RÉSUMÉ
Aflatoxin B1 (AFB1) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB1 without redox mediators. However, the use of this CotA-laccase to remove AFB1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB1, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB1, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (kcat/Km) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.
Sujet(s)
Aflatoxine B1 , Bacillus licheniformis , Protéines bactériennes , Laccase , Aflatoxine B1/métabolisme , Bacillus licheniformis/génétique , Bacillus licheniformis/métabolisme , Bacillus licheniformis/enzymologie , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Laccase/métabolisme , Laccase/génétique , Mutagenèse dirigée , Protéines recombinantes/métabolisme , Protéines recombinantes/génétique , SaccharomycetalesRÉSUMÉ
Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.
Sujet(s)
Peroxyde d'hydrogène , Laccase , Laccase/composition chimique , Peroxyde d'hydrogène/métabolisme , Rifampicine/pharmacologie , Rifampicine/métabolisme , Escherichia coli/métabolisme , Antibactériens/pharmacologie , Antibactériens/métabolismeRÉSUMÉ
Aflatoxin B1 (AFB1) contamination seriously threatens nutritional safety and common health. Bacterial CotA-laccases have great potential to degrade AFB1 without redox mediators. However, CotA-laccases are limited because of the low catalytic activity as the spore-bound nature. The AFB1 degradation ability of CotA-laccase from Bacillus licheniformis ANSB821 has been reported by a previous study in our laboratory. In this study, a Q441A mutant was constructed to enhance the activity of CotA-laccase to degrade AFB1. After the site-directed mutation, the mutant Q441A showed a 1.73-fold higher catalytic efficiency (kcat/Km) towards AFB1 than the wild-type CotA-laccase did. The degradation rate of AFB1 by Q441A mutant was higher than that by wild-type CotA-laccase in the pH range from 5.0 to 9.0. In addition, the thermostability was improved after mutation. Based on the structure analysis of CotA-laccase, the higher catalytic efficiency of Q441A for AFB1 may be due to the smaller steric hindrance of Ala441 than Gln441. This is the first research to enhance the degradation efficiency of AFB1 by CotA-laccase with site-directed mutagenesis. In summary, the mutant Q441A will be a suitable candidate for highly effective detoxification of AFB1 in the future.
RÉSUMÉ
Due to growing food safety issues, developing economic, rapid, and sensitive strategies for food spoilage monitoring has attracted significant attention. Here, a Bacillus subtilis spore-based biosensor is presented for rapid, highly sensitive, visual biogenic amines detection. The biosensor is fabricated through biogenic amines-induced pH increase which inhibits the electron transfer between Cu ion sites within CotA-laccase on the spore surface, leading to decrease in catalytic oxidation activity towards the chromogenic substrates. The developed system integrated with smartphone analysis realized the on-site monitoring of histamine with a detection range of 0.17-120 mg L-1, and a detection limit of 0.17 mg L-1 (3σ). Moreover, the color change induced by histamine is observable by the naked eye. The smart biosensor was successfully applied for food freshness evaluation in raw meat samples, showing several advantages, including eco-friendliness, low cost, and high stability, meeting the demands of on-site monitoring in the food safety field.
Sujet(s)
Techniques de biocapteur , Histamine , Histamine/analyse , Amines biogènes/analyse , Sécurité des alimentsRÉSUMÉ
Congo Red (CR) is a typical azo dye with highly toxic and carcinogenic properties. This study aimed to improve the decolorization activity of Bacillus pumilus W3 CotA-laccase for azo dye CR. This work analyzed the interaction between CotA-laccase and CR based on homology modeling and molecular docking. The three amino acids (Gly323, Thr377, Thr418) in the substrate-binding pocket were rationally modified through saturation mutation. Finally, the obtained multi-site mutants T377I/T418G and G323S/T377I/T418G decolorized 76.59% and 59.37% of CR within 24 h at pH 8.0 without a mediator, which were 3.15- and 2.44-fold higher than the wild-type CotA. The catalytic efficiency of the multi-site mutants T377I/T418G and G323S/T377I/T418G to CR were increased by 2.21- and 2.01-fold compared with the wild-type CotA, respectively. The mechanism of activity enhancement of mutants was proposed by structural analysis. This evidence suggests that the mutants T377I/T418G and G323S/T377I/T418G could be used as novel bioremediation tools.
Sujet(s)
Bacillus pumilus , Bacillus pumilus/génétique , Agents colorants , Rouge Congo , Laccase , Simulation de docking moléculaireRÉSUMÉ
Malachite green (MG) is used as fungicide/parasiticide in aquaculture, its persistence is detrimental as it exhibits carcinogenic effects to aquatic organisms. Bacterial laccase evaluated as the best enzyme at extreme condition for aquatic MG removal. Study aims to increase laccase concentration, CotA-laccase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Optimal catalysis for purified CotA-laccase were at pH 5.0, 60 °C, and 1 mM of (2,2-azino-di-[3-ethylbenzothiazoline-sulphonate-(6)]) with Km and Kcat 0.087 mM and 37.64 S-1 respectively. MG biodegradation by CotA-laccase in clam and tilapia pond wastewaters and cytotoxic effect of biodegraded products in grouper fin-1 cells were determined. MG degradation by CotA-laccase was equally efficient, exhibiting upto 90-94% decolorization at freshwater and saline conditions and treated solution was non-toxic to GF-1 cells. Thus, recombinant-CotA-laccase could be an environmentally-friendly enzyme for aquaculture to remove MG, thereby effective to reduce its accumulation in aquatic organisms and ensuring safe aquaculture products.
Sujet(s)
Laccase , Magenta I , Bacillus subtilis , Protéines bactériennes , Agents colorants , Escherichia coli/génétique , Concentration en ions d'hydrogène , Laccase/génétique , Magenta I/toxicitéRÉSUMÉ
Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5. KEY POINTS: ⢠Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis. ⢠Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively. ⢠The mechanisms of awarding enhanced activity to the mutants were supposed.
Sujet(s)
Bacillus pumilus , Laccase , Bacillus pumilus/génétique , Protéines bactériennes/génétique , Agents colorants , Laccase/génétique , Simulation de docking moléculaire , Mutagenèse , NaphtalènesulfonatesRÉSUMÉ
In the present study, the CotA protein from Bacillus licheniformis ANSB821 was cloned and expressed in Escherichia coli. Apart from the laccase activities, we found that the recombinant CotA could effectively oxidize aflatoxin B1 in the absence of redox mediators. The Km, Kcat and Vmax values of the recombinant CotA towards aflatoxin B1 were 60.62 µM, 0.03 s-1 and 10.08 µg min-1 mg-1, respectively. CotA-mediated aflatoxin B1 degradation products were purified and identified to be aflatoxin Q1 and epi-aflatoxin Q1. The treatment of human liver cells L-02 with aflatoxin Q1 and epi-aflatoxin Q1 did not suppress cell viability and induce apoptosis. Molecular docking simulation revealed that hydrogen bonds and van der Waals interaction played an important role in aflatoxin B1-CotA stability. These findings in the current study are promising for a possible application of CotA as a novel aflatoxin oxidase in degrading AFB1 in food.
RÉSUMÉ
In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.
Sujet(s)
Agents colorants/métabolisme , Laccase/métabolisme , Magenta I/métabolisme , Agents colorants/toxicité , Escherichia coli/génétique , Escherichia coli/métabolisme , Laccase/génétique , Mutation , Magenta I/toxicitéRÉSUMÉ
CotA-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel CotA-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type CotA-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7-11. Mutant S208G/F227A showed optimal temperature at 80°C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type CotA-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis.
Sujet(s)
Bacillus pumilus/génétique , Laccase/génétique , Laccase/métabolisme , Mutagenèse dirigée , Ingénierie des protéines/méthodes , Bacillus pumilus/enzymologie , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Biotransformation/génétique , Catalyse , Agents colorants/composition chimique , Agents colorants/métabolisme , Amélioration génétique/méthodes , Laccase/composition chimique , Mutation , Organismes génétiquement modifiés , TempératureRÉSUMÉ
In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a His6 tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be 50°C. Upon treatment with known laccase inhibitors, including EDTA, SDS, and NaN3, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.
Sujet(s)
Anthraquinones/métabolisme , Bacillus subtilis/enzymologie , Agents colorants/métabolisme , Laccase/métabolisme , Bacillus subtilis/génétique , Bacillus subtilis/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Dépollution biologique de l'environnement , Antienzymes/composition chimique , Expression des gènes , Cinétique , Laccase/antagonistes et inhibiteurs , Laccase/génétique , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Spores bactériens/enzymologie , Spores bactériens/génétique , Spores bactériens/métabolisme , TempératureRÉSUMÉ
Bacterial CotA-laccases exhibit higher activity in alkaline pH and salt concentration conditions compared to laccases from white-rot fungi. They are considered as green catalysts in decolorizing of industrial dyes. However, CotA-laccases are limited due to the low yield and catalytic efficiency as the spore-bound nature of CotA. A DNA shuffling strategy was applied to generate a random mutation library. To improve laccase activities, a mutant (T232P/Q367R 5E29) with two amino acid substitutions was identified. The catalytic efficiency of mutant 5E29 was 1.21 fold higher compared with that of the wild-type. The Km and kcat values of 5E29 for SGZ were of 20.3 ± 1.3 µM and 7.6 ± 2.7 s-1. The thermal stability was a slight enhancement. Indigo Carmine and Congo red were efficiently decolorized by using this mutant at pH 9.0. These results provide that 5E29 CotA-laccase is a good candidate for biotechnology applications under alkaline condition, with an effective decolorization capability.
Sujet(s)
Brassage d'ADN/méthodes , Laccase/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Biotechnologie/méthodes , Concentration en ions d'hydrogène , Laccase/génétiqueRÉSUMÉ
CotA protein from Bacillus subtilis is of laccase activity. The solubility of recombinant CotA is low, which hinders its application. In this study, histidine tag position optimization and hydrophilic engineering were applied to increase the yield and activity of CotA protein. The results showed that the protein yield of CotA with his tag at C-terminal (CH6-CotA) was four times of that of NH6-CotA (His tag at N-terminal). Then, 23 single mutants were constructed by substitutions of hydrophobic residues with hydrophilic amino acids. Among them, the protein yield of the mutant F207Y was increased by 30%; the catalytic activity (kcat/Km) of V403T and P455S was two and three times higher than that of CH6-CotA, respectively. Finally, triple mutant F2071Y/V403T/P455S with C-terminal his-tag (CH6-TSY) was constructed. When the proteins were expressed in microanaerobic condition, the activities of mutants CH6-P455S and CH6-TSY were enhanced about 48- and 42-folds compared to that of NH6-CotA in non-static culture.
Sujet(s)
Substitution d'acide aminé , Bacillus subtilis/enzymologie , Protéines bactériennes/composition chimique , Histidine/composition chimique , Laccase/composition chimique , Mutation faux-sens , Bacillus subtilis/génétique , Protéines bactériennes/génétique , Catalyse , Histidine/génétique , Laccase/génétique , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/génétiqueRÉSUMÉ
Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications.
Sujet(s)
Bacillus pumilus/enzymologie , Protéines bactériennes/métabolisme , Laccase/métabolisme , Mutagenèse dirigée , Ingénierie des protéines/méthodes , Protéines bactériennes/génétique , Catalyse , Agents colorants/composition chimique , Concentration en ions d'hydrogène , Laccase/génétique , Mutation , Stabilité protéique , SolubilitéRÉSUMÉ
In this work, a novel bacterial strain exhibiting laccase activity was isolated from black liquor and identified as Bacillus subtilis cjp3. The CotA-laccase gene was cloned from strain cjp3 and expressed in Escherichia coli. The purified recombinant laccase has a maximum activity of 7320 U/L, maintaining high stabilities under a wide pH range and high temperature conditions. Nearly no loss of laccase activity was observed even at pH 9.0 after 10 h of incubation. Reactive blue 19, reactive black 5 and indigo carmine could be efficiently decolorized by the purified laccase in the presence of a mediator ABTS. More than 86% of tested dyes were removed in 4 h at pH = 9.0. The recombinant laccase can work well in a broad range of temperatures of 20-80°C(>80% relative activity). These special properties indicated the potential use of the CotA-laccase in treating wastewater containing synthetic dyes.
Sujet(s)
Alcalis/composition chimique , Bacillus subtilis/enzymologie , Agents colorants/analyse , Laccase/composition chimique , Polluants chimiques de l'eau/analyse , Purification de l'eau/méthodes , Bacillus subtilis/génétique , Stabilité enzymatique , Escherichia coli/génétique , Concentration en ions d'hydrogène , Laccase/génétique , Phylogenèse , Protéines recombinantes/composition chimique , Protéines recombinantes/génétique , TempératureRÉSUMÉ
Laccases are green oxidases with a number of potential industrial applications. In this study, recombinant Bacillus subtilis CotA laccase was secreted by Escherichia coli via both the α-hemolysin secretion system and the YebF secretion system after microaerobic induction. Meanwhile, we discovered a much simpler approach for extracellular production of recombinant CotA laccase from E. coli, involving alternation of induction conditions to release recombinant CotA following intracellular expression. By optimizing the induction parameters, the extracellular yield of recombinant CotA laccase was improved from 157.4 to 2401.3 U/L after 24 h of induction. This strategy could be suitable for large-scale production of CotA laccase for industrial use. Recombinant CotA laccase was purified by Ni2+ affinity chromatography in a single step and showed similar biochemical properties to wild-type laccase. Purified as well as crude recombinant CotA laccase efficiently decolorized seven structurally different dyes. The decolorization capability of recombinant CotA laccase under harsh conditions was investigated by incubation of the enzyme with a simulated textile effluent (STE) with pH 11.6, 3.5 % salinity and peak absorbance of 10.42. Recombinant CotA laccase efficiently decolorized 77.0 % of STE after 48 h reaction, demonstrating the potential of this enzyme for industrial dye effluent treatment.
Sujet(s)
Bacillus subtilis/enzymologie , Laccase/génétique , Laccase/métabolisme , Protéines recombinantes/génétique , Aérobiose , Bacillus subtilis/génétique , Biotransformation , Chromatographie d'affinité , Agents colorants/métabolisme , Escherichia coli/génétique , Escherichia coli/métabolisme , Expression des gènes , Concentration en ions d'hydrogène , Laccase/isolement et purification , Protéines recombinantes/isolement et purification , Protéines recombinantes/métabolisme , Textiles , Activation de la transcriptionRÉSUMÉ
Bacterial laccases are potential enzymes for biotechnological applications because of their remarkable advantages, such as broad substrate spectrum, various reactions, high thermostability, wide pH range, and resistance to strongly alkaline environments. However, the use of bacterial laccases for industrialized applications is limited because of their low expression level and catalytic efficiency. In this study, CotA, a bacterial laccase from Bacillus pumilus, was engineered through presumptive reasoning and rational design approaches to overcome low catalytic efficiency and thermostability. L386W/G417L, a CotA double-mutant, was constructed through site-directed mutagenesis. The catalytic efficiency of L386W/G417L was 4.3 fold higher than that of wild-type CotA-laccase, but the thermostability of the former was decreased than that of the latter and other mutants. The half-life (t 1/2) of wild-type and G417L were 1.14 and 1.47 h, but the half-life of L386W/G417L was only 0.37 h when incubating the enzyme at 80 °C. Considering the high catalytic efficiency of L386W/G417L, we constructed L386W/G417L/G57F, another mutant, to improve thermostability. Results showed that the half-life of L386W/G417L/G57F was 0.54 h when incubating the enzyme at 90 °C for 2 h with about 34% residual activity, but the residual activity of L386W/G417L was less than 40% when incubating the enzyme at 90 °C for 5 min. L386W/G417L was more efficient in decolorizing various industrial dyes at pH 10 than other mutants. L386W/G417L/G57F also exhibited an efficient decolorization ability. L386W/G417L/G57F is appropriate for biotechnological applications because of its high activity and thermostability in decolorizing industrial dyes. CotA-laccase may be further subjected to molecular modification and be used as an enhancer to improve decolorization efficiency for the physical and chemical treatment of dye wastewater.
Sujet(s)
Bacillus pumilus/enzymologie , Bacillus pumilus/métabolisme , Protéines bactériennes/métabolisme , Laccase/métabolisme , Ingénierie des protéines/méthodes , Bacillus pumilus/génétique , Protéines bactériennes/génétique , Catalyse , Agents colorants/métabolisme , Température élevée , Concentration en ions d'hydrogène , Laccase/génétiqueRÉSUMÉ
Here we report the full genome sequence of Bacillus pumilus W3, which was isolated from raw gallnut honey in Nandan County, Guangxi Province of China, showing high CotA-laccase activity. The W3 strain contains 3,745,123bp with GC content of 41.39%, and contains 3695 protein-coding genes, 21 rRNAs and 70 tRNAs.