Representative Bacillus sp. AM1 from Gut Microbiota Harbor Versatile Molecular Pathways for Bisphenol A Biodegradation.

Human gut microbiota harbors numerous microbial species with molecular enzymatic potential that impact on the eubiosis/dysbiosis and health/disease balances. Microbiota species isolation and description of their specific molecular features remain largely unexplored. In the present study, we focused on the cultivation and selection of species able to tolerate or biodegrade the endocrine disruptor bisphenol A (BPA), a xenobiotic extensively found in food plastic containers. Chemical xenobiotic addition methods for the directed isolation, culturing, Whole Genome Sequencing (WGS), phylogenomic identification, and specific gene-encoding searches have been applied to isolate microorganisms, assess their BPA metabolization potential, and describe encoded catabolic pathways. BPA-tolerant strains were isolated from 30% of infant fecal microbial culture libraries analyzed. Most isolated strains were phylogenetically related to the operational taxonomic group Bacillus amyloliquefaciens spp. Importantly, WGS analysis of microbial representative strain, Bacillus sp. AM1 identified the four complete molecular pathways involved on BPA degradation indicating its versatility and high potential to degrade BPA. Pathways for Exopolysaccharide (EPS) and Polyhydroxyalkanates (PHA) biopolymer synthesis were also identified and phenotypically confirmed by transmission electronic microscopy (TEM). These microbial biopolymers could generally contribute to capture and/or deposit xenobiotics.

Analysis of single-cell microbial mass spectra profiles from single-particle aerosol mass spectrometry.

RATIONALE: Single-particle aerosol mass spectrometry is a practical method for studying microbial aerosols. However, the related mass spectral characteristics of single-cell microorganisms have not yet been studied systematically; hence, further investigations are necessary. METHODS: Different microbial cells were grown and directly aerosolized in the laboratory. These aerosols were then drawn into a single-particle mass spectrometer platform, and single-cell mass spectra profiles were obtained in real-time. The biological characteristics, ion variation trends, and microbial types were analyzed with either laser pulse energy or laser fluence. RESULTS: The single-particle mass spectra contained prominent peaks that could be attributed to the presence of biological matter, such as organic phosphate and nitrogen, amino acids, and spore-associated calcium complexes. Limited types of average mass spectral patterns were present, and a significant correlation was found between the ion intensity trend (presence and absence of peaks) and laser ionization energy (expressed by the total positive ion intensity). Although a single spectral data point does not contain all the peaks of the average spectrum, it covers most of the characteristic peaks and could be identified using a machine learning algorithm. After the analysis of single-particle mass spectra, we found that using multi-group features (e.g., peak intensity ratio of m/z +47 and +41, peak intensity ratio of 59 N(CH3 )3 + and 74 N(CH3 )4 + , and 12 peak variables) led to an identification accuracy of approximately 92.4% with the random forest algorithm. CONCLUSIONS: The results indicate that single-cell mass spectral profiles can be used to distinguish microbial aerosols and further illustrate their origin in a laboratory setting.

Analysis of disulphide bond linkage between CoA and protein cysteine thiols during sporulation and in spores of Bacillus species.

Spores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores' metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.

Scanning Ion Conductance Microscopy Reveals Differences in the Ionic Environments of Gram-Positive and Negative Bacteria.

This paper reports on the use of scanning ion conductance microscopy (SICM) to locally map the ionic properties and charge environment of two live bacterial strains: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. SICM results find heterogeneities across the bacterial surface and significant differences among the Gram-positive and Gram-negative bacteria. The bioelectrical environment of the B. subtilis was found to be considerably more negatively charged compared to E. coli. SICM measurements, fitted to a simplified finite element method (FEM) model, revealed surface charge values of -80 to -140 mC m-2 for the Gram-negative E. coli. The Gram-positive B. subtilis show a much higher conductivity around the cell wall, and surface charge values between -350 and -450 mC m-2 were found using the same simplified model. SICM was also able to detect regions of high negative charge near B. subtilis, not detected in the topographical SICM response and attributed to the extracellular polymeric substance. To further explore how the B. subtilis cell wall structure can influence the SICM current response, a more comprehensive FEM model, accounting for the physical properties of the Gram-positive cell wall, was developed. The new model provides a more realistic description of the cell wall and allows investigation of the relation between its key properties and SICM currents, building foundations to further investigate and improve understanding of the Gram-positive cellular microenvironment.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities.

Morphological and structural response of Bacillus sp. SFC 500-1E after Cr(VI) and phenol treatment.

Bacillus sp. SFC 500-1E, a bacterial strain isolated from tannery sediments, is able to remove Cr(VI) and simultaneously tolerate high concentrations of phenol. In this study, we used high-resolution microscopies, fluorescence polarization techniques, and several biochemical approaches to improve our understanding about the adaptive mechanisms of this strain to survive in the presence of Cr(VI) and phenol, both individually and simultaneously. Among adaptive strategies developed by Bacillus sp. SFC 500-1E, an increase in bacterial size, such as length, width, and height, and ultrastructural alterations, such as electron-dense precipitates, the presence of exopolymers, and cell lysis, are noteworthy. The exopolymers observed were consistent with the extensive biofilm formation and exopolysaccharides and extracellular protein quantification. At the cell membrane level, a rapid rigidity was induced in Cr(VI) + phenol treatment. This effect was counteracted after 16 h by changes at the level of phospholipids, mainly in the composition of fatty acids (FAs); in particular, an increase in the unsaturated fatty acid/saturated fatty acid ratio was detected. This study shows evidence of some adaptive responses displayed by Bacillus sp. SFC 500-1E, which allows it to survive in stressful conditions.

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles.

Light-driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS-g-PEO and diblock copolymer PBd-PEO. Activity of more than 90 % in lipid/polymer-based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long-term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer-based hybrids.

Response surface method for polyhydroxybutyrate (PHB) bioplastic accumulation in Bacillus drentensis BP17 using pineapple peel.

Polyhydroxybutyrate (PHB) is a biodegradable biopolymer which is useful for various applications including packing, medical and coating materials. An endospore-forming bacterium (strain BP17) was isolated from composted soil and evaluated for PHB production. Strain BP17, taxonomically identified as Bacillus drentensis, showed enhanced PHB accumulation and was selected for further studies. To achieve maximum PHB production, the culture conditions for B. drentensis BP17 were optimized through response surface methodology (RSM) employing central composite rotatable design (CCRD). The final optimum fermentation conditions included: pineapple peel solution, 11.5% (v/v); tryptic soy broth (TSB), 60 g/L; pH, 6.0; inoculum size, 10% (v/v) and temperature, 28°C for 36 h. This optimization yielded 5.55 g/L of PHB compared to the non-optimized condition (0.17 g/L). PHB accumulated by B. drentensis BP17 had a polydispersity value of 1.59 and an average molecular weight of 1.15x105 Da. Thermal analyses revealed that PHB existed as a thermally stable semi-crystalline polymer, exhibiting a thermal degradation temperature of 228°C, a melting temperature of 172°C and an apparent melting enthalpy of fusion of 83.69 J/g. It is evident that B. drentensis strain BP17 is a promising bacterium candidate for PHB production using agricultural waste, such as pineapple peel as a low-cost alternative carbon source for PHB production.

A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP.

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 µM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

Extracellular biosynthesis, characterization, optimization of silver nanoparticles (AgNPs) using Bacillus mojavensis BTCB15 and its antimicrobial activity against multidrug resistant pathogens.

Biosynthesis of metal nanoparticles is an area of interest among researchers because of its eco-friendly approach. Current study focuses at biosynthesis of silver nanoparticles (AgNPs) and optimization of physico-chemical conditions to obtain mono-dispersed and stable AgNPs having antimicrobial activity. Initially Bacillus mojavensis BTCB15 produced silver nanoparticles (AgNPs) of 105 nm. Silver nanoparticles (AgNPs) were characterized by particle size analyzer, UV-Vis Spectroscopy, Fourier transforms infrared spectroscopy (FTIR), Atomic force microscopy (AFM), and X-ray diffraction (XRD). Whereas, under optimal conditions of temperature 55 °C, pH 8, addition of surfactant Tween 20, and metal ion K2SO4, about 104% size reduction was achieved with average size of 2.3nm. Molecular characterization revealed 98% sequence homology with Bacillus mojavensis. AgNPs exhibited antibacterial activity at concentrations ranging from 0.5 to 2.5 µg/µl against Escherichia coli BTCB03, Klebsiella pneumonia BTCB04, Acinetobacter sp. BTCB05, and Pseudomonas aeruginosa BTCB01 but none against Staphylococcus aureus BTCB02. Highest antibacterial activity was observed at 0.27 µg/µl and lowest at 0.05 µg/µl of AgNPs indicated by zone of inhibition. Conclusively, under optimum conditions, Bacillus mojavensis BTCB15 was able to produce AgNPs of 2.3 nm size and had antibacterial activity against multi drug resistant pathogens.

Stabilizing bromelain for therapeutic applications by adsorption immobilization on spores of probiotic Bacillus.

Bromelain, a protease from pineapple plant can be applied as oral drug for the treatment of inflammation and certain diseases. Unlike most conventional supports, immobilization on edible support will make the enzyme suitable for therapeutic use. In this study, spores of probiotic Bacillus sp was used for the adsorption of bromelain. Effect of pH, temperature and enzyme concentration on bromelain immobilization was studied, followed by characterization of the enzymes. Maximum bromelain coupling (%) (50.607 ±â€¯4.194) was obtained when immobilization was carried out at pH 6.0, 24 °C for 150 min. The immobilized enzyme showed optimum activity at pH 8 and 80 °C, while the free enzyme had 6 and 60 °C as its optimum pH and temperature, respectively. Bromelain Vmax increased after immobilization while Km decreased. Activation energy, Ea was 26.513 kJ/mol and 20.942 kJ/mol for the free and immobilized enzymes, respectively. The immobilized bromelain also showed significantly greater storage and thermal stability than the free bromelain. At 80 °C, the free bromelain lost all its activity after 50 min while the immobilized enzyme lost only 46.89% activity. Bromelain was successfully immobilized on Bacillus spores with improved catalytic and non-catalytic properties and this holds great potential for its growing therapeutic applications.

Reclassification of Bacillus aryabhattai Shivaji et al. 2009 as a later heterotypic synonym of Bacillus megaterium de Bary 1884 (Approved Lists 1980).

In the present study, the taxonomic position of Bacillus aryabhattai and Bacillus megaterium was evaluated using morphological, biochemical, phylogenomic and genome analysis. The morphological and biochemical of these two species were almost similar with few exceptions. The major fatty acids in B. megaterium DSM 32T and B. aryabhattai 21047T were anteiso-C15:0 and iso-C15:0. In the phylogenomic tree, both species clade together and shared high 16S rRNA gene sequence similarity (99.6%). The average nucleotide identity values between Bacillus aryabhattai and Bacillus megaterium were above the threshold values for bacterial species delineation. Based upon morphological, biochemical, chemotaxonomic and comparative genome analysis, we propose to reclassify Bacillus aryabhattai Shivaji et al. 2009 as a later heterotypic synonym of Bacillus megaterium de Bary 1884 (Approved Lists 1980).

Augmentation of Atrazine biodegradation by two Bacilli immobilized on α-Fe2O3 magnetic nanoparticles.

In this study, a novel immobilizing carrier with α-Fe2O3 magnetic nanoparticles was developed and used for immobilization of atrazine-degrading bacterial isolates of Bacillus spp. Since the free cells of microorganisms generally not succeed to degrade pollutants; thus, extra treatments are alluring to make strides biodegradation. Scanning electron microscope (SEM) images appeared that after immobilization the bacterial cells were totally retained and entirely distributed on the surface of α-Fe2O3 magnetic nanoparticles. The performance of α-Fe2O3 immobilized cells in atrazine (ATZ) degradation was compared with the free cells, which was about 90.56% in 20 days. Experimental results exhibited that ATZ could be degraded at a broad range of physicochemical parameters viz. pH (4.0 to 9.0), temperature (20 to 45 °C), ATZ concentration (50 to 300 mg L-1) and agitation speed (50 to 300 rpm), which underlines that α-Fe2O3 immobilized cells could tolerate a higher range of ATZ concentration as compared to free cells. This research demonstrated that α-Fe2O3 could be applied as a potential carrier in cell immobilization and biodegradation of ATZ herbicide with greater efficiency.

Immobilized Cells of Bacillus circulans ATCC 21783 on Palm Curtain for Fermentation in 5 L Fermentation Tanks.

Palm curtain was selected as carrier to immobilize Bacillus circulans ATCC 21783 to produce ß-cyclodextrin (ß-CD). The influence for immobilization to CGTase activity was analyzed to determine the operation stability. 83.5% cyclodextrin glycosyltransferases (CGTase) of the 1st cycle could be produced in the 7th cycle for immobilized cells, while only 28.90% CGTase was produced with free cells. When palm curtain immobilized cells were reused at the 2th cycle, enzyme activities were increased from 5003 to 5132 U/mL, which was mainly due to physical adsorption of cells on palm curtain with special concave surface structure. Furthermore, conditions for expanded culture of immobilized cells in a 5 L fermentation tank were optimized through specific rotation speed procedure (from 350 r/min to 450 r/min with step size of 50 r/min) and fixed ventilation capacity (4.5 L/min), relations between biomass, enzyme activity, pH, and oxygen dissolution was investigated, and the fermentation periods under the two conditions were both 4 h shorter. Compared with free cell, immobilized cell was more stable, effective, and had better application potential in industries.

Secretome analysis of alkaliphilic bacterium Bacillus lehensis G1 in response to pH changes.

Bacillus lehensis G1 is an alkaliphilic bacterium that is capable of surviving in environments up to pH 11. Secretome related to bacterial acclimation in alkaline environment has been less studied compared to cytoplasmic and membrane proteome. The aim of this study was to gain better understanding of bacterial acclimation to alkaline media through analyzing extracellular proteins of B. lehensis. The pH range for B. lehensis growth was conducted, and two-dimensional electrophoresis and MALDI-TOF/TOF MS analysis were conducted to characterize changes in protein profiling in B. lehensis cultured at pH 8 and pH 11 when compared with those cultured at pH 10 (optimal growth pH). B. lehensis could grow well at pH ranging from 8 to 11 in which the bacteria showed to posses thinner flagella at pH 11. Proteomic analyses demonstrated that five proteins were up-regulated and 13 proteins were down-regulated at pH 8, whereas at pH 11, 14 proteins were up-regulated and 8 were down-regulated. Majority of the differentially expressed proteins were involved in the cell wall, main glycolytic pathways, the metabolism of amino acids and related molecules and some proteins of unknown function. A total of 40 differentially expressed protein spots corresponding to 33 proteins were identified; including GlcNAc-binding protein A, chitinase, endopeptidase lytE, flagellar hook-associated proteins and enolase. These proteins may play important roles in acclimation to alkaline media via reallocation of cell wall structure and changes to cell surface glycolytic enzymes, amino acid metabolism, flagellar hook-associated proteins and chaperones to sustain life under pH-stressed conditions.

Enhancement of bio-desulfurization capability of a newly isolated thermophilic bacterium using starch/iron nanoparticles in a controlled system.

Due to the increasing application of oil and petroleum products, increased environmental contamination has become a matter of concern. Bio-desulfurization process may be used to eliminate sulfur from fossil fuels in the moderate condition. In this study, a thermophilic bacterium was isolated that was able to desulfurize dibenzothiophene. 16S rRNA sequencing indicated that this strain is related closely to Bacillus thermoamylovorans (97%). This strain grew in Basal salt medium containing DBT (100 mgl-1) as the only sulfur source, at 55°C and showed maximum growth (OD660 = 0.850) following 72 h incubation time. 2­hydroxybiphenyl was produced at the maximal concentration (26.13 ±â€¯0.12 mgl-1) at 72 h. Bio-desulfurization and growth rate factors were optimized using response surface methodology. Starch/Fe3O4 and starch/Fe nanoparticles were used for enhancement of BDS efficiency. The size of starch/Fe3O4 and starch/Fe nanoparticles were 20 and 30-40 nm, respectively, as described by using scanning electron microscope and transmission electron microscope. The results showed that the immobilized cells by starch/Fe3O4 and starch/Fe nanoparticles had higher desulfurization capacity, about 10% and 22% more, respectively. Also, BDS in a bioreactor in the presence of nanoparticles was increased 25% with respect of the process occurred in the flask.

A Non-Enzymatic Pathway with Superoxide in Intracellular Terpenoid Synthesis.

Non-C5 -units terpenoids (norisoprenoids) with an acetonyl group are widely distributed in nature. However, studies on the biosynthesis of norisoprenoids are scarce. Now, the C33 norisoprenoid, (all-E)-farnesylfarnesylacetone, was identified from Bacillus spp. and it was elucidated for the first time that superoxide mediates the cleavage of menaquinones (vitamin K) to form norisoprenoids in saponification treatment. From in vivo experiments using gene-disrupted Bacillus subtilis strains targeted for enzymes responsible for menaquinone biosynthesis and for superoxide dismutase, it was suggested that the non-enzymatic cleavage (autoxidation) of menaquinone with superoxide resulted in norisoprenoid synthesis in Bacillus cells. Furthermore, the bioactive norisoprenoids, farnesylacetone and phytone, were produced in Bacillus cells by this novel synthesis system.

Purification of extra cellular poly-γ-glutamic acid as an antibacterial agent using anion exchange chromatography.

BLAST analysis of the 16S rRNA gene sequence for the newly isolated bacterium, revealed significant identity (99.5%) with Bacillus sonorensis [Ijadi Bajestani, M., et al., International Journal of Biological Macromolecules, 2017. 96: p. 100-110]. According to the literature review for closely related species of Bacillus sonorensis, the production of poly-γ-glutamic acid (γ-PGA) as an extra cellular biopolymer was investigated for the isolated bacteria which is deposited in IBRC (Iranian Biological Resource Center) as Bacillus sp. Strain M2 (IBRC-M11173). To determine if γ-PGA production by Bacillus sp. Strain M2 is glutamate dependent, it was grown on PGA medium, consisted of sodium glutamate. The results proved that γ-PGA production is highly dependent on glutamate component. In the following, the bioproduct has undergone different purification processes mainly consisting of dialysis, deproteinization and anion exchange chromatography. Based on the high-performance liquid chromatography (HPLC) results for ion chromatography effluents, 59% of the initial PGA in main solution was eluted via NaCl elution. Gel permeation chromatography (GPC) characterization analysis was accomplished to determine the polydispersity and γ-PGA molecular weight. Two major average molecular weights were distinguished; the heavy weight fraction of 7.7×106g/mol with polydispersity index of 1.73 and the other one with an average molecular weight number of 1.7×104g/mol and polydispersity index of 4.4. The antibacterial activity of the extracellular γ-PGA, as an anionic biopolymer, toward Staphylococcus aureus and E. coli, was assayed using the clinical and laboratory standards institute (CLSI) guidelines. For Staphylococcus aureus the minimum inhibitory concentration (MIC) value was about 34g/L while for E. coli this value reaches 53g/L.

Visualizing Bacterial Colony Morphologies Using Time-Lapse Imaging Chamber MOCHA.

Capturing microbial growth on a macroscopic scale is of great importance to further our understanding of microbial life. However, methods for imaging microbial life on a scale of millimeters to centimeters are often limited by designs that have poor environmental control, resulting in dehydration of the agar plate within just a few days. Here, we created MOCHA (microbial chamber), a simple but effective chamber that allows users to study microbial growth for extended periods (weeks) in a stable environment. Agar hydration is maintained with a double-decker design, in which two glass petri dishes are connected by a wick, allowing the lower plate to keep the upper plate hydrated. This flexible chamber allows the observation of a variety of microbiological phenomena, such as the growth and development of single bacterial and fungal colonies, interspecies interactions, swarming motility, and pellicle formation.IMPORTANCE Detailed study of microbial life on the colony scale of millimeters to centimeters has been lagging considerably behind microscopic inspection of microbes. One major reason for this is the lack of inexpensive instrumentation that can reproducibly capture images in a controlled environment. In this study, we present the design and use of a unique chamber that was used to produce several time-lapse movies that aimed to capture the diversity of microbial colony phenotypes over long periods.

High-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins.

High-throughput estimation of specific activities of an enzyme and its mutants in a group (enzyme/mutants) in cell lysates via high-throughput assay of their activities and separate immunoturbidimetric assay (ITA) of their proteins was proposed. Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) served as two models. ITA employed 0.75 mg of antisera against PAAS or BFU as the reference in 96-well microplates to measure the difference of extinction at 340 and 700 nm. According to the calibration curve, ITA quantified the reference from 0.40 to about 2.4 µg. The consistency among the abundance of enzyme/mutants through ITA of proteins in cell lysates prepared under the same conditions supported their consistent immunological reactivity to the antisera. Specific activities of PAAS/mutants or BFU/mutants in cell lysates through ITA of proteins showed excellent proportionality to those carefully determined after purification. Receiver-operating-characteristic (ROC) analysis of specific activities through ITA of proteins gave a higher area-under-curve than those for ROC analyses of other activity indices, which allowed the recognition of a PAAS/mutant of 50% higher activity after cell amplification in high-throughput mode. Therefore, ITA of enzyme/mutants as proteins is promising to estimate their specific activities in cell lysates in high-throughput mode for quantitative comparison.