Potential Enzyme Research Articles

The unique sugar conversion and complex CAZyme system of Trichoderma brev T069 during solid-state fermentation of cassava peel

Cassava peel is a lignocellulose-based agricultural by-product that can be degraded into reducing sugars as a raw material for energy. However, chemical and enzymatic treatment processes of cell wall polymers are expensive. We found that the filamentous fungus Trichoderma brev T069 can grow in a cassava peel matrix without any additional nutrient and degrade macromolecular carbohydrates, producing high levels of glucose and valuable hydrolytic enzymes. Therefore, this paper investigated the utilization strategy of T. brev T069 on cassava peels by dynamically analyzing its growth and hydrolytic enzyme system during solid fermentation, degradation of cassava peel components, and changes in reducing sugars. Results showed that T. brev T069 uses a "borrow-return" method of reducing sugar conversion, first absorbing fructose for growth for 3 days and later releasing glucose (99.56 mg/g) and cellobiose (24.69 mg/g) via hydrolases and high levels of β-glucosidase (1196.05 IU/g). Scanning electron microscopy and Fourier transform infrared spectrometry revealed the compositional breakdown and decreased carbohydrate contents of the peels. Label-free proteomics identified 257 carbohydrate-active enzymes (CAZymes), and functional annotation indicated that hemicellulases, lignin-modifying enzymes, and pectinases were induced at early stages, whereas amylases and cellulases were induced at later stages. A protein–protein interaction network revealed that protein degradation-related proteases and α-mannosidases may be involved in the realignment of enzymatic degradation strategies. This study explored the hydrolytic enzymatic potential of the Trichoderma CAZyme system and provided a practical approach for the biotransformation of tropical agricultural wastes into value-added products.

Ene-reductases: Contribution from Brazilian fungi in organic synthesis

Brazil’s territory encloses six terrestrial biomes (Amazon, Pantanal, Savannah, Atlantic Rainforest, and Caatinga-Savannah cactus) and a marine biome following the long coastline, yielding a wealth of native microbial species. Fungi found in different parts of Brazil are being studied for their enzymatic potential in biotransformation in organic synthesis processes. This review summarizes various studies on the bioreduction of activated C = C double bonds catalyzed by ene-reductases using fungi (filamentous and yeast) from the Brazilian environmental.

Isolation, screening and characterization of gelatinase and protease producing microorganisms from organic kitchen waste

The present study was aimed at isolation, screening and identification of gelatinase and protease producing bacteria from the kitchen waste. The study was also focused on the potential of isolates to produce enzyme protease and gelatinase. The total of 157 bacterial isolates were obtained from the organic kitchen waste collected from various nearby sources. The primary screening was done to screen the potential of these isolates for their enzymatic potentials on selective media. The 57 promising bacterial isolates were found to have enzymatic potentials for both gelatinase and protease. The screened isolates were subjected to secondary screening, amongst which five isolates showed maximum production of both gelatinase and protease. These five isolates viz. KW91, KW104, KW121, KW128, KW98 were the most promising isolates showing high degree of milk and gelatin hydrolysis which may be potent in faster degradation of domestic kitchen waste.

Bioprospecting for polyesterase activity relevant for PET degradation in marine Enterobacterales isolates.

Plastics have quickly become an integral part of modern life. Due to excessive production and improper waste disposal, they are recognized as contaminants present in practically all habitat types. Although there are several polymers, polyethylene terephthalate (PET) is of particular concern due to its abundance in the environment. There is a need for a solution that is both cost-effective and ecologically friendly to address this pollutant. The use of microbial depolymerizing enzymes could offer a biological avenue for plastic degradation, though the full potential of these enzymes is yet to be uncovered. The purpose of this study was to use (1) plate-based screening methods to investigate the plastic degradation potential of marine bacteria from the order Enterobacterales collected from various organismal and environmental sources, and (2) perform genome-based analysis to identify polyesterases potentially related to PET degradation. 126 bacterial isolates were obtained from the strain collection of RD3, Research Unit Marine Symbioses-GEOMAR-and sequentially tested for esterase and polyesterase activity, in combination here referred to as PETase-like activity. The results show that members of the microbial families Alteromonadaceae, Shewanellaceae, and Vibrionaceae, derived from marine sponges and bryozoans, are the most promising candidates within the order Enterobacterales. Furthermore, 389 putative hydrolases from the α/β superfamily were identified in 23 analyzed genomes, of which 22 were sequenced for this study. Several candidates showed similarities with known PETases, indicating underlying enzymatic potential within the order Enterobacterales for PET degradation.

Green tea and kombucha characterization: Phenolic composition, antioxidant capacity and enzymatic inhibition potential

The present study aimed to carry out the physical–chemical, antioxidant, and enzymatic characterization of green tea and kombucha. It was observed that kombucha had lower pH, higher acidity, and solids content compared to green tea. As for the concentration of total phenolic compounds by the Folin Ciocalteu method, there was no significant difference between the beverages. In the antioxidant analysis by the DPPH assay, it was observed that both green tea and kombucha presented significant antioxidant capacity. In the TBARS analysis with the pH of the beverages neutralized, both showed a significant reduction in lipid peroxidation; however, kombucha exhibited pro-oxidant activity when evaluated in its natural form by this method. The beverages also showed significant inhibitory activity of the α-glucosidase enzyme, however, green tea presented superior inhibitory potential.

Amylolytic activity of <i>Bacillus subtilis</i> isolates obtained from wildlife microbiota

At present, the study of the enzymatic potential of Bacillus subtilis bacteria is in demand due to the fact that probiotic and enzymatic preparations are created on their basis for their use in animal husbandry. The results of the study of amylolytic activity of 10 isolates of B. subtilis bacteria isolated from wildlife microbiota for amylopectin are presented. Determination of bacterial enzyme activity was performed by the Schomodi-Nelson method. The supernatants of the culture liquid of the 1st, 2nd, 3rd, and 7th day cultures were taken as enzyme preparation. Isolates with high qualitative indices were examined quantitatively using a spectrophotometer. The two isolates with the best performance were selected. Protein concentration was determined by the Bradford method. In addition to studying supernatants of isolates that showed extracellular enzymatic activity, studies were performed on intracellular enzymes. Cell destruction was performed using an ultrasonic disintegrator, in which the dissolved sediment was contained in a glass vial in a flask with ice. The best qualitative indicators were found in B. subtilis 2SP and B. subtilis 5SP isolates, which had high amylopectin activity from day one, so further quantitative studies were continued with them. Protein concentrations in the above isolates progressed by the days of seeding. The specific activity also increased accordingly and on day 7, the B. subtilis 2SP isolate was 2.75 units/mg, B. subtilis 5SP 2.67 units/mg. The study of the activity of intracellular enzymes in these isolates confirmed similar qualitative indicators. Two B. subtilis isolates were selected as promising for the development of enzyme preparations isolated from the microbiota of wild animals of the Far North.

A thermostable bacterial catalase-peroxidase oxidizes phenolic compounds derived from lignins.

Lignocellulosic biomass is rich in lignins, which represent a bottomless natural source of aromatic compounds. Due to the high chemical complexity of these aromatic polymers, their biological fractionation remains challenging for biorefinery. The production of aromatics from the biological valorization of lignins requires the action of ligninolytic peroxidases and laccases produced by fungi and bacteria. Therefore, identification of efficient ligninolytic enzymes with high stability represents a promising route for lignins biorefining. Our strategy consists in exploiting the enzymatic potential of the thermophilic bacteriumThermobacillus xylanilyticusto produce robust and thermostable ligninolytic enzymes. In this context, a gene encoding a putative catalase-peroxidase was identified from the bacterial genome. The present work describes the production of the recombinant protein, its biochemical characterization, and ligninolytic potential. Our results show that the catalase-peroxidase from T. xylanilyticus is thermostable and exhibits catalase-peroxidase and manganese peroxidase activities. The electrochemical characterization using intermittent pulse amperometry showed the ability of the enzyme to oxidize small aromatic compounds derived from lignins. This promising methodology allows the fast screening of the catalase-peroxidase activity towards small phenolic molecules, suggesting its potential role in lignin transformation. KEY POINTS: • Production and characterization of a new thermostable bacterial catalase-peroxidase • The enzyme is able to oxidize many phenolic monomers derived from lignins • Intermittent pulse amperometry is promising to screen ligninolytic enzyme.

Enhancing secondary metabolite production and antioxidants in Bacopa monnieri grown on tannery sludge contaminated soil

The current study focuses on the stimulation of Bacoside A content of the Bacopa plant that played a potential role in the treatment of Alzheimer’s disease and epilepsy. The objective of the study was to explore the potential of the B. monnieri plant for metal tolerance when cultivated on tannery sludge-contaminated soil without compromising its phytochemical quality. Bacoside content of the plant gets 2 times higher than the control without any chemical additives. Bacosides are the alleged bioactive component and attribute to the neuroprotective function. Export-import bank of India acknowledged Bacopa monnieri as the second most crucial medicine as it has metabolites that can cure mental disorders and amnesia. The effect of tannery sludge on enzymatic and non-enzymatic antioxidant potential along with the effect on metabolite i.e. bacoside A content of the plant. The maximum transfer (known as translocation factor) from root to shoot for Cr, Cd, Co, Ni and Pb was 0.33, 0.28, 0.75, 0.69 and 0.43 respectively. This depicts that here in tannery sludge mixed soil, B. monnieri was not behaving like a hyperaccumulator plant. Although it sequestered a large portion of metal in the root region and showed high vegetative growth in a combination of 75:25 tannery sludge and soil. Superoxide dismutase, Catalase, peroxidase, malondialdehyde, proline & ascorbic acid scaled up with increasing tannery sludge in the treatment whereas in the case of chlorophyll a, b, and carotenoid content, a sharp decline trend was observed at 100% tannery sludge treatment. A similar trend was recorded in bacoside A content of the plant, and the highest was 2% that of control in a 75:25 ratio of tannery sludge and soil. Enzymatic antioxidant along with non-enzymatic antioxidants protects the plant from oxidative stress generated by tannery sludge. Principal components confirm our finding that the translocation factor plays a direct role in the activation of the photosynthetic machinery. Principal component-I contributes 59% of the total variance and Principal component-II contributes 33% of the total variance.

Cacti as low-cost substrates to produce L-asparaginase by endophytic fungi.

This study aimed to select endophytic fungi to produce L-asparaginase and partially optimising the production of the enzyme using cacti as substrate. Seventeen endophytes were assessed for intracellular enzymatic potential in modified Czapek Dox's medium using L-proline as an inducer. The best producer was evaluated for intracellular and extracellular enzymatic activity in modified Czapek Dox's medium using flours of Opuntia ficus-indica and Nopalea cochenillifera as substrate. The biomass and L-asparaginase production profile was analysed and the best conditions for enzyme production were verified using factorial design. Penicillium decaturense URM 7966, Diaporthe ueckerae URM 8321, and Colletotrichum annellatum URM 8538 produced 0.76 U g- 1, 0.87 U g- 1, and 0.74 U g- 1L-asparaginase, respectively. Diaporthe ueckerae URM 8321 produced only intracellular L-asparaginase, using flours of N. cochenillifera (0.72 U g- 1) and O. ficus-indica (0.90 U g- 1) and the last was selected for the next steps. The ideal time for biomass and L-asparaginase production was 120h. The best conditions for enzyme production (1.67 U g- 1) were initial pH 4.0, inoculum concentration 1% and cacti flour concentration 0.2%; where was observed an increase of 46.11% in compared to the initial production. Opuntia ficus-indica flour is indicated as an alternative low-cost substrate for the production of L-asparaginase by the endophytic fungus D. ueckerae URM 8321.

Screening of Spore-Forming Bacteria with Probiotic Potential in Pristine Algerian Caves.

ABSTRACTThe interest and exploration of biodiversity in subsurface ecosystems have increased significantly during the last 2 decades. The aim of this study was to investigate the in vitro probiotic properties of spore-forming bacteria isolated from deep caves. Two hundred fifty spore-forming microbes were enriched from sediment samples from 10 different pristine caves in Algeria at different depths. Isolates showing nonpathogenic profiles were screened for their potential to produce digestive enzymes (gliadinase and beta-galactosidase) in solid and liquid media, respectively. Different probiotic potentialities were studied, including (i) growth at 37°C, (ii) survival in simulated gastric juice, (iii) survival in simulated intestinal fluid, and (iv) antibiotic sensitivity and cell surface properties. The results showed that out of 250 isolates, 13 isolates demonstrated nonpathogenic character, probiotic potentialities, and ability to hydrolyze gliadin and lactose in solution. These findings suggest that a selection of cave microbes might serve as a source of interesting candidates for probiotics.IMPORTANCE Previous microbial studies of subsurface ecosystems like caves focused mainly on the natural biodiversity in these systems. So far, only a few studies focused on the biotechnological potential of microbes in these systems, focusing in particular on their antibacterial potential, antibiotic production, and, to some extent, enzymatic potential. This study explores whether subsurface ecosystems can serve as an alternative source for microbes relevant to probiotics. The research focused on the ability of cave microbes to degrade two substrates (lactose and gliadin) that cause common digestive disorders. Since these enzymes may prove to be useful in food processing and in reducing the effect of lactose and gliadin digestion within intolerant patients, isolation of microbes such as in this study may expand the possibilities of developing alternative strategies to deal with these intolerances.

Enzymatic Potential of Filamentous Fungi as a Biological Pretreatment for Acidogenic Fermentation of Coffee Waste.

Spent coffee grounds (SCGs) are a promising substrate that can be valorized by biotechnological processes, such as for short-chain organic acid (SCOA) production, but their complex structure implies the application of a pretreatment step to increase their biodegradability. Physicochemical pretreatments are widely studied but have multiple drawbacks. An alternative is the application of biological pretreatments that include using fungi Trametes versicolor and Paecilomyces variotii that naturally can degrade complex substrates such as SCGs. This study intended to compare acidic and basic hydrolysis and supercritical CO2 extraction with the application of these fungi. The highest concentration of SCOAs, 2.52 gCOD/L, was achieved after the acidification of SCGs pretreated with acid hydrolysis, but a very similar result, 2.44 gCOD/L, was obtained after submerged fermentation of SCGs by T. versicolor. This pretreatment also resulted in the best acidification degree, 48%, a very promising result compared to the 13% obtained with the control, untreated SCGs, highlighting the potential of biological pretreatments.

Isolation, in vitro evaluation and construction of Versatile Microbial Consortia.

In this study, we constructed versatile microbial consortia (VMC) with agricultural and environmental interest. After sample and isolation procedure, the purified isolates were evaluated for their enzymatic potential such as cellulose-, xylan-, petroleum-, proteins -hydrolysis. Selected isolates were screened for other traits such as phosphate solubilization, nitrogen fixation and antimicrobial activity. Finally, the isolates were grouped into consortia according to their compatibility. The microorganisms selected for each consortium were identified by partial analysis of the 16S rRNA (bacteria) and the ITS region of the 18S RNA gene (fungi). Two microbial consortia were obtained and named VMC1 and VMC2. These two consortia are characterized by several activities of agricultural and environmental interest such as the degradation of recalcitrant and polluting organic compounds, nitrogen fixation, IAA production phosphate solubilization and antimicrobial activity. Molecular identification of the microorganisms forming the two consortia allowed us to identify two species of actinomycetes (Streptomyces sp. BM1B and Streptomyces sp. BM2B), one species of Actinobacteria (Gordonia amicalis strain BFPx) and three fungal species (Aspergillus luppii strain 3NR, Aspergillus terreus strain BVkn and Penicillium sp. BM3). The term "Versatile Microbial Consortia" is a term that we proposed in this study to establish a methodology for building multifunctional microbial groups for wide and efficient application.

New biologically potent benzimidazole‐based‐triazole derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors along with molecular docking study

AbstractA series of fifteen hybrid analogs (1–15) based on benzimidazole bearing triazole skeleton were synthesized and were evaluated for in vitro AChE and BuChE inhibition profile. All the newly afforded scaffolds showed moderate to good AChE and BuChE activity having IC50 values ranging from 0.40 ± 0.05 to 19.60 ± 0.40 μM against AChE and 1.50 ± 0.10 to 23.30 ± 0.50 μM against BuChE in comparison to Donepezil (IC50 = 0.016 ± 0.12 μM for AChE), (IC50 = 4.5 ± 0.11 μM for BuChE) as reference drug. It was concluded from structure–activity relationship (SAR) studies that scaffolds bearing groups of strong e‐withdrawing nature (–NO2, –F and –Cl) at different position of aryl ring were found to be potent inhibitors of both targeted AChE and BuChE enzymes. Therefore, compounds 7 (bearing para‐NO2), 2 (bearing para‐fluoro), 6 (bearing meta‐NO2) and 8 (bearing meta‐fluoro) were identified as the most active on screening against AChE enzyme with IC50 values of 0.40 ± 0.05, 0.60 ± 0.05 and 0.80 ± 0.05 and 0.90 ± 0.05 μM. These scaffolds 7, 2, 6 and 8 also showed superior potency against BuChE enzyme with IC50 values of 1.50 ± 0.10, 2.10 ± 0.10, 2.60 ± 0.10 and 2.30 ± 0.10 μM. It was also observed that inhibition profile of synthesized scaffolds against both these targeted enzymes was greatly affected by altering either position or number/s of substituent around aryl ring. Moreover, it was noteworthy that scaffolds that hold substituent of bulky nature are unable to interact better with active site of targeted enzymes and hence, lower the enzymatic potentials against targeted enzymes. In addition, the active scaffolds such as 7 and 2 were further subjected to molecular docking studies and result obtained revealed that these active scaffolds interacts well both with AChE and BuChE enzymes and forms several key interactions (conventional hydrogen bond, π‐cation, π‐π stacking, π‐π T‐shaped, π‐sulfur and π‐alkyl) with active site of these targeted enzymes. Furthermore, newly afforded scaffolds were characterized by using various spectroscopic tools such as HR‐EI‐MS, 13C NMR and 1HNMR spectroscopy.

Diabetic bone regeneration with nanoceria-tailored scaffolds by recapitulating cellular microenvironment: Activating integrin/TGF-β co-signaling of MSCs while relieving oxidative stress

Regenerating defective bone in patients with diabetes mellitus remains a significant challenge due to high blood glucose level and oxidative stress. Here we aim to tackle this issue by means of a drug- and cell-free scaffolding approach. We found the nanoceria decorated on various types of scaffolds (fibrous or 3D-printed one; named nCe-scaffold) could render a therapeutic surface that can recapitulate the microenvironment: modulating oxidative stress while offering a nanotopological cue to regenerating cells. Mesenchymal stem cells (MSCs) recognized the nanoscale (tens of nm) topology of nCe-scaffolds, presenting highly upregulated curvature-sensing membrane protein, integrin set, and adhesion-related molecules. Osteogenic differentiation and mineralization were further significantly enhanced by the nCe-scaffolds. Of note, the stimulated osteogenic potential was identified to be through integrin-mediated TGF-β co-signaling activation. Such MSC-regulatory effects were proven in vivo by the accelerated bone formation in rat calvarium defect model. The nCe-scaffolds further exhibited profound enzymatic and catalytic potential, leading to effectively scavenging reactive oxygen species in vivo. When implanted in diabetic calvarium defect, nCe-scaffolds significantly enhanced early bone regeneration. We consider the currently-exploited nCe-scaffolds can be a promising drug- and cell-free therapeutic means to treat defective tissues like bone in diabetic conditions.

Characterization by polyphasic approach of some indigenous halophilic archaea of Djelfa's rock salt "Hadjr el Meelh", Algeria.

Hadjr El Melh of Djelfa is an example of hypersaline ecosystems, which can harbor a wide variety of microorganisms under hostile physicochemical conditions. Given the importance of the study of halophilic microorganisms present there in terms of fundamental and applied microbiology, the purpose of this study was to characterize some halophilic archaea isolated from the brines of this environment. Eight water samples were chosen randomly and collected for physicochemical and microbiological analyses. Isolation of halophilic archaea was carried out by membrane filter technique. Ten strains were identified by polyphasic approach and tested for enzymes production. Water samples of Djelfa's rock salt were slightly acidic to neutral in pH (6.55-7.36) with salinity ranging from 258.68 g/l to 493.91 g/l. Phenotypic, biochemical, taxonomic and phylogenetic characteristics indicated that all strains were classified within the family of Halobacteiaceae. Based on the comparison of DNA sequences encoded 16S rRNA, it was determined that seven strains were affiliated to the genus Haloarcula, two strains were related to the genus Halobacterium and one strain within the genus Haloferax. Production of different enzymes such as protease, amylase, esterase, lipase, lecithinase, gelatinase and cellulase on solid medium indicated that one strain (S2-2) produced amylase, esterase, lecithinase and protease. However, no strains showed cellulolytic or lipolytic activity. Gelatinase was found in all tested strains. This report constitutes the first preliminary study of culturable halophilic archaea recovered from the brines of Djelfa's rock salt with a promising enzymatic potential in various fields of biotechnology.

Unraveling the enzymatic and antibacterial potential of rare halophilic actinomycetes from Algerian hypersaline wetland ecosystems.

The study aimed to isolate rare halophilic actinomycetes from hypersaline soils of Algerian inland Wetland Ecosystems "Sebkhas-Chotts" located in arid and hot hyperarid lands with international importance under the Ramsar Convention and to explore their enzyme-producing and antibacterial abilities. The halophilic actinomycetes were selectively isolated using agar-rich media supplemented with 5, 10, and 15% (W/V) of total salts. Thirty-one isolates were obtained and 16S rRNA gene sequencing analysis revealed the presence of members affiliated to rare halophilic actinobacterial genera (Actinopolyspora and Nocardiopsis) accounting for 74.19% (23 isolates out of 31) and 25.8% (8 isolates), respectively. Both phylotypes are alkalitolerant and halophilic thermotolerant actinomycetes displaying significant hydrolytic activities relative to (amylase, asparaginase, cellulase, esterase, glutaminase, inulinase, protease, pectinase, xylanase), and over 96% of tested isolates exhibited all common enzymes, mainly active at 10% of growing salt. In addition, high antibacterial activity was observed against Bacillus cereus, Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus. The findings showed that saline wetlands ecosystems represent a rich reservoir for the isolation of significant rare halophilic actinomycetes with potential adaptive features and valuable sources for novel bioactive metabolites and biocatalysts of biotechnological interest.

Microwave-Assisted Enzymatic Extraction, Partial Characterization, and Antioxidant Potential of Polysaccharides from Sagittaria trifolia Tuber.

Sagittaria trifolia tuber is an aquatic vegetable. In this work, microwave-assisted enzymatic extraction (MEE) was used to extract S. trifolia tuber polysaccharides (STTPs). Optimum conditions were complex enzyme of 2 %, liquid-to-solid ratio of 43 : 1 mL g-1 , microwave power of 506 W, and time of 8 min, under which STTPs yield was 36.22±0.69 %, higher than those of other methods. STTPs were sulfated polysaccharides with sulfur valence of S6+ . STTPs comprised mannose, glucose, galactose, and arabinose at a mole ratio of 3.69 : 19.33 : 6.21 : 1.00, molecular weights of 3606 kDa and 149.6 kDa, particle size of 220 nm, and zeta potential of -5.02 mV. The surface of STTPs was full of bumps and holes, and abundant in O1s and non-functionalized C1s. STTPs would scavenge reactive oxygen species with advantage. It would provide an efficient MEE method to obtain antioxidant STTPs, also a clue for extracting polysaccharides from starch-rich crops.

Phytochemical and Bioactivities of Male Flower Buds of Fruit Trees from the Southern Tunisia: Polyphenols UPLC-MS Profiles and Antioxidant Enzymatic Potential in Human Plasma of Parkinson's Disease Patients

Phytochemical and Bioactivities of Male Flower Buds of Fruit Trees from the Southern Tunisia: Polyphenols UPLC-MS Profiles and Antioxidant Enzymatic Potential in Human Plasma of Parkinson's Disease Patients

Isolasi dan Karakterisasi Molekuler Gen 16S rRNA Bakteri Lipolitik Asal Limbah Kulit Biji Jambu Mete

Lipolytic bacteria attract great attention to various biotechnology industries because of their enzymatic potential. This study aims to isolate and identify lipolytic bacteria from cashew nutshell waste using the 16S rRNA gene as a molecular marker. Lipolytic bacteria were isolated using serial dilutions and inoculated on lipolytic media. A total of 3 isolates of lipolytic bacteria were obtained from cashew nutshell waste based on screening in LA Rhodamine B. The partial sequence of 16S rRNA gene from LB15 amplified using a pair of primers 63F and 1387R having a size of 1238 bp, while BL6 and BK6 were 1283 bp, respectively. Based on genetic distance analysis and phylogenetic reconstruction, we proposed that LB15 be identified as Burkholderia sp. with 99.92% similarity. In addition, because the 16S rRNA gene sequence similarity of BL6 was 99.87% with Paraburkholderia kururiensis strain 979, BL6 was classified as Paraburkholderia kururiensis. Then, isolate BK6 was identified as Ralstonia sp. with a similarity of 99.53%. The similarity value can be used as a reference in determining the identity of bacteria. A bacterium can be categorized as the same species if it has a similarity value of more than 99%.

Identification of lipolytic enzymes using high-throughput single-cell screening and sorting of a metagenomic library

The demand for novel, robust microbial biocatalysts for use in industrial and pharmaceutical applications continues to increase rapidly. As a result, there is a need to develop advanced tools and technologies to exploit the vast metabolic potential of unculturable microorganisms found in various environments. Single-cell and functional metagenomics studies can explore the enzymatic potential of entire microbial communities in a given environment without the need to culture the microorganisms. This approach has contributed substantially to the discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples, constructing expression libraries comprising the entire microbial genome, and screening of the libraries for the presence of desired phenotypes. In this study, lipolytic enzymes from the Red Sea were targeted. A high-throughput single-cell microfluidic platform combined with a laser-based fluorescent screening bioassay was employed to discover new genes encoding lipolytic enzymes. Analysis of the metagenomic library led to the identification of three microbial genes encoding lipases based on their functional similarity and sequence homology to known lipases. The results demonstrated that microfluidics is a robust technology that can be used for screening in functional metagenomics. The results also indicate that the Red Sea is a promising, under-investigated source of new genes and gene products.