To overcome drawbacks in the conventional chemical route to synthesize eugenyl benzoate, immobilized Rhizomucor miehei lipase (RML) as the biocatalyst was proposed. The RML conjugated to a hybrid support consisting of biopolymers, chitosan (CS) and chitin nanowhiskers (CNWs). 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) was used as the crosslinker to bind the lipase. Immobilization of RML was the highest on crosslinked CS/CNWs which gave a protein loading of ∼8.12 mg/g, corresponding to specific and residual activity of 537 U/g and 137%, respectively. Fourier transform infrared spectroscopy, thermogravimetric analysis-differential thermogravimetry, field emission scanning electron and atomic force microscopy of RML-CS/CNWs revealed that RML was successfully attached to the surface of crosslinked CS/CNWs. Under an optimized condition, the highest yield of eugenyl benzoate (56.3%) was attained after 5 h using 3 mg/mL of RML-CS/CNWs with molar ratio of eugenol: benzoic acid of 3:1, as compared to only 47.3% for the free RML. Analyses of FTIR and NMR on purified eugenyl benzoate affirmed that the ester was successfully produced in the enzymatic esterification. Therefore, the use of the RML-CS/CNWs biocatalysts appears promising to afford good yields of eugenyl benzoate within a relatively shorter reaction time.
Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these PcCAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all PcCAR enzymes cluster together with the other type IV CARs. One candidate, PcCAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that PcCAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class.
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.
The sustainability in polyhydroxyalkanoates (PHA) production is drawing increasing attention as the effort to increase the economic feasibility for commercialization pursues. Oleic acid is widely preferred by bacteria but its employment for PHA production makes sustainability rather dubious. This study showed promising poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] content of 68 wt % by lipase genes-harbouring Cupriavidus malaysiensis USMAA2-4 transformant from palm olein and 1-pentanol. High oleic acid content and low oil saturation caused palm olein to outperform crude palm oil, crude palm kernel oil and soybean oil due to its preference for oleic acid shown by previous screening. The transformant showed 8-fold and 40-fold higher lipase activity compared to C. necator H16 and its wild-type respectively. The transformant was unaffected by Co2+ but the growth of C. necator H16 was inversely proportional to Co2+ concentration and the employment of 1-pentanol also ceased its growth and PHA accumulation. Although the inhibitory effect of Fe2+, Cu2+ and Zn2+ at high molarity on LipA decreased PHA content of C. malaysiensis USMAA2-4 transformant by 23-24 wt %, the lipase activity was restorable with high molarity of Ca2+, thus resulted in higher PHA content. The transformant enabled the employment of low-cost 1-pentanol as the precursor for cost-effective PHA production and its preference for palm olein contributed to higher sustainability.
Pandoraea oxalativorans DSM 23570(T) is an oxalate-degrading bacterium that was originally isolated from soil litter near to oxalate-producing plant of the genus Oxalis. Here, we report the first complete genome of P. oxalativorans DSM 23570(T) which would allow its potential biotechnological applications to be unravelled.
The attempt of this review article is to determine the impact of nuclear and mitochondrial damages on the propagation of cancer incidences. This review has advanced our understanding to altered genes and their relevant cancerous proteins. The progressive raising effects of free reactive oxygen species ROS and toxicogenic compounds contributed to significant mutation in nuclear and mitochondrial DNA where the incidence of gastric cancer is found to be linked with down regulation of some relevant genes and mutation in some important cellular proteins such as AMP-18 and CA-11. Thereby, the resulting changes in gene mutations induced the apparition of newly polymorphisms eventually leading to unusual cellular expression to mutant proteins. Reduction of these apoptotic growth factors and nuclear damages is increasingly accepted by cell reactivation effect, enhanced cellular signaling and DNA repairs. Acetylation, glycation, pegylation and phosphorylation are among the molecular techniques used in DNA repair for rectifying mutation incidences. In addition, the molecular labeling based fluorescent materials are currently used along with the bioconjugating of signal molecules in targeting disease translocation site, particularly cancers and tumors. These strategies would help in determining relevant compounds capable in overcoming problems of down regulating genes responsible for repair mechanisms. These issues of course need interplay of both proteomic and genomic studies often in combination of molecular engineering to cible the exact expressed gene relevant to these cancerous proteins.
GR15 is a short molecule or peptide composed of aliphatic amino acids and possesses to have antioxidant properties. The GR15, 1GGGAFSGKDPTKVDR15 was identified from the protein S-adenosylmethionine synthase (SAMe) expressed during the sulfur departed state of Arthrospira platensis (spirulina or cyanobacteria). The in-silico assessment and the structural features of GR15 showed its antioxidant potency. Real-time PCR analysis found the up-regulation of ApSAMe expression on day 15 against oxidative stress due to 10 mM H2O2 treatment in A. platensis (Ap). The antioxidant activity of GR15 was accessed by the cell-free antioxidant assays such as ABTS, SARS, HRAS and NO; the results showed dose-dependent antioxidant activity. The toxicity assay was performed in both in vitro and in vivo models, in which peptide does not exhibit any toxicity in MDCK cell and zebrafish embryos. The intercellular ROS reduction potential of GR15 peptide was also investigated in both in vitro and in vivo models including LDH assay, antioxidant enzymes (SOD and CAT), and fluorescent staining assay (DCFDA, Hochest and Acridine orange sting) was performed; the results showed that the GR15 peptide was effectively reduced the ROS level. Further, RT-PCR demonstrated that GR15 enhanced the antioxidant property and also up-regulated the antioxidant gene, thus reduced the ROS level in both in vitro and in vivo models. Based on the results obtained from this study, we propose that GR15 has the potential antioxidant ability; hence further research can be directed towards the therapeutic product or drug development against disease caused by oxidative stress.
The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilized enzyme microreactors (IEMR). This work is concerned with the construction of de novo designed enzyme pathways in a microreactor synthesizing chiral molecules. An interesting compound, commonly used as the building block in several pharmaceutical syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can be synthesized from simple achiral substrates using two enzymes, transketolase (TK) and transaminase (TAm). Here we describe the development of an IEMR using His6-tagged TK and TAm immobilized onto Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The kinetic parameters of both enzymes were first determined using single IEMRs evaluated by a kinetic model developed for packed bed reactors. The Km(app) for both enzymes appeared to be flow rate dependent, while the turnover number kcat was reduced 3 fold compared to solution-phase TK and TAm reactions. For the multi-step enzyme reaction, single IEMRs were cascaded in series, whereby the first enzyme, TK, catalyzed a model reaction of lithium-hydroxypyruvate (HPA) and glycolaldehyde (GA) to L-erythrulose (ERY), and the second unit of the IEMR with immobilized TAm converted ERY into ABT using (S)-α-methylbenzylamine (MBA) as amine donor. With initial 60mM (HPA and GA each) and 6mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20 min at the lowest flow rate. The ability to synthesize a chiral pharmaceutical intermediate, ABT in relatively short time proves this IEMR system as a powerful tool for construction and evaluation of de novo pathways as well as for determination of enzyme kinetics.
A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L 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 (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-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.
A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.
This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.
Pandoraea vervacti DSM 23571(T) is an oxalate metabolizing bacterium isolated from an uncultivated field soil in Mugla, Turkey. Here, we present the first complete genome sequence of P. vervacti DSM 23571(T). A complete pathway for degradation of oxalate was revealed from the genome analysis. These data are important to path new opportunities for genetic engineering in the field of biotechnology.
Pseudomonas sp. strain L10.10 (=DSM 101070) is a psychrotolerant bacterium which was isolated from Lagoon Island, Antarctica. Analysis of its complete genome sequence indicates its possible role as a plant-growth promoting bacterium, including nitrogen-fixing ability and indole acetic acid (IAA)-producing trait, with additional suggestion of plant disease prevention attributes via hydrogen cyanide production.
Planococcus rifietoensis M8(T) (=DSM 15069(T)=ATCC BAA-790(T)) is a halotolerant bacterium with potential plant growth promoting properties isolated from an algal mat collected from a sulfurous spring in Campania (Italy). This paper presents the first complete genome of P. rifietoensis M8(T). Genes coding for various potentially plant growth promoting properties were identified within its genome.
Pandoraea thiooxydans DSM 25325(T) is a thiosulfate-oxidizing bacterium isolated from rhizosphere soils of a sesame plant. Here, we present the first complete genome of P. thiooxydans DSM 25325(T). Several genes involved in thiosulfate oxidation and biodegradation of aromatic compounds were identified.
Pandoraea pnomenusa RB-38 is a bacterium isolated from a former sanitary landfill site. Here, we present the complete genome of P. pnomenusa RB38 in which an oxalate utilization pathway was identified. The genome analysis suggested the potential of this strain as an effective biocontrol agent against oxalate-producing phytopathogens.
Here, we present the first complete genome sequence of Serratia fonticola DSM 4576(T), a potential plant growth promoting (PGP) bacterium which confers solubilization of inorganic phosphate, indole-3-acetic acid production, hydrogen cyanideproduction, siderophore production and assimilation of ammonia through the glutamate synthase (GS/GOGAT) pathway. This genome sequence is valuable for functional genomics and ecological studies which are related to PGP and biocontrol activities.
Serratia multitudinisentens RB-25(T) (=DSM 28811(T) =LMG 28304(T)) is a newly proposed type strain in the genus of Serratia isolated from a municipal landfill site. Here, we present the complete genome of S. multitudinisentens RB-25(T) which contains a complete chitinase operon and other chitin and N-acetylglucosamine utilisation enzymes. To our knowledge, this is the first report of the complete genome sequence of this novel isolate and its chitinase gene discovery.
The type strain Planococcus donghaensis JH1Tis a psychrotolerant and halotolerant bacterium with starch-degrading ability. Here, we determine the carbon utilization profile of P. donghaensis JH1Tand report the first complete genome of the strain. This study revealed the strain's ability to utilize pectin and d-galacturonic acid, and identified genes responsible for degradation of the polysaccharides. The genomic information provided may serve as a fundamental resource for full exploration of the biotechnological potential of P. donghaensis JH1T.
The issue of environmental pollution has been worsened by the emergence of new contaminants whose morphology is yet to be fully understood . Several techniques have been adopted to mitigate the pollution effects of these emerging contaminants, and bioremediation involving plants, microbes, or enzymes has stood out as a cost-effective and eco-friendly approach. Enzyme-mediated bioremediation is a very promising technology as it exhibits better pollutant degradation activity and generates less waste. However, this technology is subject to challenges like temperature, pH, and storage stability, in addition to recycling difficulty as it is arduous to isolate them from the reaction media. To address these challenges, the immobilization of enzymes has been successfully applied to ameliorate the activity, stability, and reusability of enzymes. Although this has significantly increased the uses of enzymes over a wide range of environmental conditions and facilitated the use of smaller bioreactors thereby saving cost, it still comes with additional costs for carriers and immobilization. Additionally, the existing immobilization methods have their individual limitations. This review provides state-of-the-art information to readers focusing on bioremediation using enzymes. Different parameters such as: the sustainability of biocatalysts, the ecotoxicological evaluation of transformation contaminants, and enzyme groups used were reviewed. The efficacy of free and immobilized enzymes, materials and methods for immobilization, bioreactors used, challenges to large-scale implementation, and future research needs were thoroughly discussed.