Molecular determinants of azo reduction activity in the strain <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MET94

Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi–bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.     

Decolourisation of reactive textile dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by <Emphasis Type="Italic">Funalia trogii</Emphasis> ATCC 200800

Decolourisation of reactive dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by white rot fungi Funalia trogii was studied under static conditions. The effect of various conditions such as mycelial age, initial dye and glucose concentrations on decolourisation were also investigated. Decolourisation activity of F. trogii was compared with Phanerochaete chrysosporium known as test microorganism. It was found that 7-day-old cultures were more effective than 5-day-old cultures of F. trogii for decolourisation of these dyes. Decolourisations by F. trogii of both dyes were increased with glucose concentration decreasing. In contrast, decolourisations by P. chrysosporium were decreased. F. trogii decolourised 92–98% of both dyes within 4–10 h. However, P. chrysosporium partially decolourised (11–20%) these dyes during 10days incubation period under the same conditions.     

Decolorization of textile dyes by enzymatic extract and submerged cultures of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus sajor-caju PS2001 was screened in Petri dish plates to assess the dye-decolorizing ability of industrial textile dyes. P. sajor-caju PS2001 was also cultivated in solid-state fermentation containing sawdust of Pinus sp. and wheat bran to obtain the enzymatic extract, showing laccase and manganese-peroxidase activity, which was used to test the capacity to degrade the textile dyes. Additional tests of decolorization were performed in liquid cultures. Anthraquinone-type textile dyes proved to be substrates for the enzymatic system of P. sajor-caju PS2001. Cultures in Petri dish plates showed that the anthraquinone dye Reactive Blue 220 can act as a redox mediator for the enzymatic reactions involved in the decolorization process, and enables the azo dye degradation. Reactive Blue 220 and Acid Blue 280 were completely decolorized in 30 min and 60 min, respectively, during the tests with precipitated enzymatic extract, while the azo dyes showed resistance to degradation. Additionally, in submerged cultures with dyes, veratryl alcohol oxidases and lignin peroxidase activities were observed. These results suggest that the strain P. sajor-caju PS2001 has great potential for use in the bioremediation technology of recalcitrant pollutant such as textile effluents.     

Production of lignocellulose-degrading enzymes employing <Emphasis Type="Italic">Fusarium solani</Emphasis> F-552

In this work, capability of Fusarium solani F-552 of producing lignocellulose-degrading enzymes in submerged fermentation was investigated. The enzyme cocktail includes hydrolases (cellulases, xylanases, and proteinases) as well as ligninolytic enzymes: manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac). To our knowledge, this is the first report on production of MnP, LiP, and Lac together by one F. solani strain. The enzyme productions were significantly influenced by application of either lignocellulosic material or chemical inducers into the fermentation medium. Among them, corn bran significantly enhanced especially productions of cellulases and xylanases (248 and 170 U/mL, respectively) as compared to control culture (11.7 and 29.2 U/mL, respectively). High MnP activity (9.43 U/mL, control 0.45 U/mL) was observed when (+)-catechin was applied into the medium, the yield of LiP was maximal (33.06 U/mL, control 2.69 U/mL) in gallic acid, and Lac was efficiently induced by, 2,2′-azino-bis-[3-ethyltiazoline-6-sulfonate] (6.74 U/mL, not detected in control). Finally, in order to maximize the ligninolytic enzymes yields, a novel strategy of introduction of mild oxidative stress conditions caused by hydrogen peroxide into the fermentation broth was tested. Hydrogen peroxide significantly increased activities of MnP, LiP, and Lac which may indicate that these enzymes could be partially involved in stress response against H₂O₂. The concentration of H₂O₂ and the time of the stress application were optimized; hence, when 10 mmol/L H₂O₂ was applied at the second and sixth day of cultivation, the MnP, LiP, and Lac yields reached 21.67, 77.42, and 12.04 U/mL, respectively.     

Decolorization of water and oil-soluble azo dyes by <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>

The capability of Lactobacillus acidophilus and Lactobacillus fermentum to degrade azo dyes was investigated. The bacteria were incubated under anaerobic conditions in the presence of 6 μg/ml Methyl Red, Ponceau BS, Orange G, Amaranth, Orange II, and Direct Blue 15; 5 μg/ml Sudan I and II; or 1.5 μg/ml Sudan III and IV in deMann–Rogosa–Sharpe broth at 37°C for 36 h, and reduction of the dyes was monitored. Both bacteria were capable of degrading all of the water-soluble azo dyes to some extent. They were also able to completely reduce the oil-soluble diazo dyes Sudan III and IV but were unable to reduce the oil-soluble monoazo dyes Sudan I and II to any significant degree in the concentrations studied. Growth of the bacteria was not significantly affected by the presence of the Sudan azo dyes. Metabolites of the bacterial degradation of Sudan III and IV were isolated and identified by liquid chromatography electrospray ionization tandem mass spectrometry analyses and compared with authentic standards. Aniline and o-toluidine (2-methylaniline), both potentially carcinogenic aromatic amines, were metabolites of Sudan III and IV, respectively.     

Degradation of Remazol Red dye by <Emphasis Type="Italic">Galactomyces geotrichum</Emphasis> MTCC 1360 leading to increased iron uptake in <Emphasis Type="Italic">Sorghum vulgare</Emphasis> and <Emphasis Type="Italic">Phaseolus mungo</Emphasis> from soil

Removal of azo dyes from the effluent generated by textile industries is rather difficult. Azo dyes represent a major class of synthetic colorants that are both mutagenic and carcinogenic. Galactomyces geotrichum MTCC 1360, a yeast species, showed more than 96% decolorization of the azo dye Remazol Red (50 mg/L) within 36 h at 30°C and pH 11.0 under static condition with a significant reduction in the chemical oxygen demand (62%) and total organic carbon (41%). Peptone (5.0 g/L), rice husk (10 g/L extract), and ammonium chloride (5.0 g/L) were found to be more significant among the carbon and nitrogen sources used. The presence of tyrosinase, NADH-DCIP reductase, riboflavin reductase and induction in azo reductase and laccase activity during decolorization indicated their role in degradation. High performance thin layer chromatography analysis revealed the degradation of Remazol Red into different metabolites. Fourier transform infrared spectroscopy and high performance liquid chromatography analysis of samples before and after decolorization confirmed the biotransformation of dye. Atomic absorption spectroscopy analysis revealed a less toxic effect of the metabolites on iron uptake by Sorghum vulgare and Phaseolus mungo than Remazol Red dye. Remazol Red showed an inhibitory effect on iron uptake by chelation and an immobilization of iron, whereas its metabolites showed no chelation as well as immobilization of iron. Phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products which had a less toxic nature.     

Discoloration of the azo dye Congo Red by manganese-dependent peroxidase from <Emphasis Type="Italic">Pleurotus sajor caju</Emphasis>

This paper describes the production of ligninolytic enzymes by the white-rot fungus Pleurotus sajor caju under solid-state fermentation conditions using a cost-effective medium consisting of agro-industrial wastes. From the different agro-industrial wastes tested (i.e. orange, banana, mango and cantaloupe peels), banana peels led to the highest manganese-dependent peroxidase (MnP) activity (6.3 U/mL on the 10 day). MnP from banana peel cultures was purified and applied to the discoloration of the azo dye Congo Red (CR). The optimum temperature, pH and enzyme concentration for maximum discoloration (i.e. 95% in 1 h) were found to be 35°C, 4.0, and 1.4 U/mL, respectively. In addition, the phytotoxicity (with respect to Sorghum vulgare and Phaseolus radiatus seeds) of CR was considerably reduced after the treatment of plant material with MnP produced by P. sajor caju. The products obtained after discoloration of CR were characterized using GC/MS as 8-amino naphthol 3-sulfonic acid, 3-hydroperoxy 8-nitrosonaphthol, p-p'-dihydroxybiphenyl. Therefore, this approach holds promise for the production and application of MnP from P. sajor caju on a larger scale.     

Decolorization of Victoria blue by the white rot fungus, <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Synthetic textile dyes are among the most dangerous chemical pollutants released in industrial wastewater streams. Recognizing the importance of reducing the environmental impact of these dyes, the ability of the white rot fungus Phanerochaete chrysosporium to decolorize various textile dyes was investigated. This fungus decolorized 6 of the 14 structurally diverse dyes with varying efficiency (between 14% and 52%). There was no discernable pattern of decolorization even among dyes of the same chemical class, suggesting that attack on the dyes is relatively non-specific. Among the three dyes which showed >40% decolorization, Victoria Blue B (VB) was chosen for further analysis because the ability of the fungus to decolorize VB was nearly independent over a relatively broad concentration range. Blocking lignin peroxidase (LiP) and manganese peroxidase (MnP) production by the fungus did not substantially affect VB decolorization. Inhibition of laccase production by adding various inhibitors to shaken cultures reduced VB decolorization significantly suggesting a role for laccase in VB decolorization. When sodium azide and aminotriazole were used to inhibit endogenous catalase and cytochrome P-450 oxygenase activities, there was 100% and 70% reduction in VB decolorization, respectively. Adding benzoate to trap hydrogen peroxide-derived hydroxyl radicals resulted in 50% decolorization of VB. Boiling the extracellular fluid (ECF) for 30 min resulted in approximately 50% reduction in VB decolorization. Collectively, these data suggest that laccase, and/or oxygenase/oxidase and a heat-stable non-enzymatic factor, but not Lip and MnP, play a role in VB decolorization by P. chrysosporium.     

Decolorization of reactive dyes by <Emphasis Type="Italic">Clostridium bifermentans</Emphasis> SL186 isolated from contaminated soil

Decolorization of textile reactive azo dyes by a strain of bacteria (SL186) isolated from a contaminated site was investigated. SL186 was identified as Clostridium bifermentans by phenotypic characterization and 16S rDNA sequence comparison. Under anaerobic conditions, SL186 had decolorized the dyes Reactive Red 3B-A, Reactive Black 5, and Reactive Yellow 3G-P by over 90% after 36 h post-inoculation. The bacterium retained decolorizing activity over a wide range of pH values (6–12), with peak activity at pH 10. Additionally, SL186 decolorized a relatively high concentration of Reactive Red 3B-A dye (1,000 ppm) by over 80% and raw industrial effluent effectively. The addition of glucose increased the decolorization rate a little. Spectrophotometric analyses of the reactive dyes showed no distinct peak indicating aromatic amines. However, a new peak was detected between 300 and 450 nm from the decolorized raw industrial effluent. These results suggest that C. bifermentans SL186 is a suitable bacterium for the biological processing of dye-containing wastewater.     

Decolorization of Textile Dyes and Degradation of Mono-Azo Dye Amaranth by <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890

Acinetobacter calcoaceticus NCIM 2890 (A. caloaceticus) was found to decolorize 20 different textile dyes of various classes. Decolorization of an azo dye amaranth was observed effectively (91%) at static anoxic condition, whereas agitated culture grew well but showed less decolorization (68%) within 48 h of incubation. Induction of intracellular and extracellular lignin peroxidase, intracellular laccase, dichlorophenol indophenol (DCIP) reductase and riboflavin reductase represented their involvement in the biodegradation of amaranth. The products obtained after degradation of Amaranth were characterized as naphthalene sulfamide, hydroxyl naphthalene diazonium and naphthalene diazonium. The germination and growth of Sorghum vulgare and Phaseolus mungo seeds, and the growth of E. coli and Bacillus substilis were not inhibited by the metabolic products of the dye.     

Dye decolorization by <Emphasis Type="Italic">Trametes hirsuta</Emphasis> immobilized into alginate beads

Summary The present paper studies the production of laccase by Trametes hirsuta immobilized into alginate beads in an airlift bioreactor. In order to enhance laccase production fresh ammonium chloride was added, which led to the production, of high laccase activities (around 1000 U l⁻¹). The bioreactor operated for 40 days without operational problems and the bioparticles maintained their shape throughout fermentation. Dye decolorization was performed at bioreactor scale operating in the batch mode. High decolorization percentages were obtained in a short time (96% for indigo carmine and 69% for phenol red in 24 h), indicating the suitability of this process for application to synthetic dye decolorization. On the other hand, in vitro decolorization of several industrial azo dyes by crude laccase produced in the above bioreactor was also performed. It was found that some of the dyes needed the addition of 1-hydroxybenzotriazole for their decolorization.     

Biodegradation of C.I. Reactive Red 195 by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> strain YZ66

Synthetic dyes are extensively used in textile dyeing, paper, printing, colour photography, pharmaceutics, cosmetics and other industries. Among these, azodyes represents the largest and most versatile class of synthetic dyes. As high as 50% of the dyes are released into the environment during manufacture and usage. Traditional methods of treatment are found to be expensive and have operational problems. Biological decolourization has been investigated as a method to transform, degrade or mineralize azo dyes. In the present studies bacteria from soil from dye waste area, dye waste, sewage and dung were subjected to acclimatization with C.I. Reactive Red 195 an azo dye, in the basal nutrient media. The most promising bacterial isolate was used for further dye degradation studies. The 16s rRNA gene sequencing and biochemical characteristics revealed the isolated organism as Enterococcus faecalis strain YZ66. The strain showed 99.5% decolourization of the selected dye (Reactive Red 195–50 mg/l) within one and half hour in static anoxic condition. The optimum pH and temperature for the decolourization was 5.0 and 40°C respectively. The biodegradation was monitored by UV–Vis, FTIR, TLC and HPLC. The final products were characterized by Gas chromatography and Mass Spectrophotometry. Toxicity study demonstrated no toxicity of the biodegradation product. The results suggest that the isolated organism E. faecalis strain YZ 66 can be used as a useful tool to treat waste water containing reactive dyes.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.     

Biodegradation of triphenylmethane dye Malachite Green by <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis>

Triphenylmethane dyes belong to the most important group of synthetic colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. Sphingomonas paucimobilis, was isolated from the soil sample collected from contaminated sites of textile industry located in KsarHellal, Tunisia, and it was able to decolorize Malachite Green (MG) dye (50 mg/l) within 4 h under shaking condition (pH 9 and temperature 25°C). The effect of inoculum size, dye concentration, temperature and initial pH of the solution were studied. The results obtained from the batch experiments revealed the ability of the tested bacteria to remove dye. UV–Vis spectroscopy and FTIR analysis of samples before and after decolorization confirmed the ability of the tested strain to decolorize MG. In addition, the phytotoxicity study revealed the degradation of MG into non-toxic product by S. paucimobilis.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Decolorization of an industrial effluent by free and immobilized cells of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> AAP56<Emphasis Type="Italic">.</Emphasis> Implementation of efficient down flow column reactor

A bacterial strain AAP56, isolated from a polluted soil (from Kelibia city) and identified as Stentrophomonas maltophilia, was particularly interesting for its ability to decolorize recalcitrant dyes of an industrial effluent: SITEX Black. The final percentage decolorization 60% was shown by bacterial culture after incubation in LB medium at 30°C under shaking conditions. The decolorization was closely correlated with the metabolic bacterial growth. The replacement of yeast extract in LB medium composition by soya flour was clearly efficient to enhance the percentage decolorization by 20% and also to reduce the growth medium cost 60-fold. The bacteria were able to reduce 23% from the initial COD and 28% from the initial BOD₅ of the effluent. The immobilization of bacterial cells in calcium alginate beads improved by 25% the effectiveness of the biotransformation within 24 h in batch conditions. The potential of a downflow fixed column reactor (DFCR) to decolorize SITEX Black was evaluated under dilution rate. The best decolorization percentage (82%) was recorded at 0.3 h⁻¹. This bioprocess seems to be a potentially useful method to remediate the colored textile wastewater.     

Decolorization of Acid Red 27 and Reactive Red 2 by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> under a batch system

The objectives of this study were to investigate: (1) the capacity of Enterococcus faecalis on the decolorization of the azo dyes Acid Red 27 and Reactive Red 2; and (2) the growth characteristics of E. faecalis on those dyes. E. faecalis was able to decolorize Acid Red 27 and Reactive Red 2 effectively. High decolorization efficiency (95–100%) was achieved within 3 h of incubation for Acid Red 27, and 12 h for Reactive Red 2, at room temperature, neutral pH, static and non-aerated condition. Growth characteristics of E. faecalis on azo dyes, which were indicated by cell growth rate, biomass production, and growth yield, was worse than the control. E. faecalis grew better on Acid Red 27 rather than Reactive Red 2.     

High-level expression and characterization of a highly thermostable chitosanase from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was ~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides: 3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C.     

Enhanced Soluble Expression of a Thermostable Cellulase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this study, the cellulase gene celD from Clostridium thermocellum was cloned into expression vectors pET-20b(+) and pHsh. While high expression can be achieved by means of both these expression systems, only the pHsh expression system gives soluble proteins. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ± 0.3 to 6.4 ± 0.4 U ml⁻¹ in LB medium. Recombinant CelD was purified by heat treatment followed by Ni–NTA affinity. The purified CelD exhibited the highest activity at pH 5.4 and 60°C, and retained more than 50% activity after incubation at 70°C for 1 h. The cellulase activity of CelD was significantly enhanced by Ca²⁺ but inhibited by EDTA. The favorable properties of CelD offer the potential for genetic modification of strains for biomass degradation. Presently, one of the major bottlenecks for industrial cellulase users is the high cost of enzyme production. The high level expression of soluble enzymes from the pHsh expression system offers a novel approach for the production of cellulases to be used in various agro-industrial processes such as chemical, food and textile.     

High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.