Skip to main content
NCBI home page
3 Biotech logoLink to 3 Biotech
. 2020 Jun 3;10(6):288. doi: 10.1007/s13205-020-02277-3

Hyperstabilization of a thermophile bacterial laccase and its application for industrial dyes degradation

Julián E Gianolini 1,2, Claudia N Britos 1, Carlos B Mulreedy 1, Jorge A Trelles 1,2,✉
PMCID: PMC7270286  PMID: 32550107

Abstract

In the present study, a novel extracellular laccase isolated from Geobacillus stearothermophilus ATCC 10149 was entrapped in a bionanocomposite matrix consisting of copper alginate (Cu-alginate) supplemented with the nanoclay bentonite. After optimization, this nanobiocatalyst was able to degrade up to 90% of Remazol Brilliant Blue R (RBBR) without the addition of redox mediators and retained 70% of its initial activity for at least 1440 h, equivalent to more than 288 uses. The incorporation of nanoclay allowed alginate beads to be used in alkaline pH and strengthened its mechanical properties. Besides, this thermophilic laccase was able to decolorize other structurally different synthetic dyes such as Methyl Orange, Malachite Green and Indigo Carmine. These preliminary results suggested that the nanobiocatalyst could be a suitable option for dye decolorization and be further developed for large scale bioremediation of toxic dyes.

Keywords: Biodegradation, Geobacillus, Nanoclay, Bentonite, Cu-alginate, Immobilization

Introduction

It is estimated that about 10–15% of dye used in textile industry is discharged into the aqueous ecosystem due to inadequacies during the dyeing process and inappropriate liberation of the effluents (Singh et al. 2017; Maniyam et al. 2018). Since synthetic dyes have complex aromatic structures, they are stable against light, oxidizing agents, pH and thermal fluctuations, ionic strength changes and metabolism of ubiquitous microbes, so they persist in the environment and become recalcitrant molecules (Vatandoostarani et al. 2018). Furthermore, dyes have proven to be toxic to the aquatic biota and some of them have been reported to be carcinogenic and mutagenic agents (Akpor 2018).

Owing to the toxicological risk and environmental damage that dyes can present, their removal from industrial wastewater has been studied through the application of several physico-chemical methods like adsorption, coagulation, sedimentation and chemical oxidation. However, the use of physical methods in wastewater treatment implies high costs and the implementation of these methods, especially adsorbents, generates large amounts of sludge. This requires adequate disposal, which is defiant since the adsorbents are still ingrained with dyes and stay toxic, leaving the problem without a solution (Akpor 2018; Maniyam et al. 2018). Biological treatment methods are more effective due to their high efficiency, cost effectiveness, environmental sustainability and flexibility (Xie et al. 2018). There are two main enzymatic groups involved in degradation of organic pollutants: oxidoreductases and hydrolases. Laccases (EC 1.10.3.2) are multicopper oxidases that lack cofactors and catalyze the oxidation of a broad range of chemicals by reducing oxygen to water (Karigar and Rao 2011). Although these enzymes are found in many organisms, the most studied are fungal laccases. These laccases have some weaknesses, they are acid-stable, when the majority of wastewater is alkaline, rarely stable at high temperatures and also need weeks to be produced (Cardoso et al. 2018). Conversely, bacterial laccases are usually more stable at high pH and temperatures and their production process can take just a few hours (Britos et al. 2018).

Laccases are used for varied purposes in food, paper and textile industries, pharmaceuticals, chemical synthesis, biofuel production and bioremediation (Ma et al. 2018). However, the free enzyme used in those applications has presented several disadvantages, such as low stability and non-reusability, making the treatment more expensive. Thus, immobilization procedures are required to enable biocatalyst recovery and reusability, and provide higher tolerance to extreme conditions (Salami et al. 2018). Currently, there are diverse immobilization techniques applied to biocatalyst development. Among them, gel entrapment is one of the most employed in industry (Trelles and Rivero 2013). Alginate is a natural polymer hydrogel used as a carrier matrix to encapsulate molecules of biological significance, and the addition of nanocomposites can enhance its stability and mechanical properties (De Benedetti et al. 2015). Nanoclays are natural, abundant and low cost particles whose obtention implies simple processes. They have already been used in non-covalent immobilization of several enzymes such lipase, lysozyme, catalase and β-glucosidase (An et al. 2015), which allowed the biocatalyst to be recovered and resulted in the increment of its stability in a wider spectrum of pH and temperature (Serefoglou et al. 2008). Besides, it has been demonstrated that the addition of nanoclay to a natural matrix in order to stabilize a whole cell biocatalyst led to physical improvements in swelling behavior, compressive strength and fracture frequency, apart from storage stability and reusability (De Benedetti et al. 2015; Cappa et al. 2016).

In this communication, a nanobiocatalyst based on the novel Geobacillus stearothermophilus ATCC 10149 thermophile extracellular laccase was developed. The isolated laccase was immobilized in an alginate-bentonite bionanocomposite, efficiently oxidized Remazol Brilliant Blue R (RBBR) and proved remarkable stability for more than 1440 h, confirming its potential industrial application.

Materials and methods

Screening for laccase activity

Twenty bacterial strains from our laboratory collection, previously acquired from American Type Culture Collection (ATCC) or Colección Española de Cultivos Tipo (CECT) and identified as laccase producer organisms in earlier stages with the 2,2′-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid] (ABTS) reaction as described (Britos and Trelles 2016), were screened for oxidation of Remazol Brilliant Blue R. Among them, G. stearothermophilus was chosen for further exploration. Bacterial cultures were grown at optimum conditions until saturation, centrifuged at 2100 g, 4 °C for 15 min and stored at 4 °C until use. Biomass determination was performed at 600 nm and Enzyme Units of laccase were defined by the ABTS method. Briefly, one Enzyme Unit (IU) was defined as the quantity of enzyme that catalyzes the oxidation of 1 µmol of ABTS per minute. G. stearothermophilus was grown at 55 °C and 200 rpm until late exponential phase in Luria Bertani medium (10 g/L meat peptone, 5 g/L yeast extract and 5 g/L NaCl).

Synthetic dye decolorization by laccase

Crude extracellular laccase preparation (0.003 IU) was used to decolorize 10 μM Malachite Green (triarylmethane), 60 μM Indigo Carmine (indigoid), 100 μM RBBR (anthraquinone) and 50 μM Methyl Orange (azo). Reactions were conducted with 1 mL of crude extract or immobilized laccase in 2.5 mL of a 0.1 M CuSO4, 0.1 M acetate buffer pH 4.8 solution. The decolorization processes were performed at 30 °C and 200 rpm and withdrawn to measure the reduction of each dye absorbance at its maximum wavelength (Britos et al. 2018). Controls in which laccase was replaced by distilled water or a clean matrix were conducted in parallel.

Geobacillus sp. laccase purification

Three different purification and concentration methods were assayed. Firstly, cold crude extracellular laccase preparations were precipitated with ice-cold acetone or ethanol 30% (v/v), incubated 16 h at 4 °C, centrifuged at 2100 g, 4 °C for 20 min and dried. Secondly, ammonium sulfate precipitation was tested at the intervals of 0–35% and 35–70% of saturation using the standard table and protocol reported (Wingfield 2016). Then, each precipitated fraction was centrifuged and filtered with a 10 kDa membrane. All precipitation samples were resuspended in 0.1 M acetate buffer pH 4.8 for further examination. The third method involved the use of strategies to remove water from the laccase solution. According to this strategy, two options were assayed. Laccase supernatant was lyophilized and resuspended in distilled water or incubated for 16 h in a centrifugal vacuum concentrator (SpeedVac®), clarified once more by centrifugation and stored at 4 °C until use.

Laccase immobilization

Geobacillus laccase (0.005 IU) was immobilized by entrapment in three different types of gel, such as agarose 4% (w/v), polyacrylamide 18% (w/v) and alginate 8% (w/v) as Trelles and Rivero (2020) described. Protein leakage was evaluated by the adapted Bradford method (Britos and Trelles 2016). For bionanocomposites, the laccase extract was mixed with 1 mL of alginate-bentonite, 8% and 0.1% (w/v) respectively. The mixture was then added dropwise to stirred 0.1 M CuSO4 and incubated for 50 min. Alginate gel beads were filtered and washed with distilled water and kept at 4 °C in 5 mM CuSO4 and 0.1 M acetate buffer pH 4.8 solution until use. Controls and biocatalyst beads were incubated at reaction conditions for the first time before decolorization.

Nanobiocatalyst characterization

Swelling ratio, sphericity factor, fracture frequency, compressive strength and reusability parameters were used to characterize the nanobiocatalyst, as previously described (Cappa et al. 2014). Reusability was assayed through successive RBBR decolorization reactions from the second reaction. Each reuse was performed for 5 h in optimized conditions. Compressive strength of beads was measured using Universal Testing Machine TC-500 II series (MegaTest).

Temperature and pH incidence in the activity of the immobilized laccase was studied varying temperature 25–60 °C and pH 4.2–9, pH higher than 7 was achieved with 0.1 M Tris–HCl buffer. Effect of aeration was studied by insufflating the reaction with an air pump. Each condition was performed with its corresponding control.

Results and discussion

Selection of microorganism

Twenty strains were tested for RBBR degradation, a model dye commonly used in textile industry for polymeric dye production that belongs to the often recalcitrant and toxic pollutants class (Sing et al. 2017). Among several genera studied, including Aeromonas and Streptomyces, Geobacillus sp. cell-free supernatant was selected for further exploration caused by its remarkable RBBR decolorization yield and potential industrial applications owing to thermostable enzymes produced by these bacteria. Another genera such as Pseudomonas, Serratia, Flavobacterium and Arthrobacter also recognized the dye but led to minor results, and strains which gave decolorization yields below to 24% were not considered (Table 1). Then, extracellular laccase production at different stages of Geobacillus growth was analyzed using RBBR decolorization (Fig. 1). The maximum activity (3 IU/L) was achieved after 5 h (stationary phase), suggesting its constitutive production (Allison and Vitousek 2005). At death stage, similar activity was observed, but by-products derived from cell lysis might interfere with future uses. This stage was necessary for microorganism selection and characterization but this biocatalyst, as free enzyme, is not feasible for industrial purposes due to the costs associated to their production, non-reusability and environmental sensitivity. Consequently, immobilization was required.

Table 1.

Screening of microorganisms for RBBR degradation

Genera Evaluated strains RBBR degradation (%)a
Geobacillus 3 +++
Aeromonas 2 +++
Streptomyces 3 +++
Pseudomonas 2 ++
Flavobacterium 2 ++
Serratia 1 ++
Arthrobacter 2 ++
Thermonospora 2 +
Lactobacillus 2 +
Bacillus 1 +
Open in a new tab

The decolorization process was carried out with cell-free supernatant for 3 days and the maximum yield was recorded

a(+) RBBR degradation lower than 24%, (++) between 25 and 49% and (+++) higher than 50%

Fig. 1.

Fig. 1

Open in a new tab

Geobacillus growth curve (filled square) and RBBR decolorization (grey bars). Optical Density (OD) was measured at 600 nm in a Shimadzu 1603 spectrophotometer; dye decolorization was followed with a ELx800 plate reader at 595 nm

Laccase purification

Several methods were implemented to purify the laccase (Table 2). In this work, either ethanol or acetone precipitation had less than 8% laccase activity recovery. Organic solvents not only interact with the protein surface making them precipitate but also with the inner hydrophobic interactions, causing destabilization of the native structure and in some cases irreversible denaturation (McPherson and Gavira 2014). Although ammonium sulfate precipitation led to 14% laccase recovery, similar than reported (Asgher and Iqbal 2011), concentration methods like centrifugal vacuum and lyophilization with a previous freeze-defreeze precipitation led to 100% and 70% laccase recovery respectively, the later had the highest purification fold and admitted up to five times greater concentration factor so it was selected for further experiments.

Table 2.

Purification methods implemented in laccase recovery

Specific activity (IU/g protein)a Purification foldb Yield (%)c
Ethanol 30% (v/v) 0 0.0 0
Acetone 30% (v/v) 21 0.6 8
Ammonium sulphate 35% 0 0.0 0
Ammonium sulphate 70% 42 1.3 14
SpeedVac® 112 3.5 100
Lyophilization 165 5.2 70
Open in a new tab

Enzyme units (IU) were measured by ABTS oxidation method and protein content by the adapted Coomassie method (Britos and Trelles 2016)

aSpecific activity consists in total amount of active enzyme per mass of protein

bPurification fold shows the relation between the specific activity reached after the purification method and the initial specific activity

cPurification yield indicates the total amount of active enzyme remaining after the process

Laccase immobilization

Three methods to immobilize laccase were assayed. In each evaluated case, protein leakage was < 5% and laccase activity was not detected in immobilization and wash solutions, so the total amount of enzyme was loaded into each matrix (0.007 IU/g). Due to bentonite and alginate adsorption capacity, controls without enzyme were necessary. In all evaluated cases, decolorization caused by adsorption was not significant. Other authors mentioned that unmodified bentonite was not useful for RBBR decolorization (Chinoune et al. 2016). Probably by cause of the denaturing effect of acrylamide or to high reticulation degree in agarose matrix, which hindered substrate diffusion (De Benedetti et al. 2012), alginate biocatalyst was the only active after laccase immobilization, achieving 11 ± 3% of RBBR decolorization after 5 h. Clays are cheap, sustainable and resistant to microbial degradation and are obtained by simple processes. Moreover, it has previously been demonstrated that the addition of nanosized clay improves mechanical stability of gel beads (Cappa et al. 2016), so 0.1% w/v bentonite was added to stabilize and evaluate alginate matrix (Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

Schematic representation of nanobiocatalyst production. The laccase secreted during G. stearothermophilus growth was concentrated and purified. Then, the laccase solution was mixed with alginate and bentonite and entrapped by ionotropic gelation in 0.1 M CuSO4. The obtained beads were used for RBBR decolorization in a bioreactor

Alginate polymerization is driven by interactions between gel carboxylate groups and divalent cations (Ramírez-Tapias et al. 2017). Laccase is a copper containing enzyme and the incorporation of Cu ions not only increases the binding efficiency of the enzyme but also the immobilized activity (Sondhi et al. 2018). Thus, CuSO4 cross-linking solution was evaluated at 0.1 M and 0.2 M, at higher concentrations it was evinced that the biocatalyst reaction rates decreased (Britos et al. 2018). As predicted, no activity difference was observed at 5 h reaction, but operational stability was enhanced by 47%. This effect was already explained (Liu et al. 2016), high concentration of polymerizing ion causes a greater degree of cross-linking and prevents enzyme leaching during processes. As oxygen is the only co-substrate for laccases, the next step was to supply air to the reaction with an air pump. As a result, RBBR decolorization yield increased from 11 to 46% in 5 h. Then, to avoid protein denaturing (Britos et al. 2018), the alginate matrix concentration was incremented from 8 to 10% w/v. Consequently, RBBR decolorization was incremented to 71% in 5 h and reached 90% in 22 h.

The performance of the nanobiocatalyst to decolorize RBBR was evaluated in a wide range of pH (4.2–9) and temperature (25–60 °C). Both free and immobilized laccase exhibited the highest activity at 42 °C (data not shown) and their behaviour across different temperatures did not show significant differences. Nanoclay allowed the biocatalyst to be used above neutral pH (Fig. 3), where alginate matrix usually disintegrates (Phetsom et al. 2009). Free and immobilized laccase had optimal pH at 6, similar than other reported Geobacillus strains (Rai et al. 2019). Besides, free laccase deactivated at neutral and alkaline pH, while the nanobiocatalyst kept its relative activity above 80% in all the evaluated range of pH, which implies a significant stability improvement especially in alkaline conditions. This is the reason why the pH-thermal-resistant developed nanobiocatalyst represents a promising asset, since wastewaters are characterized by high temperatures and variable pH, which is predominantly alkaline (Imran et al. 2015).

Fig. 3.

Fig. 3

Open in a new tab

Relative laccase activity at different pH of free (open circle) and immobilized laccase (filled square). Activity was evaluated by RBBR decolorization at 5 h

In the following experiments, stability was studied at pH 8. The nanostabilized biocatalyst did not show activity loss due to a decrease in diffusion of the matrix as previously demonstrated (Cappa and Trelles 2017). The addition of bentonite to the alginate matrix improved five times the operational stability. The obtained nanobiocatalyst remained stable for more than 1440 h, which is equivalent to 288 reuses (Fig. 4) and 57 fold greater than previous reports (Le et al. 2016; Ma et al. 2018) to decolorize RBBR. The nanobiocatalyst evidenced a remarkable increase in terms of reusability and maintained significant activity for RBBR decolorization in a wider spectrum of pH than the free laccase. The increment of the stability of the nanobiocatalyst may be explained by a synergic stabilization of the enzyme between alginate entrapment and bentonite interaction (Table 3).

Fig. 4.

Fig. 4

Open in a new tab

Operational stability of the Cu-alginate biocatalyst (filled square) and the bentonite-nanobiocatalyst (open circle). Each use was evaluated by RBBR decolorization at 5 h and 25 °C

Table 3.

Optimization of nanobiocatalyst

Step Variable Condition Operational stability (h) RBBR decolorization yield at 5 h (%)
1 Matrix Polyacrylamide 18% (w/v) – 0
Agarose 4% (w/v) – 0
Alginate 8% (w/v) – 11
2 Concentration of ionotropic solution 0.1 M 257 11
0.2 M 377 11
3 Aeration No aeration – 11
Aeration – 46
4 Concentration of matrix 8% (w/v) – 46
10% (w/v) > 1440 71
Open in a new tab

Moreover, this nanobiocatalyst achieved similar decolorization yields than other authors who used free bacterial laccases (Liu et al. 2015), without the use of mediators yet five times swifter. This means that the cost-effectiveness relation of the developed nanobiocatalyst is notably better and advantageous for its industrial applications, summed up to its stability in a broad range of pH and temperature, which points out its suitability to be used as a strong catalyst for dye treatment.

To the best of our knowledge, the developed bionanocomposite represents the first bacterial nanostabilized biocatalyst used for RBBR oxidation with high decolorization yields and superlative stability. For that, we evaluated mechanical properties and appraised suitability of stabilized nanobiocatalyst for industrial scale-up. Sphericity factor varies from one to zero to define an elongated or a perfect sphere, respectively. Spherical and regular-shaped beads were obtained by using ionotropic gelation with an average diameter of 3 mm and sphericity factor of 0.04 ± 0.01, similar values were reported by other authors who used nanoclay alginate composites (De Benedetti et al. 2015; Cappa et al. 2014).

Fracture frequency serves as a fast approach to evaluate mechanical resistance of gel beads, where values close to 100% imply highly susceptible beads to mechanical forces and values close to 0% indicate resistant beads. In this work, the fracture frequency was not affected and stayed at zero, which indicates that the nanobiocatalyst successfully beared developed reaction conditions. These results were contrary than expected since the inclusion of nanocompounds to a polymeric matrix causes its hardening and therefore higher fragility. However, results of nule fracture frequency were coherent with those obtained by compression. Compression modulus of the beads (570 ± 20 kPa) was notably higher than previous reports (Cappa et al. 2016). The remarkable strength of beads can be explained by the high matrix concentration plus the addition of bentonite (De Benedetti et al. 2015). It was shown that the sum of ionic and dipole interactions between –OH and –COO− of alginate and O–Si–O and –OH of clay layers increment the physical stability of the nanobiocomposite (Da Silva et al. 2018).

The mayor cause of alginate polycation-based matrix rupture is osmotic swelling (Cappa et al. 2016). Conversely, the beads lost 17% of their initial mass and reached an equilibrium state in the first 24 h. During ionotropic polymerization process, the solubility of alginate decreases as a result of the solubility difference between Na+ ion replaced by Cu2+. As a result, internal water is loss to the bulk solution in a process called syneresis (Patel et al. 2017).

Finally, the biocatalyst not only recognized and degraded anthraquinone dye RBBR but also showed promising activity towards another emblematic structurally different types of dye, like azo (Methyl Orange, 44% in 120 h), triarylmethane (Malachite Green, 33% in 93 h) and indigoid (Indigo Carmine, 41% in 28 h), which indicates the great suitability of this laccase for dye treatment.

Conclusion

Laccase-assisted dye degradation is an environmental friendly remediation method where enzyme immobilization helps enhancing enzyme stability and reducing the cost of industrial processes. In this study, a novel thermophilic laccase oxidized four structurally different synthetic dyes and in its nanostabilized form proved its dye treatment potential by degrading RBBR with an extraordinary reuse increment, which drastically decreases biocatalyst production costs. Additionally, the inclusion of bentonite into the composite not only achieved substantial improvements in terms of reusability but also conferred mechanical properties which facilitate the scaling-up of processes and hence the increment of the efficiency of them. Thus, this novel bionanocomposite is a promising alternative for future industrial applications in effective dye degradation.

Acknowledgements

This research was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2013-2658 and PICT 2014-3438), Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2014-KA5-00805) and Universidad Nacional de Quilmes (PUNQ 1309/19). We are also grateful to Juan F. Delgado for his kind support in the compression tests.

Author contributions

JG, CB and JT conceived and designed the study; CM contributed in experiment design and analysis; JG performed the experiments; JG and JT wrote the paper. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

References

  1. Akpor OB. Effect of nitrogen source concentration on decolouration rates of laboratory dyes by immobilized cells of two bacterial species. World J Microbiol Biotechnol. 2018 doi: 10.1007/s11274-017-2402-7. [DOI] [PubMed] [Google Scholar]
  2. Allison SD, Vitousek PM. Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biol Biochem. 2005;37:937–944. doi: 10.1016/j.soilbio.2004.09.014. [DOI] [Google Scholar]
  3. An N, Zhou CH, Zhuang XY, et al. Immobilization of enzymes on clay minerals for biocatalysts and biosensors. Appl Clay Sci. 2015;114:283–296. doi: 10.1016/j.clay.2015.05.029. [DOI] [Google Scholar]
  4. Asgher M, Iqbal HMN. Characterization of a novel manganese peroxidase purified from solid state culture of Trametes versicolor IBL-04. BioResources. 2011;6:4317–4330. doi: 10.15376/biores.6.4.4317-4330. [DOI] [Google Scholar]
  5. Britos CN, Trelles JA. Development of strong enzymatic biocatalysts for dye decolorization. Biocatal Agric Biotechnol. 2016;7:228–233. doi: 10.1016/j.bcab.2016.06.009. [DOI] [Google Scholar]
  6. Britos CN, Gianolini JE, Portillo H, Trelles JA. Biodegradation of industrial dyes by a solvent, metal and surfactant-stable extracellular bacterial laccase. Biocatal Agric Biotechnol. 2018;14:221–227. doi: 10.1016/j.bcab.2018.03.015. [DOI] [Google Scholar]
  7. Cappa VA, Trelles JA. One-pot biosynthesis of idoxuridine using nanostabilized lactic acid bacteria. Process Biochem. 2017;62:169–173. doi: 10.1016/j.procbio.2017.07.028. [DOI] [Google Scholar]
  8. Cappa VA, Rivero CW, Britos CN, et al. An efficient biocatalytic system for floxuridine biosynthesis based on Lactobacillus animalis ATCC 35046 immobilized in Sr-alginate. Process Biochem. 2014;49:1169–1175. doi: 10.1016/j.procbio.2014.03.029. [DOI] [Google Scholar]
  9. Cappa VA, Rivero CW, Sambeth JE, Trelles JA. Bioproduction of floxuridine using nanostabilized biocatalysts. Chem Eng Technol. 2016;39:1723–1730. doi: 10.1002/ceat.201500522. [DOI] [Google Scholar]
  10. Cardoso BK, Linde GA, Colauto NB, do Valle JS. Panus strigellus laccase decolorizes anthraquinone, azo, and triphenylmethane dyes. Biocatal Agric Biotechnol. 2018;16:558–563. doi: 10.1016/j.bcab.2018.09.026. [DOI] [Google Scholar]
  11. Chinoune K, Bentaleb K, Bouberka Z, et al. Adsorption of reactive dyes from aqueous solution by dirty bentonite. Appl Clay Sci. 2016;123:64–75. doi: 10.1016/j.clay.2016.01.006. [DOI] [Google Scholar]
  12. Da Silva FR, de Moura MR, Glenn GM, Aouada FA. Thermal, microstructural, and spectroscopic analysis of Ca2+ alginate/clay nanocomposite hydrogel beads. J Mol Liq. 2018;265:327–336. doi: 10.1016/j.molliq.2018.06.005. [DOI] [Google Scholar]
  13. De Benedetti EC, Rivero CW, Britos CN, et al. Biotransformation of 2,6-diaminopurine nucleosides by immobilized Geobacillus stearothermophilus. Biotechnol Prog. 2012;28:1251–1256. doi: 10.1002/btpr.1602. [DOI] [PubMed] [Google Scholar]
  14. De Benedetti EC, Rivero CW, Trelles JA. Development of a nanostabilized biocatalyst using an extremophilic microorganism for ribavirin biosynthesis. J Mol Catal B Enzym. 2015;121:90–95. doi: 10.1016/j.molcatb.2015.08.006. [DOI] [Google Scholar]
  15. Imran M, Crowley DE, Khalid A, et al. Microbial biotechnology for decolorization of textile wastewaters. Rev Environ Sci Biotechnol. 2015;14:73–92. doi: 10.1007/s11157-014-9344-4. [DOI] [Google Scholar]
  16. Karigar CS, Rao SS. Role of microbial enzymes in the bioremediation of pollutants: a review. Enzym Res. 2011 doi: 10.4061/2011/805187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Le TT, Murugesan K, Lee CS, et al. Degradation of synthetic pollutants in real wastewater using laccase encapsulated in core-shell magnetic copper alginate beads. Bioresour Technol. 2016;216:203–210. doi: 10.1016/j.biortech.2016.05.077. [DOI] [PubMed] [Google Scholar]
  18. Liu H, Cheng Y, Du B, et al. Overexpression of a novel thermostable and chloride-tolerant laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and its application in synthetic dye decolorization. PLoS ONE. 2015 doi: 10.1371/journal.pone.0119833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu S, Li H, Tang B, et al. Scaling law and microstructure of alginate hydrogel. Carbohydr Polym. 2016;135:101–109. doi: 10.1016/j.carbpol.2015.08.086. [DOI] [PubMed] [Google Scholar]
  20. Ma HF, Meng G, Cui BK, et al. Chitosan crosslinked with genipin as supporting matrix for biodegradation of synthetic dyes: laccase immobilization and characterization. Chem Eng Res Des. 2018;132:664–676. doi: 10.1016/j.cherd.2018.02.008. [DOI] [Google Scholar]
  21. Maniyam MN, Yaacob NS, Azman HH, et al. Immobilized cells of Rhodococcus strain UCC 0004 as source of green biocatalyst for decolourization and biodegradation of methyl orange. Biocatal Agric Biotechnol. 2018;16:569–578. doi: 10.1016/j.bcab.2018.10.008. [DOI] [Google Scholar]
  22. McPherson A, Gavira JA. Introduction to protein crystallization. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2014;70:2–20. doi: 10.1107/S2053230X13033141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Patel MA, AbouGhaly MHH, Schryer-Praga JV, Chadwick K. The effect of ionotropic gelation residence time on alginate cross-linking and properties. Carbohydr Polym. 2017;155:362–371. doi: 10.1016/j.carbpol.2016.08.095. [DOI] [PubMed] [Google Scholar]
  24. Phetsom J, Khammuang S, Suwannawong P, Sarnthima R. Copper-alginate encapsulation of crude laccase from Lentinus polychorus lev. and their effectiveness in synthetic dyes decolorizations.pdf. J Biol Sci. 2009;9:573–583. doi: 10.3923/jbs.2009.573.583. [DOI] [Google Scholar]
  25. Rai R, Bibra M, Chadha BS, Sani RK. Enhanced hydrolysis of lignocellulosic biomass with doping of a highly thermostable recombinant laccase. Int J Biol Macromol. 2019;137:232–237. doi: 10.1016/j.ijbiomac.2019.06.221. [DOI] [PubMed] [Google Scholar]
  26. Ramírez-Tapias YA, Lapasset Laumann AS, Britos CN, et al. Saccharification of citrus wastes by immobilized polygalacturonase in an improved alginate matrix. 3 Biotech. 2017;7:3–8. doi: 10.1007/s13205-017-1010-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salami F, Habibi Z, Yousefi M, Mohammadi M. Covalent immobilization of laccase by one pot three component reaction and its application in the decolorization of textile dyes. Int J Biol Macromol. 2018;120:144–151. doi: 10.1016/j.ijbiomac.2018.08.077. [DOI] [PubMed] [Google Scholar]
  28. Serefoglou E, Litina K, Gournis D, et al. Smectite clays as solid supports for immobilization of β-glucosidase: synthesis, characterization, and biochemical properties. Chem Mater. 2008;20:4106–4115. doi: 10.1021/cm800486u. [DOI] [Google Scholar]
  29. Sing NN, Husaini A, Zulkharnain A, Roslan HA. Decolourisation capabilities of ligninolytic enzymes produced by Marasmius cladophyllus UMAS MS8 on Remazol Brilliant Blue R and other azo dyes. Biomed Res Int. 2017;2017:1–8. doi: 10.1155/2017/1325754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Singh K, Kumar P, Srivastava R. An overview of textile dyes and their removal techniques: Indian perspective. Pollut Res. 2017;36:790–797. [Google Scholar]
  31. Sondhi S, Kaur R, Kaur S, Kaur PS. Immobilization of laccase-ABTS system for the development of a continuous flow packed bed bioreactor for decolorization of textile effluent. Int J Biol Macromol. 2018;117:1093–1100. doi: 10.1016/j.ijbiomac.2018.06.007. [DOI] [PubMed] [Google Scholar]
  32. Trelles JA, Rivero CW. Whole cell entrapment techniques. In: Guisan JM, editor. Immobilization of enzymes and cells methods in molecular biology (methods and protocols) New York: Humana Press; 2013. pp. 365–374. [DOI] [PubMed] [Google Scholar]
  33. Trelles JA, Rivero CW. Whole cell entrapment techniques. In: Guisan J, Bolivar J, López-Gallego F, Rocha-Martín J, editors. Immobilization of enzymes and cells. Methods in molecular biology. New York: Humana Press; 2020. pp. 385–394. [Google Scholar]
  34. Vatandoostarani S, Lotfabad TB, Heidarinasab A, et al. Integrated system of multiple batches to evaluate the continuous performance of microbial cells in decolourization processes. J Environ Chem Eng. 2018;6:728–735. doi: 10.1016/j.jece.2017.12.073. [DOI] [Google Scholar]
  35. Wingfield PT. Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci. 2016;2016:A.3F.1–A.3F.9. doi: 10.1002/0471140864.psa03fs84. [DOI] [PubMed] [Google Scholar]
  36. Xie X, Liu N, Yang F, et al. Comparative study of antiestrogenic activity of two dyes after Fenton oxidation and biological degradation. Ecotoxicol Environ Saf. 2018;164:416–424. doi: 10.1016/J.ECOENV.2018.08.012. [DOI] [PubMed] [Google Scholar]

Articles from 3 Biotech are provided here courtesy of Springer

RESOURCES