ABSTRACT
Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against L-arginine as its substrate in a reaction at 20 °C, pH 9. At 10-35 °C and pH 7-9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.
Subject(s)
Basidiomycota , Saccharomyces cerevisiae , Arginase/genetics , Basidiomycota/genetics , Arginine , Escherichia coliABSTRACT
Carbon nanomaterials, due to their catalytic activity and high surface area, have potential as cell immobilization supports to increase the production of xylanase. Recombinant Kluyveromyces lactis used for xylanase production was integrated into a polymeric gel network with carbon nanomaterials. Carbon nanomaterials were pretreated before cell immobilization with hydrochloric acid (HCl) treatment and glutaraldehyde (GA) crosslinking, which contributes to cell immobilization performance. Carbon nanotubes (CNTs) and graphene oxide (GO) were further screened using a Plackett-Burman experimental design. Cell loading and agar concentration were the most important factors in xylanase production with low cell leakage. Under optimized conditions, xylanase production was increased by more than 400% compared to free cells. Immobilized cell material containing such high cell densities may exhibit new and unexplored beneficial properties because the cells comprise a large fraction of the component. The use of carbon nanomaterials as a cell immobilization support along with the entrapment method successfully enhances the production of xylanase, providing a new route to improved bioprocessing, particularly for the production of enzymes. KEY POINTS: ⢠Carbon nanomaterials (CNTs, GO) have potential as cell immobilization supports. ⢠Entrapment in a polymeric gel network provides space for xylanase production. ⢠Plackett-Burman design screen for the most important factor for cell immobilization.
Subject(s)
Kluyveromyces , Nanostructures , Nanotubes, Carbon , Enzymes, Immobilized , Kluyveromyces/genetics , Research DesignABSTRACT
Enzyme immobilization has been known to be one of the methods to improve the stability and reusability of enzyme. In this study, a strategy to optimize laccase immobilization on polyethylene terephthalate grafted with maleic anhydride electrospun nanofiber mat (PET-g-MAH ENM) was developed. The development involves the screening and optimization processes of the crucial factors that influence the immobilization yield such as enzyme concentration, pH values, covalent bonding (CV) time, CV temperature, crosslinking (CL) time, CL temperature and glutaraldehyde concentration using two-level factorial design and Box-Behnken design (BBD), respectively. It was found that laccase concentration, pH values and glutaraldehyde concentration play important role in enhancing the immobilization yield of laccase on PET-g-MAH ENM in the screening process. Subsequently, the optimization result showed at 0.28 mg/ml laccase concentration, pH 3 and 0.45% (v/v) glutaraldehyde concentrations gave the highest immobilization yield at 87.64% which was 81.2% increment from the immobilization yield before optimization. Under the optimum condition, the immobilized laccase was able to oxidize 2, 2-azino-bis 3-ethylbenzothiazoline-6- sulfonic acid (ABTS) in a broad range of pH (pH 3-6) and temperature (20- 70 °C). Meanwhile, the kinetic parameters for Km and Vmax were 1.331 mM and 0.041 mM/min, respectively. It was concluded that the optimization of immobilized laccase on PET-g-MAH ENM enhance the performance of this biocatalyst.
Subject(s)
Enzymes, Immobilized/chemistry , Laccase/chemistry , Nanofibers/chemistry , Polyethylene Terephthalates/chemistry , Benzothiazoles/chemistry , Cross-Linking Reagents/chemistry , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Kinetics , Laccase/metabolism , Maleic Anhydrides/chemistry , Sulfonic Acids/chemistry , TemperatureABSTRACT
The traditional approach of fermentation by a free cell system has limitations of low productivity and product separation that need to be addressed for production enhancement and cost effectiveness. One of potential methods to solve the problems is cell immobilization. Microbial cell immobilization allows more efficient up-scaling by reducing the nonproductive growth phase, improving product yield and simplifying product separation. Furthermore, the emergence of nanomaterials such as carbon nanotubes, graphene, and metal-based nanomaterials with excellent functional properties provides novel supports for cell immobilization. Nanomaterials have catalytic properties that can provide specific binding site with targeted cells. However, the toxicity of nanomaterials towards cells has hampered its application as it affects the biological system of the cells, which cannot be neglected in any way. This gray area in immobilization is an important concern that needs to be addressed and understood by researchers. This review paper discusses an overview of nanomaterials used for cell immobilization with special focus on its toxicological challenges and how by understanding physicochemical properties of nanomaterials could influence the toxicity and biocompatibility of the cells.
Subject(s)
Cells, Immobilized/metabolism , Escherichia coli/metabolism , Graphite/metabolism , Metals, Heavy/metabolism , Nanostructures/chemistry , Saccharomyces cerevisiae/metabolism , Biocatalysis , Cells, Immobilized/chemistry , Escherichia coli/cytology , Graphite/chemistry , Metals, Heavy/chemistry , Nanotubes, Carbon/chemistry , Saccharomyces cerevisiae/cytologyABSTRACT
Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
Subject(s)
Bacillus/enzymology , Enzymes, Immobilized/chemistry , Glycoside Hydrolases/chemistry , Oligosaccharides/chemical synthesis , Chemical Phenomena , Enzyme Stability , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Oligosaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Temperature , ThermodynamicsABSTRACT
E. coli has been engineered to produce xylitol, but the production faces bottlenecks in terms of production yield and cell viability. In this study, recombinant E. coli (rE. coli) was immobilized on untreated and treated multiwalled carbon nanotubes (MWCNTs) for xylitol production. The immobilized rE. coli on untreated MWCNTs gave the highest xylitol production (5.47â¯gâ¯L-1) and a productivity of 0.22â¯gâ¯L-1â¯h-1. The doubling time for the immobilized cells increased up to 20.40â¯h and was higher than that of free cells (3.67â¯h). Cell lysis of the immobilized cells was reduced by up to 73 %, and plasmid stability improved by up to 17 % compared to those of free cells. Xylitol production using the optimum parameters (pH 7.4, 0.005â¯mM and 29⯰C) achieved a xylitol production and productivity of 6.33â¯gâ¯L-1 and 0.26â¯gâ¯L-1â¯h-1, respectively. A seven-cycle repeated batch fermentation was carried out for up to 168â¯h, which showed maximum xylitol production of 7.36â¯gâ¯L-1 during the third cycle. Hence, this new adsorption immobilization system using MWCNTs is an alternative to improve the production of xylitol.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Nanotubes, Carbon/chemistry , Xylitol/metabolism , Cells, Immobilized/chemistry , Cells, Immobilized/metabolism , Escherichia coli/chemistry , FermentationABSTRACT
In previous studies of Lactococcus lactis, the levels of proteins secreted using heterologous signal peptides were observed to be lower than those obtained using the signal peptide from Usp45, the major secreted lactococcal protein. In this study, G1 (the native signal peptide of CGTase) and the signal peptide M5 (mutant of the G1 signal peptide) were introduced into L. lactis to investigate the effect of signal peptides on lactococcal protein secretion to improve secretion efficiency. The effectiveness of these signal peptides were compared to the Usp45 signal peptide. The highest secretion levels were obtained using the G1 signal peptide. Sequence analysis of signal peptide amino acids revealed that a basic N-terminal signal peptide is not absolutely required for efficient protein export in L. lactis. Moreover, the introduction of a helix-breaking residue in the H-region of the M5 signal peptide caused a reduction in the signal peptide hydrophobicity and decreased protein secretion. In addition, the optimization of cultivation conditions for recombinant G1-CGTase production via response surface methodology (RSM) showed that CGTase activity increased approximately 2.92-fold from 5.01 to 16.89 U/ml compared to the unoptimized conditions.
Subject(s)
Bacterial Proteins/genetics , Glucosyltransferases/drug effects , Lactococcus lactis/enzymology , Protein Sorting Signals/genetics , Bacterial Proteins/chemistry , Glucosyltransferases/biosynthesis , Glucosyltransferases/genetics , Hydrophobic and Hydrophilic Interactions/drug effects , Lactococcus lactis/genetics , Lactococcus lactis/growth & development , Protein Transport/geneticsABSTRACT
Glaciozyma antarctica PI12, is a psychrophilic yeast isolated from Antarctic sea. In this work, Expressed Sequence Tags (EST) from cells exposed to three different temperatures; 15⯰C, 0⯰C and -12⯰C were generated to identify genes associated with cold adaptation. A total of 5376 clones from each library were randomly picked and sequenced. Comparative analyses from the resulting ESTs in each condition identified several groups of genes required for cold adaptation. Additionally, 319 unique transcripts that encoded uncharacterised functions were identified in the -12⯰C library and are currently unique to G. antarctica. Gene expression analysis using RT-qPCR revealed two of the unknown genes to be up-regulated at -12⯰C compared to 0⯰C and 15⯰C. These findings further contribute to the collective knowledge into G. antarctica cold adaptation and as a resource for understanding the ecological and physiological tolerance of psychrophilic microbes in general.
Subject(s)
Adaptation, Physiological , Basidiomycota/physiology , Acclimatization , Antarctic Regions , Cold Temperature , Ice CoverABSTRACT
Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.
Subject(s)
Adaptation, Physiological/genetics , Basidiomycota/physiology , Cold Temperature , Ecosystem , Genome, Fungal , Antarctic Regions , Antifreeze Proteins/genetics , Basidiomycota/genetics , Introns , RNA, Small Nucleolar/geneticsABSTRACT
BACKGROUND: The increasing market demand for oligosaccharides has intensified the need for efficient biocatalysts. Glycosyl hydrolases (GHs) are still gaining popularity as biocatalyst for oligosaccharides synthesis owing to its simple reaction and high selectivity. PURPOSE: Over the years, research has advanced mainly directing to one goal; to reduce hydrolysis activity of GHs for increased transglycosylation activity in achieving high production of oligosaccharides. DESIGN AND METHODS: This review concisely presents the strategies to increase transglycosylation activity of GHs for oligosaccharides synthesis, focusing on controlling the reaction equilibrium, and protein engineering. Various modifications of the subsites of GHs have been demonstrated to significantly modulate the hydrolysis and transglycosylation activity of the enzymes. The clear insight of the roles of each amino acid in these sites provides a platform for designing an enzyme that could synthesize a specific oligosaccharide product. CONCLUSIONS: The key strategies presented here are important for future improvement of GHs as a biocatalyst for oligosaccharide synthesis.
Subject(s)
Hydrolases/chemistry , Oligosaccharides/chemistry , GlycosylationABSTRACT
Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.
Subject(s)
Bacterial Proton-Translocating ATPases/genetics , Bacterial Proton-Translocating ATPases/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Knockout Techniques , Models, Biological , Succinic Acid/metabolism , Fermentation , Glucose/metabolism , Glycerol/metabolism , Metabolic Networks and Pathways , MutationABSTRACT
Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H(+) conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l(-1) (41.2-fold in 7 days) from glycerol and .39 g l(-1) (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Formate Dehydrogenases/genetics , Glucose/metabolism , Glycerol/metabolism , Succinic Acid/metabolism , Computational Biology , Gene Knockout Techniques , Metabolic Networks and Pathways , Sequence DeletionABSTRACT
The structure of a novel psychrophilic ß-mannanase enzyme from Glaciozyma antarctica PI12 yeast has been modelled and analysed in detail. To our knowledge, this is the first attempt to model a psychrophilic ß-mannanase from yeast. To this end, a 3D structure of the enzyme was first predicted using a threading method because of the low sequence identity (<30%) using MODELLER9v12 and simulated using GROMACS at varying low temperatures for structure refinement. Comparisons with mesophilic and thermophilic mannanases revealed that the psychrophilic mannanase contains longer loops and shorter helices, increases in the number of aromatic and hydrophobic residues, reductions in the number of hydrogen bonds and salt bridges and numerous amino acid substitutions on the surface that increased the flexibility and its efficiency for catalytic reactions at low temperatures.
Subject(s)
Basidiomycota/enzymology , beta-Mannosidase/chemistry , Acclimatization , Amino Acid Sequence , Basidiomycota/chemistry , Basidiomycota/physiology , Cold Temperature , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Conformation , Sequence Alignment , beta-Mannosidase/metabolismABSTRACT
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.
Subject(s)
Calcium Hydroxide/chemistry , Hibiscus/chemistry , Lignin/isolation & purification , Peracetic Acid/chemistry , Polysaccharides/isolation & purification , Xylose/chemistry , Endo-1,4-beta Xylanases/chemistry , Fungal Proteins/chemistry , HydrolysisABSTRACT
Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.
Subject(s)
Bacterial Proteins/metabolism , Recombinant Proteins/metabolism , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Mutation , Protein Transport , Recombinant Proteins/geneticsABSTRACT
Two thermophilic bacteria (SK3-4 and DT3-1) were isolated from the Sungai Klah (SK) and Dusun Tua (DT) hot springs in Malaysia. The cells from both strains were rod-shaped, stained Gram positive and formed endospores. The optimal growth of both strains was observed at 55 degrees C and pH 7. Strain DT3-1 exhibited a higher tolerance to chloramphenicol (100 microg ml(-1)) but showed a lower tolerance to sodium chloride (2%, w/v) compared to strain SK3-4. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains belong to the genus Anoxybacillus. High concentrations of 15:0 iso in the fatty acid profiles support the conclusion that both strains belong to the genus Anoxybacillus and exhibit unique fatty acid compositions and percentages compared to other Anoxybacillus species. The DNA G + C contents were 42.0 mol% and 41.8 mol% for strains SK3-4 and DT3-1, respectively. Strains SK3-4 and DT3-1 were able to degrade pullulan and to produce maltotriose and glucose, respectively, as their main end products. Based on phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequences, and the DNA G + C content, we propose that strains SK3-4 and DT3-1 are new pullulan-degrading Anoxybacillus strains.