[Show abstract][Hide abstract] ABSTRACT: Malto-oligosaccharide synthesis using maltogenic amylase often struggles with product re-hydrolyzation. The malto-oligosaccharide synthesis using a maltogenic amylase (MAG1) from Bacillus lehensis G1 was enhanced using a structure-guided protein engineering approach. Mutations decreased the hydrolysis activity of the enzyme and caused various modulations in its transglycosylation properties. W359F, Y377F and M375I mutations caused a reduction in steric interference, an alteration of subsite occupation and an increase in internal flexibility to accommodate longer donor/acceptor molecules for transglycosylation, resulting in an increase in the transglycosylation to hydrolysis ratio of up to 4.0-fold. The increase in active site hydrophobicity that was caused from the W359F and M375I mutations reduced the concentration of maltotriose required for use as a donor/acceptor for transglycosylation to 100 mM and 50 mM, respectively, compared to the 200 mM needed for wild-type. An improvement of the transglycosylation to hydrolysis ratio by 4.2-fold was also demonstrated in each of the mutants. Interestingly, a reduction of steric interference and hydrolysis suppression was caused by the Y377F mutation and introduced a synergistic effect to produce malto-oligosaccharides with a higher degree of polymerization than wild-type. These findings showed that modification of the active site structure imposed various effects on MAG1 activities during malto-oligosaccharide synthesis.
PROCESS BIOCHEMISTRY 10/2015; 50(10):1572-1580. DOI:10.1016/j.procbio.2015.06.005 · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gene encoding a cellobiohydrolase (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, sub-cloned and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kD. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module (CBM). Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55°C with methylumbelliferryl-cellobioside (MUC) as the substrate and retained 85% of its activity following 24 h incubation at 50°C. Despite the lack of activity towards microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic® CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy (SEM) revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared to the non-supplemented control. CBH7B therefore has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
Biotechnology and Applied Biochemistry 08/2015; DOI:10.1002/bab.1431 · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The synthesis of small RNA (sRNA) were extracted from Glaciozyma Antarctica PII2 (G. Antarctica) yeast using the Next-Generation Sequencing (NGS) technology. Statistical approach is used in this study to analyze sRNA from G. Antarctica in order to increase the production of this yeast given in biological conditions such as temperature, time and medium of growth. Hence, this study uses the analysis of variance (ANOVA) with F-test statistic (F ANOVA) and shrinkage F-test (F S) to analyze the NGS data in identifying factors affecting the differential expression level of genes from G. Antarctica. F ANOVA statistics are computed on a gene-by-gene basis from the residual sum of squares (SSE). Whereas F S-test refers to the shrinking of variance estimators from the variance estimators of the gene-by-gene (σ g 2) F-value obtained from ANOVA. Then, the analysis results between F ANOVA-test and F S-test are compared in order to identify which statistical test is best in analyzing significantly differentially expressed gene based on accuracy value and area under the Receiver Operator Characteristics (ROC) curve. The statistical test with higher accuracy value and has a larger area under the ROC curve is the best statistical test. We found that both F ANOVA and F S tests show that the majority of genes that are significantly differentially expressed are most affected by the main effects temperature (A) and time (B) and the interaction effect between temperature and time (AB). As for the best test, we found that F S-test is the best statistical test compared to F
[Show abstract][Hide abstract] ABSTRACT: A maltogenic amylase (MAG1) from alkaliphilicBacillus lehensisG1 was cloned, expressed inEscherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of b-cyclodextrin (b-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The
thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased
specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates.
PLoS ONE 09/2014; 9(9). DOI:10.1371/journal.pone.0106481 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium–proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.
[Show abstract][Hide abstract] ABSTRACT: Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psy-chrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to pro-duce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel fil-tration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of *25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into b-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.
[Show abstract][Hide abstract] ABSTRACT: Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β' subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.
PLoS ONE 06/2014; 9(6):e99218. DOI:10.1371/journal.pone.0099218 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5 L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320g.L(-1) wet cell weight) with a cutinase production of 3,800mg.L(-1) and an activity of 434 U.mL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.
Journal of Biotechnology 06/2014; 184. DOI:10.1016/j.jbiotec.2014.05.034 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium-proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.
[Show abstract][Hide abstract] ABSTRACT: Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.
[Show abstract][Hide abstract] ABSTRACT: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) which are largely non-similar to other known, well-characterized proteins. Predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. The present study highlights the primary effort to classify and cluster 1202 HPs of Bacillus lehensis G1 alkaliphile to serve as a platform to mine and select specific HP(s) to be studied further in greater detail.
All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the betaalphabetaalphabetabeta core structure of Trx-like proteins as well as three flanking beta-sheets, a 310 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes.
We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.
[Show abstract][Hide abstract] ABSTRACT: One of the prerequisites for the functional and structural characterisation of proteins is to obtain a sufficiently high amount of the protein sample. Recombinant protein expression systems are usually employed for the overproduction of proteins in cases where isolation from their native host is difficult. For each protein, a suitable expression system must be optimised to obtain the sample in a soluble, correctly folded conformation with high production yield. The recent discovery of psychrophilic yeasts and their potential in industrial applications have sparked interest in overexpression strategies for high-level expression of psychrophilic proteins. Here, we discuss some of the recent developments in the recombinant production of cold-adapted yeast proteins, particularly those from Glaciozyma antarctica PI12, in suitable heterologous hosts. Findings from our work, as well as from recent publications, are included.
[Show abstract][Hide abstract] ABSTRACT: The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.
The Scientific World Journal 11/2013; 2013:634317. DOI:10.1155/2013/634317 · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coptotermes curvignathus Holmgren is capable of feeding on living tree. This ability is attributed to their effective digestive system that is furnished by the termite's own cellulolytic enzymes and cooperative enzymes produced by their gut microbes. In this study, the identity of an array of diverse microbes residing in the gut of C. curvignathus was revealed by sequencing the near-full-length 16S rRNA genes. A total of 154 bacterial phylotypes were found. The Bacteroidetes was the most abundant phylum and accounted for about 65% of the gut microbial profile. This is followed by Firmicutes, Actinobacteria, Spirochaetes, Proteobacteria, TM7, Deferribacteres, Plantomycetes, Verrucomicrobia and Termite Group 1. Based on the phylogenetic study, this symbiosis can be a result of long co-evolution of gut enterotypes with the phylogenic distribution, strong selection pressure in gut and other speculative pressures that determine bacterial biome to follow. The phylogenetic distribution of cloned rRNA genes in the bacterial domain that was considerably different from other termite reflects the strong selection pressures in the gut where a proportional composition of gut microbiome of C. curvignathus has established. The selection pressures could be linked to the unique diet preference of C. curvignathus which profoundly feeds on living tree. The delicate gut microbiome composition may provide available nutrients to the host as well as potential protection against opportunistic pathogen. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Recurring dengue outbreaks occur in cyclical pattern in most endemic countries. The recurrences of dengue virus (DENV) infection predispose the population to increased risk of contracting the severe forms of dengue. Understanding the DENV evolutionary mechanism underlying the recurring dengue outbreaks has important implications for epidemic prediction and disease control.
We used a set of viral envelope (E) gene to reconstruct the phylogeny of DENV-1 isolated between the periods of 1987--2011 in Malaysia. Phylogenetic analysis of DENV-1 E gene revealed that genotype I virus clade replacements were associated with the cyclical pattern of major DENV-1 outbreaks in Malaysia. A total of 9 non-conservative amino acid substitutions in the DENV-1 E gene consensus were identified; 4 in domain I, 3 in domain II and 2 in domain III. Selection pressure analyses did not reveal any positively selected codon site within the full length E gene sequences (1485 nt, 495 codons). A total of 183 (mean dN/dS = 0.0413) negatively selected sites were found within the Malaysian isolates; neither positive nor negative selection was noted for the remaining 312 codons. All the viruses were cross-neutralized by the respective patient sera suggesting no strong support for immunological advantage of any of the amino acid substitutions.
DENV-1 clade replacement is associated with recurrences of major DENV-1 outbreaks in Malaysia. Our findings are consistent with those of other studies that the DENV-1 clade replacement is a stochastic event independent of positive selection.
[Show abstract][Hide abstract] ABSTRACT: A study was conducted to optimize sugar production from oil palm empty fruit bunch fiber (EFBF). Three different pretreatments were applied to EFBF, namely steam, steam with 5% sodium hydroxide (steam + 5% NaOH) and steam with 5% acetic acid (steam + 5% acetic acid). The hydrolyzability of the pretreated EFBF was determined using cellulolytic enzyme mixture comprising of Celluclast 1.5L (C), Viscozyme L (V) and Novozyme 188 (N). The different pretreatments showed varying degree of severities to the morphology of the fiber. Steam + 5% acetic acid pretreatment was found to cause the most severe changes to the EFBF surface. The optimum combinations of the enzymes for EFBF degradation, using a fixed parameters, were determined using Simplex Lattice mixture design. For untreated, steam pretreated, steam + 5% NaOH, steam + 5% acetic acid EFBFs, the optimum combinations enzyme were 0.49C: 0.45V: 0.06N, 0.88C: 0.12N, 0.78C: 0.03V: 0.2N and 0.90C: 0.03V: 0.07N respectively. Surface morphology of EFBF appears to be the major contributing factor that affects efficiency of enzymatic hydrolysis. Among these four samples, steam + 5% acetic acid pretreated EFBF gave the highest yield of sugars (44.36% xylose; 52.03% glucose). Applying the best enzymatic combinations, saccharification of pretreated samples was optimized using RSM. saccharification of the steam + 5% acetic acid pretreated EFBF was able to yield 99.90 ± 10.92 mg g−1 xylose and 281.77 ± 28.00 mg g−1 glucose at 2.51% enzyme loading (with total protein loading 22.1 mg per gram EFBF) in 4.55% EFBF for 35.08 h of reaction. Overall, through the combination of steam + 5% acetic acid pretreatment, enzyme mixture optimization and optimization of enzymatic saccharification, EFBF has the potential to produce substantial amount of sugars (62.36% xylose; 81.84% glucose).
Biomass and Bioenergy 09/2013; 56:137–146. DOI:10.1016/j.biombioe.2013.04.021 · 3.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chitin is a structural polysaccharide which can be degraded by chitinolytic enzymes to various derivatives that can be utilised in medicine, agriculture and water treatment. The identification and characterisation of Trichoderma virens UKM1 genes encoding for enzymes involved in crustacean chitin degradation were carried out by generating expressed sequence tags (ESTs) and analysing gene expression via DNA microarray. Three cDNA libraries of T. virens UKM1 induced with chitin, glucosamine and chitosan, respectively, were constructed. A total of 1536 cDNA clones were sequenced and 1033 of high-quality ESTs were generated. Subsequently, differences in gene expression between cells grown in the presence and absence of crustacean chitin on the third and fifth days were determined. A total of 1824 cDNA clones were spotted on glass slides and co-hybridised with Cy3-or Cy5-labeled cDNA, synthesised from mRNA isolated from cells grown in medium containing crustacean chitin or glucose (control). A total of 91 and 61 genes were expressed by more than two-fold on the third and fifth day, respectively, when the fungus used crustacean chitin as the carbon source. Several genes encoding for chitinase such as ech1 and cht3 (endochitinases), nag1 (exochitinase) and nagB (glucosamine 6-P-deaminase) were found highly expressed on both days. In addition, genes encoding for hydrophobin, serine protease and several hypothetical proteins were also expressed at high levels when cells were exposed to crustacean chitin. These proteins may play significant role in the degradation of crustacean chitin.
[Show abstract][Hide abstract] ABSTRACT: 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.