochemically characterized Xyn628, a potent low molecular weight extracellular xylenolytic enzyme produced in agricultural waste (wheat) medium. This xylenolytic enzyme was suitably active at 60 ° C and under high alkali conditions (pH 11.0).

Interestingly, Xyn628 was stable over a broad range of pH 5-13, indicating an alkaline resistant xylanase negative tone. Xyn628 produced xylobiose and xylotriose as major hydrolysis products from xylan, indicating endotypic xylanase. Xyn628 has been shown to be deficient in cellulase activity, and due to its low molecular weight and various biochemical properties, the enzyme can be more suitably used in bio-bleaching and other bioindustries.

In addition, the scanning microscope analysis showed continuous degradation of corncobs and wheat bran, the agricultural wastes, by Xyn 628, which not only is useful for the production of xyloglucose with added value, but also keeps the environment pleasant due to low economic costs, As shown in FIG.

Institution name: Korea Biotechnology Research Institute
Accession number: KCTC18252
Funding date: 20130712

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<110> Chosun University Industry-Academic Cooperation Foundation <120> Alkaline resistance novel xylanase produced from Streptomyces sp.          CS628 and its use <130> pkr-455 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 12 <212> PRT <213> Streptomyces <400> 1 Ala Tyr Ile Lys Glu Val Val Ser Arg Ala Tyr Met   1 5 10

Claims (5)

delete delete delete delete Selecting Streptomyces sp. Strain CS628;
Wherein the selected strain of 2% wheat bran, 0.5% yeast extract, 1% tryptone, 0.75% of potassium phosphate _{(KH 2 PO 4), 0.15} % J. potassium phosphate (K ₂ HPO ₄₎ and 0.05% of magnesium sulfate (MgSO ₄₎ Lt; RTI ID = 0.0 &gt; 30 C &lt; / RTI &gt; and 120 rpm for 4 days in a 1 liter flask containing 250 mL of containing medium;
Centrifuging the culture obtained in the above step at 6,000 x g for 45 minutes at 4 DEG C to obtain a crude supernatant;
Precipitating the crude supernatant with 30 to 75% ammonium sulfate;
The precipitated proteins were recovered by centrifugation at 6,000 xg for 1 hour, dialyzed against 10 mM Tris / HCl (pH 7.5), and concentrated to an ultrafiltration membrane;
The enzyme solution was added to a Sepharose CL-6B column (1.7 cm x 77.5 cm), the column was equilibrated with 10 mM Tris / HCl, pH 7.5 and eluted with the same buffer at a flow rate of 5.29 cm / h (2.5 mL in each fraction) And washing,
The Streptomyces sp. CS628 is deposited with the strain Accession No. KCTC 18252P,
The resulting xylanase has a molecular weight of 18.1 kDa as measured by SDS-PAGE and xylan zymography, the first 12 amino acids of the N-terminus being of SEQ ID NO: 1, a temperature of 60 ° C and a pH of 11.0 An alkali-tolerant xylanase having activity under alkaline conditions and exhibiting activity in the range of pH 5 to 13, an endotypic xylenolytic enzyme producing xylobiose and xylotriose as hydrolysis products, Wherein the enzyme is an alkali-resistant xylenolytic enzyme.

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