HPAEC-PAD and Q-TOF-MS/MS analysis reveal a novel mode of action of endo-ß-1,3(4)-d-glucanase Eng16A from coprinopsis cinerea on barley ß-glucan.

We previously reported that an endo-ß-1,3(4)-d-glucanase, Eng16A, from C. cinerea shows a higher degradation activity toward barley ß-glucan than laminarin. HPAEC-PAD and Q-TOF-MS/MS analyses show that Eng16A-digestion products of barley ß-glucan not only contain some oligosaccharides with (1 → 3)-ß-linkage adjacent to the reducing end, which is consistent with ß-1,3(4)-glucanase-digestion products, but also include some oligosaccharides containing (1 → 4)-ß-linkage adjacent to the reducing end which is consistent with cellulase-digestion products. Thus, Eng16A possesses both cellulase and ß-1,3(4)-glucanase activities. Because Eng16A does not degrade cellulose, we propose that the insertion of a (1 → 3)-ß-linkage among the groups of (1 → 4)-ß-linkages may make these (1 → 4)-ß-linkages prone to cleavage by Eng16A. Furthermore, Eng16A also possesses transglycosylation activity which leads to some products containing one or a few consecutive (1 → 3)-ß-linkages adjacent to the non-reducing end. Therefore, HPAEC-PAD and Q-TOF-MS/MS analyses provide an efficient approach to reveal complicated modes of action of some endo-ß-1,3(4)-d-glucanases on barley ß-glucan.

Biosynthesis of lagopodins in mushroom involves a complex network of oxidation reactions.

Use of the ku70-deficient strain of Coprinopsis cinerea enabled confirmation within the native context of the central role the sesquiterpene synthase Cop6 plays in lagopodin biosynthesis. Furthermore, yeast in vivo bioconversion and in vitro assays of two cytochrome P450 monooxygenases Cox1 and Cox2 allowed elucidation of the network of oxidation steps that build structural complexity onto the α-cuprenene framework during the biosynthesis of lagopodins. Three new compounds were identified as intermediates formed by the redox enzymes.

Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/ß)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

Coprinopsis cinerea intracellular lactonases hydrolyze quorum sensing molecules of Gram-negative bacteria.

Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-ß-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle.

Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala.

The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer.

[Expression and characterization of Coprinus cinereus peroxidase].

OBJECTIVE: The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently. METHODS: We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115. RESULTS: We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol. CONCLUSION: The highly secretory expression of CIP and high special activity lay the good foundation for it' s industrial applications in waste water treatment, decolouration of dyestuffs.

Transformation of multi-component ginkgolide into ginkgolide B by Coprinus comatus.

BACKGROUND: As the strongest antagonist of the platelet activating factor, ginkgolide B (GB) possesses anti-ischemic, anti-oxidant and anti-convulsant properties, and it is used for the treatment of thrombosis in clinical practice. Till now, GB is usually obtained from extraction of Ginkgo biloba leaves through column chromatography with an extremely low yield and high cost, which can not meet clinical requirement. Therefore, it is urgent to find a new method to prepare GB. RESULTS: In the current study, we studied the ability and mechanism to transform multi-component ginkgolide into GB by Coprinus comatus in order to enhance the GB yield. Except for ginkgolide A (GA) and GB, all the other ginkgolides in the extract were transformed by the strain. In the case of culture medium containing 20 g/L glucose, the transformation product was identified as 12% GA and 88% GB by high performance liquid chromatography-Mass spectrometry (HPLC-MS), two stage mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR). Partial GA was also transformed into GB according to the yield (76%) and the content of GA in the raw ginkgolide (28.5%). Glucose was the key factor to transform ginkgolides. When glucose concentration in medium was higher than 40 g/L, all ginkgolides were transformed into the GB. Proteomic analysis showed that C. comatus transformed ginkgolide into GB by producing 5 aldo/keto reductases and catalases, and enhancing the metabolism of glucose, including Embden-Meyerhof pathway (EMP), hexose monophophate pathway (HMP) and tricarboxylic acid (TCA). CONCLUSIONS: C. comatus could transform ginkgolides into GB when the medium contained 40 g/L glucose. When the strain transformed ginkgolides, the glucose metabolism was enhanced and the strain synthesized more aldo/keto reductases and catalases. Our current study laid the groundwork for industrial production of GB.

[Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1].

The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.

Production of a lignocellulolytic enzyme system for simultaneous bio-delignification and saccharification of corn stover employing co-culture of fungi.

Aiming at improving the efficiency of transferring corn stover into sugars, an efficient lignocellulolytic enzyme system was developed and investigated by co-cultivation of the Coprinus comatus with Trichoderma reesei in a single bioreactor. The results showed that the lignocellulolytic enzyme activities of the co-culture exceeded that of the monoculture, suggesting synergistic interaction between two fungi. The highest laccase activity from the co-culture was 2.6-fold increase over that of the C. comatus monoculture and reached a peak 3days earlier. The maximum delignification obtained was 66.5% and about 82% of the original polysaccharides were converted into fermentable sugars by simultaneous bio-delignification and saccharification process. Correlation analysis showed that sugar yields were directly proportional to the lignin degradation. Our results suggested that co-fungi cultivation was a valuable technique for corn stover bioconversion, which could produce high efficiency of lignocellulolytic enzyme system as a cheaper alternative to commercial enzymes for industrial utilization.

A novel laccase with potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from mycelia of mushroom Coprinus comatus.

A novel laccase was isolated and purified from fermentation mycelia of mushroom Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2'-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, K(m) values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 µM, 4.95 µM, and 5.85 µM, respectively, signifying that it is an antipathogenic protein.

Improved selectivity of an engineered multi-product terpene synthase.

Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a kcat/KM of 0.62 mM(-1) s(-1), which is nearly identical to that of the parent Cop2, which had a kcat/KM of 0.58 mM(-1) s(-1).

Engineering the expression and characterization of two novel laccase isoenzymes from Coprinus comatus in Pichia pastoris by fusing an additional ten amino acids tag at N-terminus.

The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest Km and highest kcat/Km value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.

Induction of a laccase Lcc9 from Coprinopsis cinerea by fungal coculture and its application on indigo dye decolorization.

A fungal coculture system comprised of Coprinopsis cinerea Okayama 7 (#130) and Gongronella sp. w5 produced 900 times higher laccase activity than that in pure culture. A fungal laccase named Lcc9 was induced from C. cinerea for the first time by coculture. Lcc9 was purified, characterized, and found to have high activity toward phenolic substrates at the optimum pH of 6.5 and temperature of 60°C. The laccase was stable at alkaline pH values, and its activity was not significantly affected by cations and organic solvents. Lcc9 showed decolorization capability toward indigo dye in the presence of 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate), with 75% of indigo was decolorized by 50 U/L enzyme after 1h of incubation under optimal catalytic conditions. These results showed that fungal coculture could active silent laccase gene, and the unusual properties make Lcc9 a candidate for specific industrial and environmental applications.

Effects of rhamnolipid biosurfactant JBR425 and synthetic surfactant surfyno1465 on the peroxidase-catalyzed oxidation of 2-naphthol.

The kinetics of the recombinant Coprinus cinereus peroxidase-catalyzed 2-naphthol oxidation was investigated in the presence of rhamnolipid biosurfactant JBR425 and synthetic surfactant Surfynol465 at pH 5.5 and 250C, with concentrations of (bio)surfactants both less than critical micelle concentrations (CMC) and larger than CMC. It was shown that monomers of JBR425 as well as monomers of Surfynol465 had an enhancing effect on the conversion of 2-naphthol in dose response manner and did not influence the initial rate of 2-naphthol oxidation. The results were accounted by a scheme, which contains a stadium of enzyme inhibition by oligomeric 2-naphthol oxidation products. The action of the biosurfactant's (or synthetic surfactant's) monomers was explained by avoidance of the enzyme active center clothing with oligomers. Similar results have demonstrated the potential of rhamnolipid biosurfactant JBR425 due to its biodegradability. When biosurfactants' concentrations are larger than CMC, (bio)surfactants have an opposite effect on the oxidation of 2-naphthol by peroxidase.

Diversification of fungal specific class a glutathione transferases in saprotrophic fungi.

Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes and endogenous metabolism. The distribution of fungal-specific class A GSTs was investigated in saprotrophic fungi revealing a recent diversification within this class. Biochemical characterization of eight GSTFuA isoforms from Phanerochaete chrysosporium and Coprinus cinereus demonstrated functional diversity in saprotrophic fungi. The three-dimensional structures of three P. chrysosporium isoforms feature structural differences explaining the functional diversity of these enzymes. Competition experiments between fluorescent probes, and various molecules, showed that these GSTs function as ligandins with various small aromatic compounds, derived from lignin degradation or not, at a L-site overlapping the glutathione binding pocket. By combining genomic data with structural and biochemical determinations, we propose that this class of GST has evolved in response to environmental constraints induced by wood chemistry.

Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes.

Laccase is a blue copper oxidase with multiple copper ions and widely distributed in higher plant and fungi. To date, numerous fungal laccases have been reported by many researchers. In present work, a new laccase gene, named CcLCC5I, from Coprinus cinereus was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115 genome by electroporation. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately 56.8 kDa. Its biochemical properties was carried out using substrate 2-2(')-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was showed that the optimum pH and temperature of the laccase is 3.0 and 55 °C, respectively. Except for copper ions, most metal ions inhibited the laccase activity at a high concentration about 10 mM. Sodium sulfite can also highly inhibit laccase activity whereas EDTA had no inhibitory effect on the laccase activity. The CcLCC5I have high ability to decolor not only azo but also aryl methane dyes. The recombinant laccase decolored 44.6 % orange G, 54.8 % Crystal Violet, and 87.2 % Malachite green at about 2.6 h. The novel laccase may be a good candidate for breeding engineering strains used in the treatment of industrial effluent containing azo and aryl methane dyes.

Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus.

Two new laccase genes, named lac1 and lac2, were cloned from the edible basidiomycete Coprinus comatus. Comparison of the deduced amino acid sequences revealed two laccases showed 66.12 % identity and clustered with lac2 and lac3 from Coprinopsis cinerea in same phylogenetic group. Lac1 and lac2 encode proteins of 517 and 523 amino acids preceded by 18 and 21-residue signal peptides, respectively. Lac1 was functionally expressed in Pichia pastoris. The optimum pHs of recombinant Lac1 were 3.0, 6.0, 5.5 and 6.0 and the optimum temperatures were 65, 55, 70 and 50 °C for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The Km values of Lac1 were 34, 4,317, 7,611 and 14 µM, and the corresponding kcat values were 465.79, 7.67, 1.15 and 0.60 (s(-1) mM), for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The enzyme activity was completely inhibited by sodium azide (NaN(3)) and 1,4-dithiothreitol (DTT) at the concentration of 5 mM. Laccase activity was also inhibited by several metal ions, especially Fe(2+), while K(+) and NH(4) (+) slightly enhanced laccase activity. Twelve synthetic dyes belonging to anthraquinone, azo and triphenylmethane dyes were decolorized by the recombinant Lac1 at different extents. The recombinant Lac1 decolorized azo dye Reactive Dark Blue KR up to 90 % without any mediator and increasing to 96 % with mediator, indicating its potential in the treatment of industrial effluent containing some recalcitrant synthetic dyes.

The comparision of Coprinus cinereus peroxidase enzyme and TiO2 catalyst for phenol removal.

This article investigates phenol removal from an aqueous solution by using enzymatic and photocatalytic methods and the efficiency of these methods has been compared. In enzymatic and photocatalytic methods, Coprinus cinereus, peroxidase enzyme and commercial TiO(2) powders (Degussa P-25) in aqueous suspension were used, respectively, in ambient temperature. The effects of different operating parameters such as duration of process, catalyst dosage or enzyme concentration, pH of the solution, initial phenol concentration and H(2)O(2) concentration on both processes were examined. In enzymatic method, efficiency of degradation reached 100% within 5 min, while in the photocatalytic method, the efficiency of degradation reached approximately 70% within 60 min. In photocatalytic method, there is an optimum concentration for catalyst dosage (near 2.0 g/L) to gain 80% efficiency, while in the enzymatic method, increasing the amount of enzyme could lead to an increase in the efficiency up to 100%. Moreover, the optimum pH in enzymatic and photocatalytic methods stood at 8.0 and 7.0, respectively. In both methods, the addition of different amounts of H(2)O(2) increased the degradation efficiency to 100%.

In vitro analysis of the monolignol coupling mechanism using dehydrogenative polymerization in the presence of peroxidases and controlled feeding ratios of coniferyl and sinapyl alcohol.

In this study, dehydrogenative polymers (DHP) were synthesized in vitro through dehydrogenative polymerization using different ratios of coniferyl alcohol (CA) and sinapyl alcohol (SA) (10:0, 8:2, 6:4, 2:8, 0:10), in order to investigate the monolignol coupling mechanism in the presence of horseradish peroxidase (HRP), Coprinus cinereus peroxidase (CiP) or soybean peroxidase (SBP) with H(2)O(2), respectively. The turnover capacities of HRP, CiP and SBP were also measured for coniferyl alcohol (CA) and sinapyl alcohol (SA), and CiP and SBP were found to have the highest turnover capacity for CA and SA, respectively. The yields of HRP-catalyzed DHP (DHP-H) and CiP-catalyzed DHP (DHP-C) were estimated between ca. 7% and 72% based on the original weights of CA/SA in these synthetic conditions. However, a much lower yield of SBP-catalyzed DHP (DHP-S) was produced compared to that of DHP-H and DHP-C. In general, the DHP yields gradually increased as the ratio of CA/SA increased. The average molecular weight of DHP-H also increased with increasing CA/SA ratios, while those of DHP-C and DHP-S were not influenced by the ratios of monolignols. The frequency of ß-O-4 linkages in the DHPs decreased with increasing CA/SA ratios, indicating that the formation of ß-O-4 linkages during DHP synthesis was influenced by peroxidase type.

Amperometric sulfide detection using Coprinus cinereus peroxidase immobilized on screen printed electrode in an enzyme inhibition based biosensor.

In the present work, an amperometric inhibition biosensor for the determination of sulfide has been fabricated by immobilizing Coprinus cinereus peroxidase (CIP) on the surface of screen printed electrode (SPE). Chitosan/acrylamide was applied for immobilization of peroxidase on the working electrode. The amperometric measurement was performed at an applied potential of -150 mV versus Ag/AgCl with a scan rate of 100 mV in the presence of hydroquinone as electron mediator and 0.1M phosphate buffer solution of pH 6.5. The variables influencing the performance of sensor including the amount of substrate, mediator concentration and electrolyte pH were optimized. The determination of sulfide can be achieved in a linear range of 1.09-16.3 µM with a detection limit of 0.3 µM. Developed sensor showed quicker response to sulfide compared to the previous developed sulfide biosensors. Common anions and cations in environmental water did not interfere with sulfide detection by the developed biosensor. Cyanide interference on the enzyme inhibition caused 43.25% error in the calibration assay which is less than the amounts reported by previous studies. Because of high sensitivity and the low-cost of SPE, this inhibition biosensor can be successfully used for analysis of environmental water samples.