A novel α-amylase was isolated successfully from Glaciozyma antarctica PI12 using DNA walking and reverse transcription-polymerase chain reaction (RT-PCR) methods. The structure of this psychrophilic α-amylase (AmyPI12) from G. antarctica PI12 has yet to be studied in detail. A 3D model of AmyPI12 was built using a homology modelling approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9.9. Analysis of the AmyPI12 model revealed the presence of binding sites for a conserved calcium ion (CaI), non-conserved calcium ions (CaII and CaIII) and a sodium ion (Na). Compared with its template-the thermostable α-amylase from Bacillus stearothermophilus (BSTA)-the binding of CaII, CaIII and Na ions in AmyPI12 was observed to be looser, which suggests that the low stability of AmyPI12 allows the protein to work at different temperature scales. The AmyPI12 amino acid sequence and model were compared with thermophilic α-amylases from Bacillus species that provided the highest structural similarities with AmyPI12. These comparative studies will enable identification of possible determinants of cold adaptation.
Waterways are popular locations for the disposition of criminal evidence because the recovery of latent fingerprints from such evidence is difficult. Currently, small particle reagent is a method often used to visualize latent fingerprints containing carcinogenic and hazardous compounds. This study proposes an eco-friendly, safranin-tinted Candida rugosa lipase (triacylglycerol ester hydrolysis EC 3.1.1.3) with functionalized carbon nanotubes (CRL-MWCNTS/GA/SAF) as an alternative reagent to the small particle reagent. The CRL-MWCNTS/GA/SAF reagent was compared with the small particle reagent to visualize groomed, full fingerprints deposited on stainless steel knives which were immersed in a natural outdoor pond for 30 days. The quality of visualized fingerprints using the new reagent was similar (modified-Centre for Applied Science and Technology grade: 4; p > 0.05) to small particle reagent, even after 15 days of immersion. Despite the slight decrease in quality of visualized fingerprints using the CRL-MWCNTS/GA/SAF on the last three immersion periods, the fingerprints remained forensically identifiable (modified-Centre for Applied Science and Technology grade: 3). The possible chemical interactions that enabled successful visualization is also discussed. Thus, this novel reagent may provide a relatively greener alternative for the visualization of latent fingerprints on immersed non-porous objects.
The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 106 spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.
The human fungal pathogen Candida glabrata appears to utilise unique stealth, evasion and persistence strategies in subverting the onslaught of host immune response during systemic infection. However, macrophages actively deprive the intracellular fungal pathogen of glucose, and therefore alternative carbon sources probably support the growth and survival of engulfed C. glabrata. The present study aimed to investigate the role of the glyoxylate cycle gene ICL1 in alternative carbon utilisation and its importance for the virulence of C. glabrata. The data showed that disruption of ICL1 rendered C. glabrata unable to utilise acetate, ethanol or oleic acid. In addition, C. glabrata icl1∆ cells displayed significantly reduced biofilm growth in the presence of several alternative carbon sources. It was also found that ICL1 is crucial for the survival of C. glabrata in response to macrophage engulfment. Disruption of ICL1 also conferred a severe attenuation in the virulence of C. glabrata in the mouse model of invasive candidiasis. In conclusion, a functional glyoxylate cycle is essential for C. glabrata to utilise certain alternative carbon sources in vitro and to display full virulence in vivo. This reinforces the view that antifungal drugs that target fungal Icl1 have potential for future therapeutic intervention.
Candida albicans is a commensal yeast commonly found on the skin and in the body. However, in immunocompromised individuals, the fungi could cause local and systemic infections. The carbon source available plays an important role in the establishment of C. albicans infections. The fungi's ability to assimilate a variety of carbon sources plays a vital role in its colonization, and by extension, its fitness and pathogenicity, as it often inhabits niches that are glucose-limited but rich in alternative carbon sources. A difference in carbon sources affect the growth and mating of C. albicans, which contributes to its pathogenicity as proliferation helps the fungi colonize its environment. The carbon source also affects its metabolism and signaling pathways, which are integral parts of the fungi's fitness and pathogenicity. As a big percentage of the carbon assimilated by C. albicans goes to cell wall biogenesis, the availability of different carbon sources will result in cell walls with variations in rigidity, adhesion, and surface hydrophobicity. In addition to the biofilm formation of the fungi, the carbon source also influences whether the fungi grow in yeast- or mycelial-form. Both forms play different roles in C. albicans's infection process. A better understanding of the role of the carbon sources in C. albicans's pathogenicity would contribute to more effective treatment solutions for fungal infections.
The serological responses to Cryptococcus neoformans proteins of blood donors and HIV patients with active cryptococcosis from a tropical region were investigated in this study. Exposure to C. neoformans, an organism ubiquitous in the environment, contributes to the antibody responses observed in the blood donors. IgG responses to cryptococcal proteins were stronger than IgM responses in most sera tested in this study. A 53-kDa cryptococcal protein fragment was identified as the most immunoreactive protein on the IgM immunoblots of both blood donors and patients. Overall, there was no obvious difference in IgG responses of patients when compared with blood donors. Some immunogenic protein fragments (27.5, 76, 78 and 91.5 kDa) were detected at least two times more frequently on IgM immunoblots of patients compared with those of blood donors. It is yet to be investigated whether the proteins identified in this study may have any potential to be used as biomarker for cryptococcosis.
Immobilized Candida rugosa lipase was used for the synthesis of citronellyl laurate from citronellol and lauric acid. Screening of different types of support (Amberlite MB-1 and Celite) for immobilization of lipase and solvent (n-hexane, n-heptane, and iso-octane) and optimization of reaction conditions, such as catalyst loading, effect of substrates molar ratio and temperature, have been studied. The maximum enzyme activity was obtained at 310 K. The immobilized C. rugosa lipase onto Amberlite MB-1 support was found to be the best support with a conversion of 89% of citronellyl laurate ester in iso-octane compared to Celite 545. Deactivation of C. rugosa lipase at 313, 318 and 323 K were observed. Ordered bi bi mechanism with dead end complex of lauric acid was found to fit the initial rate data and the kinetic parameters were obtained by non-linear regression analysis.
A series of 16 oxadiazole and triazolothiadiazole derivatives were designed, synthesized and evaluated as mushroom tyrosinase inhibitors. Five derivatives were found to display high inhibition on the tyrosinase activity ranging from 0.87 to 1.49 microM. Compound 5 exhibited highest tyrosinase inhibitory activity with an IC(50) value of 0.87+/-0.16 microM. The in silico protein-ligand docking using AUTODOCK 4.1 was successfully performed on compound 5 with significant binding energy value of -5.58 kcal/mol. The docking results also showed that the tyrosinase inhibition might be due to the metal chelating effect by the presence of thione functionality in compounds 1-5. Further studies revealed that the presence of hydrophobic group such as cycloamine derivatives played a major role in the inhibition. Piperazine moiety in compound 5 appeared to be involved in an extensive hydrophobic contact and a 2.9A hydrogen bonding with residue Glu 182 in the active site.
Pleurotus pulmonarius has been reported to have a potent remedial effect on diabetic property and considered to be an alternative for type 2 diabetes mellitus treatment. This study aimed to investigate the antidiabetic properties of ammonium sulphate precipitated protein fractions from P. pulmonarius basidiocarps. Preliminary results demonstrated that 30% (NH4)2SO4 precipitated fraction (F30) inhibited Saccharomyces cerevisiae α-glucosidase activity (24.18%), and 100% (NH4)2SO4 precipitated fraction (F100) inhibited porcine pancreatic α-amylase activity (41.80%). Following RP-HPLC purification, peak 3 from F30 fraction demonstrated inhibition towards α-glucosidase at the same time with meagre inhibition towards α-amylase activity. Characterisation of proteins using MALDI-TOF/TOF MS demonstrated the presence of four different proteins, which could be implicated in the regulation of blood glucose level via various mechanisms. Therefore, this study revealed the presence of four antidiabetic-related proteins which are profilin-like protein, glyceraldehyde-3-phosphate dehydrogenase-like protein, trehalose phosphorylase-like (TP-like) protein, and catalase-like protein. Hence, P. pulmonarius basidiocarps have high potential in lowering blood glucose level, reducing insulin resistance and vascular complications.
Corynespora cassiicola is an important plant pathogenic Ascomycete causing the damaging Corynespora Leaf Fall (CLF) disease in rubber tree (Hevea brasiliensis). A small secreted glycoprotein named cassiicolin was previously described as an important effector of C. cassiicola. In this study, the diversity of the cassiicolin-encoding gene was analysed in C. cassiicola isolates sampled from various hosts and geographical origins. A cassiicolin gene was detected in 47 % of the isolates, encoding up to six distinct protein isoforms. In three isolates, two gene variants encoding cassiicolin isoforms Cas2 and Cas6 were found in the same isolate. A phylogenetic tree based on four combined loci and elucidating the diversity of the whole collection was strongly structured by the toxin class, as defined by the cassiicolin isoform. The isolates carrying the Cas1 gene (toxin class Cas1), all grouped in the same highly supported clade, were found the most aggressive on two rubber tree cultivars. Some isolates in which no Cas gene was detected could nevertheless generate moderate symptoms, suggesting the existence of other yet uncharacterized effectors. This study provides a useful base for future studies of C. cassiicola population biology and epidemiological surveys in various host plants.
The aim of this work was the synthesis of a novel hydroxyl-fatty acid derivative of kojic acid rich in kojic acid monoricinoleate (KMR) which can be widely used in the cosmetic and food industry. The synthesis of KMR was carried out by lipase-catalysed esterification of ricinoleic and kojic acids in solvent-free system. Three immobilized lipases were tested and the best KMR yields were attained with Lipozyme TL IM and Novozym 435. Since Lipozyme TL IM is the cheapest, it was selected to optimize the reaction conditions. The optimal reaction conditions were 80 °C for the temperature, 1:1 for the alcohol/acid molar ratio, 600 rpm for stirring speed and 7.8% for the catalyst concentration. Under these conditions, the reaction was scaled up in a 5×10⁻³ m³ stirred tank reactor. ¹H-¹³C HMBC-NMR showed that the primary hydroxyl group of kojic acid was regioselectively esterified. The KMR has more lipophilicity than kojic acid and showed antioxidant activity that improves the oxidation stability of biodiesel.
A study was carried out to compare the composition and thermal properties of lard (LD) and engkabang fat (EF) - canola oil (CaO) blend interesterified with Candida antartica lipase (C. antartica). A fat blend EF-4 (40% EF in CaO) was prepared and interesterified using C. antartica lipase at 60°C for different time intervals (6 h, 12 h and 24 h) with 200 rpm agitation. The fat blends before and after interesterification were compared to LD with respect to their slip melting points (SMP), fatty acid and triacyglycerol (TAG) compositions, melting, solidification and polymorphic properties. Result showed that the slip melting point (SMP) of the fat blend interesterified for 6 h was the closest to that of LD. The solid fat content (SFC) values of fat blends interesterified for 12 and 24 h were found to become equal to those of LD within the temperature range of 0 to 20°C. In addition, all three interesterified blends had SFC values similar to those of LD within the temperature range of 30-40°C. According to thermal analysis, the transition of the fat blend interesterified for 24 h appearing at -2.39°C was similar to the low melting thermal transition of LD and the transition of the fat blend interesterified for 12 h appearing at 26.25°C was similar to the high melting thermal transition of LD. However, there is no compatibility between LD and all three interesterified blends with regard to polymorphic behaviour.
The sclerotium of the "tiger's milk mushroom" (Lignosus rhinocerotis) is used as tonic and folk medicine for the treatment of cancer, fever, cough and asthma by the local and indigenous communities. It is traditionally prepared by either boiling or maceration-like methods; however, there is no attempt to understand how different processing methods might affect their efficacies as anticancer agents.
Yeast extracellular proteases play a key role in developing the taste of dry-cured ham, whereas the mechanism of yeast proteases on taste formation of dry-cured ham is not fully studied. The proteases characteristics of yeast isolated form Jinhua ham, hydrolysis capacities for myofibrillar proteins (MP), free amino acid contents, metabolite compositions, taste parameters and the relationship between metabolites and taste parameters were investigated to reveal the mechanism of Rhodotorula mucilaginosa AUMC 9298 (RM) and Candida parapsilosis d70a (CP) proteases on MP hydrolysis and taste development of dry-cured ham. The proteases of RM and CP showed high hydrolysis activities at the conditions of pH 5.0-8.0 and 30-50 °C. The proteases of RM showed higher capability to degrade myosin compared with CP proteases and Pichia kudriavzevii XS-5 (PK) proteases. The total free amino acid contents increased from 18.44 mg/100 mL in PK to 33.91 mg/100 mL in RM and 25.28 mg/100 mL in CP after 4 h hydrolysis of MP. Thirty-two metabolites were identified by LC-MS/MS, and peptides and amino acid derivatives were the key components of MP hydrolysates. The scores of umami, richness and aftertaste showed the largest values in RM among these groups. PLS-DA and correlation demonstrated that aspartic acid, N-Methyl-aspartic acid, Glu-Glu, γ-Glu-Cys, glutamic acid, γ-Glu-Glu and γ-Glu-Gln were positive correlation with the improvement of umami, richness and aftertaste.
The Sfp-type 4'-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.
Azelaic acid (AzA) and its derivatives have been known to be effective in the treatment of acne and various cutaneous hyperpigmentary disorders. The esterification of azelaic acid with lauryl alcohol (LA) to produce dilaurylazelate using immobilized lipase B from Candida antarctica (Novozym 435) is reported. Response surface methodology was selected to optimize the reaction conditions. A well-fitting quadratic polynomial regression model for the acid conversion was established with regards to several parameters, including reaction time and temperature, enzyme amount, and substrate molar ratios. The regression equation obtained by the central composite design of RSM predicted that the optimal reaction conditions included a reaction time of 360 min, 0.14 g of enzyme, a reaction temperature of 46 °C, and a molar ratio of substrates of 1:4.1. The results from the model were in good agreement with the experimental data and were within the experimental range (R² of 0.9732).The inhibition zone can be seen at dilaurylazelate ester with diameter 9.0±0.1 mm activities against Staphylococcus epidermidis S273. The normal fibroblasts cell line (3T3) was used to assess the cytotoxicity activity of AzA and AzA derivative, which is dilaurylazelate ester. The comparison of the IC50 (50% inhibition of cell viability) value for AzA and AzA derivative was demonstrated. The IC50 value for AzA was 85.28 μg/mL, whereas the IC50 value for AzA derivative was more than 100 μg/mL. The 3T3 cell was still able to survive without any sign of toxicity from the AzA derivative; thus, it was proven to be non-toxic in this MTT assay when compared with AzA.
Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.
The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia.
Dimethyl adipate (DMA) was synthesized by immobilized Candida antarctica lipase B-catalyzed esterification of adipic acid and methanol. To optimize the reaction conditions of ester production, response surface methodology was applied, and the effects of four factors namely, time, temperature, enzyme concentration, and molar ratio of substrates on product synthesis were determined. A statistical model predicted that the maximum conversion yield would be 97.6%, at the optimal conditions of 58.5 degrees C, 54.0 mg enzyme, 358.0 min, and 12:1 molar ratio of methanol to adipic acid. The R(2) (0.9769) shows a high correlation between predicted and experimental values. The kinetics of the reaction was also investigated in this study. The reaction was found to obey the ping-pong bi-bi mechanism with methanol inhibition. The kinetic parameters were determined and used to simulate the experimental results. A good quality of fit was observed between the simulated and experimental initial rates.
Immobilized Candida antarctica lipase B-catalyzed esterification of xylitol and two fatty acids (capric and caproic acid) were studied in a solvent-free system. The Taguchi orthogonal array method based on three-level-four-variables with nine experiments was applied for the analysis and optimization of the reaction parameters including time, substrate molar ratio, amount of enzyme, and amount of molecular sieve. The obtained conversion was higher in the esterification of xylitol and capric acid with longer chain length. The optimum conditions derived via the Taguchi approach for the synthesis of xylitol caprate and xylitol caproate were reaction time, 29 and 18h; substrate molar ratio, 0.3 and 1.0; enzyme amount, 0.20 and 0.05g, and molecular sieve amount of 0.03g, respectively. The good correlation between the predicted conversions (74.18% and 61.23%) and the actual values (74.05% and 60.5%) shows that the model derived from the Taguchi orthogonal array can be used for optimization and better understanding of the effect of reaction parameters on the enzymatic synthesis of xylitol esters in a solvent-free system.