Extracellular Xylanase Research Articles

Production of a thermostable and alkali resistant endoxylanase byBacillus subtilisDFR40 and its application for preparation of prebiotic xylooligosaccharides

Xylanases produced by microorganisms hydrolyze xylan, into xylose or xylooligosaccharides (XOS). Xylan is an abundant noncellulosic polysaccharide found in the plant biomass. The enzyme is of great practical importance for the conversion of xylan containing agricultural residues into XOS for use as prebiotics. A Bacillus subtilis strain DFR40 a generally recognized as safe (GRAS) organism was found to produce extracellular xylanase in a growth medium containing wheat bran, mineral salts, peptone, and yeast extract at pH 7.0. The optimum activity of the enzyme was around 100°C and pH 9.0 with stability over a broad range of pH (7.0–12.0) displaying enzyme stability of more than 70% even after pre-incubation at 55–85°C for 30 min. The enzyme hydrolyzed wheat bran into short chain XOS as revealed by TLC, HPLC, FT-IR, and NMR analysis. Practical applications The thermostable and alkali resistant characteristics of extracted xylanase along with ability to hydrolyze wheat bran for the production of xylooligosaccharides (XOS), for use as prebiotic ingredients in food, are relevant in the present health conscious worldwide scenario. XOS are emerging class of prebiotics with thermal resistance up to 100°C, and are suitable for incorporating into thermally processed food products with extended shelf life. The XOS preparation was also found suitable suggesting the prebiotic nature of XOS on known probiotic bacterial strains. The study also indicates a valuable application for wheat bran, a cheap agro residue for further cost effective scale up of XOS production. The xylanase produced is of significance as the producing bacterium is a GRAS organism for further scale-up of the processes of enzyme and XOS production.

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13.

Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6–9) and temperature (30–65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 μM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

BackgroundMetabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Experiments were performed to understand the effect of monosaccharides on gene expression during growth on the polysaccharide, WAX.ResultsMolecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were downregulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, showing a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell.ConclusionsGlycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases were not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader substrate range than that represented by the glycoside hydrolase family in which the enzymes were categorized.

Characterization of a Thermophilic Lignocellulose-Degrading Microbial Consortium with High Extracellular Xylanase Activity.

A microbial consortium, TMC7, was enriched for the degradation of natural lignocellulosic materials under high temperature. TMC7 degraded 79.7% of rice straw during 15 days of incubation at 65°C. Extracellular xylanase was effectively secreted and hemicellulose was mainly degraded in the early stage (first 3 days), whereas primary decomposition of cellulose was observed as of day 3. The optimal temperature and initial pH for extracellular xylanase activity and lignocellulose degradation were 65°C and between 7.0 and 9.0, respectively. Extracellular xylanase activity was maintained above 80% and 85% over a wide range of temperature (50-75°C) and pH values (6.0-11.0), respectively. Clostridium likely had the largest contribution to lignocellulose conversion in TMC7 initially, and Geobacillus, Aeribacillus, and Thermoanaerobacterium might have also been involved in the later phase. These results demonstrate the potential practical application of TMC7 for lignocellulosic biomass utilization in the biotechnological industry under hot and alkaline conditions.

Designing cross-linked xylanase aggregates for bioconversion of agroindustrial waste biomass towards potential production of nutraceuticals

Immobilized biocatalysts design has the potential to efficiently produce valuable bioproducts from lignocellulosic biomass. Among them, the carrier-free immobilization through the cross-linked enzyme aggregates technology is a simple and low-cost alternative. A two steps statistical approach was utilized to evaluate the synthesis of a cross-linked enzyme aggregate from a xylanolytic preparation, which was produced by Cohnella sp. AR92 grown in a peptone-based culture medium.The resulting immobilized biocatalyst, Xyl-CLEA, was significate more stable (25 to 45%) towards temperatures up to 50°C with respect to the free enzyme, and retained over 50% of its initial activity after 5 consecutive cycles of reuse. By means of infrared spectroscopy and electron microscopy, the Xyl-CLEA showed architectural features described as signature of type I and type II of protein aggregates. These, were the result of the simultaneous aggregation of a multiplicity of proteins from the crude enzymatic extract.The enzymatic activity was assessed using alkali pretreated sugar cane bagasse as substrate. Whereas the free enzymatic preparation released xylose as the main product, the immobilized xylanase produced xylooligosaccharides, thus showing that the immobilization procedure modified the potential application of the extracellular xylanase from Conhella sp. AR92.

Stable activity of extra-cellular xylanases and its phylogeny in different Bacillus species

Stable activity of extra-cellular xylanases and its phylogeny in different Bacillus species

GH11 xylanase from Aspergillus tamarii Kita: Purification by one-step chromatography and xylooligosaccharides hydrolysis monitored in real-time by mass spectrometry

The present study describes the one-step purification and biochemical characterization of an endo-1,4-β-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was obtained at 60°C and it was active over a broad pH range (4.0–9.0), with maximal activity at pH 5.5. The enzyme was thermostable at 50°C, retaining more than 70% of its initial activity for 480min. The K0.5 and Vmax values on beechwood xylan were 8.13mg/mL and 1,330.20μmol/min/mg of protein, respectively. The ions Ba2+ and Ni2+, and the compounds β-mercaptoethanol and DTT enhanced xylanase activity, while the heavy metals (Co2+, Cu2+, Hg+, Pb2+ and Zn2+) strongly inhibited the enzyme, at 5mM. Enzymatic hydrolysis of xylooligosaccharides monitored in real-time by mass spectrometer showed that the shortest xylooligosaccharide more efficiently hydrolyzed by A. tamarii Kita xylanase corresponded to xylopentaose. In agreement, HPLC analyzes did not detect xylopentaose among the hydrolysis products of xylan. Therefore, this novel GH11 endo-xylanase displays a series of physicochemical properties favorable to its application in the food, feed, pharmaceutical and paper industries.

Xylooligosaccharides production by crude microbial enzymes from agricultural waste without prior treatment and their potential application as nutraceuticals

Aspergillus fumigatus R1, on submerged fermentation using agricultural residues as carbon source produced extracellular xylanase (152IU/ml after 96h of incubation at 37°C with constant shaking at 100rpm). A maximum yield of 1gm% Xylooligosaccharides (XOS) mixture was obtained after 12h by enzymatic hydrolysis of xylan rich wheat husk without any prior pretreatment using the crude enzyme without any purification. HP-TLC data confirmed the presence of an array of XOS for its prebiotic properties by carrying out studies on ten standard probiotic cultures. Six of ten probiotic cultures were able to utilize XOS produced from agricultural wastes and showed remarkable growth in the media containing XOS as the sole source of carbon. XOS mixture also exhibited concentration dependent anti-oxidant activity. Thus, the results showed that XOS produced from agricultural residues have great prebiotic potential and good antioxidant activity; therefore, it can be used in food-related applications.

Xylanase production from marine derived Trichoderma pleuroticola 08ÇK001 strain isolated from Mediterranean coastal sediments.

Xylanases constitutes one the most important enzymes with diverse applications in different industries such as bioethanol production, animal feedstock production, production of xylo-oligosaccharides, baking industry, paper and pulp industry, xylitol production, fruit juice, and beer finishing, degumming, and agriculture. Currently, industrial xylanases are mainly produced by Aspergillus and Trichoderma members. Since the marine environments are less studied compared to terrestrial environments and harbors great microbial diversity we aimed to investigate the xylanase production of 88 marine-derived filamentous fungal strains. These strains are semi-quantitatively screened for their extracellular xylanase production and Trichoderma pleuroticola 08ÇK001 xylanase activity was further characterized. Optimum pH and temperature was determined as 5.0 and 50 °C, respectively. The enzyme preparation retained 53% of its activity at pH 5.0 after 1 h and have found resistant against several ions and compounds such as K+ , Ba2+ , Na+ , β-mercaptoethanol, Triton X-100 and toluene. This study demonstrates that marine-derived fungal strains are prolific sources for xylanase production and presents the first report about the production and characterization of xylanase from a marine derived T. pleuroticola strain. The characteristics of T. pleuroticola 08ÇK001 xylanase activity indicate possible employment in some industrial processes such as animal feed, juice and wine industries or paper pulping applications.

Isolation, screening and characterization of a novel extracellular xylanase from Aspergillus niger (KP874102.1) and its application in orange peel hydrolysis

In the present work, a potent xylanase producing fungal strain Aspergillus niger (KP874102.1) was isolated through cultural and morphological observations from soil sample of Baramura forest, Tripura west, India. 28S rDNA technique was applied for genomic identification of this fungal strain. The isolated strain was found to be phylogenetically closely related to Aspergillus niger. Kinetic constants such as Km and Vmax for extracellular xylanase were determined using various substrate such as beech wood xylan, oat spelt xylan and CM cellulose through Lineweaver–Burk plot. Km, Vmax and Kcat for beech wood xylan are found to be 2.89mg/ml, 2442U and 426178Umlmg−1 respectively. Crude enzyme did not show also CM cellulose activity. The relative efficiency of oat spelt xylan was found to be 0.819 with respect to beech wood xylan. After acid hydrolysis, enzyme was able to produce reducing sugar with 17.7, 35.5, 50.8 and 65% (w/w) from orange peel after 15, 30, 45 and 60min incubation with cellulase free xylanase and maximum reducing sugar formation rate was found to be 55.96μg/ml/min. Therefore, the Aspergillus niger (KP874102.1) is considered as a potential candidate for enzymatic hydrolysis of orange peel.

Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3

The present study investigates the production and partial biochemical characterization of an extracellular thermostable xylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phase partitioning (TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in the presence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system using the optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purification fold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of 5.0–10. The optimum temperature for xylanase activity was 55 °C and the half-life time at this temperature was of 6 h. At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist to sodium dodecyl sulfate and β-mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. The recovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% was observed. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability was observed with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested as a potential candidate for application in some industrial processes. To the best of our knowledge, this is the first report of xylanase activity and recoverey using three phase partitioning from B. oceanisediminis.

Extracellular xylanase production from a new xylanase producer Tuber maculatum mycelium under submerged fermentation and its characterization

Abstract Truffles are edible fungi that grow symbiotically with several trees. In this study, Tuber maculatum mycelia were isolated and screen qualitatively for extracellular xylanase production at a broad pH range (4.0, 7.0 and 9.0) in agar plates. Based on zone of clearance highest xylanase activity was observed at pH 7.0. Furthermore, T. maculatum mycelium was studied for effect of various carbon sources, initial medium pH, agitation and fermentation time on xylanase production in submerged fermentation. Under optimized conditions maximum xylanase activity (13.15 U/mL) was detected after 6 days of fermentation at static condition in the basal salt medium with the initial medium pH of 7.0% and 0.5% xylan. The xylanase showed maximum activity at 50 °C and pH 5.0. The Zn 2+ activated the xylanase but Co 2+ was found inhibitory towards the same. The results indicated that truffle mycelium is utilizing xylan as energy source from host plant root system.

Biobleaching of banana fibre pulp with incorporation of xylanase enzyme fromAspergillus oryzae

The potential of extracellular xylanase produced by Aspergillus oryzae through solid state fermentation was investigated on banana fibre pulp bleaching in association with conventional bleaching with chlorine dioxide. The maximum enzyme production was obtained at 30 o C after 48 hrs of incubation using wheat bran substrate. Highest enzyme activity 1136 IU/g dry substrate was found under optimized condition. Banana fibre pulp were pretreated with different dose of xylanase enzyme before the conventional bleaching sequence. Xylanase pre-treatment reduce the kappa no and enhance the optical and physical properties of pulp. The maximum reduction in kappa no were 1.3 unit at the dose of 30 IU/g. Xylanase treatment also improve burst index, tensile index and double fold by11.81 per cent, 5.7 pera cent and 2.8 per cent, respectively.

Optimization of Xylanase Production from Some Agro-lignocellulosic Wastes by Aspergillus niger Via Solid State Fermentation

Extracellular xylanase production by A. niger was studied under solid-state fermentation using six agricultural wastes individually (wheat bran, rice husk, corn cobs, rice straw, clover straw and wheat straw) as well as fifteen treatment represent different combinations of the above agro-lignocellulosic wastes. The best substrate was the combination of wheat bran and clover straw. The initial moisture content, additive carbon source, additive nitrogen source, initial pH of the culture, incubation temperature and inoculum size affected the production of xylanase. The optimal fermentation medium was at 80% of initial moisture and composed of 5 g WB+CS (1:1 w/w) supplemented with 1% (w/w) of xylan and 1% (w/w) of (NH4)3PO4 and inoculated by adding 1 ml of spore suspension containing 2x105spores/ml. Xylanase production reached a peak of 1294.15 IU/gdw after 7 days of incubation at 35 °C under SSF. The produced enzyme has maximum activity at 70 oC and pH 5.5. Addition of 10 mM FeSO4 and 5mM of KCl, CaCl2 and MnCl2 to the reaction mixture enhanced xylanase activity to 127.46, 118.86, 115.76 and 111.15%, respectively. A. niger xylanase showed superior heat and pH tolerance, it remained 86.47 and 76% of its activity after 60 min of incubation at 70 oC with and without 10 mM CaCl2.The enzyme showed more stability in a pH ranged from 4 to 5 retaining more than 75% of its original activity, therefore it may have significant applications in biofuel and paper industries.

Purification and evaluation for effects of temperature on extracellular xylanase activity from Aspergillus oryzae DSM 1863


 
 
 Xylanase was purified from the crude culture of Aspergillus oryzae DSM1863 by sephadex G200 and DEAE – cellulose ion exchange chromatography. The molecular mass of the purified xylanase determined by SDS–PAGE was 21 kDa with a specific activity of 6768 U/mg towards 1% (w/v) of birch wood xylan. The optimum temperature was observed at 60°C. The enzyme was thermostable in the temperature range of 37-50°C with a high residual activity of 62-74% (650.6-775.9 U/mg protein).
 Enzyme xylanase được tinh sạch từ dịch lên men của chủng Aspergillus oryzae DSM1863 sau khi qua cột sắc ký lọc gel sephadex G200 và sắc ký trao đổi ion DEAE – cellulose. Khối lượng phân tử của enzyme xylanase tinh sạch được xác định bằng điên di đồ SDS- PAGE. Xylanase tinh sạch có kích thước là 21 kDa với hoạt tính đặc hiệu đạt 6768 U/mg sau khi được xác định với nồng độ cơ chất là 1% birch wood xylan. Nhiệt độ tối ưu để enzyme hoạt động mạnh nhất là 60C. Enzyme xylanase khá bền nhiệt. Hoạt tính của enzyme vẫn còn duy trì 62-74% (hoạt tính đặc hiệu đạt 650.6-775.9 U/mg protein) sau khi 8 giờ ủ ở 37-50°C.
 
 

Recombinant Xylanase from Bacillus tequilensis BT21: Biochemical Characterisation and Its Application in the Production of Xylobiose from Agricultural Residues.

Bacterial strain Bacillus tequilensis BT21 isolated from marine sediments was found to produce extracellular xylanase. The xynBT21 gene encoding xylanase enzyme was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acid residues with calculated molecular mass of 23.3 kDa. Purified recombinant xylanase had optimum activity at 60 °C and pH=6. The enzyme was highly stable in alkaline pH, at pH=7 it remained 100% active for 24 h, while its activity increased at pH=8 and 9 during incubation. B. tequilensis BT21 xylanase had alkaline pI of 9.4 and belongs to glycosyl hydrolase family 11. The mode of action of XynBT21 on beechwood xylan and xylooligosaccharides was studied. It hydrolysed xylooligosaccharides and beechwood xylan yielding mainly xylobiose (X2) with a small amount of xylose (X1), indicating that XynBT21 was probably an endo-acting xylanase. Enzymatic hydrolysis using wheat bran as a substrate revealed that xylanase reported here has the potential to produce xylobiose from wheat bran. Xylooligosaccharides, especially xylobiose, have strong bifidogenic properties and are increasingly used as a prebiotic. This is the first report that describes this novel xylanase enzyme from marine B. tequilensis BT21 used for the release of xylobiose from wheat bran.

Verrucosispora sp. K2-04, Potential Xylanase Producer from Kuantan Mangrove Forest Sediment

Abstract: Xylanase is the key enzyme that involve in the hydrolysis of xylan, the main constituent of the complex hemicellulose of the plant cell wall. In this study, forty actinomycetes that were isolated from sediment of Kuantan Mangrove Forest, Malaysia, were tested for their ability to produce extracellular xylanase. At least 15 isolates were able to degrade xylan in the primary agar-based screening using marine agar containing 0.1% (v/v) azo-xylan (Birchwood). The degradation of xylan was indicated by the formation of halo zone around the colonies and the clear zone index (CZI) was calculated as a ratio of the clearing zones to the colony size. Isolate K2-04 with CZI 3.35 ± 1.91 was identified through 16S rRNA study as Verrucosispora sp. This isolate was further grown in marine broth and incubated at 30 °C, 200 rpm for 20 days. The growth of K2-04 and the xylanase activity was measured at day 2, 4, 6, 12 and 18 respectively. The highest enzyme activity for the crude enzyme was recorded at day 18 (1.836 U/mL) and exhibited stability after 20 days storage at 4°C. This study serves as a preliminary study to characterize the properties of Verrucosispora sp. K2-04, rare actinomycete of Kuantan Mangrove Forest, Malaysia.

Characterization of Xylanase and Cellulase Produced by a Newly Isolated Aspergillus fumigatus N2 and Its Efficient Saccharification of Barley Straw.

Aspergillus fumigatus N2 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest xylanase (91.9U/mL) and CMCase (5.61U/mL) activity was produced when 1% barley straw was used as the carbon source. The optimum pH and temperature for xylanase activity were 6.0 and 65°C, respectively. CMCase revealed maximum activity at pH 4.0 and in the range of 65°C. The FPase was optimally active at pH 5.0 and 60°C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that multiple enzymes were secreted into the fermentation supernatant. Five bands of proteins with xylanase activity and four bands of proteins with endoglucanase were observed in the zymogram gel. The crude enzymes were used in the barley straw saccharification; an additive effect was observed when the commercial cellulase was added as supplement.

Extracellular expression of alkali tolerant xylanase from Bacillus subtilis Lucky9 in E. coli and application for xylooligosaccharides production from agro-industrial waste

An alkali tolerant xylanase gene from Bacillus subtilis Lucky9 was cloned and extracellular expressed in E. coli BL21. Xylanase amino acid sequence showed 99% identity with xylanase sequence from Bacillus subtilis 168, and was belonged to glycoside hydrolase family 11. The recombinant E. coli (pET-pelB-xynLC9) containing pelB signal peptide produced extracellular xylanase of 436.5U/mL for 8h, which was used arabinose as extra carbon source and inducer for enhancing extracellular production. The extracellular xylanase was determined by SDS–PAGE with a relative molecular mass of 21kDa. The recombinant xylanase was optimally activity at pH 6.5 and 60°C. The xylanase exhibited 80% residual activity over a broad pH range of 6.0–9.0 for 24h. Thermostability studies showed that xylanase retained 60% residual activity after 2h at 60°C. The main end-products of hydrolysis of beech-wood xylan and corncob by the extracellular xylanase were xylobiose and xylotriose. This extracellular xylanase without purification is a suitable candidate for application in the industrial production of xylooligosaccharides from agro-industrial waste for use as prebiotics.

Sequences Analysis of a Gene Encoding Extracellular Xylanase in Streptomyces costaricanus 45I-3

Streptomyces costaricanus 45I-3 is a bacterial strain belongs to actinomycetes group isolated from peat soil. Thebacterium is known to produce extracellular xylanase. The aims of this study were to analyze DNA sequence andsub-clone gene involved in the synthesis of extracellular xylanase. Complete DNA sequence predicted to encodexylanase genes was isolated from bacterial genome using Inverse Polymerase Chain Reaction (I-PCR). Total DNAsequence of 1664 bp in size obtained from I-PCR consisted of two open reading frames (ORF) in opposite direction.ORF1 was 1029 bp and ORF2 (partial sequence) was 309 bp. Analysis sequence using BlastX indicated that ORF1was homologous with xylanase bacterium enrichment culture clone Xyl8B8 (GenBank accession No. AFH35005.1),i.e. 95% in identity and 99% in similarity. In addition, ORF2 was homologous with glyoxalase bacterium enrichmentculture clone Xyl8B8 (GenBank accession No. AFH35007.1), i.e. 95% in identity and 98% in similarity. Analysis ofamino acid sequence revealed that ORF1 consisted of 2 domains, i.e. glyco-hydrolase 11 (GH11) and CarbohydrateBinding Type 2 (CBM2). Active site was found at 130th amino acid on GH11 domain. Visualization of 3-dimensionstructure showed that 1029 bp fragment is of 19 areas.