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 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.
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.
This study investigates the effect of strategies on poly(3-hydroxybutyrate) [P(3HB)] production in bioreactor. In the production of P(3HB), urea and glucose feeding streams were developed to characterize the fed-batch culture conditions for new Cupriavidus necator NSDG-GG mutant. Feeding urea in repeated fed-batch stage (RFB-I) at 6, and 12 h in cultivation led to insignificant kinetic effect on the cell dry mass (CDM) and P(3HB) accumulation. Feeding glucose in repeated fed-batch stage (RFB-II) demonstrated that the incremental feeding approach of glucose after urea in fill-and-draw (F/D) mode at 24, 30, 36, 42, and 48 h in fermentation increased CDM and P(3HB) concentration. In the 1st cycle in RFB-II, the cumulative CDM reached the value of 26.22 g/L and then it increased with the successive repeated fed-batches to attain biomass of 145 g/L at the end of 5th cycle of RFB-II. The final cumulative P(3HB) concentration at the end of 5th cycle of RFB-II reached 111 g/L with the overall yield of 0.50 g P(3HB) g gluc- 1; the CDM productivity from the RFB-II cycles was in the range of 0.84-1.3 g/(L·h). The RFB-II of glucose in an increment mode produced nearly 2.2 times more increase in CDM and P(3HB) productivities compared to the decrement RFB-II mode. Repeated cultivation had also the advantage of avoiding extra time required for innoculum preparation, and sterilization of bioreactor during batch, thereby it increased the overall industrial importance of the process.
The polyhydroxyalkanoate (PHA) producing capability of four bacterial strains isolated from Antarctica was reported in a previous study. This study analyzed the PHA synthase genes and the PHA-associated gene clusters from the two antarctic Pseudomonas isolates (UMAB-08 and UMAB-40) and the two antarctic Janthinobacterium isolates (UMAB-56 and UMAB-60) through whole-genome sequence analysis. The Pseudomonas isolates were found to carry PHA synthase genes which fall into two different PHA gene clusters, namely Class I and Class II, which are involved in the biosynthesis of short-chain-length-PHA (SCL-PHA) and medium-chain-length-PHA (MCL-PHA), respectively. On the other hand, the Janthinobacterium isolates carry a Class I and an uncharacterized putative PHA synthase genes. No other gene involved in PHA synthesis was detected in close proximity to the uncharacterized putative PHA synthase gene in the Janthinobacterium isolates, therefore it falls into a separate clade from the ordinary Class I, II, III and IV clades of PHA synthase (PhaC) phylogenetic tree. Multiple sequence alignment showed that the uncharacterized putative PHA synthase gene contains all the highly conserved amino acid residues and the proposed catalytic triad of PHA synthase. PHA biosynthesis and in vitro PhaC enzymatic assay results showed that this uncharacterized putative PHA synthase from Janthinobacterium sp. UMAB-60 is funtional. This report adds new knowledge to the PHA synthase database as we describe scarce information of PHA synthase genes and PHA-associated gene clusters from the antarctic bacterial isolates (extreme and geographically isolated environment) and comparing with those from non-antarctic PHA-producing bacteria.
Black pepper is an important commodity crop in Malaysia that generates millions of annual revenue for the country. However, black pepper yield is affected by slow decline disease caused by a soil-borne fungus Fusarium solani. RNA sequencing transcriptomics approach has been employed in this study to explore the differential gene expression in susceptible Piper nigrum L. and resistant Piper colubrinum Link. Gene expression comparative analysis of the two pepper species has yielded 2,361 differentially expressed genes (DEGs). Among them, higher expression of 1,426 DEGs was detected in resistant plant. These DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). We selected five groups of defence-related DEGs for downstream qRT-PCR analysis. Cf-9, the gene responsible for recognizing fungal avirulence protein activity was found inexpressible in susceptible plant. However, this gene exhibited promising expression in resistant plant. Inactivation of Cf-9 could be the factor that causes susceptible plant fail in recognition of F. solani and subsequently delay activation of adaptive response to fungal invasion. This vital study advance the understanding of pepper plant defence in response to F. solani and aid in identifying potential solution to manage slow decline disease in black pepper cultivation.
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.
Jeotgalibacillus spp. are halophilic bacteria within the family Planococcaceae. No genomes of Jeotgalibacillus spp. have been reported to date, and their metabolic pathways are unknown. How the bacteria survive in hypertonic conditions such as seawater is yet to be discovered. As only few studies have been conducted on Jeotgalibacillus spp., potential applications of these bacteria are unknown. Here, we present the complete genome of J. malaysiensis D5(T) (=DSM 28777(T) =KCTC 33350(T)), which is invaluable in identifying interesting applications for this genus.
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.
Cross-linked enzyme aggregate (CLEA) is easily prepared from crude enzyme and has many advantages to the environment and it is considered as an economic method in the context of industrial biocatalysis compared to free enzyme. In this work, a highly active and stable CLEA-lipase from cocoa pod husk (CPH) which is a by-product after removal of cocoa beans, were assayed for their hydrolytic activity and characterized under the optimum condition successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of the three significant factors (concentration of ammonium sulfate, concentration of glutaraldehyde and concentration of additive) to achieve higher enzyme activity of CLEA. From 20 runs, the highest activity recorded was around 9.407U (83% recovered activity) under the condition of using 20% saturated ammonium sulfate, 60mM glutaraldehyde as cross-linker and 0.17mM bovine serum albumin as feeder. Moreover, the optimal reaction temperature and pH value in enzymatic reaction for both crude enzyme and immobilized were found to be 45°C at pH 8 and 60°C at pH 8.2, respectively. A systematic study of the stability of CLEA and crude enzyme was taken with regards to temperature (25-60°C) and pH (5-10) value and in both factors, CLEA-lipase showed more stability than free lipase. The Km value of CLEA was higher compared to free enzyme (0.55mM vs. 0.08mM). The CLEA retained more than 60% of the initial activity after six cycles of reuse compared to free enzyme. The high stability and recyclability of CLEA-lipase from CPH make it efficient for different industrial applications.
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.
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.
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.
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.
Exploring novel biological anti-quorum sensing (QS) agents to control membrane biofouling is of great worth in order to allow sustainable performance of membrane bioreactors (MBRs) for wastewater treatment. In recent studies, QS inhibitors have provided evidence of alternative route to control membrane biofouling. This study investigated the role of Piper betle extract (PBE) as an anti-QS agent to mitigate membrane biofouling. Results demonstrated the occurrence of the N-acyl-homoserine-lactone (AHL) autoinducers (AIs), correlate QS activity and membrane biofouling mitigation. The AIs production in bioreactor was confirmed using an indicator strain Agrobacterium tumefaciens (NTL4) harboring plasmid pZLR4. Moreover, three different AHLs were found in biocake using thin layer chromatographic analysis. An increase in extracellular polymeric substances (EPS) and transmembrane pressure (TMP) was observed with AHL activity of the biocake during continuous MBR operation, which shows that membrane biofouling was in close relationship with QS activity. PBE was verified to mitigate membrane biofouling via inhibiting AIs production. SEM analysis further confirmed the effect of PBE on EPS and biofilm formation. These results exhibited that PBE could be a novel agent to target AIs for mitigation of membrane biofouling. Further work can be carried out to purify the active compound of Piper betle extract to target the QS to mitigate membrane biofouling.
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.
The nanoenvironment of nanobiocatalysts, such as local hydrophobicity, pH and charge density, plays a significant role in optimizing the enzymatic selectivity and specificity. In this study, Kluyveromyces lactis β-galactosidase (Gal) was assembled onto polystyrene nanofibers (PSNFs) to form PSNF-Gal nanobiocatalysts. We proposed that local hydrophobicity on the nanofiber surface could expel water molecules so that the transgalactosylation would be preferable over hydrolysis during the bioconversion of lactose, thus improve the galacto-oligosaccharides (GOS) yield. PSNFs were fabricated by electro-spinning and the operational parameters were optimized to obtain the nanofibers with uniform size and ordered alignment. The resulting nanofibers were functionalized for enzyme immobilization through a chemical oxidation method. The functionalized PSNF improved the enzyme adsorption capacity up to 3100mg/g nanofiber as well as enhanced the enzyme stability with 80% of its original activity. Importantly, the functionalized PSNF-Gal significantly improved the GOS yield and the production rate was up to 110g/l/h in comparison with 37g/l/h by free β-galactosidase. Our research findings demonstrate that the localized nanoenvironment of the PSNF-Gal nanobiocatalysts favour transgalactosylation over hydrolysis in lactose bioconversion.
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 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.