In this study, a bacterial strain CP22 with ability to produce cellulase, xylanase and mannanase was isolated from the oil palm compost. Based on the 16S rRNA gene analysis, the strain was affiliated to genus Micromonospora. To further investigate genes that are related to cellulose and hemicellulose degradation, the genome of strain CP22 was sequenced, annotated and analyzed. The de novo assembled genome of strain CP22 featured a size of 5,856,203 bp with G + C content of 70.84%. Detailed genome analysis on lignocellulose degradation revealed a total of 60 genes consisting of 47 glycoside hydrolase domains and 16 carbohydrate esterase domains predicted to be involved in cellulolytic and hemicellulolytic deconstruction. Particularly, 20 genes encode for cellulases (8 endoglucanases, 3 exoglucanases and 9 β-glucosidases) and 40 genes encode for hemicellulases (15 endo-1,4-β-xylanase, 3 β-xylosidase, 3 α-arabinofuranosidase, 10 acetyl xylan esterase, 6 polysaccharide deacetylase, 1 β-mannanase, 1 β-mannosidase and 1 α-galactosidase). Thirty-two genes encoding carbohydrate-binding modules (CBM) from six different families (CBM2, CBM4, CBM6, CBM9, CBM13 and CBM22) were present in the genome of strain CP22. These CBMs were found in 27 cellulolytic and hemicellulolytic genes, indicating their potential role in enhancing the substrate-binding capability of the enzymes. CBM2 and CBM13 are the major CBMs present in cellulases and hemicellulases (xylanases and mannanases), respectively. Moreover, a GH10 xylanase was found to contain 3 CBMs (1 CBM9 and 2 CBM22) and these CBMs were reported to bind specifically to xylan. This genome-based analysis could facilitate the exploration of this strain for lignocellulosic biomass degradation.
A halophilic bacterium, Virgibacillus sp. strain CD6, was isolated from salted fish and its extracellular protease was characterized. Protease production was found to be highest when yeast extract was used as nitrogen source for growth. The protease exhibited stability at wide range of salt concentration (0-12.5%, w/v), temperatures (20-60 °C), and pH (4-10) with maximum activity at 10.0% (w/v) NaCl, 60 °C, pH 7 and 10, indicating its polyextremophilicity. The protease activity was enhanced in the presence of Mg2+, Mn2+, Cd2+, and Al3+ (107-122% relative activity), and with retention of activity > 80% for all of other metal ions examined (K+, Ca2+, Cu2+, Co2+, Ni2+, Zn2+, and Fe3+). Both PMSF and EDTA inhibited protease activity, denoting serine protease and metalloprotease properties, respectively. High stability (> 70%) was demonstrated in the presence of organic solvents and detergent constituents, and the extracellular protease from strain CD6 was also found to be compatible in commercial detergents. Proteinaceous stain removal efficacy revealed that crude protease of strain CD6 could significantly enhance the performance of commercial detergent. The protease from Virgibacillus sp. strain CD6 could serve as a promising alternative for various applications, especially in detergent industry.
The halophilic genus Joostella is one of the least-studied genera in the family of Flavobacteriaceae. So far, only two species were taxonomically identified with limited genomic analysis in the aspect of application has been reported. Joostella atrarenae M1-2T was previously isolated from a seashore sample and it is the second discovered species of the genus Joostella. In this project, the genome of J. atrarenae M1-2T was sequenced using NovaSeq 6000. The final assembled genome is comprised of 71 contigs, a total of 3,983,942 bp, a GC ratio of 33.2%, and encoded for 3,416 genes. The 16S rRNA gene sequence of J. atrarenae M1-2T shows 97.3% similarity against J. marina DSM 19592T. Genome-genome comparison between the two strains by ANI, dDDH, AAI, and POCP shows values of 80.8%, 23.3%, 83.4%, and 74.1% respectively. Pan-genome analysis shows that strain M1-2T and J. marina DSM 19592T shared a total of 248 core genes. Taken together, strain M-2T and J. marina DSM 19592T belong to the same genus but are two different species. CAZymes analysis revealed that strain M1-2T harbors 109 GHs, 40 GTs, 5 PLs, 9 CEs, and 6 AAs. Among these CAZymes, while 5 genes are related to cellulose degradation, 12 and 24 genes are found to encode for xylanolytic enzymes and other hemicellulases that involve majorly in the side chain removal of the lignocellulose structure, respectively. Furthermore, both the intracellular and extracellular crude extracts of strain M1-2T exhibited enzymatic activities against CMC, xylan, pNPG, and pNPX substrates, which corresponding to endoglucanase, xylanase, β-glucosidase, and β-xylosidase, respectively. Collectively, description of genome coupled with the enzyme assay results demonstrated that J. atrarenae M1-2T has a role in lignocellulosic biomass degradation, and the strain could be useful for lignocellulosic biorefining.
This study aimed at recovery of thermostable lipase from Escherichia coli BL21 using porous glass beads grafted with polyethylene glycol (PEG) in aqueous impregnated resins system (AIRS). The influencing parameters such as concentration and pH of extraction solution, concentration of NaCl, size of the beads, and pH of the desorption solution on the partition behaviour of lipase were evaluated. Smaller adsorbent (4 mm) had a 65.5% of recovery yield with approximately two-fold higher purification factor compared to that obtained with the larger adsorbent. Recombinant lipase was purified successfully using AIRS with a purification factor of 7.6 and yield of 78.4% under optimum conditions of 18% (w/w) PEG 4000, 10% (w/w) of potassium citrate at pH 9 with 3% (w/w) of NaCl. Optimum desorption was obtained with 4.0 mm of porous glass beads at pH 9.
The present study investigates the cytotoxicity of hexagonal MgO nanoparticles synthesized via Amaranthus tricolor leaf extract and spherical MgO nanoparticles synthesized via Amaranthus blitum and Andrographis paniculata leaf extracts. In vitro cytotoxicity analysis showed that the hexagonal MgO nanoparticles synthesized from A. tricolor extract demonstrated the least toxicity to both diabetic and non-diabetic cells at 600 μl/ml dosage. The viability of the diabetic cells (3T3-L1) after incubation with varying dosages of MgO nanoparticles was observed to be 55.3%. The viability of normal VERO cells was 86.6% and this stabilized to about 75% even after exposure to MgO nanoparticles dosage of up to 1000 μl/ml. Colorimetric glucose assay revealed that the A. tricolor extract synthesized MgO nanoparticles resulted in ~ 28% insulin resistance reversal. A reduction in the expression of GLUT4 protein at 54 KDa after MgO nanopaSrticles incubation with diabetic cells was observed via western blot analysis to confirm insulin reversal ability. Fluorescence microscopic analysis with propidium iodide and acridine orange dyes showed the release of reactive oxygen species as a possible mechanism of the cytotoxic effect of MgO nanoparticles. It was inferred that the synergistic effect of the phytochemicals and MgO nanoparticles played a significant role in delivering enhanced insulin resistance reversal capability in adipose cells.
Microalgae are photoautotrophic organisms in freshwater systems known to uptake and bioremediate arsenic, a heavy metal. In this study, we compared the growth and arsenic uptake of two microalgae strains, Nostoc and Chlorella, to determine their suitability for arsenic bioremediation. As compared to the control, our results showed that treatment with As (III) enhanced the Nostoc growth by approximately 15% when grown in the absence of phosphate. The highest bioconcentration factor of Nostoc at this treatment was 1463.6, whereas 0.10 mg L-1 As (V) treatment improved the Chlorella growth by 25%, in the presence of phosphate. However, arsenic uptake reduced from 175.7 to 32.3 throughout the cultivation period for Chlorella. This suggests that Nostoc has an upper advantage in the bioremediation of arsenic as compared to the Chlorella strain. To gain insights into the potential of Nostoc in arsenic bioremediation, we further conducted SEM analysis on the vegetative cell surface. The SEM results showed that As (III) disrupted the Nostoc vegetative cell surface and structure. Further to this, pathway analysis and polymerase chain reaction (PCR) were conducted to identify the potential arsenic pathway regulated by Nostoc. The primary As (III)-related pathways elucidated include the arsA transporter and arsD complex that require ATP and As (III) methylation to S-adenosylmethionine. The phosphate deficiency condition resulting in the inability to generate ATP caused As (III) could not be excreted from the Nostoc cells, potentially contributing to the high arsenic concentration accumulated under phosphate-depleted conditions. These insights contribute to understanding the efficacy of microalgae strains in freshwater arsenic bioremediation.
Rhodothermaceae bacterium RA is a halo-thermophile isolated from a saline hot spring. Previously, the genome of this bacterium was sequenced using a HiSeq 2500 platform culminating in 91 contigs. In this report, we report on the resequencing of its complete genome using a PacBio RSII platform. The genome has a GC content of 68.3%, is 4,653,222 bp in size, and encodes 3711 genes. We are interested in understanding the carbohydrate metabolic pathway, in particular the lignocellulosic biomass degradation pathway. Strain RA harbors 57 glycosyl hydrolase (GH) genes that are affiliated with 30 families. The bacterium consists of cellulose-acting (GH 3, 5, 9, and 44) and hemicellulose-acting enzymes (GH 3, 10, and 43). A crude cell-free extract of the bacterium exhibited endoglucanase, xylanase, β-glucosidase, and β-xylosidase activities. The complete genome information coupled with biochemical assays confirms that strain RA is able to degrade cellulose and xylan. Therefore, strain RA is another excellent member of family Rhodothermaceae as a repository of novel and thermostable cellulolytic and hemicellulolytic enzymes.
Cortisol is a stress hormone released from the adrenal glands and is responsible for both hyperglycemia and hypertension during pregnancy. These factors make it mandatory to detect the levels of cortisol during pregnancy to identify and treat hypoglycemia and hypertension. In this study, cortisol levels were quantified with an aptamer-conjugated gold nanorod using an electrochemical interdigitated electrode sensor. The surface uniformity was analyzed by high-power microscopy and 3D-nanoprofiler imaging. The detection limit was determined to be 0.01 ng/mL, and a linear regression indicated that the sensitivity range was in the range of 0.01-0.1 ng/mL, based on a 3σ calculation. Moreover, the specificity of the aptamer was determined by a binding analysis against norepinephrine and progesterone, and it was clearly found that the aptamer specifically recognizes only cortisol. Further, the presence of cortisol was detected in the serum in a dose-dependent manner. This method is useful to detect and correlate multiple pregnancy-related diseases by quantifying the levels of cortisol.
The storage of food grains against the fungal infection has been a great challenge to the farmers, but nanotechnology provides a solution to address this problem. The application of nanotechnology for the storage of food grains replaces synthetic fungicides in agriculture. Inorganic nanoparticles such as silver and zinc oxide are well-known for their antifungal activity. Green synthesized nanoparticles show enhanced antimicrobial activity than the chemically synthesized nanoparticles. Extracts and essential oils derived from plants exhibit very good antifungal properties. The synthesized nanoparticles can be impregnated in packaging materials, which are used to store food grains. Natural materials are having advantages like non-toxicity and easier to degrade and are suitable for food safety. This overview discusses the nanomaterials-mediated protection of food materials from mycotoxin and its releases into the open environment.
Uncontrolled disposal of feathers from the poultry industry and slaughterhouses is environmentally undesirable. The feathers are composed of approximately 90% of keratin which is an important ingredient of cosmetics, shampoos and hair treatment creams. This study aimed to determine the optimum conditions for the extraction of keratin from chicken feathers. The extraction of keratin using various reducing agents was studied using statistical experimental design. In the extraction process, pH, temperature, ratio of reducing agents, mass of chicken feathers and incubation time were analyzed. The keratin in the total extracted protein was purified by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and further characterized using amino acids profile analysis. The surface morphology and chemical composition were studied by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analysis. Sodium sulfide (Na2S) yielded 84.5% of keratin as compared to sodium hydroxide (43.8), urea mixture (50.6), mixture of sodium dodecyl sulfate (SDS) and sodium bisulfite (18.3) and a mixture of Na2S and sodium hydroxide (41.5%) under optimized conditions. The optimum yield of keratin was achieved at 80.9 °C in 9.5 h with 0.05 M sodium sulfide using response surface methodology (RSM). Among the five parameters screened, pH was found not to be significant because the p value was greater than 0.05.
Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1-12.9 µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9-8.9 indicative of intact RNA. In the improved method, the addition of 3 M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45 min at 65 °C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of Chalcone synthase gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a Dendrobium hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.
In this study, we analyzed the Rc and Rd genes that are responsible for the coloration of rice pericarps from six upland rice varieties. We also examined the association of pericarp coloration to the single nucleotide polymorphism in 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), a key gene involved in abscisic acid (ABA) biosynthesis. Our findings demonstrated that all the upland rice varieties analyzed have a Rd gene which encodes a complete dihydroflavonol-4-reductase without early translational termination codon irrespective of their pericarp colors. However, the upland rice varieties with white pericarps were found to have a defective Rc gene with a 14-base deletion at exon 7 which could disrupt the function of a positive regulator of proanthocyanidin biosynthesis. In addition, the NCED2 genes from the upland rice varieties with white pericarps in this study have a C-allele while the NCED2 genes from Pandasan Red, Tomou and Taragang varieties that bear red pericarps were found to have a T-allele which was reported to be associated with a higher ABA level in upland rice. A better understanding of the gene sequences of upland rice varieties with red pericarp may provide important information for rice breeding programs.
In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.
Pineapple peel is a potential feedstock for the extraction of essential oil due to the presence of aromatic compounds. To extract the essential oil from pineapple peels, three different methods were applied, i.e., (1) hydro-distillation (HD); (2) hydro-distillation with enzyme-assisted (HDEA); and (3) supercritical fluid extraction (SFE). SFE had successfully produced an essential oil with the yield of 0.17% (w/w) together with 0.64% (w/w) of concrete, whereby the HD and HDEA had only produced hydrosols with the yield of 70.65% (w/w) and 80.65% (w/w), respectively. Parameters' optimization for HD (substrate to solvent ratio, temperature, and extraction duration) and HDEA (cellulase loading and incubation duration) significantly affected the hydrosol yield, but did not extract out the essential oil. This is because only SFE had successfully ruptured the oil gland after observed under the scanning electron microscope. The essential oil obtained from SFE composed of mainly propanoic acid ethyl ester (40.25%), lactic acid ethyl ester (19.35%), 2-heptanol (15.02%), propanol (8.18%), 3-hexanone (2.60%), and butanoic acid ethyl ester (1.58%). In overall, it can be concluded that the SFE had successfully extracted the essential oil as compared to the HD and HDEA methods.
The biggest agricultural sector that contributes to the Malaysian economy is the oil palm industry. The effluent generated during the production of crude palm oil known as palm oil mill effluent (POME). POME undergoes anaerobic treatment that requires long retention time and produces large amount of methane that consequently contributes to global warming. In this study, an isolated bacteria was selected based on its ability to degrade kraft lignin (KL) and identified as Ochrobactrum sp. The bacteria were able to treat POME (from anaerobic pond) under the aerobic condition without addition of nutrient, resulting in a significant chemical oxygen demand (COD) removal of 71 %, removal rate of 1385 mg/l/day, and 12.3 times higher than that of the ponding system. It has also resulted in 60 % removal of ammoniacal nitrogen and 55 % of total polyphenolic after 6-day treatment period with the detection of lignocellulolytic enzymes.
In current era, majority of microRNA (miRNA) are being discovered through computational approaches which are more confined towards model plants. Here, for the first time, we have described the identification and characterization of novel miRNA in a non-model plant, Persicaria minor (P. minor) using computational approach. Unannotated sequences from deep sequencing were analyzed based on previous well-established parameters. Around 24 putative novel miRNAs were identified from 6,417,780 reads of the unannotated sequence which represented 11 unique putative miRNA sequences. PsRobot target prediction tool was deployed to identify the target transcripts of putative novel miRNAs. Most of the predicted target transcripts (mRNAs) were known to be involved in plant development and stress responses. Gene ontology showed that majority of the putative novel miRNA targets involved in cellular component (69.07%), followed by molecular function (30.08%) and biological process (0.85%). Out of 11 unique putative miRNAs, 7 miRNAs were validated through semi-quantitative PCR. These novel miRNAs discoveries in P. minor may develop and update the current public miRNA database.
Long noncoding RNAs (lncRNAs) are implicated in various biological processes, such as cell proliferation, differentiation, apoptosis, migration, and invasion. They are also key players in various biological pathways. LncRNA was considered as 'translational noise' before 1980s. It has been reported that lncRNAs are aberrantly expressed in different cancers, either as oncogene or tumor suppressor gene. Therefore, more and more lncRNAs are recognized as potential diagnostic biomarkers and/or therapeutic targets. As competitive endogenous RNA, lncRNAs can interact with microRNA to alter the expression of target genes, which may have extensive clinical implications in cancers, including diagnosis, treatment, prognosis, and chemoresistance. This review comprehensively summarizes the functions and clinical relevance of lncRNAs in digestive system cancers, especially as a potential tool to overcome chemoresistance.
The pretreatment of empty fruit bunch (EFB) was conducted using an integrated system of IL and cellulases (IL-E), with simultaneous fermentation in one vessel. The cellulase mixture (PKC-Cel) was derived from Trichoderma reesei by solid-state fermentation. Choline acetate [Cho]OAc was utilized for the pretreatment due to its biocompatibility and biodegradability. The treated EFB and its hydrolysate were characterized by the Fourier transform infrared spectroscopy, scanning electron microscopy, and chemical analysis. The results showed that there were significant structural changes in EFB after the treatment in IL-E system. The sugar yield after enzymatic hydrolysis by the PKC-Cel was increased from 0.058 g/g of EFB in the crude sample (untreated) to 0.283 and 0.62 ± 06 g/g in IL-E system after 24 and 48 h of treatment, respectively. The EFB hydrolysate showed the eligibility for ethanol production without any supplements where ethanol yield was 0.275 g ethanol/g EFB in the presence of the IL, while lower yield obtained without IL-pretreatment. Moreover, it was demonstrated that furfural and phenolic compounds were not at the level of suppressing the fermentation process.
Lignocellulosic biomasses, exhibit resistance to enzymatic hydrolysis due to the presence of lignin and hemicellulose. Ionic liquids proved their applicability in lignin degradation, however, ionic liquid removal has to be performed to proceed to hydrolysis. Therefore, this study reports an in situ hydrolysis of empty fruit bunches (EFB) that combined an ionic liquid (IL) pretreatment and enzymatic hydrolysis. For enzyme production, palm kernel cake (PKC) was used as the primary media for microbial cellulase (PKC-Cel) from Trichoderma reesei (RUTC30). The obtained enzyme exhibited a promising stability in several ionic liquids. Among few, in choline acetate [Cho]OAc, PKC-Cel retained 63.16 % of the initial activity after 6 h and lost only 10 % of its activity in 10 % IL/buffer mixture. Upon the confirmation of the PKC-Cel stability, EFB was subjected to IL-pretreatment followed by hydrolysis in a single step without further removal of the IL. The findings revealed that choline acetate [Cho]OAc and choline butyrate [Cho]Bu were among the best ILs used in the study since 0.332 ± 0.05 g glucose/g and 0.565 ± 0.08 g total reducing sugar/g EFB were obtained after 24 h of enzymatic hydrolysis. Compared to the untreated EFB, the amount of reducing sugar obtained after enzymatic hydrolysis increased by three-fold in the case of [Cho]OAc and [Cho]Bu, two-fold with [EMIM]OAc and phosphate-based ILs whereas the lowest concentration was obtained in [TBPH]OAc. Pretreatment of EFB with [Cho]OAc and [Cho]Bu showed significant differences in the morphology of EFB samples when observed with SEM. Analysis of the lignin, hemicellulose and hemicellulose showed that the total lignin content from the raw EFB was reduced from 37.8 ± 0.6 to 25.81 ± 0.35 % (w/w) upon employment of [Cho]OAc in the compatible system. The PKC-Cel from T. reesei (RUTC30) exhibited promising characteristics that need to be investigated further towards a single-step process for bioethanol production.
Microbial communities from a lake and river flowing through a highly dense urbanized township in Malaysia were profiled by sequencing amplicons of the 16S V3-V4 and 18S V9 hypervariable rRNA gene regions via Illumina MiSeq. Results revealed that Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the dominant prokaryotic phyla; whereas, eukaryotic communities were predominantly of the SAR clade and Opisthokonta. The abundance of Pseudomonas and Flavobacterium in all sites suggested the possible presence of pathogens in the urban water systems, supported by the most probable number (MPN) values of more than 1600 per 100 mL. Urbanization could have impacted the microbial communities as transient communities (clinical, water-borne and opportunistic pathogens) coexisted with common indigenous aquatic communities (Cyanobacteria). It was concluded that in urban water systems, microbial communities vary in their abundance of microbial phyla detected along the water systems. The influences of urban land use and anthropogenic activities influenced the physicochemical properties and the microbial dynamics in the water systems.
Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-020-02617-3.