Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest ...due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Cellulose-pulping requires chemicals such as Cl
2
, ClO
2
, H
2
O
2
, and O
2
. The black liquor (BL) generated exhibits a high chemical oxygen demand (COD), five-day biochemical oxygen ...demand (BOD
5
), and chlorophenol content, along with an augmented colour and increased pH. BL is often discharged into water bodies, where it has a negative impact on the environment. Towards that end, laccases are of great interest for bioremediation, since they can degrade aromatic and non-aromatic compounds while reducing O
2
to water instead of H
2
O
2
. As such, we evaluated
Pleurotus ostreatus
and
Pichia pastoris
(which produces rPOXA 1B laccase) in the treatment of synthetic BL (SBL) in an “
in vitro
” modified Kraft process followed by CuO/TiO
2
/visible light photocatalysis. Treating SBL with
P. ostreatus
viable biomass (VB) followed by CuO/TiO
2
/visible light photocatalysis resulted in 80.3% COD removal and 70.6% decolourisation. Toxic compounds such as 2-methylphenol, 4-methylphenol, and 2-methoxyphenol were eliminated. Post-treated SBL exhibited low phytotoxicity, as evidenced by a
Lactuca sativa
L seed germination index (GI) > 50%. Likewise, SBL treatment with
P. pastoris
followed by VB/CuO/TiO
2
/
visible light photocatalysis resulted in 63.7% COD removal and 46% decolourisation. Moreover, this treatment resulted in the elimination of most unwanted compounds, with the exception of 4-chlorophenol. The
Lactuca sativa
L seed GI of the post-treated SBL was 40%, indicating moderate phytotoxicity.
Different genus of bacteria has been reported with the capacity to solubilize phosphorus from phosphate rock (PR). Pseudomonas sp., (A18) and Serratia sp., (C7) isolated from soils at the ...“Departamento de Boyacá” Colombia, where Allium cepa is cultivated. Bacteria were cultured in MT11B media and evaluated as a bio-fertilizer for A. cepa germination and growth during two months at greenhouse scale. Pseudomonas sp., and Serratia sp., cultured at 30 °C, 48 h in SMRS1 agar modified with PR, (as an inorganic source of phosphorus), presented a phosphate solubilization index (SI) of 2.1 ± 0.2 and 2.0 ± 0.3 mm, respectively. During interaction assays no inhibition halos were observed, demonstrating there was no antagonism between them. In MT11B media growth curve (12 h) demonstrated that co-culture can grow in the presence of PR and glucose concentrations 7.5-fold, lower than in SMRS1 media and brewer's yeast hydrolysate; producing phosphatase enzymes with a volumetric activity of 1.3 ± 0.03 PU at 6 h of culture and 0.8 ± 0.04 PU at 12 h. Moreover, co-culture released soluble phosphorus at a rate of 58.1 ± 0.28 mg L−1 at 8 h and 88.1 ± 0.32 mg L−1 at 12 h. After five days of evaluation it was observed that germination percentage was greater than 90 % of total evaluated seeds, when placing them in contact with the co-culture in a concentration of 1 × 108 CFU mL−1. Furthermore, it was demonstrated that co-culture application (10 mL per experimental unit to complete 160 mL in two months) at 8.0 Log10 CFU mL−1 twice a week for two months increased A. cepa total dry weight (69 ± 13 mg) compared with total dry weight (38 ± 5.0 mg) obtained with the control with water.
Biofertilizer; Phosphate rock; Pseudomonas sp.; Serratia sp.; Allium cepa; Crop production; Field crops; Organic farming; Bacteria; Microbial biotechnology, Biotechnology, Plant biology, Agriculture
Laccases catalyze the oxidation of various aromatic organic compounds concomitantly with molecular oxygen reduction to water. Triphenylmethane dyes are synthetic compounds widely used in diverse ...industries. Their removal from effluents is difficult, due to their high degree of structural complexity; hence, their high concentration in effluents cause a negative impact on the environment. In the present work, molecular docking was used to evaluate interactions between rGlLCC1 or rPOXA 1B enzymes with Crystal Violet (CV) or Malachite Green (MG) dyes. In addition, removal tests of the two dyes were performed. Van der Waals interactions were obtained for only the CV dye for both GlLCC1 and POXA 1B enzymes. Nevertheless, in the GlLCC1 model, two π-π interactions were observed. For the MG dye only, Van der Waals interactions were obtained. Moreover, amino acid composition interacting in each model with each dye was similar. It is important to highlight that by molecular docking, none of the estimated ligand configurations generated hydrogen bonds. Thus, explaining the difficulty to degrade CV and MG. Regarding CV, maximum decolorization percentage was 23.6 ± 1.0% using
Ganoderma lucidum
supernatant and 5.0 ± 0.5% with
Pleurotus ostreatus
supernatant. When using recombinant laccase enzyme concentrates, decolorization percentages were 9.9 ± 0.1 and 7.5 ± 1.0% for rGlLCC1 and rPOXA 1B, respectively. On the other hand, for the MG dye, maximum decolorization percentages were 52.1 ± 5.1 and 2.3 ± 0.2% using
G. lucidum
and
P. ostreatus
concentrates, respectively. Whereas with recombinant laccase enzymatic concentrates, values of 9.4 ± 0.8% were obtained, with rGlLCC1, and 2.1 ± 0.1% when using rPOXA 1B. These findings represent an important step in bioremediation processes improvement and efficiency of industry-generated products, using environmentally friendly alternatives.
Wastewater treatment plants produce solid and semi-solid sludge, which treatment minimises secondary environmental pollution because of wastewater treatment and obtaining new bioproducts. For this ...reason, in this paper, the co-pyrolysis of biogenic biomasses recovered from a biological reactor with immobilised fungal and bacterial biomass and a tertiary reactor with
Chlorella
sp. used for dye-contaminated wastewater treatment was carried out. Biogenic biomasses mixed with pine bark allowed the production and characterisation of two types of biochar. The raw material and biochar were on the “in vitro” germination of
Lolium
sp. seeds, followed by adsorption studies for malachite green (MG) dye using the raw material and the biochar. Results showed that using 60 mg L
−1
of a cationic coagulant at pH 6.5 allowed for the recovery of more than 90% of the microalgae after 50 min of processing. Two biochar resulted: BC
300
, at pH 5.08 ± 0.08 and BC
500
, at pH 6.78 ± 0.01. The raw material and both biochars were co-inoculated with growth-promoting bacteria; their viabilities ranged from 1.7 × 10
6
± 1.0 × 10
1
to 7.5 × 10
8
± 6.0 × 10
2
CFU g
−1
for total heterotrophic, nitrogen-fixing and phosphate-solubilising bacteria. Re-use tests on
Lolium
sp. seed germination showed that with the post-coagulation effluent, the germination was 100%, while with the biochar, with and without beneficial bacteria, the germination was 98 and 99%, respectively. Finally, BC
500
adsorbed the highest percentage of malachite green at pH 4.0, obtaining
q
ecal values of 0.5249 mg g
−1
(
R
2
: 0.9875) with the pseudo-second-order model.
During and after the pandemic caused by the SARS-CoV-2 virus, the use of personal care products and disinfectants increased in universities worldwide. Among these, quaternary ammonium-based products ...stand out; these compounds and their intermediates caused substantial changes in the chemical composition of the wastewater produced by these institutions. For this reason, improvements and environmentally sustainable biological alternatives were introduced in the existing treatment systems so that these institutions could continue their research and teaching activities. For this reason, the objective of this study was to develop an improved culture medium to cultivate ammonium oxidising bacteria (AOB) to increase the biomass and use them in the treatment of wastewater produced in a faculty of sciences in Bogotá, D.C., Colombia. A Plackett Burman Experimental Design (PBED) and growth curves served for oligotrophic culture medium, and production conditions improved for the AOB. Finally, these bacteria were used with total heterotrophic bacteria (THB) for wastewater treatment in a pilot plant. Modification of base ammonium broth and culture conditions (6607 mg L
−1
of (NH
4
)
2
SO
4
, 84 mg L
−1
CaCO
3
, 40 mg L
−1
MgSO
4
·7H
2
O, 40 mg L
−1
CaCl
2
·2H
2
O and 200 mg L
−1
KH
2
PO
4
, 10% (w/v) inoculum, no copper addition, pH 7.0 ± 0.2, 200 r.p.m., 30 days) favoured the growth of
Nitrosomonas europea
,
Nitrosococcus oceani
, and
Nitrosospira multiformis
with values of 8.23 ± 1.9, 7.56 ± 0.7 and 4.2 ± 0.4 Log
10
CFU mL
−1
, respectively. NO
2
−
production was 0.396 ± 0.0264, 0.247 ± 0.013 and 0.185 ± 0.003 mg L
−1
for
Nitrosomonas europea
,
Nitrosococcus oceani
and
Nitrosospira multiformis.
After the 5-day wastewater treatment (WW) by co-inoculating the three studied bacteria in the wastewater (with their self-microorganisms), the concentrations of AOB and THB were 5.92 and 9.3 Log
10
CFU mL
−1
, respectively. These values were related to the oxidative decrease of Chemical Oxygen Demand (COD), (39.5 mg L
−1
), Ammonium ion (NH
4
+
), (6.5 mg L
−1
) Nitrite (NO
2
−
), (2.0 mg L
−1
) and Nitrate (NO
3
−
), (1.5 mg L
−1
), respectively in the five days of treatment. It was concluded, with the improvement of a culture medium and production conditions for three AOB through biotechnological strategies at the laboratory scale, being a promising alternative to bio-augment of the biomass of the studied bacteria under controlled conditions that allow the aerobic removal of COD and nitrogen cycle intermediates present in the studied wastewater.
Phosphorus (P) is an essential nutrient for plant’s development, and its deficiency restricts crop yield. To meet P requirements in agricultural settings, a low-cost culture medium (MT11B) was ...designed in which a bioinoculant was produced consisting of three bacterial isolates capable of solubilizing P from phosphoric rock (PR). Pseudomonas sp., Serratia sp., and Kosakonia sp. exhibited P solubilization in SMRS1 agar modified with PR (5.0 g L-1), as source of inorganic P. Sowings by isolation were made of the three bacteria on DNAse- and Blood-agar to rule out pathogenicity. At the interaction tests, no inhibition halos were observed; demonstrating there was no antagonism among them, thus they were used to constitute a consortium. Growth curve (12 h) in MT11B demonstrated consortium grew in presence of PR, brewer’s yeast hydrolysate, and glucose at concentrations (2.5 g L-1) fourfold lower than those in SMRS1 (10.0 g L-1); obtaining phosphate solubilizing bacteria of (10.60 ± 0.08/ log10 CFUmL-1 and, at 6 h of culture, acid and alkaline phosphatase enzyme volumetric activities of 2.3 ± 0.8 and (3.80 ± 0.13) UP, respectively. The consortium, releasing phosphorus at a rate of (45.80 ± 5.17) mg L-1 at 6 h of production, was evaluated as bioinoculant in onion plots for five months. Plants receiving a treatment that included 500 mL (10 x 107 CFU mL-1) of bioinoculant plus 100 kg ha-1 of an organic mineral fertilizer exhibited the highest determined response variables (170.1 ± 22.2) mm bulb height, (49.4 ± 6.5) mm bulb diameter, (9.0 ± 1.8) g bulb dry weight, and 15.21 mg bulb-1 total phosphorus (p < 0.05).
AbstractGanoderma lucidum’s potential to decolorize and detoxify malachite green (MG) was studied. A Plackett-Burman experimental design to select operational factors and conditions favoring ...decolorization associated with laccase activity was performed. The most influential variables based on this statistical optimization were temperature, rpm for flask agitation, inoculum percentage, and dye concentration. Based on these features, removal experiments for 96 h were performed. Following thr 96-h removal assays, it was demonstrated that viable biomass at different glucose concentrations was able to remove over 90% MG at a 10 mg/L concentration, evidencing a positive correlation with laccase activity. Adsoprtion studies allowed to evidence acid pHs favored MG adsorption, with the Elovich model best describing the chimioadsorption phenomenon. Finally, it was demonstrated that MG (10 mgL−1) without treatment was highly toxic for Lactuca sativa seed germination index (GI) (<50%). This index increased when seeds were exposed to MG solution after it was treated with G. lucidum viable biomass (VB).
The original version of this article unfortunately contained a mistake. The replacement image of Fig. 4 provided by the first corresponding author, Aura M. Pedroza-Rodríguez, is incorrect and that ...the originally submitted Fig. 4 should have been retained. The original article has been corrected.
Fungal laccases Rivera-Hoyos, Claudia M.; Morales-Álvarez, Edwin David; Poutou-Piñales, Raúl A. ...
Fungal biology reviews,
December 2013, 2013-12-00, 20131201, Letnik:
27, Številka:
3-4
Journal Article
Recenzirano
Laccases are enzymes widely distributed in plants, fungi, bacteria, and insects. They are multicopper oxidases that catalyze the transformation of aromatic and non-aromatic compounds with reduction ...of molecular oxygen to water. These enzymes participate in processes such as biosynthesis and lignin degradation, morphogenesis, and pigment biosynthesis, among others. In this review we discuss relevant aspects of fungal laccases regarding the existence of fungal laccases gene families, the growing interest in investigating mechanisms of their molecular regulation, and factors that influence the production of laccases, due to their potential biotechnological applications. In addition we comparatively analyzed some structural similarities and differences depicting general features of laccases' active site, demonstrating their frequency as monomeric proteins with highly conserved cupredoxine type domains. Although inter- and intra-specific differences have been determined, structural differences encountered between fungal laccases remain unclear based on Crystallography and X-ray diffraction.
•Exist several lacasses genes as a consequence of physiological function diversity.•Crystallography or X-ray resolutions influence the structural differences encountered.•Not all recombinant lacasses combines attributes to promote production processes.•There are intra-specific differences, among laccases of the same fungus.•We discuss the diversity of fungal laccases with crystallography structures.
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