Gene Cloning,Overexpression, and Characterization of a Xylanase from <Emphasis Type="Italic">Penicillium</Emphasis> sp. CGMCC 1669

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml⁻¹. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The specific activity, K _m, and V _max for oat spelt xylan substrate was 7,988 U mg⁻¹, 22.2 mg ml⁻¹, and 15,105.7 μmol min⁻¹ mg⁻¹, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.     

Purification and Characterization of a Low Molecular Weight Endo-xylanase from Mushroom <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

A low molecular weight endo-xylanase (EC 3.2.1.8) was purified from an edible mushroom Termitomyces clypeatus grown in submerged medium with oat spelt xylan. Xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. Its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kDa. The enzyme was found to be most active at 50°C and pH 5.0, being most stable at pH 6.5. The K_m for oat spelt xylan was determined to be 10.4 mg/ml. The specificities of the enzyme was observed to be highly specific towards oat spelt xylan and was inhibited by mercuric chloride (HgCl₂), N-bromosuccinimide, and trans-1,2-diaminocyclohexane-N′,N′,N′,N′-tetraacetic acid strongly. The inhibitory action of N-bromosuccinimide on enzyme confirmed the presence of one tryptophan residue in its substrate-binding site. Amino acid analysis for xylanase showed the presence of high amount of hydrophobic serine, glycine, threonine, and alanine residues. The N-terminal sequencing study for the previously purified and characterized 56 kDa xylanolytic amyloglucosidase reveal the presence of 33.30% identity with glucoamylase chain A from Aspergillus awamori. The N-terminal sequence analysis of the present 12 kDa enzyme showed highest similarity (72.22% identity) towards xylanase from Neurospora crassa.     

Purification and Characterization of Two Extracellular Xylanases from <Emphasis Type="Italic">Penicillium sclerotiorum</Emphasis>: A Novel Acidophilic Xylanase

Two xylanases from the crude culture filtrate of Penicillium sclerotiorum were purified to homogeneity by a rapid and efficient procedure, using ion-exchange and molecular exclusion chromatography. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 23.9 and 33.1 kDa for xylanase I and II, respectively. The native enzymes’ molecular masses of 23.8 and 30.8 kDa were estimated for xylanase I and II, respectively, by molecular exclusion chromatography. Both enzymes are glycoproteins with optimum temperature and pH of 50 °C and pH 2.5 for xylanase I and 55 °C and pH 4.5 for xylanase II. The reducing agents β-mercaptoethanol and dithio-treitol enhanced xylanase activities, while the ions Hg²⁺ and Cu²⁺ as well the detergent SDS were strong inhibitors of both enzymes, but xylanase II was stimulated when incubated with Mn²⁺. The K _m value of xylanase I for birchwood xylan and for oat spelt xylan were 6.5 and 2.6 mg mL⁻¹, respectively, whereas the K _m values of xylanase II for these substrates were 26.61 and 23.45 mg mL⁻¹. The hydrolysis of oat spelt xylan by xylanase I released xylobiose and larger xylooligosaccharides while xylooligosaccharides with a decreasing polymerization degree up to xylotriose were observed by the action of xylanase II. The present study is among the first works to examine and describe an extracellular, highly acidophilic xylanase, with an unusual optimum pH at 2.5. Previously, only one work described a xylanase with optimum pH 2.0. This novel xylanase showed interesting characteristics for biotechnological process such as feed and food industries.     

A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization

A newly isolated bacterial strain, Bacillus sp. MX47, was actively producing extracellular xylanase only in xylan-containing medium. The xylanase was purified from the culture broth by two chromatographic steps. The xylanase had an apparent molecular weight of 26.4?kDa with an NH₂-terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct from that of reported proteins, implying it is a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0 and 40?°C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5?mM. The xylanase was highly specific to beechwood and oat spelt xylan, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it is an endo-??-1,4-xylanase. It hydrolyzed xylan to xylobiose as the end product. The K _m and V _max values toward beechwood xylan were 3.24?mg?ml^?1 and 58.21???mol?min^?1?mg^?1 protein, respectively.     

Purification and Biochemical Characterization of a Highly Thermostable Xylanase from <Emphasis Type="Italic">Actinomadura</Emphasis> sp. Strain Cpt20 Isolated from Poultry Compost

An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption–ionization time-of-flight mass spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase activity were pH 10 and 80 °C, respectively. This xylanase was stable within a pH range of 5–10 and up to a temperature of 90 °C. It showed high thermostability at 60 °C for 5 days and half-life times at 90 °C and 100 °C were 2 and 1 h, respectively. The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan. This enzyme obeyed the Michaelis–Menten kinetics, with the K _m and k _cat values being 1.55 mg soluble oat-spelt xylan/ml and 388 min⁻¹, respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn²⁺, Ca²⁺, and Cu²⁺, it was, strongly inhibited by Hg²⁺, Zn²⁺, and Ba²⁺. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.     

Cloning of a New Xylanase Gene from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TN119 Using a Modified Thermal Asymmetric Interlaced-PCR Specific for GC-Rich Genes and Biochemical Characterization

A bacterial strain, Streptomyces sp. TN119, was isolated from the gut of Batocera horsfieldi larvae and showed xylanolytic activity. A degenerate primer set was designed based on the base usage of G and C in Actinobacteria xylanase-coding sequences belonging to the glycosyl hydrolases family 10 (GH 10), and used to clone the partial xylanase gene from Streptomyces sp. TN119. A modified thermal asymmetric interlaced (TAIL)-PCR specific for high-GC genes, named GC TAIL-PCR, was developed to obtain the full-length xylanase gene (xynA119; 1089 bp). Rich in GC content (67.8%), xynA119 encodes a new GH 10 xylanase (XynA119), which shares highest identity (48.8%) with an endo-1,4-β-xylanase from Cellulosimicrobium sp. HY-12. Recombinant XynA119 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 6.5 and 60 °C, was stable at pH 4.0 to 10.0 and 50 °C, was resistant to most chemicals (except for Cu²⁺, Mn²⁺, Ag⁺, Hg²⁺ and SDS) and trypsin, and produced simple products. The specific activity, K _m, V _max, and k _cat using oat-spelt xylan as substrate were 57.9 U mg⁻¹, 1.0 mg ml⁻¹, 74.8 μmol min⁻¹ mg⁻¹, and 49.2 s^–1, respectively.     

Purification and Properties of a Psychrotrophic <Emphasis Type="Italic">Trichoderma</Emphasis> sp. Xylanase and its Gene Sequence

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K _m values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.     

Cellulases and Hemicellulases from Endophytic <Emphasis Type="Italic">Acremonium</Emphasis> Species and Its Application on Sugarcane Bagasse Hydrolysis

The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing l-arabinose, d-xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using d-xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.     

Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6

An extracellular, endo-??-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS?CPAGE with an apparent molecular weight of 30?kDa. The xylanase activity was optimal at pH?3.0 and 65?°C. The specific activity measured for oat spelt xylan was 2,368?U?mg^?1. The apparent K _m and V _max values were 8.33?mg?ml^?1 and 58.82???mol?min^?1?ml^?1, respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962?bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88?kDa.     

Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K _m and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K _m of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.     

Production of extracellular xylanases byPenicillium janthinellum

Xylanase production byPenicillium janthinellum using 10–100 mM of 2,2-dimethylsuccinate (DMS) buffer, in a range of pH 4.5-6.0 was studied. The enzyme activity was enhanced using oat xylan as the carbon source. Under these conditions a culture produced 1.14 Μmol/ min (11.4 U/mL or 84.4 U/mg) of Β-xylanase after 5 d of growth in a 10-mM buffer solution at pH 4.5. Protease was absent in the DMS buffer except when 100 mM phosphate buffer at pH 6.0 was used (4 U/mL). Β-Xylosidase was only found at a pH of 4.5 in all the buffer concentrations. At a 50 mM DMS buffer concentration at pH 4.5 Β- xylanases were induced by both oat and birch xylans, having a greater effect with oat spelt xylans. Electrophoretic analyses showed that the birchwood xylan induction exhibited different proteins profiles. No Β-xylosidase or Β- glucosidase was induced until d 5. The Β-xylanases were rapidly inactivated at 50‡C, however, birch xylanase appeared to be more stable than oat xylanase. Using oat xylan as an inductor, theΒ-xylosidase andΒ-glucosidase were 85 and 91 U/L, respectively, on d 7. The xylanase produced by induction from sugar cane bagasse hydrolyzate was used for pulp biobleaching. A 20% decrease on the Kappa value in Kraft pulp using the culture extract was obtained. These selective growth conditions led us to modulate the xylanase production for pulp delignification.     

A Temperature and Salt-Tolerant <Emphasis Type="SmallCaps">l</Emphasis>-Glutaminase from Gangotri Region of Uttarakhand Himalaya: Enzyme Purification and Characterization

Purification and characterization of halotolerant, thermostable alkaline l-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH₄)₂SO₄ precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. l-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0–11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1–20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. l-Glutaminase is almost active with K⁺, Zn²⁺, and Ni²⁺ ions and K _m and V _max values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance, and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived l-glutaminase reported so far.     

Gene Cloning,Expression, and Characterization of a Family 51 α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S9

An α-l-arabinofuranosidase gene, abf51S9, was cloned from Streptomyces sp. S9 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene consisted of 1,506 bp and encoded 501 amino acids with a calculated mass of 55.2 kDa. The deduced amino acid sequence was highly homologous with the α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by Ni-NTA affinity chromatography and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.0 and 60∼65 °C, respectively. The enzyme showed a broad pH range of stability, retaining over 75% of the maximum activity at pH 5.0 to 11.0. The specific activity, K _m, and V _max with p-nitrophenyl-α-l-arabinofuranoside as substrate were 60.0 U mg⁻¹, 1.45 mM, and 221 μmol min⁻¹ mg⁻¹, respectively. Abf51S9 showed a mild but significant synergistic effect in combination with xylanase on the degradation of oat-spelt xylan and soluble wheat arabinoxylan substrates with a 1.19- and 1.21-fold increase in the amount of reducing sugar released, respectively. These favorable properties make Abf51S9 a good candidate in various industrial applications.     

Purification and Biochemical Characterization of Two Xylanases from <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> SBS 45

Two xylanases were isolated and purified from crude culture filtrate of Aspergillus sydowii SBS 45 after 9 days of growth on wheat bran containing 0.5% (w/v) birch wood xylan as the carbon source under solid-state fermentation. After a three-step purification scheme involving ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-200), and anion exchange chromatography (DEAE-Sephadex A-50), xylanase I was purified 93.41 times, and xylanase II was purified 77.40 times with yields of 4.49 and 10.46, respectively. Molecular weights of xylanase I and II were 20.1 and 43 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature was 50 degrees C, and optimum pH was 10.0 for both xylanase I and II. The Km value of xylanase I for birch wood xylan was 3.18 mg ml(-1) and for oat spelt xylan 6.45 mg ml(-1), while the Km value of xylanase II for birch wood xylan was 6.51 mg ml(-1) and for oat spelt xylan 7.69 mg ml(-1). Metal ions like Al3+, Ba2+, Ca2+, Na+, and Zn2+ enhanced the activity of xylanase I and II at 10 mM concentration. Among the additives, L-tryptophan enhanced the activity of xylanase I and II at 10-, 20-, and 30-mM concentrations. Both xylanases appeared to be glycoproteins.     

Isolation,Purification and Characterisation of Low Molecular Weight Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> SSP-34

Low molecular weight endo-xylanase from Bacillus pumilus SSP-34 was purified to homogeneity using ion exchange and size exclusion chromatographies. Xylanases were isolated by novel purification protocol which includes the use of anion exchange matrix such as DEAE Sepharose CL 6B with less affinity towards enzyme protein. The purified B. pumilus SSP-34 have a molecular weight of 20 kDa, with optimum pH and temperature at 6.0 and 50 °C, respectively. The enzyme was stable at 50 °C for 30 min. It showed remarkable stability at pH values ranging from 4.5 to 9 when the reaction was carried out at 50 °C. K _m and V _max values, determined with oats spelts xylan were 6.5 mg ml⁻¹ and 1,233 μmol min⁻¹ mg⁻¹ protein, respectively, and the specific activity was 1,723 U mg⁻¹     

Cloning,Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg⁻¹ at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K _m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na⁺, NH₄⁺, Mg²⁺, Fe²⁺, Fe³⁺, Co²⁺, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.     

Keratinase Production by Endophytic <Emphasis Type="Italic">Penicillium</Emphasis> spp. Morsy1 Under Solid-State Fermentation Using Rice Straw

Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g⁻¹, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 10⁵ spores ml⁻¹, and an average particle size of the substrate 0.6 mm (3,560 U g⁻¹, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA. These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases.     

Controlled Production of Fructose by an Exoinulinase from <Emphasis Type="Italic">Aspergillus Ficuum</Emphasis>

An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg⁻¹(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography. The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4 and 50 °C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation for 12 h and 72 h respectively. The respective K _m and V _max values were 4.75 mM and 833.3 μmol min⁻¹ ml⁻¹, respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 °C, 150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 °C, 150% inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose from inulin hydrolysis is biotechnologically feasible.     

High Level Expression of an Acid-Stable Phytase from <Emphasis Type="Italic">Citrobacter freundii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni²⁺–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg⁻¹, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg⁻¹ min⁻¹, respectively. The enzyme activity was significantly improved by 1 mM of K⁺, Ca²⁺, and Mg²⁺. These characteristics contribute to its potential application in feed industry.