Green Biocatalyst Research Articles

Immobilized cells of Rhodococcus strain UCC 0004 as source of green biocatalyst for decolourization and biodegradation of methyl orange

Abstract The bulk presence of methyl orange is contributed by anthropogenic sources which poses substantial threat to health and the environment. Locally isolated Rhodococcus strains were used as alternatives to develop greener and cost effective methyl orange removal technology. Rhodococcus strain UCC 0004 in particular demonstrated complete removal of 0.5 g/L methyl orange after 2 days of incubation and this strain exhibited 513% higher methyl orange removal efficiency under static mode in comparison to agitated mode. Adopting the technology of immobilization enabled the decolourization of higher concentration of methyl orange which resulted in 66% greater methyl orange removal activity as opposed to the non-immobilized cells. Optimization of immobilization parameters at 0.4% (w/v) alginate concentration, 0.3 M CaCl2, 3 mm bead diameter and 50 beads led to improved methyl orange-decolourizing activity and reduced the time taken to achieve complete decolourization by 67%. The immobilized beads could be reused up to nine batches without significant loss of methyl orange-decolourizing ability which is advantageous economically. The immobilized cells demonstrated higher substrate affinity (Km = 0.3771 g/L) and accelerated rate of disappearance of methyl orange (Vmax = 0.05903 g/L/h) compared to free cells. No germination inhibition was observed on Triticum aestivum and Vigna radiata indicating complete removal of dye toxicity. The decolourization of methyl orange occurred through biodegradation as supported by complete disappearance of the major peak from UV–vis spectrum. Therefore, this strain has a huge potential to be developed as biological tool for efficient removal of wastewater containing methyl orange.

Utilization of one novel deep-sea microbial protease sin3406-1 in the preparation of ethyl (S)-3-hydroxybutyrate through kinetic resolution.

One novel protease sin3406-1 was identified from Streptomyces niveus SCSIO 3406, which was isolated from the deep sea of the South China Sea, and heterologously expressed in E. coli BL21(DE3). Protease sin3406-1 was further used as a green biocatalyst in the kinetic resolution of racemic ethyl-3-hydroxybutyrate. After careful process optimization, chiral product ethyl (S)-3-hydroxybutyrate was generated with an enantiomeric excess of over 99% and a conversion rate of up to 50% through direct hydrolysis of inexpensive racemic ethyl-3-hydroxybutyrate catalyzed by sin3406-1. Interestingly, protease sin3406-1 exhibited the same enantio-preference as that of esterase PHE21 during the asymmetric hydrolysis of the ester bonds of racemic ethyl-3-hydroxybutyrate. Through mutation studies and molecular docking, we also demonstrated that the four residues close to the catalytic center, S85, A86, Q87 and Y254, played key roles in both the hydrolytic activity and the enantioselectivity of protease sin3406-1, possibly through forming hydrogen bonds between the enzyme and the substrates. Deep-sea microbial proteases represented by sin3406-1 are new contributions to the biocatalyst library for the preparation of valuable chiral drug intermediates and chemicals through enzymatic kinetic resolution.

A sustainable and cleaner speedy tanning system based on condensed tannins catalyzed by laccase

Abstract Condensed tannins, a sustainable and environmental-friendly vegetable tanning agent, is confined in the wide application of leather industry owing to its poor shrinkage temperature (Ts). In this work, the tanning effect of condensed tannins was enhanced by the green biocatalyst laccase. Meanwhile, the molecular weight and chemical structure of condensed tannins catalyzed by laccase were observed. Its polydispersity index (PDI) was decreased from 2.88 to 1.77 and the Mw from 6354 to 4041. The changes of relative molecular mass were confirmed by Gel permeation chromatography (GPC) analysis. The UV–visible spectroscopy (UV–vis) and X-ray photoelectron spectroscopy (XPS) spectra provided evidence for the catalyzing change from o-hydroxylation to o-quinones. From the aspects of the thermal stability, tensile strength, breaking elongation, tear strength, thickening percentage and morphological analysis, the leather tanned by catalyzed condensed tannins (BA) was proved to have more excellent physical properties contrast with that of the leather tanned by untreated BA. This indicates that the physical properties of leather tanned by BA were improved significantly, whilst the catalyzed sustainable agent can be independently used as a promising quick tanning agent of sheepskin light leather.

Utilization of deep-sea microbial esterase PHE21 to generate chiral sec-butyl acetate through kinetic resolutions.

We previously identified and characterized 1 novel deep-sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)-3-hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)-sec-butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)-sec-butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)-sec-butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)-sec-butyl acetate reached 64% and 43%, respectively, after process optimization. Deep-sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols.

Optimization and up scaling of ionic liquid tolerant and thermo-alkali stable laccase from a marine Staphylococcus arlettae S1-20 using tea waste

Abstract Laccase are versatile oxidoreductase enzyme having a capability of degrading variety of phenolic and non phenolic compounds. Therefore, in this work cost effective process is developed for the higher production of extracellular, thermo-alkali stable laccase (S1-20LAC) from Staphylococcus arlettae S1-20 using tea waste. To enhance the enzyme yield, multiple sequential statistical tools i.e. RSM and ANN linked GA are employed which led to 16 fold enhancement in enzyme yield. Further, a pilot scale bioprocess is developed using optimum conditions shown by GA i.e., 2.50% tea waste, 4.95 mM NaCl, 37 °C Temperature and 5.65 mM L -DOPA which led to 72 fold (879 nkatml−1) improve in the enzyme yield . The optimum temperature and pH for S1-20LAC is 85 °C and 9.0. S1-20 retained significant amount of activity even in the presence of 20% (v/v) Ionic liquids (ILs). The detailed kinetic and thermodynamic studies revealed that the overall quality of laccase is spontaneous process at high temperature. The outcome of this study shows that high production and remarkable properties of S1-20LAC offers a significant opportunity for the degradation and synthesis of wide range of compounds, which makes it an attractive green biocatalyst for the future aspects.

Recent advances in enzyme immobilization

Enzyme is an efficient and green biocatalyst, and widely used in many areas. Immobilized enzyme is superior to its free form in a variety of properties. Enzyme immobilization studies started in the 1970s in China. Till now, immobilized enzymes are widely applied in the fields of food, medical, energy, environmental management, among others. However, there are still some defects such as no universal method and the high cost. Therefore, based on the relatively mature traditional immobilization technologies, efforts have been made to innovate immobilization technologies. As a result, many new immobilization technologies focusing on new carriers and methods are continuously generated. Coupling with more than ten years' study on enzyme immobilization, we present here recent development and application of new immobilization technologies, as well as suggestions to future development of immobilization technology.

Functional Characterization of a New Antarctic Microbial Esterase EST112-2 and Its Use in the Preparation of Chiral Tertiary Alcohol (S)-Linalool

Chiral tertiary alcohols (TAs) are key building blocks for the synthesis of many crucial flavor compounds and pharmaceuticals. The two enantiomers of tertiary alcohol, linalool, differ in odor. So, sustainable strategies for the manufacture of optically pure TAs represented by linalool, are highly desirable. But the enzymatic synthesis of chiral tertian' alcohols through kinetic resolution was not easily achieved, possibly because of the steric hindrance from the chemical structures of tertiary alcohols. Herein, we identified and functionally characterized a new microbial esterase EST112-2 from the antarctic sediments and utilized esterase EST112-2 as a green biocatalyst in the synthesis of chiral tertiary alcohol (S)-linalool through asymmetric hydrolysis of racemic linalyl acetate. Parameters such as pH, temperature, co-solvents, substrate concentrations, enzyme loading and reaction time were optimized for the kinetic resolutions. Desired chiral product (S)-linalool was finally obtained with an enantiomeric excess of over 66% and a yield of over 72% after process optimization. The enantiomeric excess of (S)-linalool prepared by esterase EST112-2 was much higher than that from previous reports.

Nanomagnetically modified vitamin B3 (Fe3O4@Niacin): An efficient and reusable green biocatalyst for microwave‐assisted rapid synthesis of 2‐amino‐3‐cyanopyridines in aqueous medium

Superparamagnetic nanoparticles of modified vitamin B3 (Fe3O4@Niacin) represent a new, efficient and green biocatalyst for the one‐pot synthesis of 2‐amino‐3‐cyanopyridine derivatives via four‐component condensation reaction between aldehydes, ketones, malononitrile, and ammonium acetate under microwave irradiation in water. This new magnetic organocatalyst was easily isolated from the reaction mixture by magnetic decantation using an external magnet and reused at least six times without significant degradation in the activity. The catalyst was fully characterized by FT‐IR, XRD, SEM, VSM, UV–Vis, DLS and EDS. Excellent yield, very short reaction time (7–10 min), operational simplicity, easy work‐up procedure, avoidance of hazardous or toxic catalysts and organic solvents are the main advantages of this green methodology which makes it more economic than the other conventional methods.

Keratinolytic protease: a green biocatalyst for leather industry.

Depilation/unhairing is the crucial but heavy pollution process in leather industry. Traditional inorganic sulfide treatment was the most widely used depilation technique in the past decades, which was usually detrimental to leather quality and resulted in serious environmental pollution. Using biocatalysts to substitute inorganic sulfide showed great advantages in environment protection and unhairing efficiency. Keratinolytic protease is one of the excellent biocatalysts to hydrolyze disulfide bond-rich proteins of hair and has little damage to leather. Biological treatment with keratinolytic proteases could largely reduce the quantity and toxicity of wastewater effluent from the leather industry. But low thermostability and substrate specificity or specific activity of these enzymes limited their practical application. Therefore, recent progresses on protein engineering strategies (site-directed mutagenesis, protein fusion, N/C-terminus truncation, and domain swapping) used to enhance the keratinolytic enzyme performance were presented.

Chitosan based metal-chelated copolymer nanoparticles: Laccase immobilization and phenol degradation studies

Immobilization improves the stability and reusability of enzymes. In this study, laccase, which is a green biocatalyst, was immobilized onto Cu (II)-chelated chitosan nanoparticles via adsorption and successfully applied to remove phenol from aqueous solution. Cu (II)-chelated chitosan-graft-poly (glycidyl methacrylate) nanoparticles were prepared using poly (ethylene imine), PEI, which is employed as both a spacer arm and metal chelator and used to immobilize laccase by coordination. Properties of chitosan based nanoparticles were characterized using FTIR, TGA, SEM and zeta-sizer analysis. The maximum laccase loading capacity of Cu (II)-chelated chitosan based nanoparticles (CHT-PGMA-PEI-Cu (II) NPs) was calculated as 65.75 ± 2.51 mg/g. This immobilized system exhibited broad pH and temperature profiles, and better stability and reusability than free enzyme; after eight cycles of continuous use, the activity of the immobilized enzyme remained above 50 ± 0.62%. The Km and Vmax values of free and immobilized laccase were 0.055 mM, 0.070 mM, and 0.19 U/mg, 0.14 U/mg, respectively. The catalytic efficiencies (Vmax/KM) of the free and immobilized laccase were 3.45 and 2.0, respectively. Feasibility of the laccase immobilized system in the removal of phenol was investigated in a batch system. The results showed that the CHT-PGMA-PEI-Cu (II) NPs have great potential for industrial applications. More than 96% of phenol was removed with laccase immobilized metal chelated NPs in the presence of mediator, ABTS, indicating that the metal-chelated chitosan based NPs is a promising support for both laccase immobilization and further applications in the removal of phenolic compounds.

Characterization of a novel deep-sea microbial esterase EstC10 and its use in the generation of (R)-methyl2-chloropropionate

A novel esterase EstC10 from Bacillus sp. CX01 isolated from the deep sea of the Western Pacific Ocean and the functionalities of EstC10 was characterized. At present, the reports about the kinetic resolution of racemic methyl 2-chloropropionate were quite rare. So we developed deep-sea microbial esterase EstC10 as a novel biocatalyst in the kinetic resolution of racemic methyl 2-chloropropionate and generate (R)-methyl 2-chloropropionate with high enantiomeric excess (>99%) after the optimization of process parameters such as pH, temperature, organic co-solvents, surfactants, substrate concentration and reaction time. Notably, the optimal substrate concentration (80 mmol/L) of esterase EstC10 was higher than the kinetic resolution of another esterase, Est12-7 (50 mmol/L). The novel microbial esterase EstC10 identified from the deep sea was a promising green biocatalyst in the generation of (R)-methyl 2-chloropropionate as well of many other valuable chiral chemicals in industry.

Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris

Background: Laccases are copper-containing enzymes which have been used as green biocatalysts for many industrial processes. Although bacterial laccases have high stabilities which facilitate their application under harsh conditions, their activities and production yields are usually very low. In this work, we attempt to use a combinatorial strategy, including site-directed mutagenesis, codon and cultivation optimization, for improving the productivity of a thermo-alkali stable bacterial laccase in Pichia pastoris . Results: A D500G mutant of Bacillus licheniformis LS04 laccase, which was constructed by site-directed mutagenesis, demonstrated 2.1-fold higher activity when expressed in P. pastoris . The D500G variant retained similar catalytic characteristics to the wild-type laccase, and could efficiently decolorize synthetic dyes at alkaline conditions. Various cultivation factors such as medium components, pH and temperature were investigated for their effects on laccase expression. After cultivation optimization, a laccase activity of 347 ± 7 U/L was finally achieved for D500G after 3 days of induction, which was about 9.3 times higher than that of wild-type enzyme. The protein yield under the optimized conditions was about 59 mg/L for D500G. Conclusions: The productivity of the thermo-alkali stable laccase from B. licheniformis expressed in P. pastoris was significantly improved through the combination of site-directed mutagenesis and optimization of the cultivation process. The mutant enzyme retains good stability under high temperature and alkaline conditions, and is a good candidate for industrial application in dye decolorization. Normal 0 false false false EN-US ZH-CN X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.5pt; mso-bidi-font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-font-kerning:1.0pt; mso-fareast-language:ZH-CN;}

Baker’s yeast: An efficient, green, and reusable biocatalyst for the one-pot synthesis of biologically important N-substituted decahydroacridine-1,8-dione derivatives

ABSTRACTA green approach for one-pot three-component synthesis of N-substituted decahydroacridine-1,8-diones is offered for the first time using baker’s yeast (Saccharomyces cerevisiae) as a biocatalyst under ultrasonication. Due to growing safety and environmental concerns, enzymatic methods were constantly investigated as an attractive alternative to toxic and nonspecific chemical approaches. This method is relatively simple, efficient, inexpensive, and environment-friendly. The catalyst was recovered and reused and also the recyclability of baker’s yeast resulted in excellent yields of products without loss of any catalytic activity.

Characterization of a Highly Thermostable and Organic Solvent-Tolerant Copper-Containing Polyphenol Oxidase with Dye-Decolorizing Ability from Kurthia huakuii LAM0618T.

Laccases are green biocatalysts that possess attractive advantages for the treatment of resistant environmental pollutants and dye effluents. A putative laccase-like gene, laclK, encoding a protein of 29.3 kDa and belonging to the Cu-oxidase_4 superfamily, was cloned and overexpressed in Escherichia coli. The purified recombinant protein LaclK (LaclK) was able to oxidize typical laccase substrates such as 2,6-dimethoxyphenol and l-dopamine. The characteristic adsorption maximums of typical laccases at 330 nm and 610 nm were not detected for LaclK. Cu2+ was essential for substrate oxidation, but the ratio of copper atoms/molecule of LaclK was determined to only be 1:1. Notably, the optimal temperature of LaclK was 85°C with 2,6-dimethoxyphenol as substrates, and the half-life approximately 3 days at 80°C. Furthermore, 10% (v/v) organic solvents (methanol, ethanol, isopropyl alcohol, butyl alcohol, Triton x-100 or dimethyl sulfoxide) could promote enzymatic activity. LaclK exhibited wide-spectrum decolorization ability towards triphenylmethane dyes, azo dyes and aromatic dyes, decolorizing 92% and 94% of Victoria Blue B (25 μM) and Ethyl Violet (25 μM), respectively, at a concentration of 60 U/L after 1 h of incubation at 60°C. Overall, we characterized a novel thermostable and organic solvent-tolerant copper-containing polyphenol oxidase possessing dye-decolorizing ability. These unusual properties make LaclK an alternative for industrial applications, particularly processes that require high-temperature conditions.

Characterization of a novel marine microbial esterase and its use to make D-methyl lactate

A novel marine microbial esterase PHE14 was cloned from the genome of Pseudomonas oryzihabitans HUP022 isolated from the deep sea of the western Pacific Ocean. Esterase PHE14 exhibited very good tolerance to most organic solvents, surfactants and metal ions tested, thus making it a good esterase candidate for organic synthesis that requires an organic solvent, surfactants or metal ions. Esterase PHE14 was utilized as a biocatalyst in the asymmetric synthesis of D-methyl lactate by enzymatic kinetic resolution. D-methyl lactate is a key chiral chemical. Contrary to some previous reports, the addition of an organic solvent and surfactants in the enzymatic reaction did not have a beneficial effect on the kinetic resolution catalyzed by esterase PHE14. Our study is the first report on the preparation of the enantiomerically enriched product D-methyl lactate by enzymatic kinetic resolution. The desired enantiomerically enriched product D-methyl lactate was obtained with a high enantiomeric excess of 99% and yield of 88.7% after process optimization. The deep sea microbial esterase PHE14 is a green biocatalyst with very good potential in asymmetric synthesis in industry and can replace the traditional organic synthesis that causes pollution to the environment.

Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization

Laccase has been emerged as a valuable green biocatalyst in cleaner production, which requires vast amount of laccase at low production cost and with good properties. The current work evaluated a wild strain Pycnoporus sp. SYBC-L3 as an efficient microbial cell factory for laccase production. High-cell density cultivation (HCDC) of L3 was developed in a 5-L bioreactor by adjusting the ratio of inoculum to medium, achieving over 4-fold of both laccase activity (240 U/mL) and fungal biomass (20 g/L) than those obtained under routine procedures. Laccase productivity was further assessed at pilot-scale in a 500-L and a 5-ton bioreactor, where the highest activity of around 60 U/mL and 80 U/mL were produced, respectively. Cost percentage analysis for the 5-ton reactor demonstrated that labor was the highest portion (50%) in total cost for industrial production in this case. Using a one-step purification scheme, laccase was purified to homogeneity from the culture broth with yield of 44% and 97% purity. Circular dichroism (CD) analyses suggested that the secondary structure of laccase was mainly composed of β-sheet that played a vital role in maintaining laccase activity, and also revealed the potential mechanisms for the changes in laccase activities under varying conditions. Powdered laccase was efficiently prepared from cell-free culture broth using spray drying technique with above 74% activity retained. This study provides important information for an efficient and scalable production of laccase.

Co-immobilization of laccase and mediator through a self-initiated one-pot process for enhanced conversion of malachite green

Laccase is a green biocatalyst. It works with molecular oxygen and produces water as the only by-product. However, its practical application is far less than satisfactory due to the low stability/poor reusability of free laccase and the potential secondary pollution caused by dissolved mediators. To address those bottlenecks in laccase-based catalysis, a novel biocatalyst (Immo-LMS) was fabricated by simultaneously immobilizing both laccase and a mediator (acetylacetone, abbreviated as AA) into a hydrogel through the laccase-AA initiated polymerization. This self-initiated immobilization process avoided the forced conformational change of laccase in the passive embedding to pre-existing carriers. Resulting from the effective cooperation of laccase and AA, the Immo-LMS had the highest substrate conversion quantity to malachite green, followed by the sole immobilized laccase and the immobilized laccase with an external mediator. Besides the improved activity, the Immo-LMS showed enhanced stability. The good performance of the Immo-LMS suggests that the co-immobilization of laccase and mediator through the self-initiated one-pot process was a promising strategy for the immobilization of laccase, which is expected to be helpful to cut down the running cost as well as the potential toxicity that come from mediators in the practical application of laccase.

The green metric evaluation and synthesis of diesel-blend compounds from biomass derived levulinic acid in supercritical carbon dioxide

Levulinate compounds are the biomass derived important energy products which are primarily used as diesel-blending components. These levulinates are the oxygenated fuel additive which promote complete combustion of fuel and reduces emission of pollutants like particulate matters and nitrogen oxides. In the present study, we have investigated sustainable synthesis of biomass derived levulinate compounds catalyzed by Cal B lipase in supercritical carbon dioxide (SC-CO2) as a green biocatalyst in green reaction media which provided an excellent mass yield. Various reaction parameters were optimized in details such as butanol, levulinic acid, biocatalyst, co-solvent, temperature and pressure to obtain 99% mass yield of the desired product. Moreover various green metric parameters (e.g. E-factor, carbon efficiency and mass productivity) of present methodology were evaluated. The green metrics evaluation suggested that developed procedure is a promising and a greener alternative as compared to various reported conventional synthetic protocols. The biocatalyst was efficiently recycled up to the five cycles. Moreover, the developed protocol can be used for synthesis of various industrially important levulinate compounds which also provided excellent mass yield. Thus, the present protocol demonstrated (i) robust biocatalytic application for transformation of renewable biomass derived levulinic acid (LVA) into value added chemicals and (ii) various green metric evaluation study for the sustainability.

Monascus kaoliang CBS 302.78 immobilized in tailor-made agars as catalyst for reduction of ketones: On the quest for a green biocatalyst

After a hierarchical screening Monascus kaoliang CBS 302.78 was selected due to its high activity in the reduction of cyclic ketones. Several tailor made agars and agaroses were used in the immobilization of the fungus. The best-immobilized biocatalyst was obtained after immobilization in low methoxylated agar (A27/03) at 2.5% (w/v). The biocatalyst shape was optimized as microplates (1.27 cm × 1.27 cm × 0.31 cm). The observable modulus for external mass transfer, Ω ⋘ 1, indicates the absence of external mass transfer phenomena. The internal effectiveness factor η < 1 and the high value of the Thiele module ( ϕ > 3), indicates that there is an intra-particle mass transfer restriction according to the Weisz criteria. As consequence, the kinetics of the reduction of ketones follows a formal first order kinetic model. The catalysts deactivation follows a simple exponential decay expression. Due to the renewal characteristics of the agar, the biocatalysts can be considered as green catalysts.