Expression And Characterization Of Recombinant Thermostable L2 Lipase In Pichia Pastoris

The gene encoding mature thermostable L2 lipase from Bacillus sp. L2 was cloned into Pichia pastoris expression vectors and placed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and methanol inducible alcohol oxidase (AOX) promoter. In inducible system, recombinant L2 lipase was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence, compared to the constitutive system. The optimization of the recombinant L2 lipase production (from inducible system) in 100 mL culture was done for the best clones pPαS3 and pPαG2 from Pichia strains SMD1168H and GS115, respectively. The effect of media formulation, methanol concentration and induction time on L2 lipase production from inducible system was evaluated. A time course profile of recombinant lipase production in 500-mL flasks with the optimized conditions was performed and 15.3 mg/mL and 14.25 mg/mL of dry cell weight were produced after 144 h of induction time from recombinant pPαS3 and pPαG2, respectively. The lipase activities detected from both clones were 91 U/mL and 125 U/mL for pPαS3 and pPαG2, respectively. The recombinant L2 lipase was purified to 1.8-fold with 63.2% yield and with specific activity of 458.1 U/mg using affinity chromatography. The enzyme was in a monomeric form, non-glycosylated with a molecular weight of 44.5 kDa. The optimum pH and temperature were 8.0 and 70ºC, respectively. The enzyme was stable in the pH range of 8.0-9.0 and at 65ºC for 60 min where it retained more than 70% of its residual activity. The metal ions Ca2+, Na+, Cu2+ and Mn2+ activated the lipase at 1 mM, whereas Mg2+and Zn2+ inhibited it. Lipase showed a notable preference for medium to long chain triacylglycerols (C10–C16), with the highest activity toward tripalmitin (C16). It hydrolyzed all the natural oil tested, with the highest hydrolysis rate on corn oil and the least was on sunflower oil. L2 lipase was inhibited by EDTA, PMSF, pepstatin A and all the surfactants tested. It showed random positional specificity towards triolein. CD spectral analysis of L2 lipase revealed a Tm of around 67.2°C

The CRISPR growth spurt: from bench to clinic on versatile small RNAs

Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) in association with CRISPR associated protein (Cas) is an adaptive immune system, playing a pivotal role in the defense of bacteria and archaea. Ease of handling and cost effectiveness make CRISPR-Cas system an ideal programmable nuclease tool. Recent advances in understanding the CRISPR-Cas system have tremendously improved its efficiency. For instance, it is possible to recapitulate the chronicle CRISPR-Cas from its infancy and inaugurate a developed version by generating novel variants of Cas proteins, subduing off-target effects and optimization of innovative strategies. In summary, CRISPR-Cas system could be employed in a number of applications including providing model systems, rectification of detrimental mutations, and antiviral therapies

Antifungal activity of dichloromethane and hexane extracts of four Malaysian seaweed species against Ganoderma boninense

Ganoderma boninense is a major oil palm fungal pathogen that caused basal stem rot disease and serious efforts are required to identify alternative methods to control this disease. To date, little attempt was done to explore the antifungal potential of bioactive compounds in seaweeds. This study reported the antifungal activity of seaweed extracts against G. boninense. Seaweeds cfSargassum oligocystum, Caulerpa racemosa var. lamourouxii, Caulerpa racemosa and cfHalimeda macrophysa were collected from Port Dickson, Malaysia and extracted using dichloromethane and hexane. The antifungal activity assay towards G. boninense was carried out using the poisoned food technique followed by gas chromatography-mass spectrometry (GC-MS) to screen the compounds in the seaweed extracts. Our findings revealed that C. racemosa var. lamourouxiidichloromethane extract exhibited the highest antifungal activity at a concentration of 0.25 mg/mL with 46.82% inhibition of G. boninense’s growth followed by C. racemosa var. lamourouxii-hexane extract with 36.43% inhibition. Phytol and tetradecanoic acid were found to be the dominant compounds in the extracts and further analysis of phytol standard proved its antifungal activity. This study highlights the potential of local Malaysian seaweed species as a source of natural and powerful antifungal compounds which could be useful for alternative oil palm disease control in Malaysia

The aMAZEing Brain

Trillions of neural network are connected to one another in the brain. “The aMAZEing Brain” is the product of the thoughts generated in a whimsical way by these connections. This model appears to be a brain figure with many partitions representing different brain regions

Humanizing glycosylation pathways in eukaryotic expression systems

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system

Humanizing glycosylation pathways in eukaryotic expression systems

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system

Probiotic properties of Bacillus strains isolated from stingless bee (Heterotrigona itama) honey collected across Malaysia

This study aimed to isolate, identify, and evaluate the probiotic properties of Bacillus species from honey of the stingless bee Heterotrigona itama. Bacillus spp. were isolated from five different H. itama meliponicultures, and the isolates were characterized through Gram-staining and a catalase test. Tolerance to acidic conditions and bile salt (0.3%), hydrophobicity, and autoaggregation tests were performed to assess the probiotic properties of the selected isolates, B. amyloliquefaciens HTI-19 and B. subtilis HTI-23. Both Bacillus isolates exhibited excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria and possessed significantly high survival rates in 0.3% bile solution for 3 h. Their survival rates in acidic conditions were also comparable to a commercial probiotic strain, Lactobacillus rhamnosus GG. Interestingly, the hydrophobicity and autoaggregation percentage showed no significant difference from L. rhamnosus GG, a commercial probiotic strain. The results from this study suggest that B. amyloliquefaciens HTI-19 and B. subtilis HTI-23 isolated from stingless bee honey have considerably good probiotic properties. Therefore, more studies should be done to investigate the effects of these bacteria cultures on gastrointestinal health

Polyunsaturated fatty acids in marine bacteria and strategies to enhance their production

Polyunsaturated fatty acids (PUFAs) play an important role in human diet. Despite the wide-ranging importance and benefits from heart health to brain functions, humans and mammals cannot synthesize PUFAs de novo. The primary sources of PUFA are fish and plants. Due to the increasing concerns associated with food security as well as issues of environmental contaminants in fish oil, there has been considerable interest in the production of polyunsaturated fatty acids from alternative resources which are more sustainable, safer, and economical. For instance, marine bacteria, particularly the genus of Shewanella, Photobacterium, Colwellia, Moritella, Psychromonas, Vibrio, and Alteromonas, are found to be one among the major microbial producers of polyunsaturated fatty acids. Recent developments in the area with a focus on the production of polyunsaturated fatty acids from marine bacteria as well as the metabolic engineering strategies for the improvement of PUFA production are discussed

Genome sequence of Photobacterium sp. strain J15: an insight view for conservation genetics and molecular engineering

The genus Photobacterium was one of the earliest known bacterial taxa (Beijerinck 1889) and it comes from the family Vibrionaceae, one of the most widely distributed groups of prokaryotes that have radiated into hundreds of existing niches in the environment. However, the precise evolutionary history of this bacteria could not be infer due to the lack of whole genome sequencing data on Photobacterium available publicly. Photobacterium sp. strain J15 is a marine bacterium which was isolated from Tanjung Pelepas, Johor and it initially was studied for its lipase and asparaginase producing properties. Lipases has been used widely in the production of food, detergent and also in pharmaceutical industry while asparaginase on the other hand is on the World Health Organization’s List of Essential Medicines. Recently, it is also found out that Photobacterium species have the ability to produce DHAs, which is recommended for infant’s consumption. However, there is a limitation in the industrial use of these enzymes and it is mainly due to the high production cost which could be overcome by molecular technologies. Here we present the draft genome sequence of this Photobacterium sp. It was sequenced using Illumina technology and the genome sequence was then being assembled and the assembly produced 23 scaffolds with a final reading of 5,647,128 bp. However, there are 15,246 of unsequenced nucleotides which would be closed by using Sanger sequencing. The whole genome sequence and the future genome scale metabolic model of the Photobacterium sp. strain J15 is hope to uncover the mechanisms of this bacterium which will ease the molecular engineering applications besides being important in analysing the evolutionary biology and future genetics conservation of this interesting group of bacteria

Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis

This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 30 RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and β-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future