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Fulltext Application of response surface methodology for optimization of decolorization and mineralization of triazo dye Direct Blue 71 by Pseudomonas aeruginosa

Hafshejani MK, Ogugbue CJ, Morad N

3 Biotech, 2014 Dec;4(6):605-619.
PMID: 28324306 DOI: 10.1007/s13205-013-0192-7

The decolorization and degradation of Direct Blue 71 were investigated using a mono culture of Pseudomonas aeruginosa. The bacterium was able to decolorize the dye medium to 70.43 % within 48 h under microaerophilic conditions. The medium was then aerated for 24 h to promote the biodegradation of the aromatic amines generated from azo bond cleavage. Reduction in total organic carbon in dye medium was 42.58 % in the microaerophilic stage and 78.39 % in the aerobic stage. The degradation metabolites formed were studied using UV-vis techniques, high performance liquid chromatography, Fourier transform infra red spectroscopy and nuclear magnetic resonance spectroscopy analysis. Data obtained provide evidence for the formation of aromatic amines and their subsequent oxidative biodegradation by a single strain of P. aeruginosa during successive microaerophilic/aerobic stages in the same flask. The influence of incubation temperature (20-45 °C), medium pH (5-10) and initial dye concentration (25-150 mg/L) on decolorization was evaluated to greatly influence decolorization extent. The optimal decolorization conditions were determined by response surface methodology based on three-variable central composite design to obtain maximum decolorization and to determine the significance and interaction effect of the variables on decolorization. The optimal conditions of response were found to be 35.15 °C, pH 8.01 and 49.95 mg/L dye concentration giving an experimental decolorization value of 84.80 %. Very high regression coefficient between the variables and the response (R(2) = 0.9624) indicated a good evaluation of experimental data by polynomial regression model.
Fulltext Cross-amplification of microsatellite markers across agarwood-producing species of the Aquilarieae tribe (Thymelaeaceae)

Pern YC, Lee SY, Ng WL, Mohamed R

3 Biotech, 2020 Mar;10(3):103.
PMID: 32099744 DOI: 10.1007/s13205-020-2072-2

Tree species in the Aquilarieae tribe of the Thymelaeaceae family produce agarwood, a natural product highly valued for its fragrance, but the species are under threat due to indiscriminate harvesting. For conservation of these species, molecular techniques such as DNA profiling have been used. In this study, we assessed cross-amplification of microsatellite markers, initially developed for three Aquilaria species (A.crassna, A.malaccensis, and A.sinensis), on ten other agarwood-producing species, including members of Aquilaria (A.beccariana, A.hirta, A.microcarpa, A.rostrata, A.rugosa, A.subintegra, and A.yunnanensis) and Gyrinops (G.caudata, G.versteegii, and G.walla), both from the Aquilarieae tribe. Primers for 18 out of the 30 microsatellite markers successfully amplified bands of expected sizes in 1 sample each of at least 10 species. These were further used to genotype 74 individuals representing all the 13 studied species, yielding 13 cross-amplifiable markers, of which only 1 being polymorphic across all species. At each locus, the number of alleles ranged from 7 to 23, indicating a rather high variability. Four markers had relatively high species discrimination power. Our results demonstrated that genetic fingerprinting can be an effective tool in helping to manage agarwood genetic resources by potentially supporting the chain-of-custody of agarwood and its products in the market.
Highly divergent regions in the complete plastome sequences of Aquilaria are suitable for DNA barcoding applications including identifying species origin of agarwood products

Hishamuddin MS, Lee SY, Syazwan SA, Ramlee SI, Lamasudin DU, Mohamed R

3 Biotech, 2023 Mar;13(3):78.
PMID: 36761338 DOI: 10.1007/s13205-023-03479-1

Members of Aquilaria Lam. (Thymelaeaceae) are evergreen trees that are widely distributed in the Indomalesia region. Aquilaria is highly prized for its unique scented resin, agarwood, which is often the subject of unlawful trade activities. Survival of the tree is heavily threatened by destructive harvesting and agarwood poaching, leading to its protection under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Unfortunately, an efficient species identification method, which is crucial to aid in the conservation efforts of Aquilaria is lacking. Here, we described our search for a suitable specific DNA barcode for Aquilaria species using eight complete plastome sequences. We identified five highly variable regions (HVR) (matK-rps16, ndhF-rpl32, psbJ-petA, trnD, and trnT-trnL) in the plastomes. These regions were further analyzed using the neighbor-joining (NJ) method to assess their ability at discriminating the eight species. Coupled with in silico primer design, two potential barcoding regions, psbJ-petA and trnT-trnL, were identified. Their strengths in species delimitation were evaluated individually and in combination, via DNA barcoding analysis. Our findings showed that the combined dataset, psbJ-petA + trnT-trnL, effectively resolved members of the genus Aquilaria by clustering all species into their respective clades. In addition, we demonstrated that the newly proposed DNA barcode was capable at identifying the species of origin of six commercial agarwood samples that were included as unknown samples. Such achievement offers a new technical advancement, useful in the combat against illicit agarwood trades and in assisting the conservation of these valuable species in natural populations.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03479-1.
Gut microbiota of healthy Asians and their discriminative features revealed by metagenomics approach

Taha SFM, Bhassu S, Omar H, Raju CS, Rajamanikam A, Govind SKP, et al.

3 Biotech, 2023 Aug;13(8):275.
PMID: 37457869 DOI: 10.1007/s13205-023-03671-3

This study is conducted to identify the microbial architecture and its functional capacity in the Asian population via the whole metagenomics approach. A brief comparison of the Asian countries namely Malaysia, India, China, and Thailand, was conducted, giving a total of 916 taxa under observation. Results show a close representation of the taxonomic diversity in the gut microbiota of Malaysia, India, and China, where Bacteroidetes, Firmicutes, and Actinobacteria were more predominant compared to other phyla. Mainly due to the multi-racial population in Malaysia, which also consists of Malays, Indian, and Chinese, the population tend to share similar dietary preferences, culture, and lifestyle, which are major influences that shapes the structure of the gut microbiota. Moreover, Thailand showed a more distinct diversity in the gut microbiota which was highly dominated by Firmicutes. Meanwhile, functional profiles show 1034 gene families that are common between the four countries. The Malaysia samples are having the most unique gene families with a total of 67,517 gene families, and 51 unique KEGG Orthologs, mainly dominated by the metabolic pathways, followed by microbial metabolism in diverse environments. In conclusion, this study provides some general overview on the structure of the Asian gut microbiota, with some additional highlights on the Malaysian population.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03671-3.
Fulltext Treatment of landfill leachate using ASBR combined with zeolite adsorption technology

Lim CK, Seow TW, Neoh CH, Md Nor MH, Ibrahim Z, Ware I, et al.

3 Biotech, 2016 Dec;6(2):195.
PMID: 28330267 DOI: 10.1007/s13205-016-0513-8

Sanitary landfilling is the most common way to dispose solid urban waste; however, improper landfill management may pose serious environmental threats through discharge of high strength polluted wastewater also known as leachate. The treatment of landfill leachate to fully reduce the negative impact on the environment, is nowadays a challenge. In this study, an aerobic sequencing batch reactor (ASBR) was proposed for the treatment of locally obtained real landfill leachate with initial ammoniacal nitrogen and chemical oxygen demand (COD) concentration of 1800 and 3200 mg/L, respectively. ASBR could remove 65 % of ammoniacal nitrogen and 30 % of COD during seven days of treatment time. Thereafter, an effective adsorbent, i.e., zeolite was used as a secondary treatment step for polishing the ammoniacal nitrogen and COD content that is present in leachate. The results obtained are promising where the adsorption of leachate by zeolite further enhanced the removal of ammoniacal nitrogen and COD up to 96 and 43 %, respectively. Furthermore, this combined biological-physical treatment system was able to remove heavy metals, i.e. aluminium, vanadium, chromium, magnesium, cuprum and plumbum significantly. These results demonstrate that combined ASBR and zeolite adsorption is a feasible technique for the treatment of landfill leachate, even considering this effluent's high resistance to treatment.
Microwave-assisted solvent-free extraction of essential oil from Coleus aromaticus: anti-phytopathogenic potential for fruit post-harvesting

Gunny AAN, Fang LP, Misnan NM, Gopinath SCB, Salleh NHM, Hashim RHR, et al.

3 Biotech, 2021 Apr;11(4):166.
PMID: 33816043 DOI: 10.1007/s13205-021-02701-2

This work evaluates the fungicidal effect of essential oil from Coleus aromaticus (C. aromaticus) by solvent-free microwave-assisted extraction with a yield of 0.54%. Fourier-transform infrared spectroscopy was utilised to identify the functional groups, which were O-H, C-O, C-H, and C=C. Gas chromatography-mass spectrometry analysis was performed to determine the primary essential oil components, namely, thymol (92.62%), thymoquinone (2.64%), creosol (1.77%), linalool (1.68%), p-Cymene-2,5-diol (0.73%), and p-Cymene (0.56%). The inhibitory effect of essential oil extracted from C. aromaticus against the isolated fungi, Aspergillus niger from mango, was investigated. The mycelial growth inhibition of the extracted essential oil by the poisoned food test and disc diffusion assay showed the reduction at 79.63 ± 1.7 and 70.45 ± 6.54%, respectively. In vivo experiment was conducted with artificially wounded and unwounded mangoes, applying the extracted essential oil to the wounded mangoes inoculated with A. niger that could decrease the disease incidence from 100 to 58.33 ± 14.43%. Meanwhile, the treatment of the extracted essential oil did not affect the quality of the mango and it also shows improvement in weight loss reduction of the mango in comparison to the chemical fungicide and untreated mango. Hence, essential oil from C. aromaticus by solvent-free microwave-assisted extraction could be used as an effective control for the fruit spoilage and potential source of fruit preservative.
Fulltext A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

Badai SS, Rasid OA, Parveez GKA, Masani MYA

3 Biotech, 2020 Dec;10(12):530.
PMID: 33214977 DOI: 10.1007/s13205-020-02514-9

Cetyltrimethylammonium bromide (CTAB) is the preferred detergent in RNA extraction of oil palm tissues. However, the CTAB-based protocol is time-consuming. In this study, a combination of the CTAB-based method and silica-based purification reduced the extraction time from two days to five hours. Quality of total RNA from 27 different tissues of oil palm was shown to have an RNA integrity number (RIN) value of more than seven. The extracted RNA was evaluated by RT-qPCR using three reference oil palm genes (GRAS, CYP2, and SLU7) and three putative mesocarp-specific transcripts annotated as WRKY DNA-binding protein 70 (WRKY-70), metallothionein (MT) and pentatricopeptide repeat (PPR) genes. Tissue-specific expression profiling across complete developmental stages of mesocarp and vegetative tissues was determined in this study. Overall, the RNA extraction protocol described here is rapid, simple and yields good quality RNAs from oil palm tissues.
Fulltext Targeted DNA complementation on a 1,1'-carbonyldiimidazole-functionalized surface for identifying Mycobacterium tuberculosis

Xu S, Xue Y, Guo F, Xu M, Gopinath SCB, Mao X

3 Biotech, 2020 May;10(5):227.
PMID: 32373419 DOI: 10.1007/s13205-020-02216-2

Herein, a rapid and sensitive current-volt measurement was developed for identifying the IS6110 DNA sequence to diagnose Mycobacterium tuberculosis (TB). An aminated capture probe was immobilized on a 1,1'-carbonyldiimidazole-functionalized interdigitated electrode (IDE) silica substrate, and the target sequence was detected by complementation. It was found that all tested concentrations displayed a higher response in current changes than the control, and the limit of detection was 10 fM. The sensitivity ranged from 1 to 10 fM. The control sequences with single-, triple-mismatch and noncomplementary sequences showed great discrimination. This rapid and easy DNA detection method helps to identify M. tuberculosis for early-stage diagnosis of TB.
Fulltext Bioremediation of palm industry wastes using vermicomposting technology: its environmental application as green fertilizer

Rupani PF, Embrandiri A, Ibrahim MH, Shahadat M, Hansen SB, Mansor NNA

3 Biotech, 2017 Jul;7(3):155.
PMID: 28623493 DOI: 10.1007/s13205-017-0770-1

Several technologies are being applied for treatment of palm oil mill wastes. Among them, the biological treatments (vermicomposting) have widely been recognized as one of the most efficient and eco-friendly methods for converting organic waste materials into valuable products. The present study focuses on vermicomposting of acidic palm oil mill effluent (POME) mixed with the palm pressed fibre (PPF) which are found difficult to decompose in the environment. The industrial waste (POME) was vermicomposted using Lumbricus rubellus under laboratory conditions for a period of 45 days. A significant improvement in nitrogen, phosphorus, and potassium content was monitored during vermicomposting process. In addition, the decline in C:N ratio of vermicompost (up to 17.20 ± 0.60) reflects the degree of stabilization of POME-PPF mixture. Different percentages of the vermicompost extract obtained from POME-PPF mixture were also examined for the germination of mung bean (Vigna radiata) seed. The results showed that 75% vermicompost extract demonstrated better performance for the seed germination. On the basis of significant findings, POME-PPF mixture can be successfully used as a feeding material for the earthworms, while on the other hand, it can also be used as a cost-effective fertilizer for the germination and the proper growth of mung bean.
Fulltext Electrospun polyurethane nanofibrous composite impregnated with metallic copper for wound-healing application

Jaganathan SK, Mani MP

3 Biotech, 2018 Aug;8(8):327.
PMID: 30073112 DOI: 10.1007/s13205-018-1356-2

In this study, a wound dressing based on polyurethane (PU) blended with copper sulphate nanofibers was developed using an electrospinning technique. The prepared PU and PU nanocomposites showed smooth fibers without any bead defects. The prepared nanocomposites showed smaller fiber (663 ± 156.30 nm) and pore (888 ± 70.93 nm) diameter compared to the pristine PU (fiber diameter 1159 ± 147.48 nm and pore diameter 1087 ± 62.51 nm). The interaction of PU with copper sulphate was evident in the infrared spectrum through hydrogen-bond formation. Thermal analysis displayed enhanced weight residue at higher temperature suggesting interaction of PU with copper sulphate. The contact angle measurements revealed the hydrophilic nature of the prepared nanocomposites (71° ± 2.309°) compared with pure PU (100° ± 0.5774°). The addition of copper sulphate into the PU matrix increased the surface roughness, as revealed in the atomic force microscopy (AFM) analysis. Mechanical testing demonstrated the enhanced tensile strength behavior of the fabricated nanocomposites (18.58 MPa) compared with the pristine PU (7.12 MPa). The coagulation assays indicated the enhanced blood compatibility of the developed nanocomposites [activated partial thromboplastin time (APTT)-179 ± 3.606 s and partial thromboplastin time (PT)-105 ± 2.646 s] by showing a prolonged blood clotting time compared with the pristine PU (APTT-147.7 ± 3.512 s and PT-84.67 ± 2.517 s). Furthermore, the hemolysis and cytotoxicity studies suggested a less toxicity nature of prepared nanocomposites by displaying low hemolytic index and enhanced cell viability rates compared with the PU membrane. It was observed that the fabricated novel wound dressing possesses better physicochemical and enhanced blood compatibility properties, and may be utilized for wound-healing applications.
Fulltext Cladosporium cladosporioides from the perspectives of medical and biotechnological approaches

AlMatar M, Makky EA

3 Biotech, 2016 Jun;6(1):4.
PMID: 28330073 DOI: 10.1007/s13205-015-0323-4

Fungi are important natural product sources that have enormous potential for the production of novel compounds for use in pharmacology, agricultural applications and industry. Compared with other natural sources such as plants, fungi are highly diverse but understudied. However, research on Cladosporium cladosporioides revealed the existence of bioactive products such as p-methylbenzoic acid, ergosterol peroxide (EP) and calphostin C as well as enzymes including pectin methylesterase (PME), polygalacturonase (PG) and chlorpyrifos hydrolase. p-Methylbenzoic acid has ability to synthesise 1,5-benzodiazepine and its derivatives, polyethylene terephthalate and eicosapentaenoic acid. EP has anticancer, antiangiogenic, antibacterial, anti-oxidative and immunosuppressive properties. Calphostin C inhibits protein kinase C (PKC) by inactivating both PKC-epsilon and PKC-alpha. In addition, calphostin C stimulates apoptosis in WEHI-231 cells and vascular smooth muscle cells. Based on the stimulation of endoplasmic reticulum stress in some types of cancer, calphostin C has also been evaluated as a potential photodynamic therapeutic agent. Methylesterase (PME) and PG have garnered attention because of their usage in the food processing industry and significant physiological function in plants. Chlorpyrifos, a human, animal and plant toxin, can be degraded and eliminated by chlorpyrifos hydrolase.
Fulltext Proteome of rice roots treated with exogenous proline

Teh CY, Ho CL, Shaharuddin NA, Lai KS, Mahmood M

3 Biotech, 2019 Mar;9(3):101.
PMID: 30800612 DOI: 10.1007/s13205-019-1615-x

Proteomic analysis was conducted to identify the rice root proteins induced by exogenous proline and their involvement in root growth. Proteins were extracted from the root tissues grown under two conditions, T1 (control) and T2 (10 mM proline), and profiled by two-dimensional polyacrylamide gel electrophoresis. Seventeen of 30 differentially expressed proteins were identified by mass spectrometry. Proline-treated rice roots showed up-regulation and down-regulation of nine and eight proteins, respectively, when compared to those in the control. Among the differentially expressed proteins, the down-regulation of glutathione reductase and peroxidase could be involved in the regulation of cellular hydrogen peroxide and reactive oxygen species levels that modulate the root cell wall structure. Differentially expressed proteins identified as pathogenesis-related proteins might be related to stress adaptive mechanisms in response to exogenous proline treatment. In addition, differentially expressed protein identified as the fructose-bisphosphate aldolases and cytochrome c oxidase might be associated with energy metabolism, which is needed during root developmental process. This is the first attempt to study the changes in rice root proteome treated with proline. The acquired information could open new avenues for further functional studies on the involvement of proline in modulating root development and its relation to stress adaptation of plants.
Fulltext Pseudogene product YqiG is important for pflB expression and biohydrogen production in Escherichia coli BW25113

Zakaria MA, Mohd Yusoff MZ, Zakaria MR, Hassan MA, Wood TK, Maeda T

3 Biotech, 2018 Oct;8(10):435.
PMID: 30306004 DOI: 10.1007/s13205-018-1461-2

Pseudogenes in the Escherichia coli genome are assumed to be non-functional. In this study, Keio collection BW25113∆yqiG and YqiG-producing strain (BW25113/pCA24N-YqiG) were used to evaluate the importance of pseudogene yqiG in hydrogen metabolism. Our results show pseudogene protein YqiG was identified as an essential protein in the production of biohydrogen from glucose. The mutant yqiG decreased biohydrogen production from 37 µmol mg-1 protein to 6 µmol mg-1 protein compared to the wild-type strain, and glucose consumption was reduced by 80%. Through transcriptional analysis, we found that the yqiG mutation represses pflB transcription tenfold; pflB encodes pyruvate-formate lyase, one of the key enzymes in the anaerobic metabolism of E. coli. Moreover, production of YqiG stimulated glycolysis and increased biohydrogen productivity 1.5-fold compared to that of the wild-type strain. Thus, YqiG is important for the central glycolysis reaction and is able to influence hydrogen metabolism activity in E. coli.
Encapsulation of in vitro Plectranthus amboinicus (Lour.) Spreng. shoot apices for propagation and conservation

Arumugam G, Sinniah UR, Swamy MK, Lynch PT

3 Biotech, 2019 Aug;9(8):298.
PMID: 31328080 DOI: 10.1007/s13205-019-1831-4

This investigation demonstrates an efficient method of propagation, short-term conservation, and germplasm exchange for Plectranthus amboinicus (Lour.) Spreng. encapsulated propagules. In vitro-derived shoot apices (shoot tips and nodal segments) which showed 100% survival on MS medium supplemented with 0.4 mg/L 6-benzylaminopurine were selected for encapsulation studies. Shoot apices measuring about 3-5 mm in size showed the ability to break the beads and exhibited 100% survival and regrowth. The combination of 3% (w/v) sodium alginate and 100 mM CaCl2 was found to be ideal for forming uniformally spherical beads, and successive preservation of encapsulated shoot apices into plantlets. The encapsulated shoot tips were relatively more effective than the nodal segments in terms of shoot growth and multiplication. Encapsulated shoot tips retained the ability to regrow (63.3%) for up to 40 days when maintained at 4 °C. Encapsulated shoot tips effectively converted into plantlets on agar medium (78%) and peat moss (58%) under in vitro conditions. Encapsulated shoot tips on agar medium showed a higher shoot regeneration (9.91 ± 0.15 shoots per explant) ability than the peat moss (5.71 ± 0.34 shoots per explant), while the highest rooting (12.16 ± 0.23 roots per explant) was observed on peat moss. Thus, calcium alginate encapsulation holds latent qualities that could be explored to develop a future alternative method of propagation, short-term storage and germplasm distribution for elite genotypes of Plectranthus sp.
Physiological and metabolic responses of Scenedesmus quadricauda (Chlorophyceae) to nickel toxicity and warming

Yong WK, Sim KS, Poong SW, Wei D, Phang SM, Lim PE

3 Biotech, 2019 Aug;9(8):315.
PMID: 31406637 DOI: 10.1007/s13205-019-1848-8

An ecologically important tropical freshwater microalga, Scenedesmus quadricauda, was exposed to Ni toxicity under two temperature regimes, 25 and 35 °C to investigate the interactive effects of warming and different Ni concentrations (0.1, 1.0 and 10.0 ppm). The stress responses were assessed from the growth, photosynthesis, reactive oxygen species (ROS) generation and metabolomics aspects to understand the effects at both the physiological and biochemical levels. The results showed that the cell densities of the cultures were higher at 35 °C compared to 25 °C, but decreased with increasing Ni concentrations at 35 °C. In terms of photosynthetic efficiency, the maximum quantum yield of photosystem II (Fv/Fm) of S. quadricauda remained consistent across different conditions. Nickel concentration at 10.0 ppm affected the maximum rate of relative electron transport (rETRm) and saturation irradiance for electron transport (Ek) in photosynthesis. At 25 °C, the increase of non-photochemical quenching (NPQ) values in cells exposed to 10.0 ppm Ni might indicate the onset of thermal dissipation process as a self-protection mechanism against Ni toxicity. The combination of warming and Ni toxicity induced a strong oxidative stress response in the cells. The ROS level increased significantly by 40% after exposure to 10.0 ppm of Ni at 35 °C. The amount of Ni accumulated in the biomass was higher at 25 °C compared to 35 °C. Based on the metabolic profile, temperature contributed the most significant differentiation among the samples compared to Ni treatment and the interaction between the two factors. Amino acids, sugars and organic acids were significantly regulated by the combined factors to restore homeostasis. The most affected pathways include sulphur, amino acids, and nitrogen metabolisms. Overall, the results suggest that the inhibitory effect of Ni was lower at 35 °C compared to 25 °C probably due to lower metal uptake and primary metabolism restructuring. The ability of S. quadricauda to accumulate substantial amount of Ni and thrive at 35 °C suggests the potential use of this strain for phycoremediation and outdoor wastewater treatment.
Fulltext Anxiety determination by antibody-conjugated nanoparticles on an interdigitated electrode sensor

Wang X, Gopinath SCB, Li J

3 Biotech, 2020 Sep;10(9):377.
PMID: 32802719 DOI: 10.1007/s13205-020-02370-7

This work focused on the detection of cortisol on an interdigitated electrode sensor surface using an anti-cortisol antibody. To improve immobilization, antibodies were conjugated with silver nanoparticles and attached to the surface of the sensor. Cortisol interacted in a dose-dependent manner on the antibody-immobilized sensor surface, and current changes were observed. Linear regression analysis was performed by a 3σ calculation, and the limit of detection fell into the range of 0.01 and 0.1 ng/mL. The sensitivity of cortisol was calculated to be 0.01 ng/mL and the sensor discriminated against other hormones, namely norepinephrine and progesterone, with higher selectivity for cortisol. This result represented the selective detection of cortisol with high performance, which can help to determine anxiety disorders.
Fulltext In silico design of potentially functional artificial metallo-haloalkane dehalogenase containing catalytic zinc

Ang TF, Salleh AB, Normi YM, Leow TC

3 Biotech, 2018 Jul;8(7):314.
PMID: 30023146 DOI: 10.1007/s13205-018-1333-9

Artificial metalloenzymes are unique as they combine the good features of homogeneous and enzymatic catalysts, and they can potentially improve some difficult catalytic assays. This study reports a method that can be used to create an artificial metal-binding site prior to proving it to be functional in a wet lab. Haloalkane dehalogenase was grafted into a metal-binding site to form an artificial metallo-haloalkane dehalogenase and was studied for its potential functionalities in silico. Computational protocols regarding dynamic metal docking were studied using native metalloenzymes and functional artificial metalloenzymes. Using YASARA Structure, a simulation box covering template structure was created to be filled with water molecules followed by one mutated water molecule closest to the metal-binding site to metal ion. A simple energy minimization step was subsequently run using an AMBER force field to allow the metal ion to interact with the metal-binding residues. Long molecular dynamic simulation using YASARA Structure was performed to analyze the stability of the metal-binding site and the distance between metal-binding residues. Metal ions fluctuating around 2.0 Å across a 20 ns simulation indicated a stable metal-binding site. Metal-binding energies were predicted using FoldX, with a native metalloenzyme (carbonic anhydrase) scoring 18.0 kcal/mol and the best mutant model (C1a) scoring 16.4 kcal/mol. Analysis of the metal-binding site geometry was performed using CheckMyMetal, and all scores for the metalloenzymes and mutant models were in an acceptable range. Like native metalloenzymes, the metal-binding site of C1a was supported by residues in the second coordination shell to maintain a more coordinated metal-binding site. Short-chain multihalogenated alkanes (1,2-dibromoethane and 1,2,3-trichloropropane) were able to dock in the active site of C1a. The halides of the substrate were in contact with both the metal and halide-stabilizing residues, thus indicating a better stabilization of the substrate. The simple catalytic mechanism proposed is that the metal ion interacted with halogen and polarized the carbon-halogen bond, thus making the alpha carbon susceptible to attack by nucleophilic hydroxide. The interaction between halogen in the metal ion and halide-stabilizing residues may help to improve the stabilization of the substrate-enzyme complex and reduce the activation energy. This study reports a modified dynamic metal-docking protocol and validation tests to verify the metal-binding site. These approaches can be applied to design different kinds of artificial metalloenzymes or metal-binding sites.
X-ray crystallography of mutant GDSL esterase S12A of Photobacterium marinum J15

Rahman NNA, Sharif FM, Kamarudin NHA, Ali MSM, Aris SNAM, Jonet MA, et al.

3 Biotech, 2023 May;13(5):128.
PMID: 37064003 DOI: 10.1007/s13205-023-03534-x

GDSL esterase is designated as a member of Family II of lipolytic enzymes known to catalyse the synthesis and hydrolysis of ester bonds. The enzyme possesses a highly conserved motif Ser-Gly-Asn-His in the four conserved blocks I, II, III and V respectively. The enzyme characteristics, such as region-, chemo-, and enantioselectivity, help in resolving the racemic mixture of single-isomer chiral drugs. Recently, crystal structure of GDSL esterase from Photobacterium J15 has been reported (PDB ID: 5XTU) but not in complex with substrate. Therefore, GDSL in complex with substrate could provide insights into the binding mode of substrate towards inactive form of GDSL esterase (S12A) and identify the hot spot residues for the designing of a better binding pocket. Insight into molecular mechanisms is limited due to the lack of crystal structure of GDSL esterase-substrate complex. In this paper, the crystallization of mutant GDSL esterase (S12A) (PDB ID: 8HWO) and its complex with butyric acid (PDB ID: 8HWP) are reported. The optimized structure would be vital in determining hot spot residue for GDSL esterase. This preliminary study provides an understanding of the interactions between enzymes and hydrolysed p-nitro-phenyl butyrate. The information could guide in the rational design of GDSL esterase in overcoming the medical limitations associated with racemic mixture.
Insight on recently discovered PET polyester-degrading enzymes, thermostability and activity analyses

Buhari SB, Nezhad NG, Normi YM, Shariff FM, Leow TC

3 Biotech, 2024 Jan;14(1):31.
PMID: 38178895 DOI: 10.1007/s13205-023-03882-8

The flexibility and the low production costs offered by plastics have made them crucial to society. Unfortunately, due to their resistance to biological degradation, plastics remain in the environment for an extended period of time, posing a growing risk to life on earth. Synthetic treatments of plastic waste damage the environment and may cause damage to human health. Bacterial and fungal isolates have been reported to degrade plastic polymers in a logistic safe approach with the help of their microbial cell enzymes. Recently, the bacterial strain Ideonella sakaiensis (201-F6) was discovered to break down and assimilate polyethylene terephthalate (PET) plastic via metabolic processes at 30 °C to 37 °C. PETase and MHETase enzymes help the bacterium to accomplish such tremendous action at lower temperatures than previously discovered enzymes. In addition to functioning at low temperatures, the noble bacterium's enzymes have amazing qualities over pH and PET plastic degradation, including a shorter period of degradation. It has been proven that using the enzyme PETase, this bacterium hydrolyzes the ester linkages of PET plastic, resulting in production of terephthalic acid (TPA), nontoxic compound and mono-2-hydroxyethyl (MHET), along with further depolymerization of MHET to release ethylene glycogen (EG) and terephthalic acid (TPA) by the second enzyme MHETase. Enzymatic plastic degradation has been proposed as an environmentally friendly and long-term solution to plastic waste in the environment. As a result, this review focuses on the enzymes involved in hydrolyzing PET plastic polymers, as well as some of the other microorganisms involved in plastic degradation.
Fulltext Synthesis and characterization of reduced graphene oxide using the aqueous extract of Eclipta prostrata

Chuah R, Gopinath SCB, Anbu P, Salimi MN, Yaakub ARW, Lakshmipriya T

3 Biotech, 2020 Aug;10(8):364.
PMID: 32832325 DOI: 10.1007/s13205-020-02365-4

In this study, biological deoxygenation of graphene oxide (GO) using an Eclipta prostrata phytoextract was performed via the infusion method. The presence of oxide groups on the surface of graphene and removal of oxides groups by reduction were characterized through morphological and structural analyses. Field emission scanning electron microscopy images revealed that the synthesized GO and rGO were smooth and morphologically sound. Transmission electron microscopy images showed rGO developing lattice fringes with smooth edges and transparent sheets. Atomic force microscopy images showed an increase in the surface roughness of graphite oxide (14.29 nm) compared with that of graphite (1.784 nm) due to the presence of oxide groups after oxidation, and the restoration of surface roughness to 2.051 nm upon reduction. Energy dispersive X-ray analysis indicated a difference in the carbon/oxygen ratio between GO (1.90) and rGO (2.70). Fourier-transform infrared spectroscopy spectrum revealed peak stretches at 1029, 1388, 1578, and 1630 cm-1 for GO, and a decrease in the peak intensity after reduction that confirmed the removal of oxide groups. X-ray photoelectron microscopy also showed a decrease in the intensity of oxygen peak after reduction. In addition, thermogravimetric analysis suggested that rGO was less thermally stable than graphite, graphite oxide, and GO, with rGO decomposing after heating at temperatures ranging from room temperature to 600 °C.

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