Linked Enzyme Aggregates Research Articles

Food processing applications of protease activity identified in the methotrexate degrading enzyme of Variovorax paradoxus

A 46 kDa dimeric methotrexate (MTX) degrading enzyme was reported from Variovorax paradoxus. This enzyme also showed caseinolytic protease activity. The optimum pH (5.5), and temperature (35 ℃) of protease activity were different from those of MTX degrading activity of the enzyme. Cross linked enzyme aggregates (CLEAs) of the purified soluble protease showed higher activity, higher thermostability, and lower Michaelis–Menten constant (Km) value on casein compared to the soluble enzyme. The soluble enzyme showed casein hydrolysis capacity in the zymogram reactions. The CLEAs could lead to faster milk clotting, lower bread hardness, shorten dough forming time, and increased dough and bread volumes compared to the soluble enzyme, which could be due to the higher affinity of CLEAs to the proteins over soluble enzyme. Majority of the panellists given ‘excellent’ in the five-point scale starting from ‘not satisfactory’ to ‘excellent’ to the CLEAs treated cheese, and bread samples compared to the soluble enzyme treated samples with respect to flavor, texture, and taste. These results suggest that CLEAs are advantageous over soluble enzyme during food processing applications.

Preparation, characterization and stability of cross linked nitrilase aggregates (nitrilase-CLEAs) for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid.

Nitrilases capable of performing hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid were screened, and ES-NIT-102 was the best nitrilase for said biotransformation. Nitrilase was immobilized as cross linked enzyme aggregates (nitrilase-CLEAs) by fractional precipitation with iso-propanol, and cross linked with glutaraldehyde. The nitrilase-CLEAs prepared with optimized 35mM glutaraldehyde for 120min cross linking time had 82.36 ± 4.45% residual activity, and displayed type-II structural CLEAs formation as confirmed by particle size, SEM, FTIR, and SDS-PAGE analysis. Nitrilase-CLEAs had superior pH and temperature stability, showed a shift in optimal temperature by 5°C, and retained nearly 1.5 to 1.7 folds activity over free nitrilase at 50°C and 55°C after more than 9h incubation. Nitrilase-CLEAs showed reduced affinity and decreased conversion of substrate as indicated by slightly higher Km values by 5.19% and reduced Vmax by 17%. Furthermore, these nitrilase-CLEAs showed 98% conversion, 94.72g/L product formation, and 83.30% recovery after 24h when used for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid. Nitrilase-CLEAs were catalytically active for 3 cycles showcasing 81% conversion, 75.53g/L product formation and 66.42% yield. The recovered product was confirmed by HPLC, FTIR, LC-MS, and 1H NMR, and displayed > 99% purity.

Preparation of enantiopure pregabalin intermediate using cross linked enzyme aggregates (CLEAs) in basket reactor

Route to the synthesis of enantiomerically pure ethyl (S)-3-cyano-5-methylhexanoate, (S)-5, a key chiral intermediate for Pregabalin has been improved. The racemic β-cyano diester, 3 was prepared in 98% purity via gelatine catalysed Knoevenagel condensation of diethylmalonate with isovaleraldehyde followed by hydrocyantion of α,β-unsaturated diester 14 using acetone cyanohydrin and K2CO3. Racemic diethyl 2-(1-cyano-3-methylbutyl)malonate, rac-3, has been resolved using lipase from Thermomyces lanuginosus immobilised in form of crosslinked enzyme aggregates, CLEAs. The CLEAs were made by employing commercial soymilk as an additional protein source and a reaction was carried out in a moving basket reactor. The immobilised enzyme was found to be stable in many organic solvents and temperature up to 50 °C. The resolution reaction was studied in a basket reactor at 50% substrate loading in calcium acetate buffer, pH 7.5 at 30 °C by using 20% w/w enzyme loading. The apparent kinetic parameters were V max,app = (8.74 ± 0.43) mM/h/g and K m,app = (1.5 ± 0.07) M (correlation coeff. r = 0.98). The desired ethyl (S)-3-cyano-5-methylhexanoate, (S)-5 is obtained in 90-92% theoretical yield and e.e > 99%. The advantages of this improved process are mild reaction conditions; an alternate method for hydrocyanation step avoiding the use of highly toxic potassium cyanide at large scale operation and an immobilised enzyme that can be reused for at least 11 recycles.

Magnetic combi CLEA for inhibition of bacterial biofilm: A green approach

In the present study different enzymes (α- amylase, trypsin, cellulase, horse-radish peroxidase and pectinex ultra clear) were studied for bacterial biofilm inhibition and Pectinex ultra clear showed best inhibition. So, m-combi-CLEA of Pectinex ultra clear was developed by cross linked enzyme aggregate (CLEA) formation on APTES (3-aminopropyltriethoxysilane) modified iron oxide nanoparticles. Different parameters were optimized and it was observed that 0.4 mg/ml of protein (containing 25 U/mg cellulase activity), 0.5 mg/ml BSA and 10 mM glutaraldehyde when incubated for 3 h gives 100% enzyme activity using ethanol as the precipitant. The CLEA formed were thermally more stable as compared to free enzyme. m-combi-CLEA of Pectinex ultra clear shows 75–78% biofilm inhibition of E. coli and S. aureus. Furthermore, m-combi-CLEA can be reused till 4 cycles with same efficiency. The carbohydrate contents of E. coli biofilm decreased from 64.629 μg to 6.23 μg and for S. aureus biofilm, it decreased from 58.46 μg to 5.52 μg when treated with m-combi CLEA in comparison to untreated biofilms. FTIR, darkfield illumination Fluorescence Microscopy, and Scanning Electron Microscopy was further used for characterization.

A porous-cross linked enzyme aggregates of maltogenic amylase from Bacillus lehensis G1: Robust biocatalyst with improved stability and substrate diffusion

Enzymatic synthesis of maltooligosaccharides is hampered due to lack of stability of soluble enzyme. This limitation can be tackled by cross linked enzyme aggregates (CLEAs) immobilization approach. However, substrate diffusion is a major bottleneck in cross linking technology. Herein, CLEAs of maltogenic amylase from Bacillus lehensis G1 (Mag1) was developed with addition of porous agent (Mag1-p-CLEAs). Comparison of thermal, pH and kinetic analysis with CLEAs without porous agent (Mag1-CLEAs) and free Mag1 was performed. Mag1-p-CLEAs with porous structure prepared at 0.8% (w/v) of citrus pectin (porous agent), 0.25% (w/v) of chitosan (cross linker) and cross linked for 1.5 h yielded 91.20% activity. 80% of activity is retained after 30 min of incubation at 40 °C and showed longer half-life than free Mag1 and Mag1-CLEAs. Mag1-p-CLEAs also showed pH stability at acidic and alkaline pH. The 1.68-fold increase in Vmax value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin accessibility. The increase in high catalytic efficiency (Kcat/Km) value, 1.90-fold and 1.05-fold showed that it also has better catalytic efficiency than free Mag1 and Mag1-CLEAs, respectively. Mag1-p-CLEAs not only improved substrate diffusibility of CLEAs, but also leads to higher thermal and pH stability of Mag1.

Construction of CLEAs-lipase on magnetic graphene oxide nanocomposite: An efficient nanobiocatalyst for biodiesel production

In the present work, cross linked enzyme aggregates of Lipase (CLEAs-lip) was synthesized and immobilized on the magnetic amino functionalized graphene oxide (maGO-CLEAs-lip) nanocomposites. The immobilized lipase showed a broad range of temperature activity about 40–60 °C, as compared to free enzyme. In the case of maGO-CLEAs-lip nanocomposite, the observed lower Km value state 2.25 folds affinity for the p-nitrophenyl palmitate. Enzyme activity of maGO-CLEAs-lip nanocomposite was the highest up to 5 cycles. Storage stability results displayed maGO-CLEAs-lip retained about 75% of its original activity after 30 days of incubation. Remarkably, maGO-CLEAs-lip formed the highest biodiesel construction (78%) from R. communisoil after 24 h of incubation. The biodiesel yield of this nanocomposite was 3.0 folds higher than free enzyme, making it talented as an excellent nanobiocatalyst for efficient production of biodiesel.

Synthesis and characterization of cross linked enzyme aggregates of serine hydroxyl methyltransferase from Idiomerina leihiensis

A thermo-stable purified serine hydroxymethyltransferase (SHMT; 418 AA) was used for the carrier free immobilization using pectin as a coach molecule and formaldehyde as a cross-linker. The purified protein was cross linked with formaldehyde in the presence of pectin to form stable and active aggregates. The cross-linked enzyme aggregates [CLEAs] of SHMT showed improved catalytic properties and reusability. The SHMT-CLEAs showed a noteworthy change in the thermo-stability and activity compared to its free counterpart. The optimum activity for free SHMT was reported at 55 °C and pH 7.5 which SHMT CLEAs showed maximum activity at 60 °C and pH 8.0. Similarly, the CLEAs were noticed to increase the thermo-stability in comparison to free enzyme. The divalent salt ion Ca2+ and Ba2+ were found to enhance the activity at 1 and 5 mM of concentrations while Ni+, Co2+ and Zn2+ strongly inhibited the activity of both free as well as CLEAs. The Vmax and km values for free SHMT were recorded to be 1.21 μM s−1 and 272 μM while for CLEAs Vmax 1.42 μM s−1 and km 248.6 μM was recorded. Thus, a 120% increase in the Vmax was recorded for SHMT-CLEAs. The CLEAs were also found to be more stable at pH 6.5 and 8.5 pHs and retained 50% of its original activity for 180 and 200 min respectively. The CLEAs also retained 72% of its activity after 12 repetitive cycles of d-phenylserine hydrolysis. Also, the synthesized CLEAs retained more than 60% of its original activity after 10 days of incubation at 25 °C in comparison to free enzyme which loses more than 90% of its residual activity. Thus, with improved thermostability and activity the CLEAs of SHMT can be used repetitively at industrial scale for the synthesis of commercially important amino acids.

Development and characterization of cross-linked enzyme aggregates of thermotolerant alkaline protease from Bacillus licheniformis

An alkaline protease was produced by B. licheniformis with 132.43±3.4U/mL activity in LSF which was further enhanced by optimizing culture conditions. The optimum enzyme activity (148.9±4.1U/mL) was harvested at pH7.5; temperature, 40°C and inoculum, 1.5mL after 48h incubation. Alkaline protease was immobilization by forming cross linked enzyme aggregates (CLEAs) and the processes of CLEAs formation was also optimized. The protease CLEAs developed using 80% ammonium sulfate, 65mM glutaraldehyde and 0.11mM BSA showed best activity recovery (39.76%). Free protease and CLEAs were characterized and compared. It was observed that CLEAs of protease exhibited broad pH range with best activity at pH10. The immobilized protease was also thermo-tolerant with optimum activity at 65°C temperature. The Vmax and Km of protease-CLEAs were 125.5U/mL and 18.97μM, respectively as compared to 104.9U/mL and 29μM, respectively for free protease. It was concluded that immobilized enzyme in the form of CLEAs is a valuable catalyst for potential industrial applications.

Cocoa pod husk: A new source of CLEA-lipase for preparation of low-cost biodiesel: An optimized process

Enzymatic reactions involving lipases as catalyst in transesterification can be an excellent alternative to produce environmental-friendly biodiesel. In this study, lipase extracted from Cocoa Pod Husk (CPH) and immobilized through cross linked enzyme aggregate (CLEA) technology catalysed the transesterification of Jatropha curcas oil successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of 3% (w/w) enzyme loading, 4h reaction time and 1:6 oil/ethanol ratio to achieve the highest conversion of free fatty acid and glycerides into biodiesel (93%). The reusability of CLEA-lipase was tested and after seven cycles, the conversion percentage reduced to 58%. The results revealed that CLEA lipase from CPH is a potential catalyst for biodiesel production.

Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase

This work illustrates the preparation of magnetic macromolecular glucoamylase CLEAs using dialdehydic pectin, as a cross linker instead of traditional glutaraldehyde. The effect of precipitators type and amount, cross linker concentration, cross linking time and amount of amino functionalized magnetic nanoparticles (AFMNs) on glucoamylase activity was studied. Glucoamylase magnetic macromolecular CLEAs prepared by precipitation in presence of AFMNs by ammonium sulfate were subsequently cross linked by dialdehydic pectin. After cross-linked by pectin, 95.4% activity recovery was achieved in magnetic macromolecular CLEAs, whereas in case of glutaraldehyde cross linker, 85.3% activity recovery was achieved. Magnetic macromolecular CLEAs showed 2.91 and 1.27 folds higher thermal stability as compared to free and magnetic glutaraldehyde CLEAs. In kinetics study, magnetic macromolecular CLEAs retained same Km values, whereas magnetic glutaraldehyde CLEAs showed higher Km value than free enzyme. The porous structure of magnetic macromolecular CLEAs was not only enhanced mass transfer toward macromolecular substrates, but also showed compression resistance for 5 consecutive cycles which was checked in terms of effectiveness factor. At the end, in reusability study; magnetic macromolecular CLEAs were retained 84% activity after 10th cycle without leaching of enzyme which is 22% higher than traditional magnetic CLEAs.

Synthesis of the antitumoral nucleoside capecitabine through a chemo-enzymatic approach

Capecitabine is a 5′-deoxynucleoside endowed with antitumoral activity. We planned a new approach to its synthesis: a cross linked enzyme aggregate subtilisin (Alcalase CLEA)-catalyzed alcoholysis allowed the selective deprotection of primary acetyl ester of the N1-(2′,3′,5′-tri-O-acetyl-β-d-ribofuranosyl)-5-fluoro-N4-(n-pentyloxycarbonyl)cytosine affording the corresponding 5′-hydroxyderivative; the 5′-alcohol was transformed into the methyl group of capecitabine after a careful investigation about the best reducing agent and reaction conditions.

Solid state fermentation with recovery of Amyloglucosidase from extract by direct immobilization in cross linked enzyme aggregate for starch hydrolysis

Abstract Main limitations in application of Amyloglucosidase (AMG), an important industrial enzyme for starch hydrolysis, areits high production cost and limited reusability. Immobilization techniques have been tried to address the issue of reusability, however, these techniques either increase the cost of enzyme o rare difficult to scale up owing to special requirements for synthesis of carriers. We tried to address the issue of production cost and reusability for enzyme application by forming carrier free cross-linked enzyme aggregate (CLEA) of AMG from crude SSF extract of Aspergillus niger . CLEA recovered 65 percent activity from extract and retained 90% even after 25 cycles of reuse. CLEA showed improved affinity toward substrate; higher storage, thermal and pH stability. Use of cheap SSF and direct immobilization of AMG from extract can reduce the cost of starch hydrolysis.

Strategic optimization of xylanase–mannanase combi-CLEAs for synergistic and efficient hydrolysis of complex lignocellulosic substrates

Cost-effective application of lignocellulolytic enzymes holds the key towards commercialization of enzymatic hydrolysis of lignocellulosic biomass. Carrier free immobilization of enzyme(s) offers a lucrative prospect. Combined-cross linked enzyme aggregates (combi-CLEAs) are a novel prospective and this present study addresses the preparation, characterization and application of xylanase–mannanase combi-CLEAS on lime-preteated sugarcane bagasse and milled corn stover. X6-CLEAs, X7-CLEAs, L1-CLEAs and L7-CLEAs were prepared after elaborative optimization of the precipitating agent and glutaraldehyde concentration. The highest activity after precipitation was observed with acetone but following cross-linking with glutaraldehyde less than 60% activity was retained, while more than 60% activity was retained after precipitation with ammonium sulphate and cross-linking with glutaraldehyde. Accessory enzyme activities including α-arabinofuranosidase, β-xylosidase, esterases, β-mannosidase, α-galactosidase and β-glucosidase were also determined. More than an 1.5 fold increase in thermostability compared to the free enzyme was observed over a broad temperature range (50–70°C). Tri-synergy studies and quad synergy studies were used to generate combi-CLEAs with different protein ratios. Hydrolysis of lime pre-treated bagasse with combi-CLEAs at protein ratios corresponding to X6 (33.0%):X7 (17.0%):L1 (17.0%):L7 (33.0%) resulted in a 1.68 fold higher sugar release compared to the quad synergy model using free enzymes. Similarly, hydrolysis of corn stover with combi-CLEAs at protein ratios corresponding to X6 (40.0%):X7 (10.0%):L1 (10.0%):L7 (40.0%) resulted in an 1.58 fold higher sugar release compared to the sugar release observed with the quad synergy model using free enzymes. Monomeric sugars constituted 70–75% of reducing sugars released during hydrolysis. The role of accessory enzymes in improving enzyme synergy was clearly shown. The efficiency of combi-CLEAs compared to free enzymes makes them ideal candidates for the prudent and cost-effective commercialization of lignocellulolytic enzymes.

Preparation of stable cross-linked enzyme aggregates (CLEAs) of NADH-dependent nitrate reductase and its use for silver nanoparticle synthesis from silver nitrate

The NADH-dependent nitrate reductase from Fusarium oxysporum cell extract was directly immobilized as cross linked enzyme aggregates (CLEAs) and investigated for the synthesis of silver nanoparticles by a reduction of silver nitrate. The effects of precipitant type and cross-linking on activity recovery of enzyme in CLEAs were studied. After aggregation of enzyme with ammonium sulfate followed by cross-linking formed aggregates for 4h with 8mM glutaraldehyde, 93% activity recovery was achieved in CLEAs with enhanced thermal stability at 50°C and 40°C. Scanning electron microscopy analysis showed that immobilized NADH-dependent nitrate reductase was of spherical structure. CLEAs showed 90% catalytic yield even after 4cycles of repeated use in silver nanoparticle synthesis at pH7.2 and temperature 35°C.

Magnetic cross-linked laccase aggregates — Bioremediation tool for decolorization of distinct classes of recalcitrant dyes

The increasing use of laccase in waste water industries is useful to explore the high benefit/cost ratio of insolubilization technologies like cross linked enzyme aggregates (CLEAs) for the decolorization and detoxification of distinctive classes of recalcitrant dyes. Amino-functionalized magnetic nanoparticles bonded to CLEAs increased the potential of laccase-based CLEAs and are applicable for commercial implementation of this technology in environmental applications. The activity recovery obtained from the stable rigid structure of magnetic CLEAs was around 32%. High volumetric activity, increased in thermal and operational stability of laccase and its resistance to extreme conditions were the properties provided by these magnetic CLEAs. Kinetic studies show that the catalytic efficiency of the enzyme, based on the kcat/km value, changed significantly upon CLEAs and magnetic CLEA formations. When 0.2U/mL of magnetic CLEAs was used, the biocatalyst rapidly decolorized 61–96% of remazol brilliant blue R, malachite green and reactive black 5 initially at 50mgL−1 at 20°C and pH7.0. Investigation of dye degradation using both active and heat denatured CLEAs revealed a slight adsorption of dyes on inactivated biocatalysts. A laboratory scale perfusion basket reactor (BR) was used to study the continuous decolorization of dyes. The efficient decolorization (>90%) of remazol brilliant blue R and slight decrease in CLEA activity were measured over a 10h period of continuous operation, which illustrates the potential of CLEAs for the wastewater treatment. The present findings will advance the understanding of dye decolorization mechanism by CLEA laccase, which could provide useful references for developing industrial wastewater treatment.

Baeyer–Villiger Oxidation of Cyclohexanone in Aqueous Medium with In Situ Generation of Peracid Catalyzed by Perhydrolase CLEA

A perhydrolase, immobilized as a cross linked enzyme aggregate (CLEA), was employed to catalyze the in situ formation of peracetic acid (PAA) from ethylene glycol diacetate (EGDA) and hydrogen peroxide. The produced PAA was used for the Baeyer–Villiger oxidation of cyclohexanone, which afforded caprolactone in 63 % yield. The effect of type and amount of acyl donor, solvent, pH, temperature and ratio of cyclohexanone to hydrogen peroxide on the production of caprolactone was studied. The highest caprolactone yield was obtained with 100 mM EGDA as the acyl donor at pH 6 and room temperature using a ratio of cyclohexanone to hydrogen peroxide ratio of 1:4. Interestingly, the perhydrolase CLEA exhibited the highest activity in aqueous medium in contrast to the well studied lipase B from Candida antarctica. The perhydrolase CLEA proved to be a very efficient catalyst; the K m and Vmax values were 118 mM and 56.3 μmol min−1, respectively.

Highly active biocatalyst for transesterification: Cross linked enzyme aggregates of Thermomyces lanuginosus and Candida antarctica B

The preparation of cross‐linked enzyme aggregates (CLEAs) with albumin as an additive is demonstrated successfully with lipase Thermomyces lanuginosus (TL) and lipase B of Candida antarctica (CalB). Investigations regarding the albumin admixture and the temperature optimum are employed. Compared to non‐cross‐linked lipase, CalB delivers the best results at a mass ratio of 1:16 lipase/albumin, which leads to a 24‐fold increase of activity; TL at a mass ratio of 1:12, which leads to a eightfold increased activity. A transesterification yield of 61% m/m ethyl ester can be reached with 2.5% m/m CLEAs of lipase TL applied on rapeseed oil within 1.5 h reaction time. A catalyst mass of 10% m/m yields 94% m/m ethyl ester in the same time. Within six cycles of reuse, the catalyst remains stable. CLEAs of CalB and Novozym 435 (a commercial immobilized lipase B of C. antarctica) show comparable rates in ethanolysis and glycerolysis reaction.Practical applications: CLEAs have become of increasing interest in terms of synthesis of organic compounds via bio‐catalytic (green) approaches. The findings presented here are targeting at transesterification of fats and oils into corresponding alkyl esters applicable, e.g., as alternative fuel substitutes. High conversion rates coupled with hyperactivation effects as well as highly appealing reusability behavior indicate the practical benefits even for technical applications. Conversion of lipids into derivatives by CLEAs as demonstrated here, might extend utilization and conversion possibilities of biomass.

Cross linked enzyme aggregates of Prunus armeniaca hydroxynitrile lyase for synthesis of enantiopure (R)-cyanohydrins

Cross linked enzyme aggregates of Prunus armeniaca hydroxynitrile lyase for synthesis of enantiopure (R)-cyanohydrins

Carrier free co-immobilization of glucoamylase and pullulanase as combi-cross linked enzyme aggregates (combi-CLEAs)

Herein we report the successful preparation of carrier free co-immobilized form of glucoamylase and pullulanase using combi-CLEAs method from commercial multi-enzyme preparation OPTIMAX® 7525 HP. Glucoamylase and pullulanase were precipitated using ammonium sulfate, followed by cross-linking for 8 h with 2% (v/v) glutaraldehyde to produce insoluble catalytically active co-immobilized form of enzymes. The effects of precipitant type and cross-linking were studied and the biocatalyst was characterized. Scanning electron microscopy analysis showed that co-immobilized form of enzymes was of spherical structure. For starch hydrolytic activity, shift in optimum pH from 5 to 7 and temperature from 60 to 70 °C were observed after co-immobilization of enzymes. After starch hydrolysis reaction in batch mode, 100, 80 and 30% conversions were obtained with co-immobilized enzymes, mixture of separate CLEAs and free enzymes, respectively. Co-immobilization also enhanced the thermal stability and storage stability. After eight reuses with 30 min reaction time, co-immobilized glucoamylase and pullulanase retained 90 and 85% of its initial activity, respectively.

Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs)

In the past couple of decades, cross linked enzyme aggregates (CLEAs) have emerged as a novel and versatile carrier free immobilization technique. The immobilization of enzymes as cross linked enzyme aggregates (CLEAs) involves precipitation of an enzyme from aqueous solution followed by cross linking with a bi-functional reagent. It is worth noting that many parameters alter the enzyme precipitation and the aggregate cross linking and hence affect the activity and stability of CLEAs. Therefore to endorse CLEAs for industrial application, each newly synthesised CLEA is characterized. This review intends to investigate the effects of various parameters, such as the nature and purity of the enzyme, the nature and amount of precipitant, the nature and amount of cross linker, the cross linking time, the pH and temperature during CLEA preparation and washing and separation techniques on the activity and stability of CLEAs. The major parameters such as catalytic properties, particle size and morphology, stability and reusability required for approval of industrial applicability of newly synthesized CLEAs are critically reviewed. Furthermore the scope of CLEAs in non-aqueous solvent, the development of one pot cascade processes and the design of different types of enzyme reactors is also discussed.