Extracellular Esterase Research Articles

Isolation and characterization of a novel bacterium that promotes the degradation of poly(glycolic acid) by its extracellular esterase under thermophilic conditions

Poly(glycolic acid) (PGA) is widely utilized in the shale oil and gas industry owing to its biodegradable nature, as well as superior mechanical and barrier properties. However, PGA degradability is limited by environmental conditions such as temperature and pH, and using acids to optimize the degradation can have adverse environmental impact. Thus, it is essential to identify methods that can effectively promote the degradation of PGA under mild conditions, such as microbial degradation. In the present study, strain DB14, a bacterium that promotes PGA degradation, was isolated from drain water of a steam pipeline. Sequence analysis of the 16S rRNA gene of the bacterium revealed that it is mostly closely related to Geobacillus icigianus; however, it also differed from Geobacillus icigianus in several physiological properties. To investigate PGA degradation by strain DB14, a PGA film and disc were incubated with the strain. The residual weight of the PGA film (thickness: 170 μm) significantly reduced after incubation, whereas the decrease in the thickness of the PGA disc (thickness: 3 mm) was relatively small. The penetration of water, the bacterium, and the extracellular enzymes into the interior from the reaction-erosion front of the PGA disc may be inhibited by the high barrier performance of PGA. Strain DB14 was also found to change the pH of the surrounding environment to approximately 8–9. To investigate the effect of pH on PGA degradability, degradation tests with crude extracellular enzymes derived from strain DB14 were conducted in various buffers. The results showed that the degradation activity was highest at pH 8, which implied that DB14 efficiently maximized the hydrolytic capacity of its enzyme for degrading PGA. Thus, this study provides a basis for developing environmentally friendly technologies that can promote the degradation of PGA molding articles, especially those used in wellbores for oil and gas recovery.

Biodegradation of polybutylene succinate by an extracellular esterase from Pseudomonas mendocina

An extracellular esterase (HP) with polybutylene succinate (PBS)-degrading ability was identified from Pseudomonas mendocina SA-1503. The HP also had the ability to degrade poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and polycaprolactone. This HP had optimal activity at pH 9.0 and 40 °C and remained stable at pH 8.0–9.0 and temperatures of 30–40 °C. Mn2+ promoted the enzyme activity. HP could hydrolyze all p-NP fatty acid ester substrates containing even numbers of carbon atoms from C2 to C18 and had the highest catalytic activity for the p-NP C6 substrate. After 60 h of HP-catalyzed degradation, PBS films experienced a weight loss of more than 60%. Butanedioic acid, 1,4-butanediol, and a series of oligomers were detected in the degradation products of PBS by HP. Further structural analysis of HP revealed that it could be classified as a microbial esterase of α/β hydrolase superfamily and contained a conserved catalytic triad structure (Ser-148, Asp-198, and His-228) with a relatively exposed active site.

Insights into the molecular mechanisms of lipid transformation in sturgeon fillets: Interplay between specific spoilage and dominant bacteria

This study investigates spoilage bacteria's impact on lipid metabolism in sturgeon fillets using UHPLC-Q-Orbitrap-MS/MS-based untargeted lipidomic analysis. A total of 4041 lipid molecules across five classes and 42 subclasses were identified, including glycerophospholipids (GPs, 50.88%), glycerolipids (GLs, 36.08%), sphingolipids (SPs, 10.47%), fatty acyls (FAs, 2.45%), and sterol lipids (STs, 0.12%). Aeromonas sobria, a specific spoilage bacterium, reduced GPs and FAs while increasing GLs, SPs, and STs via extracellular lipases and esterases. Acinetobacter albensis, the dominant bacterium, mainly elevated SPs and FAs. Their interaction promoted lipid metabolism and oxidation while producing volatile organic compounds (VOCs). Ethyl isobutyrate, ethyl propionate, isobutyl formate, pentan-2-one, propan-2-one, 2-butanone, 3-methyl-3-buten-1-ol, and dimethyl sulfide were mainly associated with Acinetobacter albensis, while 1-hexanol, 1-pentanol, 1-penten-3-ol, 1-hydroxypropan-2-one, 3-methyl-1-butanol, 2-methylbutanal, 3-hydroxy-2-butanone, and propionaldehyde were mainly related to Aeromonas sobria. This work unveils the mechanism of lipid transformation in sturgeon fillets during refrigerated storage, offering insights for aquatic products quality control.

Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum

BackgroundXylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster.ResultsGenetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD’EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw.ConclusionsThis study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

Multiple strategies to improve extracellular secretion and activity of feruloyl esterase

Feruloyl esterase has a wide range of applications, but there are still problems with low enzyme yield and activity, and complex purification steps. Our previous research found Lactobacillus amylovorus feruloyl esterase could be secreted extracellular in Escherichia coli. In this study, multiple strategies were implemented to maximize the extracellular production of feruloyl esterase with improved activity in E. coli. Firstly, codon-optimized feruloyl esterase was obtained based on the preference of E. coli, resulting in 41.97 % increase in extracellular secretion. Furthermore, by cascading T7 promoters, replacing the 5′ UTR, randomly mutating the N-terminal sequence, and co-expressing secretory cofactors, the extracellular secretion was increased by 36.46 %, 31.25 %, 20.66 % and 25.75 %, respectively. Moreover, the feruloyl esterase were mutated to improve the substrate affinity and activity. The catalytic efficiency of Fae-Q134T and Fae-Q198A increased by 4.62-fold and 5.42-fold. Combining above strategies, extracellular feruloyl esterase activity was increased from 2013.70 U/L to 10,349.04 U/L. These results indicated that the activity and yield of feruloyl esterase secreted by E. coli were significantly increased, which laid a foundation for its industrial application.

Polyurethane degradation by extracellular urethanase producing bacterial isolate Moraxella catarrhalis strain BMPPS3

Plastic waste has become a global issue and a threat to the ecosystem. The present study isolated polyurethane (PU) degrading bacterial species from soil dumped with plastic wastes. Four bacterial isolates, RS1, RS6, RS9 and RS13 were obtained and their ability to degrade PU in a synthetic medium with PU as a sole source of carbon was assessed individually. After thirty days of incubation, the highest PU weight loss of 67.36 ± 0.32% was recorded in the medium containing RS13 isolate. The results of FTIR revealed the occurrence of carbonyl peaks. The putative isolate RS13 confirmed with the genus Moraxella according to 16S rRNA gene sequencing and the isolate was specified as Moraxella catarrhalis strain BMPPS3. The restriction analysis of Moraxella catarrhalis strain BMPPS3 revealed that the GCAT content to 51% and 49% correspondingly. Moraxella catarrhalis strain BMPPS3 was able to colonize on PU surface and form a biofilm as revealed by SEM investigation. Fatty acids and alkanes were found to be the degradation products by GC-MS analysis. The presence of these metabolites facilitated the growth of strain RS13 and suggested that ester hydrolysis products had been mineralized into CO2 and H2O. Extracellular biosurfactant synthesis has also been found in Moraxella catarrhalis strain BMPPS13 inoculated with synthetic media and mineral salt media containing PU and glucose as carbon sources, respectively with a significant level of cell-surface hydrophobicity (32%). The production and activity of extracellular esterase showed consistent increase from day 1–15 which peaked (1.029 mM/min/mg) on day 24 significantly at P < 0.001. Crude biosurfactants were lipopeptide-based, according to the characteristic investigation. According to this study findings, Moraxella catarrhalis produces biosurfactants of the esterase, urethanase and lipase (lipopeptide) types when carbon source PU is present.

Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Novel perspective on the spoilage metabolism of refrigerated sturgeon fillets: Nonspecific spoilage dominant organisms play an important role

Aeromonas sobria, a specific spoilage organism (SSO) in refrigerated (4 °C) sturgeon fillets, is considered as the main contributor for sturgeon spoilage. However, the spoilage metabolism of A. sobria is influenced by bacterial interactions, and the effect of nonspecific spoilage dominant organisms on the spoilage metabolism of A. sobria during storage is still unknown. In this study, the spoilage characteristics and metabolite changes of different inoculation groups under chilled storage were investigated through a co-cultivation model. The results of spoilage indicators indicated that the microbial interactions changed the spoilage characteristic of the fish model, although A. sobria played the most important role. Based on metabolome results, 549 differential metabolites were identified in two ion modes, which involved 9 chemical categories and 4 main metabolic pathways. The nonspecific spoilage dominant bacteria M. caseolyticus in the early stage of refrigeration affects the amino acid metabolism pathways of fish model by secreting extracellular proteases and peptidases. However, the nonspecific spoilage dominant bacteria A. albensis in the later period of refrigeration accelerate the lipid metabolism, and amino acid metabolism via secreting extracellular peptidases, lipase and esterase. This work may provide insights on controlling the spoilage of aquatic products by intervening degradation pathways.

Communities of culturable freshwater fungi present in Antarctic lakes and detection of their low-temperature-active enzymes.

We evaluated the diversity and enzymatic activities of culturable fungi recovered from cotton baits submerged for 2years in Hennequin Lake, King George Island, and from benthic biofilms in Kroner Lake, Deception Island, South Shetland Islands, maritime Antarctica. A total of 154 fungal isolates were obtained, representing in rank abundance the phyla Ascomycota, Basidiomycota and Mortierellomycota. Thelebolus globosus, Goffeauzyma sp., Pseudogymnoascus verrucosus and Metschnikowia australis were the most abundant taxa. The fungal community obtained from the biofilm was more diverse and richer than that recovered from the cotton baits. However, diversity indices suggested that the lakes may harbour further fungal diversity. The capabilities of all cultured fungi to produce the extracellular enzymes cellulase, protease, lipase, agarase, carrageenase, invertase, amylase, esterase, pectinase, inulinase and gelatinase at low temperature were evaluated. All enzymes were detected, but the most widely produced were protease and pectinase. The best enzymatic indices were obtained from Holtermanniella wattica (for invertase, esterase), Goffeauzyma sp. (amylase), Metschnikowia australis (protease), Mrakia blollopis (cellulase, pectinase), Pseudogymnoascus verrucosus (agarase, carrageenase) and Leucosporidium fragarium (inulinase). The detection of multiple enzymes reinforces the ecological role of fungi in nutrient cycling in Antarctic lakes, making nutrients available to the complex aquatic food web. Furthermore, such low-temperature-active enzymes may find application in different biotechnological processes, such as in the textile, pharmaceutical, food, detergent and paper industries, as well as environmental application in pollutant bioremediation processes.

Biodegradation of Polyester Polyurethane by Embarria clematidis.

Polyester urethanes (PUR) are widely used in industries and have led to a worldwide plastic waste problem. Thus, novel solutions for PUR degradation are required to reduce environmental pollution. This work investigates the PUR biodegradation efficiency of 33 fungal species using a polyester-polyurethane colloid branded Impranil DLN (Impranil) compared to Aspergillus niger, which served as the positive control. The biodegradation is evaluated based on its ability to clear Impranil in media. Eleven fungi can clear Impranil in both solid- and liquid-medium assays. The highest degradation was attributed to Embarria clematidis cultured with Impranil as a carbon source. The degradation was confirmed by the Sturm test, Fourier-transform infrared (FTIR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). From the Sturm test, CO2 at a concentration of 0.85 g/L was found in E. clematidis cultured with 150 mL of Impranil solution after a 2-week incubation period while the CO2 at a concentration of 0.53 g/L was detected from A. niger in the same conditions. The biodegradation was further confirmed by evaluating the clearance percentage of supernatant of E. clematidis and A. niger culturing with Impranil from the Sturm test. The clearance percentage of E. clematidis and A. niger supernatant was 88.84 and 48.97%, respectively. Moreover, the degradation of soft segment and breakdown of ester linkages were observed, as evidenced by the decrease of the carbonyl (1,715 cm–1) and N-H stretching (1,340 cm–1 and 1,020 cm–1) FTIR spectral peaks, respectively. GC-MS detected 3Z-heptenol, 5Z-octenol, 2E,4E-hexadienol acetate, and 3E,6Z-nonadienol as degradation products from the E. clematidis culture supernatant. This fungus was screened for its ability to produce extracellular esterase, protease, and urease enzymes. Extracellular esterase, very low urease, and no protease activities were detected in the culture supernatant of E. clematidis in the presence of Impranil. E. clematidis can degrade Impranil partially via hydrolysis of ester linkages by cell-bound esterases at a considerable rate without any prior treatment. This fungus not only degraded Impranil but also mineralized them into CO2 and H2O. E. clematidis can be applied in the process of biochemical depolymerization of PUR for the pure monomers recycling.

Concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities during Impranil biodegradation by Alicycliphilus denitrificans BQ1.

The concerted action of commercial esterases, proteases and amidases has been demonstrated to be relevant in polyurethane (PU) degradation by in vitro experiments. However, the spatial and temporal dynamics of these activities during PU biodegradation by PU-degrading bacteria have not been addressed. Here, we examined the capability of Alicycliphilus denitrificans BQ1 to biodegrade the polyester (PS)-PU Impranil, analyzed the temporal and spatial coordination between the extracellular and cytoplasmic esterase and urethane-cleaving activities, and their independent and combined effects on Impranil biodegradation. A. denitrificans BQ1 grew in Impranil, and its clearing was correlated with the cleavage of ester and urethane groups since early times, with decrements of some Impranil compounds and the appearance of biodegradation products. While extracellular esterase was active at early times with its maximum at 18h, urethanase appeared at this time and increased up to the end of the analysis (48h), with the cytoplasmic activities behaving similarly but with lower levels than the extracellular ones. Both enzymatic activities exhibited distinct substrate specificity depending on their cellular localization and cultivation times, suggesting they cleave differentially located groups. As the urethane cleavage occurred since early times, when no urethane-cleaving activity was detected, different proteins should be acting at early and late times. In vitro experiments with independent or combined cellular protein fractions supported the previous deduction and confirmed the concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities. A two-stage process for Impranil degradation by A. denitrificans BQ1 is proposed.

Arabidopsis GELP7 functions as a plasma membrane-localized acetyl xylan esterase, and its overexpression improves saccharification efficiency.

Acetyl substitution on the xylan chain is critical for stable interaction with cellulose and other cell wall polymers in the secondary cell wall. Xylan acetylation pattern is governed by Golgi and extracellular localized acetyl xylan esterase (AXE). We investigated the role of Arabidopsis clade Id from the GDSL esterase/lipase or GELP family in polysaccharide deacetylation. The investigation of the AtGELP7 T-DNA mutant line showed a decrease in stem esterase activity and an increase in stem acetyl content. We further generated overexpressor AtGELP7 transgenic lines, and these lines showed an increase in AXE activity and a decrease in xylan acetylation compared to wild-type plants. Therefore, we have named this enzyme as AtAXE1. The subcellular localization and immunoblot studies showed that the AtAXE1 enzyme is secreted out, associated with the plasma membrane and involved in xylan de-esterification post-synthesis. The cellulose digestibility was improved in AtAXE1 overexpressor lines without pre-treatment, after alkali and xylanases pre-treatment. Furthermore, we have also established that the AtGELP7 gene is upregulated in the overexpressor line of AtMYB46, a secondary cell wall specific transcription factor. This transcriptional regulation can drive AtGELP7 or AtAXE1 to perform de-esterification of xylan in a tissue-specific manner. Overall, these data suggest that AtGELP7 overexpression in Arabidopsis reduces xylan acetylation and improves digestibility properties of polysaccharides of stem lignocellulosic biomass.

Statistical optimisation of esterase from Salinicoccus roseus strain RF1H and its potential application in synthetic dye decolorisation

Halophilic bacteria isolated from Indian Sundarban Wetland, identified as Salinicoccus roseus strain RF1H, were screened for extracellular esterase production. Media components for esterase production under submerged fermentation were optimised using Plackett–Burman Design followed by Box-Behnken Design of Response Surface Methodology (RSM) at pH 4.0, 3.5 g/L of MgSO4, and tributyrin concentration of 1(%v/v). The regression model was significant with a determination coefficient (R 2) value of 0.826. Using RSM, the experimental value of maximum enzyme activity (2.442 UmL−1) was 1.35 fold increased. The kinetic parameters K m and V max for hydrolysis of p-nitrophenyl acetate were 9.98 mM and 2.26 µmol/min/mg respectively, establishing a high affinity for the substrate. Further, the enzyme conferred promising results in decolorisation of synthetic dyes -Malachite Green, Congo Red, Brilliant Yellow, and Orange G. Molecular docking was used to investigate the binding pockets of the esterase, enzyme-ligand interactions, and amino acids which played a key role in dye decolorisation. Docking score results and decolorisation percentage were found in good agreement. This is the first report on the biotransformation of synthetic dyes to non-toxic products using esterase from Salinicoccus roseus.

Exploring the polyurethanolytic activity and microbial composition of landfill microbial communities.

The microbial composition of polyurethane degrading communities has been barely addressed, and it is unknown if microenvironmental conditions modify its composition, affecting its biodegradative capacity. The polyurethanolytic activity and taxonomic composition of five microbial communities, selected by enrichment in the polyether-polyurethane-acrylic (PE-PU-A) coating PolyLack®, from deteriorated PU foams collected at different microenvironments in a municipal landfill (El Bordo Poniente, BP) were explored. All BP communities grew similarly in PolyLack® as the sole carbon source, although BP1, BP4, and BP5 showed better performance than BP2 and BP7. FTIR spectroscopy showed that ester, urethane, ether, aromatic and aliphatic groups, and the acrylate component were targets of the biodegradative activity. Extracellular esterase activity was higher at 5days of cultivation and decreased at 21days, while urease activity showed the opposite. Microbial composition analysis, assessed by 16S rDNA V3 region PCR-DGGE, revealed a preponderance of Rhizobiales and Micrococcales. The reported PU-degrading genera Paracoccus, Acinetobacter, and Pseudomonas were identified. In contrast, Advenella, Bordetella, Microbacterium, Castellaniella, and Populibacterium, some of them xenobiotics degraders, can be considered potentially PU-degrading genera. Correspondence analysis identified independent groups for all communities, except the BP4 and BP5. Although partial taxonomic redundancy was detected, unique OTUs were identified, e.g., three members of the Weeksellaceae family were present only in the BP4/BP5 group. These results suggest that the microenvironmental conditions where the landfill microbial communities were collected shaped their taxonomical composition, impacting their PE-PU biodegradative capacities. These BP communities represent valuable biological material for the treatment of PU waste and other xenobiotics. KEY POINTS: • Landfill microbial communities display slightly different capacities for growing in polyether-polyurethane-acrylic. • Ester, urethane, ether, aromatic, aliphatic, and acrylate groups were attacked. • Esterase activity was more significant at early culture times while urease activity at latter. • Landfill microenvironments shape partial taxonomical redundancy in the communities. • Best communities' performance seems to be related to unique members' composition.

Optimization of culture conditions by response surface methodology for production of extracellular esterase from Serratia sp. EST-4

ObjectiveThe aim of present study was to determine the effect of interaction among different production variables on esterase production from Serratia sp. EST-4. MethodThe optimum and combined effect of independent parameters were determined using Plackett-Burman and Central Composite Design (CCD) (Design Expert 11). ResultsThe effect of six parameters-cotton seed oil, peptone and maltose concentrations, pH, temperature and inoculum volume for production of esterase was determined by Plackett-Burman design. Four factors i.e. inoculum size, pH, peptone concentration and cotton seed oil concentration had significant effect on yield of esterase as suggested by the Pareto chart. Under optimized conditions, (inoculum size (1.0%, v/v), pH (8.0), peptone concentration (1.5%, w/v) and cotton seed oil concentration (4.0%, v/v)), the maximum esterase production was 9.77 U/ml. Analysis of variance (ANOVA) of the Central Composite Design (CCD)-based experiment exhibited the model F-value of 44.24 and P-value of <0.0001 which implies that model was significant and adequate to represent the system. ConclusionInoculum size 1.0%, v/v, pH 8.0, concentration of peptone 1.5%, w/v and concentration of cotton seed oil 4.0%, v/v had positive effect on esterase production and provided 1.52-fold increase in esterase production than that obtained by optimizing one factor at one time.

Purification of high molecular weight thermotolerant esterase from Serratia sp. and its characterization.

In the present study, an extracellular esterase from Serratia sp. was purified 24.46 fold using an initial ammonium sulphate precipitation step (optimized concentration of 30-40%), followed by Diethylaminoethyl cellulose (DEAE-cellulose) chromatography and size exclusion Sephadex G-200 column chromatography steps. The molecular weight of the esterase using native polyacrylamide gel electrophoresis (PAGE) was determined to be 236kDa and by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was found to be 60kDa suggesting that the enzyme was a tetramer of 4 subunits. The purified esterase was able to catalyze the hydrolysis of p-nitrophenyl esters, especially p-nitrophenyl acetate. Maximum esterase activity was achieved in 0.15M Tris-HCl buffer of pH 8.5 at 50°C after 10min. The enzyme was stable for at least 8h at 4 and 35°C but the half-life was determined to be 4.5h at 50°C and 3h at 60°C. The esterase activity was inhibited by detergents (1mM) (Triton X-100, Tween 60, Tween 80, ethylenediamine tetraacetic acid and SDS) except Tween 20. The esterase activity was inhibited by organic solvents (1mM) such as ethanol, methanol, acetone, acetonitrile and was stable in the presence of glycerol, isopropanol but the organic solvent dimethyl sulfoxide (DMSO) significantly (p < 0.05) enhanced esterase activity. The matrix-assisted laser desorption ionization-time of flight mass spectrometry showed that the enzyme exhibited similarity with the pimeloyl-[acyl carrier protein] methyl ester esterase of Serratia marcescens.

SELECTION OF AUTOCHThONOUS sACCHAROMYCES AND NON-sACCHAROMYCES YEASTS STRAINS ACCORDING TO THEIR EXTRACELLULAR ENZIMATIC ACTIVITY

The aim of the present study was to investigate the production of extracellular enzymes in a number of twenty six autochtonous Saccharomyces and nonSaccharomyces strains selected in Dealu Mare region for wine production. The strains were screened for the production of extracellular b-glucosidase, esterase, pectinase and protease activity by inoculation the yeast strains onto selective media. All Saccharomyces tested strains showed at least two enzymatic activities while nonSaccharomyces strains showed activity at least for one enzyme. The weakest activity was recorded in case of β-glucosidase. Most of the tested strains exhibit more or less intense activity for polygalacturonase/pectinase and protease. This study put into evidence the potential of autochtonous and especially of non-Saccharomyces strains as source of production of secondary compounds which can play an important role in improving the quality of wines.

Complete genome sequence of Sphingobium sp. strain PAMC 28499 reveals a potential for degrading pectin with comparative genomics approach.

Spingobium sp. PAMC 28499 is isolated from the glaciers of Uganda. Uganda is a unique region where hot areas and glaciers coexist, with a variety of living creatures surviving, but the survey on them is very poor. The genetic character and complete genome information of Sphingobium strains help with environmental studies and the development of better to enzyme industry. In this study, complete genome sequence of Spingobium sp. PAMC 28499 and comparative analysis of Spingobium species strains isolated from variety of the region. Genome sequencing was performed using PacBio sequel single-molecule real-time (SMRT) sequencing technology. The predicted gene sequences were functionally annotated and gene prediction was carried out using the program NCBI non-redundant database. And using dbCAN2 and KEGG data base were degradation pathway predicted and protein prediction about carbohydrate active enzymes (CAZymes). The genome sequence has 64.5% GC content, 4432 coding protein coding genes, 61 tRNAs, and 12 rRNA operons. Its genome encodes a simple set of metabolic pathways relevant to pectin and its predicted degradation protein an unusual distribution of CAZymes with extracellular esterases and pectate lyases. CAZyme annotation analyses revealed 165 genes related to carbohydrate active, and especially we have found GH1, GH2, GH3, GH38, GH35, GH51, GH51, GH53, GH106, GH146, CE12, PL1 and PL11 such as known pectin degradation genes from Sphingobium yanoikuiae. These results confirmed that this Sphingobium sp. strain PAMC 28499 have similar patterns to RG I pectin-degrading pathway. In this study, isolated and sequenced the complete genome of Spingobium sp. PAMC 28499. Also, this strain has comparative genome analysis. Through the complete genome we can predict how this strain can store and produce energy in extreme environment. It can also provide bioengineered data by finding new genes that degradation the pectin.

Purification and Properties of an Esterase from Bacillus licheniformis and it’s Application in Synthesis of Octyl Acetate

Background: Esterase plays a major role in the degradation of natural materials, industrial pollutants and also provides an immense contribution to the eco-friendly approaches in various industrial applications. Objective: In the present study, extracellular esterase from bacterial isolate Bacillus licheniformis was purified, characterized and used in the synthesis of octyl acetate. Methods: Purification of esterase from Bacillus licheniformis was achieved using Sephadex G-75 column chromatography. Gas chromatography was used to analyze the octyl acetate synthesis. Results: The enzyme was salted out using ammonium sulphate precipitation and 60-70% saturation gave maximum specific activity of the enzyme during precipitation. A purification fold of 6.46 and yield of 9.69% was achieved when esterase from Bacillus licheniformis was purified using Sephadex G-75 column chromatography. Native as well as SDS-PAGE analysis gave a single band of 42 kDa. This showed that the enzyme was purified to homogeneity and it was a monomer with molecular weight of 42 kDa. Biochemical characterization of the enzyme revealed that it had optimum temperature of 45°C in 0.1 M Tris-HCl buffer of pH 8.0. On optimizing different parameters, such as molar ratio of reactants, incubation time, temperature, and amount of protein, the % yield of octyl acetate was found to be 77.3%. Conclusion: In this work, simple method was used to purify esterase and the enzyme was further used in producing esters/products of commercial value within a reasonably short period of 12 h with a maximum yield of 77.3%.

Influence of Culture Conditions on the Production of Extracellular Esterase from Bacillus licheniformis and Its Characterization.

Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. In the present study, thirty-seven bacterial isolates were isolated from soil contaminated with waste cooking oil, dairy waste etc. from Shimla and Solan district of H.P. Out of 37 isolates, the isolate RL-1, which gave maximum activity, was identified as Bacillus licheniformis MH061919. The optimization of various production parameters resulted in maximum activity at inoculum age of 24 h and inoculum size of 1.5% (v/v). Esterase gave considerable activity in production medium containing sodium chloride (0.5 % w/v), galactose (1%, w/v), coconut oil (2.0%, v/v) and beef extract (0.3%, w/v) at a temperature of 45℃ and pH 8.5.The enzyme production was enhanced by 3-fold after optimization of production parameters. Further, on optimizing reaction conditions, enzyme gave maximum activity at a temperature of 45℃ and pH 8.5. The para-nitrophenyl acetate (p-NPA) was found to be optimum substrate and metal ions and detergents have inhibitory effect on esterase activity.