Immobilized Rhodotorula mucilaginosa: A Novel Urethanase-Producing Strain for Degrading Ethyl Carbamate

Rhodotorula mucilaginosa, producing the ethyl carbamate (EC)-degrading enzyme, urethanase, was newly isolated from the Chinese rice wine making process. It removed 80 % of EC when it was incubated with 5.0 g/L EC. It grew and stably produced urethanase, with pH ranging from 7.0 to 3.0. In addition, urethanase production by R. mucilaginosa was systematically optimized. Glucose, yeast extract, peptone, and inoculum size were selected with the Plackett–Burman design. They were further optimized via uniform design and determined to be 24.6 g/L, 2.5 g/L, 23.1 g/L, and 65.8 mL/500 mL, respectively. Urethanase activity reached 4,340.0 U/L in the optimal fermentation condition. Furthermore, cell immobilization of R. mucilaginosa in calcium alginate/chitosan was applied to improve cell resistance to environmental stresses. The immobilized cells removed 51.6 % of EC in commercial rice wine, which was 10 times more than that of the free cells. It indicated that the immobilized R. mucilaginosa was effective for degrading EC.     

Purification and Characterization of a Urethanase from Penicillium variabile

Urethanase produced by Penicillium variabile was purified through ultrasonication, concentration by polyethylene glycol 20,000, and Superdex G-200 gel filtration chromatography. The molecular weight of urethanase was determined to be around 96 kDa by gel filtration. The purified enzyme showed a single band in SDS-PAGE with the molecular weight of ~13.7 kDa, which suggests that the enzyme has a multimeric structure composed of the same subunits. Peptide map fingerprinting analysis was then carried out by MALDI/TOF-TOF MS. Within the known sequences in NCBI, glucosamine-6-phosphate deaminase and 6-phosphogluconate dehydrogenase get high score as compared with urethanase. Sequence analysis informs that N-terminal sequence of urethanase is GTNTADNDAA. The Minchaelis constant (K _m) and maximum reaction rate (V _m) of urethanase are 27.2 mmol/L and 156.25 μmol/L min, respectively.     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

Isolation,Purification and Characterisation of an Organic Solvent-Tolerant Ca2+-Dependent Protease from Bacillus megaterium AU02

A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl₂, and it retained 90 % activity at 50 °C for 3 h. The K _m and V _max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH₂) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.     

Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90 % activity when incubated for 2 h at 60 °C and remain about 75 % and 44 % activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98 % with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.     

Improvement of Highly Thermostable Xylanases Production by Talaromyces thermophilus for the Agro-industrials Residue Hydrolysis

A newly isolated thermophilic fungal strain from Tunisian soil samples was identified as Talaromyces thermophilus and was selected for its ability to produce extracellular hemicellulases when grown on various lignocellulosic substrates. Following the optimization of carbon source, nitrogen source, and initial pH of the growth medium in submerged liquid cultures, yields as high as 10.00?±?0.15 and 0.21?±?0.02 U/ml were obtained for xylanase and β-xylosidase, respectively. In fact, wheat bran was found to be a good inducer of hemicellulase enzymes, mainly β-xylosidase. The optimal temperature and pH of the xylanase activity were 75°C and 8.0, respectively. This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for 7 days at pH?7.0–8.0. The half-lives of the enzyme were 4 h at 80°C, 2 h at 90°C, and 1 h at 100°C. T. thermophilus could therefore be considered as a satisfactory and promising producer of thermostable xylanases. Crude enzyme of T. thermophilus rich in xylanase and β-xylosidase was established for the hydrolysis of lignocellulosic materials as wheat bran.     

Evaluation of Enhanced Thermostability and Operational Stability of Carbonic Anhydrase from Micrococcus Species

Carbonic anhydrase (CA) was purified from Micrococcus lylae and Micrococcus luteus with 49.90 and 53.8 % yield, respectively, isolated from calcium carbonate kilns. CA from M. lylae retained 80 % stability in the pH and temperature range of 6.0–8.0 and 35–45 °C, respectively. However, CA from M. luteus was stable in the pH and temperature range of 7.5–10.0 and 35–55 °C, respectively. Cross-linked enzyme aggregates (CLEAs) raised the transition temperature of M. lylae and M. luteus CA up to 67.5 and 74.0 °C, while the operational stability (T _1/2) of CA at 55 °C was calculated to be 7.7 and 12.0 h, respectively. CA from both the strains was found to be monomeric in nature with subunit molecular weight and molecular mass of 29 kDa. Ethoxozolamide was identified as the most potent inhibitor based on both IC₅₀ values and inhibitory constant measurement (K _i). The K _m and V _max for M. lylae CA (2.31 mM; 769.23 μmol/mg/min) and M. luteus CA (2.0 mM; 1,000 μmol/mg/min) were calculated from Lineweaver–Burk plots in terms of esterase activity. Enhanced thermostability of CLEAs alleviates its role in operational stability for application at an on-site scrubber. The characteristic profile of purified CA from Micrococcus spp. advocates its effective application in biomimetic CO₂ sequestration.     

Partial Characterization of Xylanase Produced by Caldicoprobacter algeriensis,a New Thermophilic Anaerobic Bacterium Isolated from an Algerian Hot Spring

To date, xylanases have expanded their use in many processing industries, such as pulp, paper, food, and textile. This study aimed the production and partial characterization of a thermostable xylanase from a novel thermophilic anaerobic bacterium Caldicoprobacter algeriensis strain TH7C1^T isolated from a northeast hot spring in Algeria. The obtained results showed that C. algeriensis xylanase seems not to be correlated with the biomass growth profile whereas the maximum enzyme production (140.0 U/ml) was recorded in stationary phase (18 h). The temperature and pH for optimal activities were 70 °C and 11.0, respectively. The enzyme was found to be stable at 50, 60, 70, and 80 °C, with a half-life of 10, 9, 8, and 4 h, respectively. Influence of metal ions on enzyme activity revealed that Ca⁺² enhances greatly the relative activity to 151.3 %; whereas Hg²⁺ inhibited significantly the enzyme. At the best of our knowledge, this is the first report on the production of xylanase by the thermophilic bacterium C. algeriensis. This thermo- and alkaline-tolerant xylanase could be used in pulp bleaching process.     

Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH₂-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca²⁺ and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5–10 and at temperatures below 45 °C.     

Ligase-Independent Cloning of Amylase Gene from a Local Bacillus subtilis Isolate and Biochemical Characterization of the Purified Enzyme

Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5–7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co⁺², Cu²⁺, Fe²⁺, Fe³⁺, Ni⁺², and Zn⁺² caused significant inhibition in enzyme activity, which was not affected by Na⁺, Mg²⁺, Li⁺, and Ba²⁺. The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca²⁺ ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.     

Molecular and Kinetic Characterization of Two Extracellular Xylanases Isolated from Leucoagaricus gongylophorus

In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 °C, optimum pH between 8.5 and 10.5, and Km?=?14.7?±?7.6 mg mL^?1. Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 °C, and Km?=?2.2?±?0.5 mg mL^?1. XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 °C) and pH (4.0), as well as the kinetic parameters (Km?=?7.4?±?2.0 mg mL^?1) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.     

Genome Mining for New α-Amylase and Glucoamylase Encoding Sequences and High Level Expression of a Glucoamylase from Talaromyces stipitatus for Potential Raw Starch Hydrolysis

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD₆₀₀?>?200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ～8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71–87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.     

Characterization and High Level Expression of Acidic Endoglucanase in Pichia pastoris

Bioconversion of cellulosic material into glucose needs cellulase enzymes. One of the most important organisms that produces cellulases is Trichoderma reesei, whose cellulose enzymes are probably the most widely used in the industry. However, these enzymes are not stable enough at high pH and temperatures. The optimized synthetic endoglucanase II gene with Pichia pastoris codon preferences was secretary expressed in P. pastoris. Recombinant enzyme characterization showed maximum activity at pH 4.8 and temperature 75 °C, and it demonstrated increasing thermal stability in high temperature. The enzyme maintained its activity in a wide pH range from 3.5 to 6.5. The optimization of fermentation medium was carried out in shaking flasks. Recombinant protein expression at optimum conditions (pH 7, temperature 25 °C, and 1 % methanol induction) for 72 h demonstrated 2,358.8 U/ml endoglucanase activity units. To our knowledge, this is the highest acidic thermophilic endoglucanase activity that is reported in crude intracellular medium in P. pastoris. We conclude that P. pastoris is an appropriate host for high-level expression of optimized endoglucanase gene with improved thermal stability.     

Production and Partial Characterization of Cellulases and Xylanases from Trichoderma atroviride 676 Using Lignocellulosic Residual Biomass

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL^?1 for xylanase, 1.9 U mL^?1 for endoglucanase, 0.25 U mL^?1 for FPase, and 0.17 U mL^?1 for β-glucosidase) after 3–4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50–60 °C and pH?4.0–5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.     

Purification and Characterization of Hyaluronate Lyase from <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> A152

A hyaluronate lyase was obtained by cultivating Arthrobacter globiformis strain A152. The enzyme was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q Sepharose Fast Flow, and Sephadex G-100 chromatography. The purification resulted in a 32.78-fold increase in hyaluronate lyase activity with specific activity of 297.2 U/mg. The molecular weight of the enzyme determined by SDS-PAGE was approximately 73.7 kDa. Using hyaluronic acid (HA) as a substrate, the maximal reaction rate (V_max) and the Michaelis–Menten constant (K_m) of hyaluronate lyase were found to be 4.76 μmol/min/ml and 0.11 mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42 °C, respectively. This enzyme was stable at pH 4–10, 5–7, and 5–7 at 4, 37, and 42 °C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30–37 °C and also showed high activity at 37 °C. The enzymatic activity was enhanced by Ca²⁺ and was strongly inhibited by Cu²⁺ and SDS. These properties suggested that the hyaluronate lyase in this study could bring promising prospects in medical and industry applications.     

Production and Characterization of a Novel Yeast Extracellular Invertase Activity Towards Improved Dibenzothiophene Biodesulfurization

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml^?1) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5–6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5–6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l^?1 sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l^?1 sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μ_max from 0.035 to 0.070 h^?1 and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.     

Characterization of a Novel Organic Solvent Tolerant Protease from a Moderately Halophilic Bacterium and Its Behavior in Ionic Liquids

An extracellular protease was purified from a novel moderately halophilic bacterium Salinivibrio sp. strain MS-7 by the combination of an acetone precipitation (40–80 %) step and a DEAE-cellulose anion exchange column chromatography. Kinetic parameters of the enzyme exhibited V _max and K _m of 130 U/mg and 1.14 mg/ml, respectively, using casein as a substrate. The biochemical properties of the enzyme revealed that the 21-kDa protease had a temperature and pH optimum of 50 °C and 8.0, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, Pefabloc SC, chymostatin, and also EDTA, indicating that it belongs to the class of serine metalloproteases. Interestingly, Ba²⁺ and Ca²⁺ (2 mM) strongly enhanced the enzyme activity, while Fe²⁺ and Mg²⁺ activated moderately and Zn²⁺, Ni²⁺, and Hg²⁺ decreased the enzyme activity. The effect of organic solvents with different logP on the purified protease revealed complete stability in toluene, ethyl acetate, chloroform, and n-hexane at 10 and 50 % (v/v) and moderate stability even in 50 % of DMSO and ethanol. The behavior of the MS-7 protease in three imidazolium-based ionic liquids exhibited suitable activity in these green solvent systems, especially in 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]). Comparison of the purified protease with other previously reported proteases suggests that strain MS-7 secrets a novel organic solvent-tolerant protease with outstanding activity in organic solvents and imidazolium-based ionic liquids, which could be applied in low water synthetic section of industrial biotechnology.     

Efficient Production of γ-GABA Using Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ～47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg^?1. The purified cellulase retained its activity over a wide range of temperature (50–70 °C) and pH (3–7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.     

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-β-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 °C and remained stable at weakly acidic to alkaline pH (pH 5.0–11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 °C for 1 h and more than 40% of the activity at 90 °C for 15 min. The K _m and V _max values for oat spelt xylan and birchwood xylan were 16.08 mg ml^?1 and 45.66 μmol min^?1 mg^?1 and 9.22 mg ml^?1 and 16.05 μmol min^?1 mg^?1, respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.     

Production of Xylooligosaccharides by Immobilized His-tagged Recombinant Xylanase from Penicillium occitanis on Nickel-Chelate Eupergit C

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.