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.
Streptomyces sp. strain CFMR 7, which naturally degrades rubber, was isolated from a rubber plantation. Whole genome sequencing and assembly resulted in 2 contigs with total genome size of 8.248 Mb. Two latex clearing protein (lcp) genes which are responsible for rubber degrading activities were identified.
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.
Pursuing the current trend, the "green-polymers", polyhydroxyalkanoates (PHAs) which are degradable and made from renewable sources have been a potential substitute for synthetic plastics. Due to the increasing concern towards escalating crude oil price, depleting petroleum resource and environmental damages done by plastics, PHAs have gained more and more attractions, both from industry and research. From the view point of Escherichia coli, a microorganism that used in the biopolymer large scale production, this paper describes the backgrounds of PHA and summarizes the current advances in PHA developments. In the short-chain-length (scl) PHAs section, the study of poly[(R)-3-hydroxybutyrate] [P(3HB)] as model polymer, ultra-high-molecular-weight P(3HB) which rarely discussed, and P(3HB-co-3HV), another commercialized PHA polymer are included. Other than that, this review also shed some light on the new members of PHA family, lactate-based PHAs and P(3HP) with topics such as block copolymers and invention of novel biopolymers. Flexibility of microorganisms in utilizing different carbon sources to accumulate medium-chain-length (mcl) PHAs and lastly, the promising scl-mcl-PHAs with interesting properties are also discussed.
Expanded bed adsorption chromatography (EBAC) is a single pass operation that has been used as primary capture step in various protein purifications. The most common problem in EBAC is often associated with successful formation of a stable fluidized bed during the absorption stage, which is critically dependent on parameters such as liquid velocity, bed height, particle (adsorbent) size and density as well as design of column and type of flow distributor. In this study, residence time distribution (RTD) test using acetone as non-binding tracer acetone was performed to evaluate liquid dispersion characteristics of the EBAC system. A high B(o) number was obtained indicating the liquid dispersion in the system employed is very minimal and the liquid flow within the bed was close to plug flow, which mimics a packed bed chromatography system. Evaluation on the effect of flow velocities and bed height on the performance of Streamline DEAE using feedstock containing heat-treated crude Escherichia coli homogenate of different biomass concentrations was carried out in this study. The advantages and disadvantages as well as the problems encountered during recovery of HBcAg with aforementioned parameters are also discussed in this paper.
Bacterial pigments are potential substitute of chemical photosensitizer for dye-sensitized solar cell (DSSC) due to its non-toxic property and cost-effective production from microbial fermentation. Serratia nematodiphila YO1 was isolated from waterfall in Malaysia and identified using 16S ribosomal RNA. Characterization of the red pigment produced by the bacteria has confirmed the pigment as prodigiosin. Prodigiosin was produced from the fermentation of the bacteria in the presence of different oil substrates. Palm oil exhibited the best performance of cell growth and equivalent prodigiosin yield compared to olive oil and peanut oil. Prodigiosin produced with palm oil supplementation was 93 mg/l compared to 7.8 mg/l produced without supplementation, which recorded 11.9 times improvement. Specific growth rate of the cells improved 1.4 times when palm oil was supplemented in the medium. The prodigiosin pigment produced showed comparable performance as a DSSC sensitizer by displaying an open circuit voltage of 336.1 mV and a maximum short circuit current of 0.098 mV/cm2. This study stands a novelty in proving that the production of prodigiosin is favorable in the presence of palm oil substrate with high saturated fat content, which has not been studied before. This is also among the first bacterial prodigiosin tested as photosensitizer for DSSC application.
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.
The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.
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.
Increasing levels of antibiotic resistance in pathogens, including Staphylococcus aureus, remains a serious problem for public health, leading to the need for better alternative antimicrobial strategies. The antimicrobial proteins produced by Lactobacillus plantarum USM8613 attributed to its anti-staphylococcal activity were identified as extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase (GADPH), both with different mechanisms of action. Extracellular transglycosylase, which contains a LysM domain, exerts a cell wall-mediated killing mechanism, while GADPH penetrates into S. aureus cells and subsequently induces the overexpression of autolysis regulators, resulting in S. aureus autolysis. Both extracellular transglycosylase and GADPH exert anti-inflammatory effects in S. aureus-infected HaCaT cells by reducing the expression and production of TLR-2, hBDs and various pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α, and IL-8). Taken together, extracellular transglycosylase and GADPH produced by L. plantarum USM8613 could potentially be applied as an alternative therapeutic agent to treat S. aureus skin infections and promote skin health.
The study reports the preparation of a composite consisting of magnetite coated with nanosilica extracted from oil palm leaves (OPL) ash as nanosupports for immobilization of Candida rugosa lipase (CRL) and its application for the synthesis of butyl butyrate. Results of immobilization parameters showed that ∼ 80% of CRL (84.5 mg) initially offered was immobilized onto the surface of the nanosupports to yield a maximum protein loading and specific activity of 67.5 ± 0.72 mg/g and 320.8 ± 0.42 U/g of support, respectively. Surface topography, morphology as well as information on surface composition obtained by Raman spectroscopy, atomic force microscopy, field emission scanning electron microscopy and transmission electron microscopy showed that CRL was successfully immobilized onto the nanosupports, affirming its biocompatibility. Under optimal conditions (3.5 mg/mL protein loading, at 45 ℃, 3 h and molar ratio 2:1 (1-butanol:n-butyric acid) the CRL/Gl-A-SiO2-MNPs gave a maximum yield of 94 ± 0.24% butyl butyrate as compared to 84 ± 0.32% in the lyophilized CRL. CRL/Gl-A-SiO2-MNPs showed an extended operational stability, retaining 50% of its initial activity after 17 consecutive esterification cycles. The results indicated that OPL derived nanosilica coated on magnetite can potentially be employed as carrier for lipase immobilization in replacement of the non-renewable conventionalsilica sources.
Biofuels from microalgae have promising potential for a sustainable bioeconomy. Algal strains' oil content and biomass yield are the most influential cost drivers in the fourth generation biofuel (FGB) production. Genetic modification is the key to improving oil accumulation and biomass yield, consequently developing the bioeconomy. This paper discusses current practices, new insights, and emerging trends in genetic modification and their bioeconomic impact on FGB production. It was demonstrated that enhancing the oil and biomass yield could significantly improve the probability of economic success and the net present value of the FGB production process. The techno-economic and socioeconomic burden of using genetically modified (GM) strains and the preventive control strategies on the bioeconomy of FGB production is reviewed. It is shown that the fully lined open raceway pond could cost up to 25% more than unlined ponds. The cost of a plastic hoop air-supported greenhouse covering cultivation ponds is estimated to be US 60,000$ /ha. The competitiveness and profitability of large-scale cultivation of GM biomass are significantly locked to techno-economic and socioeconomic drivers. Nonetheless, it necessitates further research and careful long-term follow-up studies to understand the mechanism that affects these parameters the most.
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 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.
Co-culture of microalgae and microorganisms, supported with the resulting synergistic effects, can be used for wastewater treatment, biomass production, agricultural applications and etc. Therefore, this study aimed to explore the role of Bacillus subtilis (B. subtilis) in tolerance against the harsh environment of seafood wastewater, at which these microalgal-bacterial flocs were formed by microalgae cultivation. In this present study, B. subtilis isolated from the cultivation medium of Chlorella vulgaris and exposed to different salinity (0.1-4% w/v sodium chloride) and various pH range to determine the tolerant ability and biofilm formation. Interestingly, this bacteria strain that isolated from microalgae cultivation medium showed the intense viability in the salt concentration exceeding up to 4% (w/v) NaCl but demonstrated the decrease in cell division as environmental culture undergoing over pH 10. Cell viability was recorded higher than 71% and 92% for B. subtilis inoculum in media with salt concentration greater than 20 gL-1 and external pH 6.5-9, respectively. This showed that B. subtilis isolated from microalgal-bacteria cocultivation exhibited its tolerant ability to survive in the extremely harsh conditions and thus, mitigating the stresses due to salinity and pH.
The cost of polyhydroxyalkanoates (PHAs) can be reduced by improving their productivity and recovery. In this study, we attempted to obtain a high cell density culture from a 13 L bioreactor and subsequently improved the recently developed biological recovery process using mealworms to obtain the PHA granules. A cell dry weight of 161 g/L containing 68-70 wt% P(3HB) was obtained. The freeze-dried cells contained a significant amount of mineral salts from the culture medium which reduced the cells' palatability for the mealworms. A simple washing procedure with water was sufficient to remove the residual mineral salts and this improved the cells' consumption by up to 12.5% of the mealworms' body weight. As a result, one kilogram of mealworms consumed 125 g of the washed cells daily and 87.2 g of feacal pellets were recovered, which was almost twice the weight of the unwashed cells. In addition, it also improved the purity of the PHA in the faecal pellets to a value <90% upon washing with water to remove the water-soluble compounds. This study has demonstrated a significant improvement in the production and recovery of PHA. In addition, the resulting mealworms showed a significant increase in protein content up to 79% and a decrease in fat content down to 8.3% of its dry weight.
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.
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.
Biodiesel, as a renewable and eco-friendly energy source that can be produced through algae oil esterification, has recently received much attention. Maximization of algal biomass and lipid content is crucial for commercial biodiesel production. In this study, Chlorella sp. PG96, a microalgal strain isolated from urban wastewater, was identified considering its morphological and molecular characteristics. Fractional factorial design (211-7) was employed to screen medium and environmental factors for achieving high lipid productivity. The effects of eleven factors including light intensity, light spectrum, aeration rate, temperature, salinity, NaHCO3, CO2, NaNO3, NH4Cl, MgSO4.7H2O, and K2HPO4 and their interactions on growth characteristics of Chlorella sp. PG96 (biomass and lipid production) were statistically assessed. Based on the experimental results, lipid productivity was at its maximum (54.19 ± 8.40 mglipid L-1 day-1) under a combination of high levels of all factors. The analysis also showed that physical parameters of light intensity and temperature were more effective on algal growth compared to nutritional parameters. Furthermore, nitrogen source of ammonium and carbon source of bicarbonate played more significant roles in biomass and lipid production, compared with nitrate and CO2, respectively. Although the effect of sulfur limitation on cellular growth was similar to phosphorus deficiency, S-limitation had a greater impact on lipid accumulation. The interaction between NaHCO3 and NH4Cl was the most prominent interaction affecting all responses. It is concluded that Chlorella sp. PG96 at a high level of light intensity and temperature (22500 Lux and 32 °C, respectively) can be a prospective candidate for biodiesel production.