Purification and characterization of polygalacturonase produced by thermophilic <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Heterologous expression and characterization of a novel halotolerant,thermostable, and alkali-stable GH6 endoglucanase from <Emphasis Type="Italic">Thermobifida halotolerans</Emphasis>

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462^T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 % of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.     

<Emphasis Type="Italic">Talaromyces heiheensis</Emphasis> and <Emphasis Type="Italic">T. mangshanicus</Emphasis>, two new species from China

Two new species, Talaromyces heiheensis from rotten wood and T. mangshanicus isolated from soil, are illustrated and described as new to science in sections Trachyspermi and Talaromyces. The phylogenetic positions of the two new species inferred from the internal transcribed spacer, beta-tubulin, calmodulin and RNA polymerase II second largest subunit regions were carried out. Talaromyces heiheensis is phylogenetically closely related to T. albobiverticillius, T. rubrifaciens, T. solicola and T. erythromellis, and characterised by slow growth on Czapek yeast autolysate agar at 25 °C, orange conidia en masse on malt extract agar at 25 °C, biverticillate and terverticillate conidiophores, acerose phialides and subglobose to ellipsoidal, smooth-walled conidia. Talaromyces mangshanicus is related to T. kendrickii, T. qii and T. thailandensis, and characterised by slow-growing colonies with absent or sparse sporulation on CYA agar at 25 °C, conidia en masse greyish purple, purplish red soluble pigment on yeast extract agar (YES) at 25 °C, biverticillate conidiophores, ampulliform phialides and subglobose to ellipsoidal conidia with echinulate walls. They are distinguished from the known species in culture characteristics on four standard media, microscopic features and sequence data.     

A new source of resistance to 2-furaldehyde from <Emphasis Type="Italic">Scheffersomyces</Emphasis> (<Emphasis Type="Italic">Pichia</Emphasis>) <Emphasis Type="Italic">stipitis</Emphasis> for sustainable lignocellulose-to-biofuel conversion

Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuel production. Development of robust next-generation biocatalyst is a key for a low-cost biofuel production industry. Scheffersomyces (Pichia) stipitis is a naturally occurring C-5 sugar utilization yeast; however, little is known about the genetic background underlying its potential tolerance to biomass conversion inhibitors. We investigated and identified five uncharacterized putative aryl-alcohol dehydrogenase genes (SsAADs) from this yeast as a new source of resistance against biomass fermentation inhibitor 2-furaldehyde (furfural) by gene expression, gene cloning, and direct enzyme assay analysis using partially purified proteins. All five proteins from S. stipitis showed furfural reduction using cofactor NADH. An optimum active temperature was observed at 40 °C for SsAad1p; 30 °C for SsAad3p, SsAad4p, and SsAad5p; and 20 °C for SsAad2p. SsAad2p, SsAad3p, and SsAad4p showed tolerance to a wide range of pH from 4.5 to 8, but SsAad1p and SsAad5p were sensitive to pH changes beyond 7. Genes SsAAD2, SsAAD3, and SsAAD4 displayed significantly enhanced higher levels of expression in response to the challenge of furfural. Their encoding proteins also showed higher levels of specific activity toward furfural and were suggested as core functional enzymes contributing aldehyde resistance in S. stipitis.     

Optimized expression of prolyl aminopeptidase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its characteristics after glycosylation

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL^?1, an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L^?1.     

Decolorization of textile dye RB19 using volcanic rock matrix immobilized <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> cells with surface displayed laccase

A triplicate volcanic rock matrix–Bacillus thuringiensis–laccase WlacD (VRMs–Bt–WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn)₂ as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn)₂–WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L ₉(3⁴)-orthogonal test, Plackett–Burman test, steepest ascent method, and Box–Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L^?1, which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs–Bt–WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs–Bt–WlacD toward an initial concentration of 500 mg L^?1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g–100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs–Bt–WlacD and have the potential for large-scale or continuous operations.     

Surface Display of Bacterial Laccase CotA on <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells and its Application in Industrial Dye Decolorization

Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/10⁸ cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.     

Seasonal and altitudinal changes of culturable bacterial and yeast diversity in Alpine forest soils

The effect of altitude and season on abundance and diversity of the culturable heterotrophic bacterial and yeast community was examined at four forest sites in the Italian Alps along an altitude gradient (545–2000 m). Independently of altitude, bacteria isolated at 0 °C (psychrophiles) were less numerous than those recovered at 20 °C. In autumn, psychrophilic bacterial population increased with altitude. The 1194 bacterial strains were primarily affiliated with the classes Alpha-, Beta-, Gammaproteobacteria, Spingobacteriia and Flavobacteriia. Fifty-seven of 112 operational taxonomic units represented potential novel species. Strains isolated at 20 °C had a higher diversity and showed similarities in taxa composition and abundance, regardless of altitude or season, while strains isolated at 0 °C showed differences in community composition at lower and higher altitudes. In contrast to bacteria, yeast diversity was season-dependent: site- and altitude-specific effects on yeast diversity were only detected in spring. Isolation temperature affected the relative proportions of yeast genera. Isolations recovered 719 strains, belonging to the classes Dothideomycetes, Saccharomycetes, Tremellomycetes and Mycrobotryomycetes. The presence of few dominant bacterial OTUs and yeast species indicated a resilient microbial population that is not affected by season or altitude. Soil nutrient contents influenced significantly abundance and diversity of culturable bacteria, but not of culturable yeasts.     

Impact of Preservation Conditions on Fatty Acids,Xanthan Gum Production and Other Characteristics of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">mangiferaeindicae</Emphasis> IBSBF 2103

The conditions of storage, cultivation and maintenance of microbial cultures should preserve the microbiological homogeneity, phenotypic and genotypic characteristics to ensure better reproducibility of metabolic production. To evaluate the influence of the storage condition on the composition of cell fatty acids, genetic profile and biochemical characteristics of Xanthomonas campestris pv. mangiferaeindicae IBSBF 2103, as well as, to identify its relationship with the yielding and viscosity of the xanthan gum produced, this study monitored the strain preserved in two simple and widely used conditions, ultra-freezer (?80 °C) and refrigeration (3–8 °C) during 5 months. Were identified and quantified 13 fatty acids. The cells preserved at ?80 °C showed more stable concentration of all fatty acids, producing more xanthan gum and with higher viscosity. The chromosomal analysis obtained with the enzyme XbaI revealed 17 distinct fragments with maximum size of 485 kilobases, without variations among the subcultures maintained in both storage conditions. The X. campestris pv. mangiferaeindicae subcultures preserved at ?80 °C showed less pronounced phenotypic variations, which had positive influence in the qualitative and quantitative characteristics of the xanthan gum produced.     

<Emphasis Type="Italic">Haloquadratum walsbyi</Emphasis> Yields a Versatile,NAD<Superscript>+</Superscript>/NADP<Superscript>+</Superscript> Dual Affinity,Thermostable, Alcohol Dehydrogenase (<Emphasis Type="Italic">Hw</Emphasis>ADH)

This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.     

Functional Expression of a Thermostable Endoglucanase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> RCKK in <Emphasis Type="Italic">Pichia pastoris</Emphasis> X-33 and Its Characterization

Thermostable cellulases offer several advantages like higher rates of substrate hydrolysis, lowered risk of contamination, and increased flexibility with respect to process design. In the present study, a thermostable native endoglucanase nEG (EC 3.2.1.4) was purified and characterized from T. aurantiacus RCKK. Further, it was cloned in P. pastoris X-33 and processed for over expression. Expression of recombinant endoglucanase (rEG) of molecular size ~?33 kDa was confirmed by SDS-PAGE and western blotting followed by in gel activity determination by zymogram analysis. Similar to nEG, the purified rEG was characterized to harbor high thermostability while retaining 50% of its initial activity even after 6- and 10-h incubation at 80 and 70 °C, respectively, and exhibited considerable stability in pH range 3.0–7.0. CD spectroscopy revealed more than 20% β-sheets in protein structure consistently when incubated upto 85 °C as a speculated reason for protein high thermostability. Interestingly, both nEG and rEG were found tolerant up to 10% of the presence of 1-ethyl-3-methylimidazolium acetate [C2mim][OAc]. Values of the catalytic constants K_m and V_max for rEG were recorded as 2.5 mg/ml and 303.4 µmol/mg/min, respectively. Thermostability, pH stability, and resistance to the presence of ionic liquid signify the potential applicability of present enzyme in cellulose hydrolysis and enzymatic deinking of recycled paper pulp.     

A novel thermostable and organic solvent-tolerant lipase from <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> YB103: screening,purification and characterization

Thermostable lipases offer major biotechnological advantages over mesophilic lipases. In this study, an intracellular thermostable and organic solvent-tolerant lipase-producing strain YB103 was isolated from soil samples and identified taxonomically as Xanthomonas oryzae pv. oryzae. The lipase from X. oryzae pv. oryzae YB103 (LipXO) was purified 101.1-fold to homogeneity with a specific activity of 373.9 U/mg. The purified lipase showed excellent thermostability, exhibiting 51.1 % of its residual activity after incubation for 3 days at 70 °C. The enzyme showed optimal activity at 70 °C, suggesting it is a thermostable lipase. LipXO retained 75.1–154.1 % of its original activity after incubation in 20 % (v/v) hydrophobic organic solvents at 70 °C for 24 h. Furthermore, LipXO displayed excellent stereoselectivity (e.e._p >99 %) toward (S)-1-phenethyl alcohol in n-hexane. These unique properties of LipXO make it promising as a biocatalyst for industrial processes.     

Cloning,purification and characterization of novel Cu-containing nitrite reductase from the <Emphasis Type="Italic">Bacillus firmus</Emphasis> GY-49

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K _m and K _cat of NirK on sodium nitrite were 0.27 mM and 0.36?×?10³ s^?1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.     

Heterologous expression and characterisation of a laccase from <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis> and decolourisation of different synthetic dyes

Laccases have received considerable attention in recent decades because of their ability to oxidise a large spectrum of phenolic and non-phenolic organic substrates and highly recalcitrant environmental pollutants. In this research, a laccase gene from Colletotrichum lagenarium was chemically synthesised using yeast bias codons and expressed in Pichia pastoris. The molecular mass of the recombinant laccase was estimated to be 64.6 kDa by SDS–PAGE, and the enzyme exhibited maximum activity at pH 3.6–4.0 but more stability in buffer with higher pH (>pH 3.6). The optimal reaction temperature of the enzyme was 40 °C, beyond which stability significantly decreased. By using 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as a substrate, K _m and V _max values of 0.34 mM and 7.11 mM min^?1 mg^?1, respectively, were obtained. Using ABTS as a mediator, the laccase could oxidise hydroquinone to p-benzoquinone and decolourise the synthetic dyes malachite green, crystal violet and orange G. These results indicated that the laccase could be used to treat industrial effluents containing artificial dyes.