In this study, an artificial neural network (ANN) trained by backpropagation algorithm, Levenberg-Marquadart, was applied to predict the yield of enzymatic synthesis of dioctyl adipate. Immobilized Candida antarctica lipase B was used as a biocatalyst for the reaction. Temperature, time, amount of enzyme, and substrate molar ratio were the four input variables. After evaluating various ANN configurations, the best network was composed of seven hidden nodes using a hyperbolic tangent sigmoid transfer function. The correlation coefficient (R2) and mean absolute error (MAE) values between the actual and predicted responses were determined as 0.9998 and 0.0966 for training set and 0.9241 and 1.9439 for validating dataset. A simulation test with a testing dataset showed that the MAE was low and R2 was close to 1. These results imply the good generalization of the developed model and its capability to predict the reaction yield. Comparison of the performance of radial basis network with the developed models showed that radial basis function was more accurate but its performance was poor when tested with unseen data. In further part of the study, the feedforward backpropagation model was used for prediction of the ester yield within the given range of the main parameters.
A biotechnological route via enzymatic esterification was proposed as an alternative way to synthesize the problematic anti-oxidant eugenyl benzoate. The new method overcomes the well-known drawbacks of the chemical route in favor of a more sustainable reaction process. The present work reports a Box-Behnken design (BBD) optimization process to synthesize eugenyl benzoate by esterification of eugenol and benzoic acid catalyzed by the chitosan-chitin nanowhiskers supported Rhizomucor miehei lipase (RML-CS/CNWs). Effects of four reaction parameters: reaction time, temperature, substrate molar ratio of eugenol: benzoic acid and enzyme loading were assessed. Under optimum conditions, a maximum conversion yield as high as 66% at 50°C in 5h using 3mg/mL of RML-CS/CNWs, and a substrate molar ratio (eugenol: benzoic acid) of 3:1. Kinetic assessments revealed the RML-CS/CNWs catalyzed the reaction via a ping-pong bi-bi mechanism with eugenol inhibition, characterized by a Vmax of 3.83mMmin-1. The Michaelis-Menten constants for benzoic acid (Km,A) and eugenol (Km,B) were 34.04 and 138.28mM, respectively. The inhibition constant for eugenol (Ki,B) was 438.6mM while the turnover number (kcat) for the RML-CS/CNWs-catalyzed esterification reaction was 40.39min-1. RML-CS/CNWs were reusable up to 8 esterification cycles and showed higher thermal stability than free RML.
The contribution of chitosan/nanocellulose (CS-NC) to the enzymatic activity of Candida rugosa lipase covalently bound on the surface of CS-NC (CRL/CS-NC) was investigated. Cellulosic material from oil palm frond leaves (OPFL) were bleached, alkaline treated and acid hydrolyzed to obtain the purified NC and used as nano-fillers in CS. XRD, Raman spectroscopy and optical fluorescence microscopic analyses revealed existence of strong hydrogen bonds between CS and the NC nanofillers. The CRLs were successfully conjugated to the surface of the CS-NC supports via imine bonds that occurred through a Schiff's based mechanism. Process parameters for the immobilization of CRL were assessed for factors temperature, concentration of glutaraldehyde and pH, to afford the highest enzyme activity to achieve maximum conversion of butyl butyrate within 3h of incubation. Conversion as high as 88% was reached under an optimized condition of 25°C, 0.3% glutaraldehyde concentration and buffer at pH7. Thermal stability of CRL/CS-NCs was 1.5-fold greater than that of free CRL, with biocatalysts reusability for up to 8 successive esterification cycles. This research provides a promising approach for expanding the use of NC from OPFL for enhancing enzyme activity in favour of an alternative eco-friendly means to synthesize butyl butyrate.
A statistical approach with D-optimal design was used to optimize the process parameters for polycaprolactone (PCL) synthesis. The variables selected were temperature (50°C-110°C), time (1-7 h), mixing speed (50-500 rpm) and monomer/solvent ratio (1:1-1:6). Molecular weight was chosen as response and was determined using matrix-assisted laser desorption/ionization time of flight (MALDI TOF). Using the D-optimal method in design of experiments, the interactions between parameters and responses were analysed and validated. The results show a good agreement with a minimum error between the actual and predicted values.
Due to environmental concern, the research to date has tended to focus on how textile dye removal can be carried out in a greener manner. Therefore, this study aims to evaluate the decolorization and biotransformation pathway of Mordant Orange-1 (MO-1) by Cylindrocephalum aurelium RY06 (C. aurelium RY06). Decolorization study was conducted in a batch experiment including the investigation of the effects of physio-chemical parameters. Enzymatic activity of C. aurelium RY06 during the decolorization was also investigated. Moreover, transformation and biodegradation of MO-1 by C. aurelium RY06 were observed using the gas chromatography-mass spectrometry. Manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase enzymes were detected during the decolorization. In general, the present work concluded that the MO-1 was successfully degraded by C. aurelium RY06 and transformed to be maleic acid and to be isophtalic acid.
The fungal insect pathogen Beauveria bassiana produces a range of insecticidal metabolites and enzymes, including chitinases and proteases, which may assist the disease progression. The enzymes often play a predominant role in the pathogenicity pathway and both chitinases and proteases have previously been shown to be important in host infection. Spray application of supernatants of B. bassiana broth cultures of an isolate from New Zealand caused significant mortality in the green peach aphid, Myzus persicae, within 24 h, demonstrating an apparent contact toxicity. Three-day-old broth cultures were the most effective, with less insect mortality seen using six-day-old broth. However, aphicidal activity increased again when treating aphids with seven-day-old broth. Cultures grew substantially better and produced more potent aphicidal cultures when cultured in media with an initial pH above 5.5. Chitinase was produced a day earlier than the serine protease Pr1, but the peak production periods of these enzymes did not correlate with the aphicidal activities of three- or six-day-old cultures. Cultures treated with EDTA or heated to inactivate the enzymes still showed strong insecticidal activity. Neither beauvericin nor bassianolide, two known insecticidal metabolites, were detected in the supernatants. Therefore the key aphicidal components of B. bassiana cultures were not associated with chitinase nor Pr1 and are yet to be identified.
Laccases, oxidative copper-enzymes found in fungi and bacteria were used as the basis in the design of nona- and tetrapeptides. Laccases are known to be excellent catalysts for the degradation of phenolic xenobiotic waste. However, since solvent extraction of laccases is environmentally-unfriendly and yields obtained are low, they are less preferred compared to synthetic catalysts. The histidine rich peptides were designed based on the active site of laccase extracted from Trametes versicolor through RCSB Protein Data Bank, LOMETS and PyMol software. The peptides were synthesized using Fmoc-solid phase peptide synthesis (SPPS) with 30-40% yield. These peptides were purified and characterized using LC-MS (purities >75%), FTIR and NMR spectroscopy. Synthesized copper(II)-peptides were crystallized and then analyzed spectroscopically. Their structures were elucidated using 1D and 2D NMR. Standards (o,m,p-cresol, 2,4-dichlorophenol) catalysed using laccase from Trametes versicolor (0.66 U/mg) were screened under different temperatures and stirring rate conditions. After optimizing the degradation of the standards with the best reaction conditions reported herein, medications with phenolic and aromatic structures such as ibuprofen, paracetamol (acetaminophen), salbutamol, erythromycin and insulin were screened using laccase (positive control), apo-peptides and copper-peptides. Their activities evaluated using GC-MS, were compared with those of peptide and copper-peptide catalysts. The tetrapeptide was found to have the higher degradation activity towards salbutamol (96.8%) compared with laccase at 42.8%. Ibuprofen (35.1%), salbutamol (52.9%) and erythromycin (49.7%) were reported to have the highest degradation activities using Cu-tetrapeptide as catalyst when compared with the other medications. Consequently, o-cresol (84%) was oxidized by Tp-Cu while the apo-peptides failed to oxidize the cresols. Copper(II)-peptides were observed to have higher catalytic activity compared to their parent peptides and the enzyme laccase for xenobiotic degradation.
Isozymes of 23 cultures of the anaerobic rumen fungi and seven cultures of aerobic chytridiomycete fungi were analysed by PAGE. A total of 14 isozyme loci were successfully typed by PAGE. They were peptidase A & C-1, peptidase A & C-2, peptidase D-1, peptidase D-2, malate dehydrogenase-1, malate dehydrogenase-2, esterase-1, esterase-2, malic enzyme-1, malic enzyme-2, isocitrate dehydrogenase, shikimate dehydrogenase, phosphoglucomutase and 6-phosphogluconate dehydrogenase. Isozyme analysis can be used for studying the genetic relationships among the different anaerobic rumen fungi and the aerobic chytridiomycete fungi and the isozyme characteristics can serve as additional taxonomic criteria in the classification of the anaerobic rumen fungi. A dendrogram based on the isozyme data demonstrated that the anaerobic rumen fungi formed a cluster, indicating a monophyletic group, distinctly separated from the aerobic chytridiomycete fungi. Piromyces communis and P. minutus showed a close relationship but P. spiralis showed a more distant relationship to both P. communis and P. minutus. Piromyces as a whole was more related to Caecomyces than to Neocallimastix. Orpinomyces was also found to be more related to Piromyces and Caecomyces than to Neocallimastix. Orpinomyces intercalaris C 70 from cattle showed large genetic variation from O. joyonii, indicating that it is a different species.
In this study, laccase was immobilized on nylon 6,6/Fe(3+) composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80 nm (diameter) and 2.70 MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe(3+) particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11 weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33 V and the catalytic current was found to be 30 µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97 %) cell inhibition at 75 µM L(-1) within 24 h. The cytotoxicity of DMOB was significantly decreased to 78 % after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.
Isolates of anaerobic fungi obtained from the rumen, duodenum and faeces of sheep were identified as Piromyces mae based on their morphological characteristics observed using light microscopy. There was no significant morphological variation among the isolates of P. mae from the rumen, duodenum and faeces. Isozymes of 12 isolates of P. mae (one each from the rumen, duodenum and faeces from 4 different sheep) were analysed by PAGE. A total of 12 isozymes were studied and 5 isozyme loci were successfully typed. They were malic enzyme, malate dehydrogenase, shikimate dehydrogenase, alpha-esterase and beta-esterase. All the isolates of P. mae regardless of whether they were from the rumen, duodenum or faeces or from different animals produced very similar isozyme banding patterns for each of the enzyme systems. The similar isozyme profiles of the isolates indicate that they are of the same species although they exist in different regions of the alimentary tract.
The present work describesthe development of highly potent mushroom tyrosinase inhibitor better than the standard kojic acid. Carvacrol derivatives 4a-f and 6a-d having substituted benzoic acid and cinnamic acidresidues were synthesized with the aim to possess potent tyrosinase inhibitory activity.The structures of the synthesized compounds were ascertained by their spectroscopic data (FTIR, 1HNMR, 13CNMR and Mass Spectroscopy).Mushroom tyrosinase inhibitory activity of synthesized compounds was determined and it was found that one of the derivative 6c possess higher activity (IC50 0.0167μM) than standard kojic acid (IC50 16.69μM). The derivatives 4c and 6b also showed good tyrosinase inhibitory activity with (IC50 16.69μM) and (IC50 16.69μM) respectively.Lineweaver-Burk and Dixon plots were used for the determination of kinetic mechanism of the compounds 4c and 6b and 6c. The kinetic analysis revealed that compounds 4c and 6b showed mixed-type inhibition while 6c is a non-competitive inhibitor having Ki values19 μM, 10 μM, and 0.05 μMrespectively. The enzyme inhibitory kinetics further showed thatcompounds 6b and 6c formed irreversible enzyme inhibitor complex while 4c bind reversibly with mushroom tyrosinase.The docking studies showed that compound 6c have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (-7.90 kcal/mol) as compared to others.The 2-hydroxy group in compound 6c interacts with amino acid HIS85 which is present in active binding site. The wet lab results are in good agreement with the dry lab findings.Based upon our investigation we may propose that the compound 6c is promising candidate for the development of safe cosmetic agent.
Proper management of agricultural disease is important to ensure sustainable food security. Staple food crops like rice, wheat, cereals, and other cash crops hold great export value for countries. Ensuring proper supply is critical; hence any biotic or abiotic factors contributing to the shortfall in yield of these crops should be alleviated. Rhizoctonia solani is a major biotic factor that results in yield losses in many agriculturally important crops. This paper focuses on genome informatics of our Malaysian Draft R. solani AG1-IA, and the comparative genomics (inter- and intra- AG) with four AGs including China AG1-IA (AG1-IA_KB317705.1), AG1-IB, AG3, and AG8. The genomic content of repeat elements, transposable elements (TEs), syntenic genomic blocks, functions of protein-coding genes as well as core orthologous genic information that underlies R. solani's pathogenicity strategy were investigated. Our analyses show that all studied AGs have low content and varying profiles of TEs. All AGs were dominant for Class I TE, much like other basidiomycete pathogens. All AGs demonstrate dominance in Glycoside Hydrolase protein-coding gene assignments suggesting its importance in infiltration and infection of host. Our profiling also provides a basis for further investigation on lack of correlation observed between number of pathogenicity and enzyme-related genes with host range. Despite being grouped within the same AG with China AG1-IA, our Draft AG1-IA exhibits differences in terms of protein-coding gene proportions and classifications. This implies that strains from similar AG do not necessarily have to retain similar proportions and classification of TE but must have the necessary arsenal to enable successful infiltration and colonization of host. In a larger perspective, all the studied AGs essentially share core genes that are generally involved in adhesion, penetration, and host colonization. However, the different infiltration strategies will depend on the level of host resilience where this is clearly exhibited by the gene sets encoded for the process of infiltration, infection, and protection from host.
Cladosporium sphaerospermum is one of the most widely distributed allergens causing serious problems in patients with respiratory tract disease. We report the 26,644,473-bp draft genome sequence and gene annotation of C. sphaerospermum UM843. Analysis of the genome sequence led to the finding of genes associated with C. sphaerospermum's melanin biosynthesis, allergens, and antifungal drug resistance.
Candida albicans is an opportunistic pathogen that causes candidiasis in humans. In recent years, metabolic pathways in C. albicans have been explored as potential antifungal targets to treat candidiasis. The glyoxylate cycle, which enables C. albicans to survive in nutrient-limited host niches and its. Key enzymes (e.g., isocitrate lyase (ICL1), are particularly attractive antifungal targets for C. albicans. In this study, we used a new screening approach that better reflects the physiological environment that C. albicans cells experience during infection to identify potential inhibitors of ICL. Three compounds (caffeic acid (CAFF), rosmarinic acid (ROS), and apigenin (API)) were found to have antifungal activity against C. albicans when tested under glucose-depleted conditions. We further confirmed the inhibitory potential of these compounds against ICL using the ICL enzyme assay. Lastly, we assessed the bioavailability and toxicity of these compounds using Lipinski's rule-of-five and ADMET analysis.
The production and characterization of a bioflocculant, IH-7, by Aspergillus flavus was investigated. About 0.4 g of purified bioflocculant with an average molecular weight of 2.574 × 10(4)Da could be obtained from 1L of fermentation medium. The bioflocculant mainly consisted of protein (28.5%) and sugar (69.7%), including 40% of neutral sugar, 2.48% of uronic acid and 1.8% amino sugar. The neutral sugar components are sucrose, lactose, glucose, xylose, galactose, mannose and fructose at a molar ratio of 2.4:4.4:4.1:5.8:9.9:0.8:3.1. Fourier-transform infrared spectroscopy analysis revealed that purified IH-7 contained hydroxyl, amide, carboxyl and methoxyl groups. The elemental analysis of purified IH-7 showed that the weight fractions of the elements C, H, O, N and S were 29.9%, 4.8%, 34.7%, 3.3%, and 2.0%, respectively. IH-7 had good flocculating rate in kaolin suspension without cation addition and stable over wide range of pH and temperature.
Aureobasidium pullulans AY4 is an opportunistic pathogen that was isolated from the skin of an immunocompromised patient. We present here the draft genome of strain AY4, which reveals an abundance of genes relevant to bioindustrial applications, including biocontrol and biodegradation. Putative genes responsible for the pathogenicity of strain AY4 were also identified.
Mushroom industries generate a virtually in-exhaustible supply of a co-product called spent mushroom substrate (SMS). This is the unutilised substrate and the mushroom mycelium left after harvesting of mushrooms. As the mushroom industry is steadily growing, the volume of SMS generated annually is increasing. In recent years, the mushroom industry has faced challenges in storing and disposing the SMS. The obvious solution is to explore new applications of SMS. There has been considerable discussion recently about the potentials of using SMS for production of value-added products. One of them is production of lignocellulosic enzymes such as laccase, xylanase, lignin peroxidase, cellulase and hemicellulase. This paper reviews scientific research and practical applications of SMS as a readily available and cheap source of enzymes for bioremediation, animal feed and energy feedstock.
This review tracks a decade of dynamic kinetic resolution developments with a biocatalytic inclination using enzymatic/microbial means for the resolution part followed by the racemization reactions either by means of enzymatic or chemocatalyst. These fast developments are due to the ability of the biocatalysts to significantly reduce the number of synthetic steps which are common for conventional synthesis. Future developments in novel reactions and products of dynamic kinetic resolutions should consider factors that are needed to be extracted at the early synthetic stage to avoid inhibition at scale-up stage have been highlighted.
Recently, the increased demand of fructooligosaccharides (FOS) as a functional food has alarmed researchers to screen and identify new strains capable of producing fructosyltransferase (FTase). FTase is the enzyme that converts the substrate (sucrose) to glucose and fructose. The characterization of complex sugar such as table sugar, brown sugar, molasses, etc. will be carried out and the sugar that contained the highest sucrose concentration will be selected as a substrate. Eight species of macro-fungi will be screened for its ability to produce FTase and only one strain with the highest FTase activity will be selected for further studies. In this work, neural networks (NN) have been chosen to model the process based on their excellent 'resume' in coping with nonlinear process. Bootstrap re-sampling method has been utilized in re-sampling the data in this work. This method has successfully modeled the process as shown in the results.
Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.