Effective and rapid methods for RNA extraction from conidia, germinating conidia and appressoria of the fungal plant pathogen, Colletotrichum gloeosporioides is reported in this study. The procedure for the RNA extraction from conidia and germinating conidia was carried out using TRI REAGENT® solution (Molecular Research Center, USA) and can be completed in less than one and a half hours. The procedure for RNA extraction from appressoria was carried out using a modified protocol employing guanidine isothiocyanate and mechanical cell disruption by glass beads. The efficiency of the RNA extraction procedures was evaluated by several measures to determine RNA integrity, purity and applicability in RT-PCR. RNA integrity was assessed by observing the integrity of the major RNA species (18S and 28S rRNA) on denaturing agarose gel electrophoresis. The ethidium bromide-staining pattern of intact total RNA extracted from the three fungal morphogenetic cells showed clearly defined 28S and 18S rRNA bands and no genomic DNA contamination. Spectrophotometric assessment of RNA from each sample indicated relatively high purity and absence of carbohydrate contamination. Finally, we have demonstrated that the methods used for RNA extraction of conidia, germinating conidia and appressoria produced RNA of sufficient quality suitable for RT-PCR in detecting the expression of protein kinase A regulatory subunit gene in C. gloeosporioides.
The ability of a locally isolated clinical strain of C. albicans to adapt and response towards oxidative stress were investigated in this study. Treatment of C. albicans with hydrogen peroxide (H2O2) will exert an oxidative stress to C. albicans cells and affect the growth rate of this opportunistic pathogen. Cells that were grown in Yeast Peptone Dextrose (YPD) medium with the presence of 4.0 mM H2O2 gave a doubling time of 51 min/generation compared to 44 min/generation for those grown in 0.4 mM H2O2 and without H2O2. In order to determine the resistance level of C. albicans towards oxidative stress, cells were exposed to different concentration of H2O2. Results showed that percentage of cells viability decreased with the increased concentration of H2O2. These data indicated that C. albicans could overcome oxidative stress to a certain level before they were killed. To determine whether this strain exhibited a stress induce protection, cells were treated with mild oxidative stress for one hour before exposed to a stronger oxidative stress. Data obtained shows that cells that were pre-treated with mild oxidative stress showed higher percentage of survival compared to non pre-treated cells. This strongly suggested that stress induce protection was present in this C. albicans. Finally, in order to determine whether oxidative stress can induce yeast to hyphal morphogenesis in C. albicans, cells were then exposed to 0.4 mM H2O2 at 37 ºC and the number of hyphal formed were compared to hyphal formation when the cells were grown at 37 ºC only. However the results showed that oxidative stress failed to induce yeast to hyphal morphogenesis in this clinically isolated C. albicans strain.
Keywords: Candida albicans; oxidative stress
The present study describes the new record of Dicyathifer mannii under the family Teredinidae Rafinesque, 1815. Sampling was conducted in the mangrove area of Kuala Penyu and sample was collected from dead wood debris. The pallets of Dicyathifer is half-conical in shape and 8mm in length. The cone measured 3.9mm in length and 3.6mm in width. The cavity is 1.2mm deep; the curve of the opening on the cone is about 98% of the depth of the cone. Inside the cone cavity, from the center, a ridge with rib-like feature runs down the length of the cavity. Only one species of Dicyathifer is recorded and the present species is the first new record described in Malaysia with some additional measurement metrics for future taxonomic identification purposes.
Maltogenic amylase (MAG1) from Bacillus lehensis G1 displayed the highest hydrolysis activity on β-cyclodextrin (β-CD) to produce maltose as a main product and exhibited high transglycosylation activity on malto-oligosaccharides with polymerization degree of three and above. These substrate and product specificities of MAG1 were elucidated from structural point of view in this study. A three-dimensional structure of MAG1 was constructed using homology modeling. Docking of β-CD and malto-oligosaccharides was then performed in the MAG1 active site. An aromatic platform in the active site was identified which is responsible in substrate recognition especially in determining the enzyme's preference toward β-CD. Molecular dynamics (MD) simulation showed MAG1 structure is most stable when docked with β-CD and least stable when docked with maltose. The docking analysis and MD simulation showed that the main subsites for substrate stabilization in the active site are -2, -1, +1 and +2. A bulky residue, Trp359 at the +2 subsite was identified to cause steric interference to the bound linear malto-oligosaccharides thus prevented it to occupy subsite +3, which can only be reached by a highly bent glucose molecule such as β-CD. The resulted modes of binding from docking simulation show a good correlation with the experimentally determined hydrolysis pattern. The subsite structure generated from this study led to a possible mode of action that revealed how maltose was mainly produced during hydrolysis. Furthermore, maltose only occupies subsite +1 and +2, therefore could not be hydrolyzed or transglycosylated by the enzyme. This important knowledge has paved the way for a novel structure-based molecular design for modulation of its catalytic activities.
In this study, the pyrG gene which encodes for orotidine 5-monophosphate decarboxylase (OMP decarboxylase) of Aspergillus oryzae strain S1 was cloned and analysed. This 1.8kb A. oryzae pyrG encompasses the 5’-regulatory flanking region (465 bp), open reading frame (899 bp) and 3’-regulatory region (475 bp). The pyrG contained one intron at position 623-687 bp based on the AUGUSTUS and FGENESH (SoftBerry) analysis corresponding to the intron present in the pyrG of A. oryzae (Accession Number: Y13811). In silico analysis showed that the enzyme encoded by the A. oryzae S1 pyrG gene has a theoretical molecular weight of 30.28 kDa and theoretical pI value of 5.92. This enzyme is hydrophilic, located in a region outside of the transmembrane and it functions in the cytoplasm. Five motives such as N-glycosylation site, protein kinase C (PKC) phosphorylation site, casein kinase II (CK-2) phosphorylation site, N-myristolation site and orotidine 5-monophoshate decarboxylase active site have been identified in the pyrG amino acid sequence. The three dimensional structure of this enzyme generated via protein homology modeling using the bioinformatic software, Swiss Model, shows that OMP decarboxylase is a protein with an α/ß barrel structure possessing 8 ß-strands surrounded by 9 α-helices. The amino acid residues involved in the active site have been identified and it is located on one of the ß-strands. The pyrG DNA sequence will be used for the complementation of a pyrG auxotroph mutant of A. oryzae.
Delta 6-asid lemak desaturase dan delta 12-asid lemak desaturase merupakan enzim yang diperlukan bagi langkah desaturasi semasa proses biosintesis asid gamma-linolenik (GLA) oleh kulat oleaginus. Objektif kajian ini ialah untuk menganalisis profil pengekspresan gen mengekod enzim delta 6-asid lemak desaturase (des6) dan delta 12-asid lemak desaturase (des12) kulat oleaginus Cunninghamella bainieri semasa penghasilan GLA. Jujukan gen separa bersaiz 1372 pb bagi des6 dan 1008 pb bagi des12 telah dipencil daripada C. bainieri. Analisis pengekspresan gen menggunakan kaedah tindak balas berantai polimerase kuantitatif masa sebenar (RT-qPCR) menunjukkan perubahan kadar pengekspresan des6 adalah lebih tinggi berbanding kadar pengekspresan des12 semasa penghasilan GLA. Pengekspresan des6 adalah tertinggi selepas 24 jam dikultur dalam medium penghasilan GLA. Namun, kadar pengekspresannya menurun hingga jam ke-96 pertumbuhan tetapi meningkat semula pada jam ke-120. Bagi des12, kadar pengekspresannya adalah lebih sekata dengan pengekspresan tertinggi dikesan pada jam ke-120. Analisis penghasilan GLA menunjukkan jumlah GLA dalam sel berkolerasi dengan kadar pengekspresan des6. Hasil kajian mencadangkan bahawa aras pengekspresan des6 adalah penting dalam menentukan aras GLA dalam C. bainieri.
Mekanisme pengambilan dan penghasilan asid amino bagi mikroorganisma psikrofil yang bermandiri dan berpoliferasi
pada persekitaran sejuk melampau masih belum difahami sepenuhnya. Objektif kajian ini ialah untuk mengenal pasti
gen yang terlibat dalam penjanaan asid amino bagi yis psikrofil, Glaciozyma antarctica serta menentukan pengekspresan
gen tersebut semasa kehadiran dan kekurangan asid amino dalam medium pertumbuhan. Pengenalpastian gen telah
dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) daripada dua perpustakaan cDNA yang dibina daripada
sel yang dikultur dalam medium pertumbuhan kompleks dan medium pertumbuhan minimum tanpa asid amino. Sebanyak
3552 klon cDNA daripada setiap perpustakaan dipilih secara rawak untuk dijujuk menghasilkan 1492 transkrip unik
(medium kompleks) dan 1928 transkrip unik (medium minimum). Analisis pemadanan telah mengenl pasti gen mengekod
protein yang terlibat di dalam pengambilan asid amino bebas, biosintesis asid amino serta gen yang terlibat dengan
kitar semula asid amino berdasarkan tapak jalan yang digunakan oleh yis model, Saccharomyces cerevisiae. Analisis
pengekspresan gen menggunakan kaedah RT-qPCR menunjukkan pengekspresan gen mengekod protein yang terlibat di
dalam pengambilan asid amino bebas iaitu permease adalah tinggi pada medium kompleks manakala pengekspresan
kebanyakan gen mengekod protein yang terlibat dalam kitar semula dan biosintesis asid amino adalah tinggi di dalam
medium minimum. Kesimpulannya, gen yang terlibat dalam penjanaan dan pengambilan asid amino bagi mikroorganisma
psikrofil adalah terpulihara seperti mikroorganisma mesofil dan pengekspresan gen-gen ini adalah diaruh oleh kehadiran
atau ketiadaan asid amino bebas pada persekitaran.
Yeasts with GRAS (Generally Regarded as Safe) status are commonly used as hosts for heterologous protein production. Yeasts are suitable expression hosts as they have been extensively characterised genetically. The objective of this project was to isolate yeasts from Malaysian food sources and subsequently to develop these as alternative hosts for heterologous protein production. Yeasts were isolated from Malaysian traditional fermented food namely ‘tapai’, ‘tuak’ and ‘ragi’. A total of 23 isolates were obtained and subjected to molecular identification by amplification and sequencing of the universally conserved ribosomal internal transcribed spacer (ITS), 26S rDNA and 18S rDNA sequences. We identified three species of yeasts, Saccharomyces cerevisiae, Hanseniaspora guilliermondii and Pichia anomala, which have a long tradition of usage in food production and have no adverse effects on humans. To test the feasibility of the yeasts as heterologous expression hosts, we have constructed an integrative vector, p1926Zeo containing the yeast 26S rDNA and Zeocin® resistance cassette. The p1926Zeo vector was linearised and transformed into both P. anomala and H. guilliermondii isolates via electroporation. Both hosts were successfully transformed at a relatively high efficiency. The transformants obtained had a growth profile similar to the respective wild type, indicating that integration of the plasmids into the host chromosome did not affect growth. These transformants were stable as they exhibited resistance to Zeocin even after 20 generations. Thus, both P. anomala and H. guilliermondii isolates exhibited the potential to be further developed as alternative heterologous protein expression hosts.
Kitin merupakan polisakarida struktur yang dapat dicurai oleh enzim kitinolisis kepada pelbagai terbitan yang boleh digunakan dalam bidang perubatan, pertanian dan rawatan air. Pengenalpastian dan pencirian gen-gen Trichoderma virens UKM1 mengekod enzim terlibat dalam pencuraian kitin krustasea telah dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) dan analisis pengekspresan gen menggunakan mikroatur DNA. Sebanyak tiga perpustakaan cDNA T. virens UKM1 yang masing-masing diaruh oleh kitin, glukosamina dan kitosan telah dibina. Sejumlah 1536 klon cDNA telah dijujuk dan sebanyak 1033 ESTs berkualiti telah dijana. Seterusnya, perbezaan pengekspresan gen apabila pertumbuhan kulat diaruh dengan kehadiran kitin krustasea dan tanpa kitin pada hari ketiga dan kelima telah ditentukan. Sebanyak 1824 klon cDNA telah dititik ke atas slaid kaca dan dihibrid bersama dengan cDNA terlabel Cy3 atau Cy5 yang disintesis daripada mRNA yang dipencil daripada kulat yang ditumbuhkan dalam medium mengandungi kitin krustasea atau glukosa (kawalan). Sebanyak 91 dan 61 gen, masing-masing bagi hari ketiga dan kelima didapati terekspres melebihi dua gandaan apabila kulat menggunakan kitin krustasea sebagai sumber karbon. Beberapa gen mengekod kitinase seperti ech1 dan cht3 (endokitinase), nag1 (eksokitinase) dan nagB (glukosamina 6-P-deaminase) didapati terekspres dengan tinggi pada kedua-dua hari. Selain daripada itu, gen mengekod protein hidrofobin, protease serina dan beberapa protein hipotetik juga terekspres dengan tinggi dengan kehadiran kitin krustasea. Protein-protein ini dijangka memainkan peranan penting dalam membantu pencuraian kitin krustasea.
The enhancement of lignocellulose hydrolysis using enzyme complexes requires an efficient pretreatment process to obtain susceptible conditions for the enzyme attack. This study focuses on removing a major part of the lignin layer from kenaf (Hibiscus cannabinus) while simultaneously maintaining most of the hemicellulose. A two-stage pretreatment process is adopted using calcium hydroxide, Ca(OH)₂, and peracetic acid, PPA, to break the recalcitrant lignin layer from other structural polysaccharides. An experimental screening of several pretreatment chemicals, concentrations, temperatures and solid-liquid ratios enabled the production of an optimally designed pretreatment process for kenaf. Our results showed that the pretreatment process has provide 59.25% lignin removal while maintaining 87.72% and 96.17% hemicellulose and cellulose, respectively, using 1g of Ca(OH)₂/L and a 8:1 (mL:g) ratio of liquid-Ca(OH)₂ at 50 °C for 1.5 h followed by 20% peracetic acid pretreatment at 75 °C for 2 h. These results validate this mild approach for aiding future enzymatic hydrolysis.
The cyclic AMP- (cAMP-) dependent protein kinase A signaling pathway is one of the major signaling pathways responsible for regulation of the morphogenesis and pathogenesis of several pathogenic fungi. To evaluate the role of this pathway in the plant pathogenic fungus, Colletotrichum gloeosporioides, the gene encoding the catalytic subunit of cAMP-dependent protein kinase A, CgPKAC, was cloned, inactivated, and the mutant was analyzed. Analysis of the Cgpkac mutant generated via gene replacement showed that the mutants were able to form appressoria; however, their formation was delayed compared to the wild type. In addition, the mutant conidia underwent bipolar germination after appressoria formation, but no appressoria were generated from the second germ tube. The mutants also showed reduced ability to adhere to a hydrophobic surface and to degrade lipids localized in the appressoria. Based on the number of lesions produced during a pathogenicity test, the mutant's ability to cause disease in healthy mango fruits was reduced, which may be due to failure to penetrate into the fruit. These findings indicate that cAMP-dependent protein kinase A has an important role in regulating morphogenesis and is required for pathogenicity of C. gloeosporioides.
Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.
Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward 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 spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
Dehydroquinase or 3-dehydroquinate dehydratase (DHQD) reversibly cleaves 3-dehydroquinate to form 3-dehydroshikimate. Here, we describe the functional and structural features of a cold active type II 3-dehydroquinate dehydratase from the psychrophilic yeast, Glaciozyma antarctica PI12 (GaDHQD). Functional studies showed that the enzyme was active at low temperatures (10-30 °C), but displayed maximal activity at 40 °C. Yet the enzyme was stable over a wide range of temperatures (10-70 °C) and between pH 6.0-10.0 with an optimum pH of 8.0. Interestingly, the enzyme was highly thermo-tolerant, denaturing only at approximately 84 °C. Three-dimensional structure analyses showed that the G. antarctica dehydroquinase (GaDHQD) possesses psychrophilic features in comparison with its mesophilic and thermophilic counterparts such as higher numbers of non-polar residues on the surface, lower numbers of arginine and higher numbers of glycine-residues with lower numbers of hydrophobic interactions. On the other hand, GaDHQD shares some traits (i.e. total number of hydrogen bonds, number of proline residues and overall folding) with its mesophilic and thermophilic counterparts. Combined, these features contribute synergistically towards the enzyme's ability to function at both low and high temperatures.
We report a detailed structural analysis of the psychrophilic exo-β-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-β-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-β-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-β-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.
Bacillus lehensis G1 is an alkaliphilic bacterium that is capable of surviving in environments up to pH 11. Secretome related to bacterial acclimation in alkaline environment has been less studied compared to cytoplasmic and membrane proteome. The aim of this study was to gain better understanding of bacterial acclimation to alkaline media through analyzing extracellular proteins of B. lehensis. The pH range for B. lehensis growth was conducted, and two-dimensional electrophoresis and MALDI-TOF/TOF MS analysis were conducted to characterize changes in protein profiling in B. lehensis cultured at pH 8 and pH 11 when compared with those cultured at pH 10 (optimal growth pH). B. lehensis could grow well at pH ranging from 8 to 11 in which the bacteria showed to posses thinner flagella at pH 11. Proteomic analyses demonstrated that five proteins were up-regulated and 13 proteins were down-regulated at pH 8, whereas at pH 11, 14 proteins were up-regulated and 8 were down-regulated. Majority of the differentially expressed proteins were involved in the cell wall, main glycolytic pathways, the metabolism of amino acids and related molecules and some proteins of unknown function. A total of 40 differentially expressed protein spots corresponding to 33 proteins were identified; including GlcNAc-binding protein A, chitinase, endopeptidase lytE, flagellar hook-associated proteins and enolase. These proteins may play important roles in acclimation to alkaline media via reallocation of cell wall structure and changes to cell surface glycolytic enzymes, amino acid metabolism, flagellar hook-associated proteins and chaperones to sustain life under pH-stressed conditions.
Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (β-CD) (Km = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.
β-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for β-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding β-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 β-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site's narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 β-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 β-glucosidases following relevant in-silico analyses.
Carboxylic acid reductases (CARs) are well-known for their eminent selective one-step synthesis of carboxylic acids to aldehydes. To date, however, few CARs have been identified and characterized, especially from fungal sources. In this study, the CAR from the white rot fungus Pycnoporus cinnabarinus (PcCAR2) was expressed in Escherichia coli. PcCAR2's biochemical properties were explored in vitro after purification, revealing a melting temperature of 53 °C, while the reaction temperature optimum was at 35 °C. In the tested buffers, the enzyme showed a pH optimum of 6.0 and notably, a similar activity up to pH 7.5. PcCAR2 was immobilized to explore its potential as a recyclable biocatalyst. PcCAR2 showed no critical loss of activity after six cycles, with an average conversion to benzaldehyde of more than 85% per cycle. Immobilization yield and efficiency were 82% and 76%, respectively, on Ni-sepharose. Overall, our findings contribute to the characterization of a thermotolerant fungal CAR, and established a more sustainable use of the valuable biocatalyst.