Purification and Immobilization of Superoxide Dismutase Obtained from Saccharomyces cerevisiae TBRC657 on Bacterial Cellulose and Its Protective Effect against Oxidative Damage in Fibroblasts.

Superoxide dismutase (SOD) is an essential enzyme that eliminates harmful reactive oxygen species (ROS) generating inside living cells. Due to its efficacities, SOD is widely applied in many applications. In this study, the purification of SOD produced from Saccharomyces cerevisiae TBRC657 was conducted to obtain the purified SOD that exhibited specific activity of 513.74 U/mg with a purification factor of 10.36-fold. The inhibitory test revealed that the purified SOD was classified as Mn-SOD with an estimated molecular weight of 25 kDa on SDS-PAGE. After investigating the biochemical characterization, the purified SOD exhibited optimal activity under conditions of pH 7.0 and 35 °C, which are suitable for various applications. The stability test showed that the purified SOD rapidly decreased in activity under high temperatures. To overcome this, SOD was successfully immobilized on bacterial cellulose (BC), resulting in enhanced stability under those conditions. The immobilized SOD was investigated for its ability to eliminate ROS in fibroblasts. The results indicated that the immobilized SOD released and retained its function to regulate the ROS level inside the cells. Thus, the immobilized SOD on BC could be a promising candidate for application in many industries that require antioxidant functionality under operating conditions.

Pretreatment of Cellulose from Sugarcane Bagasse with Xylanase for Improving Dyeability with Natural Dyes.

In this study, cellulose was obtained from sugarcane bagasse (SCB) and treated with xylanase to remove residual noncellulosic polymers (hemicellulose and lignin) to improve its dyeability. The cellulose fibers were dyed with natural dye solutions extracted from the heart wood of Ceasalpinia sappan Linn. and Artocarpus heterophyllus Lam. Fourier-transform infrared (FTIR) spectroscopy, Raman analysis, and whiteness index (WI) indicated successful extraction of cellulose by eliminating hemicellulose and lignin. The FTIR analysis of the dyed fibers confirmed successful interaction between natural dyes and cellulose fibers. The absorption (K) and scattering (S) coefficient (K/S) values of the dyed fibers increased in cellulose treated with xylanase before dyeing. Scanning electron microscopy (SEM) analysis showed that the surface of alkaline-bleached fibers (AB-fibers) was smoother than alkaline-bleached xylanase fibers (ABX-fibers), and the presence of dye particles on the surface of dyed fibers was confirmed by energy-dispersive spectrometry (EDS) analysis. The X-ray diffraction (XRD) revealed a higher crystallinity index (CrI), and thermal gravimetric analysis (TGA) also presented higher thermal stability in the dyed fibers with good colorfastness to light. Therefore, xylanase treatment and natural dyes can enhance dyeability and improve the properties of cellulose for various industrial applications.

Effect of xylanase-assisted pretreatment on the properties of cellulose and regenerated cellulose films from sugarcane bagasse.

A two-stage pretreatment process using alkaline and xylanase-assisted pretreatments was studied and compared to one-step alkaline pretreatment to investigate the effect of xylanase-assisted pretreatments on the properties of fibers and regenerated cellulose films. The alkaline-xylanase bleached fibers (AXB-fibers) could reduce bleaching time from 6 to 4 times to obtain an 83.3% whiteness index. A substantial proportion of the cellulose content (83%) was successfully extracted from sugarcane bagasse using the two-step process. Moreover, the fiber had an increased crystallinity index and thermal stability. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) revealed that hemicellulose and lignin were removed from the sugarcane bagasse (SCB) structure during the cellulose extraction process. The alkaline bleached fibers (AB-fibers) and AXB-fibers were dissolved in 1-butyl-3-methylimidazolium chloride, and regenerated cellulose films, AB-films and AXB-films, respectively, were prepared from the solutions. SEM images showed that both cellulose films were homogeneous and had a smooth surface. FTIR and X-ray diffraction (XRD) analyses corroborated that the transition from cellulose I to cellulose II occurred during the dissolution and regeneration process. Furthermore, the AXB-films displayed higher thermal stability and mechanical properties (258 °C and 90.43 MPa for the onset temperature and tensile strength, respectively) than those of the AB-films.

Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-ß-1,4-xylanase for applications in pulp bleaching.

In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the kcat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pKa and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry.

Heterologous expression of Aspergillus aculeatus endo-polygalacturonase in Pichia pastoris by high cell density fermentation and its application in textile scouring.

BACKGROUND: Removal of non-cellulosic impurities from cotton fabric, known as scouring, by conventional alkaline treatment causes environmental problems and reduces physical strength of fabrics. In this study, an endo-polygalacturonase (EndoPG) from Aspergillus aculeatus produced in Pichia pastoris was evaluated for its efficiency as a bioscouring agent while most current bioscouring process has been performed using crude pectinase preparation. RESULTS: The recombinant EndoPG exhibited a specific activity of 1892.08 U/mg on citrus pectin under the optimal condition at 50 °C, pH 5.0 with a V max and K m of 65,451.35 µmol/min/mL and 15.14 mg/mL, respectively. A maximal activity of 2408.70 ± 26.50 U/mL in the culture supernatant was obtained by high cell density batch fermentation, equivalent to a 4.8 times greater yield than that from shake-flask culture. The recombinant enzyme was shown to be suitable for application as a bioscouring agent, in which the wettability of cotton fabric was increased by treatment with enzyme at 300 U/mL scouring solution at 40 °C, pH 5.0 for 1 h. The bio-scoured fabric has comparable wettability to that obtained by conventional chemical scouring, but has higher tensile strength. CONCLUSION: The work has demonstrated for the first time functions of A. aculeatus EndoPG on bioscouring in eco-textile processing. EndoPG alone was shown to possess effective scouring activity. High expression level and homogeneity could be achieved in bench-scale bioreactor.

Alkaliphilic endoxylanase from lignocellulolytic microbial consortium metagenome for biobleaching of eucalyptus pulp.

Enzymatic pre-bleaching by modification of pulp fibers with xylanases is an attractive approach to reduce the consumption of toxic bleaching chemicals in the paper industry. In this study, an alkaliphilic endoxylanase gene was isolated from metagenomic DNA of a structurally stable thermophilic lignocellulose-degrading microbial consortium using amplification with conserved glycosyl hydrolase family 10 primers and subsequent genome walking. The full-length xylanase showed 78% sequence identity to an endo-beta-1,4-xylanase of Clostridium phytofermentans and was expressed in a mature form with an N-terminal His6 tag fusion in Escherichia coli. The recombinant xylanase Xyn3F was thermotolerant and alkaliphilic, working optimally at 65-70 degrees C with an optimal pH at 9- 10 and retaining >80% activity at pH 9, 60 degrees C for 1 h. Xyn3F showed a Vmax of 2,327 IU/mg and Km of 3.5 mg/ml on birchwood xylan. Pre-bleaching of industrial eucalyptus pulp with no prior pH adjustment (pH 9) using Xyn3F at 50 IU/g dried pulp led to 4.5-5.1% increase in final pulp brightness and 90.4-102.4% increase in whiteness after a single-step hypochlorite bleaching over the untreated pulp, which allowed at least 20% decrease in hypochlorite consumption to achieve the same final bleaching indices. The alkaliphilic xylanase is promising for application in an environmentally friendly bleaching step of kraft and soda pulps with no requirement for pH adjustment, leading to improved economic feasibility of the process.

Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes.

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from 50 degrees C to 55 degrees C. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

Insights into the phylogeny and metabolic potential of a primary tropical peat swamp forest microbial community by metagenomic analysis.

A primary tropical peat swamp forest is a unique ecosystem characterized by long-term accumulation of plant biomass under high humidity and acidic water-logged conditions, and is regarded as an important terrestrial carbon sink in the biosphere. In this study, the microbial community in the surface peat layer in Pru Toh Daeng, a primary tropical peat swamp forest, was studied for its phylogenetic diversity and metabolic potential using direct shotgun pyrosequencing of environmental DNA, together with analysis of 16S rRNA gene library and key metabolic genes. The community was dominated by aerobic microbes together with a significant number of facultative and anaerobic microbial taxa. Acidobacteria and diverse Proteobacteria (mainly Alphaproteobacteria) constituted the major phylogenetic groups, with minor representation of archaea and eukaryotic microbes. Based on comparative pyrosequencing dataset analysis, the microbial community showed high metabolic versatility of plant polysaccharide decomposition. A variety of glycosyl hydrolases targeting lignocellulosic and starch-based polysaccharides from diverse bacterial phyla were annotated, originating mostly from Proteobacteria, and Acidobacteria together with Firmicutes, Bacteroidetes, Chlamydiae/Verrucomicrobia, and Actinobacteria, suggesting the key role of these microbes in plant biomass degradation. Pyrosequencing dataset annotation and direct mcrA gene analysis indicated the presence of methanogenic archaea clustering in the order Methanomicrobiales, suggesting the potential on partial carbon flux from biomass degradation through methanogenesis. The insights on the peat swamp microbial assemblage thus provide a valuable approach for further study on biogeochemical processes in this unique ecosystem.

Screening of tropical fungi producing polyethylene terephthalate-hydrolyzing enzyme for fabric modification.

Microfungi were selectively isolated for production of polyethylene terephthalate (PET) fiber-degrading enzymes potentially to be used to modify the surface of polyester fabric. A range of fungi were isolated from plant surfaces and soil samples using a polycaprolactone (PCL) plate-clearing assay technique, and screened for cutinolytic esterase (cutinase) activity. Twenty-two of 115 isolates showed clearing indicating the production of cutinase. The ability of the fungi to produce cutinase in mineral medium (MM) using either potato suberin or PET (1 cm of untreated pre-washed PET fiber) fiber as substrates was assessed based on the hydrolysis of p-nitrophenyl butyrate (p-NPB). All isolates exhibited activity towards p-NPB, isolate PBURU-B5 giving the highest activity with PET fiber as an inducer. PBURU-B5 was identified as Fusarium solani based on its conidial morphology and also nucleotide sequencing from internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS). Enzymatic modification of PET cloth material properties using crude enzyme from strain PBURU-B5 showed hydrolysis of ester bonds of the PET fiber. The modification of the PET fabric resulted in increase of water and moisture absorption, and general enhancement of hydrophilicity of the fabric, properties that could facilitate processing of fabric ranging from easier dyeing while also yielding a softer feeling fabric for the user.

Comparison of the hydrolysis of polyethylene terephthalate fibers by a hydrolase from Fusarium oxysporum LCH I and Fusarium solani f. sp. pisi.

The hydrolysis of polyethylene terephthalate (PET) fibers by two fungal hydrolases was investigated. The hydrolase from a newly isolated Fusarium oxysporum strain (LCH 1) was more efficient in releasing terephthalic acid from PET fibers compared to the enzyme from F. solani f. sp. pisi DSM 62420 when equal amounts of p-nitrophenyl butyrate-hydrolyzing activity were employed. PET fabrics treated under the same conditions with the enzyme from F. oxysporum LCH 1 also showed a considerably higher increase in hydrophilicity compared to fabrics treated with the enzyme from F. solani f. sp. pisi DSM 62420.