The potential for using agricultural and industrial by-products as substrate for the production of the edible mushroom, Auricularia polytricha, was evaluated using several formulations of selected palm oil wastes mixed with sawdust and further supplemented with selected nitrogen sources. The best substrate formulations were sawdust (SD) mixed with oil palm frond (OPF; 90:10) added with 15% spent grain (SG) and sawdust mixed with empty fruit bunch (EFB; 50:50) added with 10% spent grain (SG) with mycelia growth rate of 8 mm/day and 7 mm/day respectively. These two substrate formulations were then subjected to different moisture content levels (65%, 75% and 85%). Highest total fresh sporophore yield at 0.43% was obtained on SD+OPF (90:10)+15% SG at 85% moisture content, followed closely by SD+EFB (50:50)+10% SG with 0.40% total yield, also at 85% moisture content. Each of the substrate formulations at 85% moisture content gave the highest biological efficiency (BE) at 288.9% and 260.7%, respectively. Both yield and biological efficiency of A. polytricha on these two formulations were almost three times higher when compared to sawdust substrate alone, thus proving the potential of these formulations to improve yield of this mushroom.
Psychrophiles are cold-living microorganisms synthesizing enzymes that are permanently active at almost near-zero temperatures. Psychrozymes are supposed to be structurally more flexible than their homologous proteins. This structural flexibility enables these proteins to undergo conformational changes during catalysis and improve catalytic efficiency at low temperatures. The outstanding characteristics of the psychrophilic enzymes have attracted the attention of the scientific community to utilize them in a wide variety of industrial and pharmaceutical applications. In this review, we first highlight the current knowledge of the cold-adaptation mechanisms of the psychrophiles. In the sequel, we describe the potential applications of the enzymes in different biotechnological processes specifically, in the production of industrial and pharmaceutical products. KEY POINTS: • Methods that organisms have evolved to survive and proliferate at cold environments. • The economic benefits due to their high activity at low and moderate temperatures. • Applications of the psychrophiles in biotechnological and pharmaceutical industry.
Viruses have spread throughout the world and cause acute illness or death among millions of people. There is a growing concern about methods to control and combat early-stage viral infections to prevent the significant public health problem. However, conventional detection methods like polymerase chain reaction (PCR) requires sample purification and are time-consuming for further clinical diagnosis. Hence, establishing a portable device for rapid detection with enhanced sensitivity and selectivity for the specific virus to prevent further spread becomes an urgent need. Many research groups are focusing on the potential of the electrochemical sensor to become a key for developing point-of-care (POC) technologies for clinical analysis because it can solve most of the limitations of conventional diagnostic methods. Herein, this review discusses the current development of electrochemical sensors for the detection of respiratory virus infections and flaviviruses over the past 10 years. Trends in future perspectives in rapid clinical detection sensors on viruses are also discussed. KEY POINTS: • Respiratory related viruses and Flavivirus are being concerned for past decades. • Important to differentiate the cross-reactivity between the virus in same family. • Electrochemical biosensor as a suitable device to detect viruses with high performance.
In this study, the bioelectrical power generation potential of four tropical marine microalgal strains native to Malaysia was investigated using BPV platforms. Chlorella UMACC 258 produced the highest power density (0.108 mW m-2), followed by Halamphora subtropica UMACC 370 (0.090 mW m-2), Synechococcus UMACC 371 (0.065 mW m-2) and Parachlorella UMACC 245 (0.017 mW m-2). The chlorophyll-a (chl-a) content was examined to have a linear positive relationship with the power density (p
This study focuses on sirtuins, which catalyze the reaction of NAD+-dependent protein deacetylase, for riboflavin production in A. gossypii. Nicotinamide, a known inhibitor of sirtuin, made the color of A. gossypii colonies appear a deeper yellow at 5 mM. A. gossypii has 4 sirtuin genes (AgHST1, AgHST2, AgHST3, AgHST4) and these were disrupted to investigate the role of sirtuins in riboflavin production in A. gossypii. AgHST1∆, AgHST3∆, and AgHST4∆ strains were obtained, but AgHST2∆ was not. The AgHST1∆ and AgHST3∆ strains produced approximately 4.3- and 2.9-fold higher amounts of riboflavin than the WT strain. The AgHST3∆ strain showed a lower human sirtuin 6 (SIRT6)-like activity than the WT strain and only in the AgHST3∆ strain was a higher amount of acetylation of histone H3 K9 and K56 (H3K9ac and H3K56ac) observed compared to the WT strain. These results indicate that AgHst3 is SIRT6-like sirtuin in A. gossypii and the activity has an influence on the riboflavin production in A. gossypii. In the presence of 5 mM hydroxyurea and 50 µM camptothecin, which causes DNA damage, especially double-strand DNA breaks, the color of the WT strain colonies turned a deeper yellow. Additionally, hydroxyurea significantly led to the production of approximately 1.5 higher amounts of riboflavin and camptothecin also enhanced the riboflavin production even through the significant difference was not detected. Camptothecin tended to increase the amount of H3K56ac, but the amount of H3K56ac was not increased by hydroxyurea treatment. This study revealed that AgHst1 and AgHst3 are involved in the riboflavin production in A. gossypii through NAD metabolism and the acetylation of H3, respectively. This new finding is a step toward clarifying the role of sirtuins in riboflavin over-production by A. gossypii.Key points• Nicotinamide enhanced the riboflavin production in Ashbya gossypii.• Disruption of AgHST1 or AgHST3 gene also enhanced the riboflavin production in Ashbya gossypii.• Acetylation of H3K56 led to the enhancement of the riboflavin production in Ashbya gossypii.
Anaerobic digestion treatments have often been used for biological stabilization of solid wastes. These treatment processes generate biogas which can be used as a renewable energy sources. Recently, anaerobic digestion of solid wastes has attracted more interest because of current environmental problems, most especially those concerned with global warming. Thus, laboratory-scale research on this area has increased significantly. In this review paper, the summary of the most recent research activities covering production of biogas from solid wastes according to its origin via various anaerobic technologies was presented.
Preservation of leptospiral cultures is tantamount to success in leptospiral diagnostics, research, and development of preventive strategies. Each Leptospira isolate has imperative value not only in disease diagnosis but also in epidemiology, virulence, pathogenesis, and drug development studies. As the number of circulating leptospires is continuously increasing and congruent with the importance to retain their original characteristics and properties, an efficient long-term preservation is critically needed to be well-established. However, the preservation of Leptospira is currently characterized by difficulties and conflicting results mainly due to the biological nature of this organism. Hence, this review seeks to describe the efforts in developing efficient preservation methods, to discover the challenges in preserving this organism and to identify the factors that can contribute to an effective long-term preservation of Leptospira. Through the enlightenment of the previous studies, a potentially effective method has been suggested. The article also attempts to evaluate novel strategies used in other industrial and biotechnological preservation efforts and consider their potential application to the conservation of Leptospira spp.
Prodigiosin, a red linear tripyrrole pigment and a member of the prodiginine family, is normally secreted by the human pathogen Serratia marcescens as a secondary metabolite. Studies on prodigiosin have received renewed attention as a result of reported immunosuppressive, antimicrobial and anticancer properties. High-level synthesis of prodigiosin and the bioengineering of strains to synthesise useful prodiginine derivatives have also been a subject of investigation. To exploit the potential use of prodigiosin as a clinical drug targeting bacteria or as a dye for textiles, high-level synthesis of prodigiosin is a prerequisite. This review presents an overview on the biosynthesis of prodigiosin from its natural host Serratia marcescens and through recombinant approaches as well as highlighting the beneficial properties of prodigiosin. We also discuss the prospect of adopting a synthetic biology approach for safe and cost-effective production of prodigiosin in a more industrially compliant surrogate host.
Thiocyanate (SCN-) forms as a by-product of cyanidation during gold ore processing and can be degraded by a variety of microorganisms utilizing it as an energy, nitrogen, sulphur and/or carbon source. In complex consortia inhabiting bioreactor systems, a range of metabolisms are sustained by SCN- degradation; however, despite the addition or presence of labile carbon sources in most bioreactor designs to date, autotrophic bacteria have been found to dominate key metabolic functions. In this study, we cultured an autotrophic SCN--degrading consortium directly from gold mine tailings. In a batch-mode bioreactor experiment, this consortium degraded 22 mM SCN-, accumulating ammonium (NH4+) and sulphate (SO42-) as the major end products. The consortium consisted of a diverse microbial community comprised of chemolithoautotrophic members, and despite the absence of an added organic carbon substrate, a significant population of heterotrophic bacteria. The role of eukaryotes in bioreactor systems is often poorly understood; however, we found their 18S rRNA genes to be most closely related to sequences from bacterivorous Amoebozoa. Through combined chemical and phylogenetic analyses, we were able to infer roles for key microbial consortium members during SCN- biodegradation. This study provides a basis for understanding the behaviour of a SCN- degrading bioreactor under autotrophic conditions, an anticipated approach to remediating SCN- at contemporary gold mines.
Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.
Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.
This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.
Efficient approaches for the utilization of waste sewage sludge have been widely studied. One of them is to use it for the bioenergy production, specifically methane gas which is well-known to be driven by complex bacterial interactions during the anaerobic digestion process. Therefore, it is important to understand not only microorganisms for producing methane but also those for controlling or regulating the process. In this study, azithromycin analogs belonging to macrolide, ketolide, and lincosamide groups were applied to investigate the mechanisms and dynamics of bacterial community in waste sewage sludge for methane production. The stages of anaerobic digestion process were evaluated by measuring the production of intermediate substrates, such as protease activity, organic acids, the quantification of bacteria and archaea, and its community dynamics. All azithromycin analogs used in this study achieved a high methane production compared to the control sample without any antibiotic due to the efficient hydrolysis process and the presence of important fermentative bacteria and archaea responsible in the methanogenesis stage. The key microorganisms contributing to the methane production may be Clostridia, Cladilinea, Planctomycetes, and Alphaproteobacteria as an accelerator whereas Nitrosomonadaceae and Nitrospiraceae may be suppressors for methane production. In conclusion, the utilization of antibiotic analogs of macrolide, ketolide, and lincosamide groups has a promising ability in finding the essential microorganisms and improving the methane production using waste sewage sludge.
This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future.
Marine microbes provide an important resource to discover new chemical compounds with biological activities beneficial to drug discovery. In our study, two new polyene macrolides, pyranpolyenolides A (1) and B (2), and one new natural cyclic peptide (9), together with two known polyenes (7 and 8) and three known cyclic peptides (10-12), were isolated from a culture of the marine Streptomyces sp. MS110128. In addition, four new polyene macrolides, pyranpolyenolides C-F (3-6), were identified as olefin geometric isomers that were most likely produced by photochemical conversion during the cultivation or isolation procedures. The pyranpolyenolides are 32-membered macrolides endowed with a conjugated tetraene and several pairs of 1,3-dihydroxyl groups. Pyranpolyenolides that contain a hydropyran group have not been previously reported. Four cyclic peptides (9-12) showed significant activities against Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant S. aureus with supporting MIC values ranging from 0.025 to 1.25 μg/mL. These cyclic peptides containing piperazic moieties showed moderate activities with MIC values of 12.5 μg/mL against Bacille Calmette Guerin (BCG), an attenuated form of the bovine. Additionally, cyclic peptide 12 showed moderate antifungal activity against Candida albicans with an MIC value of 12.5 μg/mL. KEY POINTS: • Discovery of new polyenes and cyclic peptides from a marine-derived Actinomycete. • Cyclic peptides containing piperazic moieties exhibited good antibacterial activity.
Microbial transglutaminase (mTGase) is commonly known in the food industry as meat glue due to its incredible ability to "glue" meat proteins together. Aside from being widely exploited in the meat processing industries, mTGase is also widely applied in other food and textile industries by catalysing the formation of isopeptide bonds between peptides or protein substrates. The advancement of technology has opened up new avenues for mTGase in the field of biomedical engineering. Efforts have been made to study the structural properties of mTGase in order to gain an in-depth understanding of the structure-function relationship. This review highlights the developments in mTGase engineering together with its role in biomedical applications including biomaterial fabrication for tissue engineering and biotherapeutics.
Fungal immunomodulatory proteins (FIPs) are bioactive proteins with immunomodulatory properties. We previously reported the heterologous production in Escherichia coli of FIP-Lrh from Tiger milk mushroom (Lignosus rhinocerus) with potent cytotoxic effect on cancer cell lines. However, protein produced in E. coli lacks post-translational modifications and may be contaminated with lipopolysaccharide (LPS) endotoxin. Therefore, in this study, yFIP-Lrh produced in Pichia pastoris was functionally compared with eFIP-Lrh produced in E. coli. Expression construct of FIP-Lrh cDNA in pPICZα was generated, transformed into P. pastoris X-33 and Mut+ transformants were verified by colony PCR. Induction with 0.5% or 1% methanol resulted in a secreted 13.6 kDa yFIP-Lrh which was subsequently purified and verified using LCMS/MS analysis. Size exclusion chromatography confirmed eFIP-Lrh as a homodimer whereas the larger size of yFIP-Lrh may indicate post-translational modification despite negative for glycoproteins staining. At lower concentration (4-8 μg/mL), yFIP-Lrh induced significantly higher Th1 (IFN-γ, TNF-α) and Th2 (IL-6, IL-4, IL-5, IL-13) cytokines production in mice splenocytes, whereas 16 μg/mL eFIP-Lrh induced significantly higher pro-inflammatory cytokines (TNF-α, IL-6, IL-10), possibly due to higher residual LPS endotoxin (0.082 EU/mL) in eFIP-Lrh compared to negligible level in yFIP-Lrh (0.001 EU/mL). Furthermore, yFIP-Lrh showed higher cytotoxic effect on MCF-7 and HeLa cancer cells. Since both recombinant proteins of FIP-Lrh have the same peptide sequence, besides glycosylation, other post-translational modifications in yFIP-Lrh may account for its enhanced immunomodulatory and anti-proliferative activities. In conclusion, P. pastoris is preferred over E. coli for production of a functionally active yFIP-Lrh devoid of endotoxin contamination. KEY POINTS: • FIP-Lrh can induced production of Th1 and Th2 cytokines by mouse splenocytes. • Higher cytotoxic effect on cancer cells observed for yeast compared to E. coli produced FIP-Lrh. • P. pastoris allows production of an endotoxin-free and functionally active recombinant FIP-Lrh.
Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.
Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed. KEY POINTS: • The dynamics of lipases contributes to their non-covalent interactions and structural stability. • Thermostability can be enhanced by well-established genetic tools for improved kinetic efficiency. • Molecular dynamics greatly provides structure-function insights on thermodynamics of lipase.
Lipase biocatalysts offer unique properties which are often impaired by low thermal and methanol stability. In this study, the rational design was employed to engineer a disulfide bond in the protein structure of Geobacillus zalihae T1 lipase in order to improve its stability. The selection of targeted disulfide bond sites was based on analysis of protein spatial configuration and change of Gibbs free energy. Two mutation points (S2C and A384C) were generated to rigidify the N-terminal and C-terminal regions of T1 lipase. The results showed the mutant 2DC lipase improved methanol stability from 35 to 40% (v/v) after 30 min of pre-incubation. Enhancement in thermostability for the mutant 2DC lipase at 70 °C and 75 °C showed higher half-life at 70 °C and 75 °C for 30 min and 52 min, respectively. The mutant 2DC lipase maintained the same optimum temperature (70 °C) as T1 lipase, while thermally induced unfolding showed the mutant maintained higher rigidity. The kcat/Km values demonstrated a relatively small difference between the T1 lipase (WT) and 2DC lipase (mutant). The kcat/Km (s-1 mM-1) of the T1 and 2DC showed values of 13,043 ± 224 and 13,047 ± 312, respectively. X-ray diffraction of 2DC lipase crystal structure with a resolution of 2.04 Å revealed that the introduced single disulfide bond did not lower initial structural interactions within the residues. Enhanced methanol and thermal stability are suggested to be strongly related to the newly disulfide bridge formation and the enhanced compactness and rigidity of the mutant structure. KEY POINTS: • Protein engineering via rational design revealed relative improved enzymatic performance. • The presence of disulfide bond impacts on the rigidity and structural function of proteins. • X-ray crystallography reveals structural changes accompanying protein modification.