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.
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.
As the world's population grows, it is necessary to rethink how countries throughout the world produce food in order to replace the conventional and unsustainable agricultural techniques. Microalgae cultivation using a nutrient-rich solution from hydroponic systems not only presents a novel approach to solving problems pertaining to the impact of the discharges on the natural environment but also provides a plethora of other biotechnological applications particularly in the productions of high value-added products and plants growth stimulants, which can be potentially assimilated into the circular bioeconomy (CBE) in the hydroponic sector. In this review, the potential and practicability of microalgae to be merged into hydroponics CBE are reviewed. Overall, the integration of microalgal biorefineries in hydroponics systems can be realized after considering their Technology Readiness Level and System Readiness Level beforehand. Several suggestions on strains and hydroponics system improvement using existing biotechnological tools, Artificial Intelligence (AI) and nanobiotechnology in support of the CBE will be covered.
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.
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.
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.
An intensified process was developed that enables high level production of recombinant core streptavidin (cSAV), a non-glycosylated tetrameric protein utilised in a wide range of applications. A pH-stat fed-batch feeding strategy was employed to achieve high-cell-density and improve volumetric yield of cSAV which was expressed as inclusion bodies (IBs). The effect of induction at different cell densities (OD 20, 60 and 100) on volumetric and specific yield were then studied. Highest volumetric yield of cSAV (1550 mg L-1) was obtained from induction at OD 100 without significant reductions in specific yield. To recover active cSAV from IBs, the possibility of refolding using a temperature-based refolding method was investigated. Refolded cSAV obtained from temperature-based refolding were then compared against cSAV refolded with conventional dialysis and dilution methods using quantitative and qualitative metrics. The temperature-based refolding method was found to improve the yield of cSAV by 6-18% in comparison to conventional methods without compromising quality. Intensification was achieved by reductions in process volumes and a more concentrated product stream. Using the newly developed process, the volumetric yield of cSAV IBs was improved by thirty-six fold in comparison to low-cell-density shake flask cultivation, and 33% of cSAV can be recovered from IBs at 90% purity.
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.
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.
The glycoprotein (G) of Nipah virus (NiV) is important for virus infectivity and induction of the protective immunity. In this study, the extra-cellular domain of NiV G protein was fused with hexahistidine residues at its N-terminal end and expressed in Escherichia coli. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 8 M urea and the protein was purified to homogeneity under denaturing conditions using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the urea. Light scattering analysis of the purified protein showed primarily monodispersity. The purified protein showed significant reactivity with the antibodies present in the sera of NiV-infected swine, as demonstrated in Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Taken together, the data indicate the potential usefulness of the purified G protein for structural or functional studies and the development of immunoassay for detection of the NiV antibodies.
Natural antioxidants from sustainable sources are favoured to accommodate worldwide antioxidant demand. In addition to bioprospecting for natural and sustainable antioxidant sources, this study aimed to investigate the relationship between the bioactives (i.e. carotenoid and phenolic acids) and the antioxidant capacities in fucoxanthin-producing algae. Total carotenoid, phenolic acid, fucoxanthin contents and fatty acid profile of six species of algae (five microalgae and one macroalga) were quantified followed by bioactivity evaluation using four antioxidant assays. Chaetoceros calcitrans and Isochrysis galbana displayed the highest antioxidant activity, followed by Odontella sinensis and Skeletonema costatum which showed moderate bioactivities. Phaeodactylum tricornutum and Saccharina japonica exhibited the least antioxidant activities amongst the algae species examined. Pearson correlation and multiple linear regression showed that both carotenoids and phenolic acids were significantly correlated (p<0.05) with the antioxidant activities, indicating the influence of these bioactives on the algal antioxidant capacities.
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.
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.
Sulfanilic acid (4-aminobenzenesulfonic acid) is a sulfonated aromatic amine widely used in chemical industries for synthesis of various organic dyes and sulfa drugs. There are quite a few microbial co-cultures or single isolates capable of completely degrading this compound. Novosphingobium resinovorum SA1 was the first single bacterium which could utilize sulfanilic acid as its sole carbon, nitrogen and sulfur source. The strain has versatile catabolic routes for the bioconversion of numerous other aromatic compounds. Here, the complete genome sequence of the N. resinovorum SA1 strain is reported. The genome consists of a circular chromosome of 3.8 Mbp and four extrachromosomal elements between 67 and 1 759.8 kbp in size. Three alternative 3-ketoadipate pathways were identified on the plasmids. Sulfanilic acid is decomposed via a modified 3-ketoadipate pathway and the oxygenases involved form a phylogenetically separate branch on the tree. Sequence analysis of these elements might provide a genetic background for deeper insight into the versatile catabolic metabolism of various aromatic xenobiotics, including sulfanilic acid and its derivatives. Moreover, this is also a good model strain for understanding the role and evolution of multiple genetic elements within a single strain.
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.
We isolated fifty-two strains from the marine aquaculture ponds in Malaysia that were evaluated for their lipid production and ammonium tolerance and four isolates were selected as new ammonium tolerant microalgae with high-lipid production: TRG10-p102 Oocystis heteromucosa (Chlorophyceae); TRG10-p103 and TRG10-p105 Thalassiosira weissflogii (Bacillariophyceae); and TRG10-p201 Amphora coffeiformis (Bacillariophyceae). Eicosapentenoic acid (EPA) in three diatom strain was between 2.6 and 18.6 % of total fatty acids, which were higher than in O. heteromucosa. Only A. coffeiformi possessed arachidonic acid. Oocystis heteromucosa naturally grew at high ammonium concentrations (1.4-10 mM), whereas the growth of the other strains, T. weissflogii and A. coffeiformi, were visibly inhibited at high ammonium concentrations (>1.4 mM-NH4). However, two strains of T. weissflogii were able to grow at up to 10 mM-NH4 by gradually acclimating to higher ammonium concentrations. The ammonium tolerant strains, especially T. weissflogii which have high EPA contents, were identified as a valuable candidate for biomass production utilizing NH4-N media, such as ammonium-rich wastewater.
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.
Enzymatic reactions involving lipases as catalyst in transesterification can be an excellent alternative to produce environmental-friendly biodiesel. In this study, lipase extracted from Cocoa Pod Husk (CPH) and immobilized through cross linked enzyme aggregate (CLEA) technology catalysed the transesterification of Jatropha curcas oil successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of 3% (w/w) enzyme loading, 4h reaction time and 1:6 oil/ethanol ratio to achieve the highest conversion of free fatty acid and glycerides into biodiesel (93%). The reusability of CLEA-lipase was tested and after seven cycles, the conversion percentage reduced to 58%. The results revealed that CLEA lipase from CPH is a potential catalyst for biodiesel production.
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.