Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions

A novel coccoid, anaerobic, Fe²⁺-oxidizing archaeum was isolated from a shallow submarine hydrothermal system at Vulcano, Italy. In addition to ferrous iron, H₂ and sulfide served as electron donors. NO₃ ^– was used as electron acceptor. In the presence of H₂, also S₂O₃ ^2– could serve as electron acceptor. The isolate was a neutrophilic hyperthermophile that grew between 65° C and 95° C. It represents a novel genus among the Archaeoglobales that we name Ferroglobus. The type species is Ferroglobus placidus (DSM 10642). Received: 7 March 1996 / Accepted: 4 September 1996     

Characterization of novel thermostable dextranase from Thermotoga lettingae TMO

Biochemical properties of a putative thermostable dextranase gene from Thermotoga lettingae TMO were determined in a recombinant protein (TLDex) expressed in Escherichia coli and purified to sevenfold apparent homogeneity. The 64-kDa protein displayed maximum activity at pH 4.3, and enzyme activity was stable from pH 4.3–10. The optimal temperature was 55–60°C during 15 min incubation, and the half-life of the enzyme was 1.5 h at 65°C. The enzyme showed higher activity against α-(1 → 6) glucan and released isomaltose and isomaltotriose as main products from dextran T2000. An unusual kinetic feature of TLDex was the negative cooperative behavior on the reaction of dextran T2000 cleavage. Enzyme activity was not significantly affected by the presence of metal ions, except for the strong inhibited by 1 mM Fe²⁺ and Ag²⁺. TLDex may prove useful as an enzyme for high temperature sugar milling processes.     

Food-Grade Expression and Characterization of a Dextranase from Chaetomium gracile Suitable for Sugarcane Juice Clarification

The microbial production of dextranase using cheap carbon sources is beneficial to solve the economic loss caused by the accumulation of dextran in syrup. A food-grade microbial cell factory was constructed by introducing the dextranase encoding gene DEX from Chaetomium gracile to the chromosome of Bacillus subtilis, and the antibiotic resistance marker gene was subsequently deleted via the Cre/loxP strategy. The dual-promoter system with a sequentially arranged constitutive P43 promoter resulted in an 85 % increase in DEX expression. Under the optimal fermentation conditions of 10 g/L maltose, 15 g/L casein, 1 g/L Na₂HPO₄, 1 g/L FeSO₄ and 8 g/L NaCl, DEX activity was increased from 2.625 to 64.34 U/mL. Recombinant DEX was purified 5.98-fold with a recovery ratio of 26.67 % and specific activity of 3935.02 U/mg. Enzyme activity was optimal at 55 °C and pH 5.0 and remained 80.34 % and 71.36 % of the initial activity at 55 °C and pH 4.0 after 60 min, respectively. The enzyme possessed high activity in the presence of Co²⁺, while Ag⁺ showed the strongest inhibition ability. The optimal substrate was 20 g/L dextran T-2000. The findings could facilitate the low-cost, large-scale production of food-grade DEX for use in the sugar industry.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Summary A metalloprotease from sorghum malt variety KSV8-I was purified by a combination of 4-M sucrose fractionation, ion-exchange chromatography on Q-Sepharose (Fast flow), gel-filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The enzyme was purified 7.9-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2128.7 U mg⁻¹ protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 35 kDa. The purified enzyme had optimal activity at 60 °C and maximal temperature stability between 40 and 60 °C but retained over 77% of its initial activity after incubation at 70 °C for 30 min. Both pH optimum and maximal stability were at 7.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. Using 0.2 ml of 5 mM solution of each metal ion, the purified protease was slightly (P<0.05) inhibited by Zn²⁺, appreciably (P<0.01) inhibited by Ca²⁺ and Co²⁺ and highly significantly (P<0.001) inhibited by Ag⁺, Ba²⁺, Hg²⁺, Mn²⁺ and Pb²⁺. The enzyme was equally highly significantly (P<0.001) inhibited by EDTA and hydrolysed casein to give the following kinetic constants: K_m = 21.0 mg ml⁻¹; V_max = 8.2 μmol ml¹ min⁻¹ and K_i = 0.390 mM.     

Purification and Characterization of an Esterase from <Emphasis Type="Italic">Acinetobacter lwoffii</Emphasis> I6C-1

EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at temperatures higher than 50°C. The Michaelis constant (K _m ) and V _max for p-nitrophenyl butyrate were 11 μM and 131.6 μM min⁻¹ mg of protein-1, respectively. The enzyme was strongly inhibited by Hg²⁻, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP). Received: 20 August 2001 / Accepted: 20 September 2001     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Purification and Characterization of an Extracellular β-Glucosidase from the Wood-Grown Fungus Xylaria regalis

Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K _m was 1.72 mM and V _max was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca²⁺, Mg²⁺, Mn²⁺, Cd²⁺ and β-mercaptoethanol, and inhibited by Ag⁺, Hg²⁺, SDS, and p-chloromercuribenzoate (PCMB). Received: 30 March 1996 / Accepted: 3 May 1996     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.     

Desulforhopalus vacuolatus gen. nov., sp. nov., a new moderately psychrophilic sulfate-reducing bacterium with gas vacuoles isolated from a temperate estuary

A new type of gas-vacuolated, sulfate-reducing bacterium was isolated at 10° C from reduced mud (E₀ < 0) obtained from a temperate estuary with thiosulfate and lactate as substrates. The strain was moderately psychrophilic with optimum growth at 18–19° C and a maximum growth temperature of 24° C. Propionate, lactate, and alcohols served as electron donors and carbon sources. The organism grew heterotrophically only with hydrogen as electron donor. Propionate and lactate were incompletely oxidized to acetate; traces of lactate were fermented to propionate, CO₂, and possibly acetate in the presence of sulfate. Pyruvate was utilized both with and without an electron acceptor present. The strain did not contain desulfoviridin. The G+C content was 48.4 mol%. The differences in the 16S rRNA sequence of the isolate compared with that of its closest phylogenetic neighbors, bacteria of the genus Desulfobulbus, support the assignment of the isolate to a new genus. The isolate is described as the type strain of the new species and genus, Desulforhopalus vacuolatus. Received: 4 March 1996 / Accepted: 17 June 1996     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Characterization of trehalose synthase from <Emphasis Type="Italic">Corynebacterium nitrilophilus</Emphasis> NRC

Trehalose synthase (TSII) from Corynebacterium nitrilophilus NRC was successively purified by ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-100 columns. The specific activity of the trehalose synthase was increased ~200-fold, from 0.14 U mg⁻¹ protein to 28.3 U mg⁻¹ protein. TSII was found to be a monomeric protein with a molecular weight of 67–69 kDa. Characterization of the enzyme exhibited optimum pH and temperature were 7.5 and 35°C, respectively. The purified enzyme was stable from pH 6.6 to 7.8 and able to prolong its thermal stability up to 35°C. The enzyme activity was inhibited strongly by Zn²⁺, Hg²⁺ and Cu²⁺ and moderately by Ba²⁺, Fe²⁺, Pb²⁺ and Ni²⁺. Other metal ions Ca²⁺, Mg²⁺, Co²⁺, Mn²⁺ and EDTA had almost no effect.     

FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

 Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse, bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D₂O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54  °C in horse and at 57  °C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35  °C, followed by a second amide II transition at 50  °C revealing a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50  °C (tuna) and 54  °C (horse) are also observed during the thermal titration of the CN^–-ligated cytochromes (where CN^– displaces the Met80 ligand), but a well-defined 35  °C amide II transition is absent from the titration curve of the CN^–adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5  °C range with T _m values at 74  °C (bovine c), 70  °C (horse c) and 65  °C (tuna c), as monitored by changes in the amide I′ bands. The FTIR spectra were also used to compare the secondary structures of the ferricytochromes c at 25  °C. Curve fitting of the amide I (H₂O) and amide I′ (D₂O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the α-helical absorption of tuna c indicates the presence of less-stable helical structures. CN^– adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D₂O is a powerful tool in protein conformational analysis. Received: 1 April 1999 / Accepted: 24 August 1999     

Purification and characterization of a highly selective sucrose isomerase from Erwinia rhapontici NX-5

A highly selective sucrose isomerase (SIase) was purified to homogeneity from the cell-free extract of Erwinia rhapontici NX-5 with a recovery of 27.7% and a fold purification of 213.6. The purified SIase showed a high specific activity of 427.1 U mg⁻¹ with molecular weight of 65.6 kDa. The K _m for sucrose was 222 mM while V _max was 546 U mg⁻¹. The optimum pH and temperature for SIase activity were 6.0 and 30 °C, respectively. The purified SIase was stable in the temperature range of 10–40 °C and retained 65% of the enzyme activity after 2 weeks’ storage at 30 °C. The SIase activity was enhanced by Mg²⁺ and Mn²⁺, inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and Co²⁺, completely inhibited by Hg²⁺ and Ag²⁺. The purified SIase was strongly inhibited by SDS, while partially inhibited by dimethylformamide, tetrahydrofuran, and PMSF. Additionally, glucose and fructose acted as competitive inhibitors for purified SIase.     

Cloning,Expression, Purification,and Characterization of Cold-Adapted α-Amylase from <Emphasis Type="Italic">Pseudoalteromonas arctica</Emphasis> GS230

A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7–8.5 at 30 °C for 1 h. ParAmy was activated by Mn²⁺, K⁺ and Na⁺, and inhibited by Hg²⁺, Cu²⁺, and Fe³⁺. N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K _m and V _max values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry.     

Enzymatic detection of heavy metal ions in aqueous solution from vegetable wastes by immobilizing pumpkin (<Emphasis Type="Italic">Cucumis melo</Emphasis>) urease in calcium alginate beads

Enzyme urease is extracted from the discarded seeds of pumpkin. Urease was purified to apparent homogeneity (5.2 fold) by heat treatment at 48 ± 1°C and gel filtration through Sephadex G-200. Effect of model metal ions on the activity of the homogeneous enzyme preparation (sp. activity 353 U/mg protein, A₂₈₀/A₂₆₀ = 1.12) of soluble as well as immobilized enzyme was investigated. The soluble and immobilized urease has been used for the quantitative estimation of general water pollution with heavy metal ions like Hg²⁺, Cu²⁺, Cd²⁺, and Co²⁺. The measurements of the urease residual activity have been carried out in tris-acetate buffer after pre-incubation of model metal salt. The inhibition was found to be biphasic with an initial rapid loss of activity and remainder in slow phase of 10∼15 min. The immobilization was done in 3.5% alginate beads leading to 86% of entrapment. There was no leaching of the enzyme over a period of 15 days at 4°C. The beads were fairly stable up to 50°C and exhibited activity even at −10°C. The inhibition by these ions was non-competitive and irreversible, hence could not be restored by dialysis. Based on the values of inhibition constant Ki the heavy-metal ions were found to inhibit urease in the following order Hg²⁺ > Cu²⁺ > Cd²⁺ > Co²⁺.     

Production of β-galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg⁻¹) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg⁻¹). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co²⁺, Mn²⁺, Fe²⁺ and Zn²⁺ had strong inhibitory effects on the enzyme activity. The K _m and V _max for p-nitrophenyl-β- _D-galactopyranoside and o-nitrophenyl-β - _D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min⁻¹ mg⁻¹ respectively. The K _m and V _max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min⁻¹ mg⁻¹. Received 10 March 1997/ Accepted in revised form 17 July 1997     

Stability of metal ion complexes formed with methyl phosphate and hydrogen phosphate

 The acidity constants of methyl phosphoric acid, CH₃OPO(OH)₂, and orthophosphoric acid, HOPO(OH)₂, and the stability constants of the 1 : 1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, or Cd²⁺ and methyl phosphate, CH₃OPO₃ ^2–, or hydrogen phosphate, HOPO₃ ^2–, were determined by potentiometric pH titration in aqueous solution (25  °C;I = 0.1 M, NaNO₃). On the basis of previously established log K versus pK _a straight-line plots for the complexes of simple phosphate monoesters and phosphonate derivatives, R-PO₃ ^2–, where R is a noncoordinating residue, it is shown that the stability of the M(CH₃OPO₃) complexes is solely determined (as one might expect) by the basicity of the –PO₃ ^2– residue. It is emphasized that the mentioned reference lines may also be used to reveal increased complex stabilities, for example, for certain complexes formed with 8-quinolyl phosphate the occurrence of 7-membered chelates can be proven in this way; the same procedure is also applicable to complexes of nucleotides, etc. The M(HOPO₃) complexes are slightly more stable (on average by 0.08 log unit) than it is expected from the basicity of HPO₄ ^2–; this observation is attributed to a more effective solvation, including hydrogen bonding, than is possible with CH₃OPO₃ ^2– species. Received: 9 November 1995 / Accepted: 5 February 1996     

De novo synthesis,constitutive expression of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> β-xylanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and partial enzymic characterization

The endo-β-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an α-mating factor in a constitutive expression vector pGAPzαA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast–peptone–dextrose liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed with a yield of 120 units/mL under the flask culture at 28°C for 3 days. The enzyme showed optimal activity at 50°C and pH 2.4–3.4. Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80°C for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60°C for 30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na₂HPO₄-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na⁺, Mg²⁺, Ca²⁺, K⁺, Ba²⁺, Zn²⁺, Fe²⁺, and Mn²⁺) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.