In-silico structural analysis affecting thermostability in recombinant psychrophilic chitinase (CHI II) from Glaciozyma antarctica PI12

Cold-adapted enzymes are significant with structure flexibility and high catalytic activity at low temperature. High structural flexibility could be due to combination of several features such as weak intramolecular bonds, decreased compactness of hydrophobic core and reduced number of proline and arginine residues. However, to compensate the structural flexibility, cold-adapted enzymes are also thermolabile which causes them to be easily inactivated at elevated temperature. Therefore, it would be more interesting and beneficial if more stable cold-adapted enzymes are produced to fulfill the industrial needs. In this study, a novel cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was rationally designed to improve their thermostability thus make them more resistant to increased temperature. Four CHI II mutants were designed through rational design named as A157Q, I134P, mutant Loop and Y257R by manipulating the structural hydrophobicity, introduction of proline in the loop regions, introduction of arginine salt bridges and loop shortening. Mutant Loop was designed by removing 9 residues in loop regions thus makes loop involved became shorter. Stability of all mutants was first predicted through a computational approach where all structures were subjected to 10 ns molecular dynamics simulation at three temperatures; 273 K, 288 K and at 300 K. Based on the simulation, it was found that mutants I134P, mutant Loop and Y257R exhibited structural stability at 300 K. This conclusion was made based on low and stable root-mean square deviation (RMSD) value at 300 K in comparison to RMSD values at 288 K and 273 K. Low RMSD values indicated mutant structure experienced low structural deviation throughout the simulation. Besides, this observation is correlated with reduction of structure compactness (radius of gyration), reduced solvent accessible surface area and increased numbers of hydrogen and salt bridges. However, mutant A157Q experienced structure destabilization at 300 K. Substitution of helix-preferred residue, alanine with a thermolabile residue, glutamine had caused A157Q structure become loosely packed at 300 K indicating a thermal denaturation. To support the theoretical model, CHI II and all mutants were then cloned into Pichia pastoris expression vector pPICZαC and expressed in P. pastoris (GS115)

Molecular and bioinformatics characterization of fruit bromelain from ananas comosus

Pineapple scientifically known as Ananas comosus, has several available cultivars in Malaysia, including Moris cv, N36 cv, and Sarawak cv. Bromelain has been identified as an active component and a major protease of A. comosus and has gained wide acceptance and compliance as a phototherapeutic drug. Although a considerable level of research has been devoted to bromelain from A. comosus, less attention has been paid to the fruit bromelain compared to the stem bromelain. Therefore, the purpose of this research is to reveal an in-depth information regarding fruit bromelain from A. comosus. Until recently, the three-dimensional (3D) structure of bromelain remained to be elucidated. A comprehensive information on the thorough structural organisation of bromelain is vital for therapeutic application and in the understanding of their role in cells and in other related molecular mechanisms. In this study, the screening of fruit bromelain from the local pineapple cultivars (Morris cv, N36 cv. and Sarawak cv) was implemented, followed by the isolation and cloning of the fruit bromelain from the best cultivar with the highest proteolytic activity for sequence analysis. Additionally, a comparison of the fruit and stem bromelain was performed using bioinformatics tools, including both amino acids and structural comparisons. From the screening results, the highest proteolytic activity (0.8220 U/mL) was observed from the fruit bromelain of Morris cv, followed by N36 cv (0.7695 U/mL) and Sarawak cv (0.6942 U/mL). A gene encoding for pineapple fruit bromelain was successfully isolated from Morris cv. using Reverse Transcription -Polymerase Chain Reaction (RT-PCR) techniques. The amino acid sequence and domain analysis of the fruit and stem bromelains demonstrated several differences and similarities of the cysteine protease family members. Additionally, an analysis of the modelled fruit (BAA21848) and stem (CAA08861) bromelains revealed the presence of unique properties of the predicted structures Cys-148, His-281, Gln-174 and Asn-275 are the catalytic residues of fruit bromelain whereas stem bromelain Cys-147, His-281, His-141 and Asn-302. This play crucial roles in chemical catalysis as general acid/base catalysts. The sequence analysis and structural prediction of the stem and fruit bromelain from A. comosus, along with the comparison of both structures provided a new insight on their distinct properties for industrial application. From the analysis, stem bromelain is more hydrophobic than fruit bromelain. The knowledge of the structure of these proteolytic enzymes from A. comosus is expected to increase the understanding of their functions and mechanism

Temperature and pH Profiling of Amylase from Antarctic and Arctic Soil Microfungi

While diversity studies and screening for enzyme activities are important elements of understanding fungal roles in the soil ecosystem, extracting and purifying the target enzyme from the fungal cellular system is also required to characterize the enzyme. This is, in particular, necessary before developing the enzyme for industrial-scale production. In the present study, partially purified α-amylase was obtained from strains of Pseudogymnoascus sp. obtained from Antarctic and Arctic locations. Partially purified α-amylases from these polar fungi exhibited very similar characteristics, including being active at 15 °C, although having a small difference in optimum pH. Both fungal taxa are good candidates for the potential application of cold-active enzymes in biotechnological industries, and further purification and characterization steps are now required. The α-amylases from polar fungi are attractive in terms of industrial development because they are active at lower temperatures and acidic pH, thus potentially creating energy and cost savings. Furthermore, they prevent the production of maltulose, which is an undesirable by-product often formed under alkaline conditions. Psychrophilic amylases from the polar Pseudogymnoascus sp. investigated in the present study could provide a valuable future contribution to biotechnological applications

Cold shock induction of recombinant Arctic environmental genes

Published Version, also available at http://dx.doi.org/10.1186/s12896-015-0185-1Background: Heterologous expression of psychrophilic enzymes in E. coli is particularly challenging due to their intrinsic instability. The low stability is regarded as a consequence of adaptation that allow them to function at low temperatures. Recombinant production presents a significant barrier to their exploitation for commercial applications in industry. Methods: As part of an enzyme discovery project we have investigated the utility of a cold-shock inducible promoter for low-temperature expression of five diverse genes derived from the metagenomes of marine Arctic sediments. After evaluation of their production, we further optimized for soluble production by building a vector suite from which the environmental genes could be expressed as fusions with solubility tags. Results: We found that the low-temperature optimized system produced high expression levels for all putatively cold-active proteins, as well as reducing host toxicity for several candidates. As a proof of concept, activity assays with one of the candidates, a putative chitinase, showed that functional protein was obtained using the lowtemperature optimized vector suite. Conclusions: We conclude that a cold-shock inducible system is advantageous for the heterologous expression of psychrophilic proteins, and may also be useful for expression of toxic mesophilic and thermophilic proteins where properties of the proteins are deleterious to the host cell growth

Marine Fungus

Most of the available studies on marine fungi are based on the isolation and identification of fungi from different surfaces (e.g., submerged wood, sediments, macrophytes), mostly in coastal benthic environments. However, recent evidence suggests that fungi are also present in the oceanic water column, most likely mainly associated to particles, with the genomic potential to significantly contribute to marine biogeochemical cycles. Still, we lack even basic information on the ecology of the oceanic mycobiome, precluding us from determining the ecological role of this enigmatic kingdom in our oceans. The aim of this book and Special Issue was to focus on the ecology of marine fungi. Topics include, fungal abundance, distribution, activity, and phylogenetic and/or functional diversity in coastal to open ocean environments, including seawater column and sediments, derived both from laboratory and field studies

Darkening of the Greenland Ice Sheet: Fungal Abundance and Diversity Are Associated With Algal Bloom

Recent studies have highlighted the importance of ice-algal blooms in driving darkening and therefore surface melt of the Greenland Ice Sheet (GrIS). However, the contribution of fungal and bacterial communities to this microbially driven albedo reduction remains unconstrained. To address this significant knowledge gap, fungi were isolated from key GrIS surface habitats (surface ice containing varying abundance of ice algae, supraglacial water, cryoconite holes, and snow), and a combination of cultivation and sequencing methods utilized to characterize the algal-associated fungal and bacterial diversity and abundance. Six hundred and ninety-seven taxa of fungi were obtained by amplicon sequencing and more than 200 fungal cultures belonging to 46 different species were isolated through cultivation approaches. Basidiomycota dominated in surface ice and water samples, and Ascomycota in snow samples. Amplicon sequencing revealed that bacteria were characterized by a higher diversity (883 taxa detected). Results from cultivation as well as ergosterol analyses suggested that surface ice dominated by ice algae and cryoconite holes supported the highest fungal biomass (104–105 CFU/100 ml) and that many fungal taxa recognized as endophytes and plant pathogens were associated with dark ice characterized by a high abundance of ice algae. This paper significantly advances this field of research by investigating for the first time the fungal abundance and diversity associated with algal blooms causing the darkening of the GrIS. There is a strong association between the abundance and diversity of fungal species and the blooming of algae on the surface ice of the Greenland Ice Sheet

Reaction control and protein engineering of bacillus lehensis G1 maltogenic amylase for higher malto-oligosaccharide synthesis

A multi-functional maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 exhibited remarkable hydrolysis and transglycosylation activity to produce malto-oligosaccharides of various lengths. MAG1 demonstrated hydrolysis activity over wide range of substrates. Kinetic analysis revealed that the enzyme hydrolyzed small substrate more efficiently than the larger substrate. This was shown by lower Michaelis constant (Km) value and higher turnover number (kcat) and second order rate constant (kcat/Km) values for β-cyclodextrin compared to that of soluble starch. Malto-oligosaccharide synthesis by transglycosylation activity of MAG1 faces problem of product re-hydrolyzation due to the hydrolysis activity of the enzyme. An equilibrium-control reaction approach has been successfully employed to improve malto-oligosaccharides production by decreasing hydrolysis activity. A yield of 38% transglycosylation products was obtained with the presence of malto-oligosaccharides longer than maltoheptaose. The addition of organic solvents demonstrated an increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15. The transglycosylation activity of MAG1 was also successfully enhanced by using structure-guided protein engineering approach. A molecular modeling and substrate docking was performed to study the structure-function relationship for rational design. A unique subsite structure which has not been reported in other maltogenic amylases was revealed and the information was used to design mutants that have active sites with reduced steric interference and higher hydrophobicity properties to increase the transglycosylation activity. Mutations decreased the hydrolysis activity of the enzyme and caused various modulations in its transglycosylation property. W359F, Y377F and M375I mutations caused reductions in steric interference and alteration of subsite occupation. In addition, the mutations increased internal flexibility to accommodate longer donor/acceptor molecule for transglycosylation, resulted in increased transglycosylation to hydrolysis ratio of up to 4.0-fold. The increase of the active site hydrophobicity from W359F and M375I mutations reduced concentration of maltotriose used as donor/acceptor for transglycosylation to 100 mM and 50 mM, respectively compared to 200 mM of the wild-type. The improvement of the transglycosylation to hydrolysis ratio by 4.3-fold was also demonstrated by both mutants. Interestingly, reductions of both steric interference and hydrolysis by Y377F and W359F mutations caused a synergistic effect to produce malto-oligosaccharides with higher degree of polymerization than the wild-type. These findings showed that the transglycosylation activity of MAG1 was successfully improved by controlling water activity and modification of the active site structure. The high transglycosylation activity of MAG1 and mutants offers a great advantage for synthesizing malto-oligosaccharides and rare carbohydrates

Genomic signatures of optimal growth temperature in the family Colwelliaceae

Thesis (M.S.) University of Alaska Fairbanks, 2020The temperature range supporting growth defines a complex physiological phenotype that depends on interactions between an organism's genome and its environment. Its implications are widespread since small changes in optimal growth temperature (OGT) can alter an organism's ability to inhabit an ecological niche. Thus, organisms with extreme thermal growth traits (e.g., psychrophilic, with OGT < 15℃, or thermophilic, with OGT 60 -80℃) may be useful for identifying promising targets when searching for life on other planets, as well as predicting population dynamics in a warming Arctic. We performed comparative genomic analyses of bacteria newly isolated from Arctic sea ice that were affiliated with Colwelliaceae, a family of Gammaproteobacteria that contains many psychrophilic strains, to identify genomic factors that might be used to predict OGT. A phylogenomic analysis of 67 public and 39 newly-sequenced strains, was used to construct an updated phylogenetic tree of Colwelliaceae, of which at least two genera were well represented. To augment the previously reported OGTs of 26 strains, we measured growth rates at −1, 4, 11, and 17 ℃ to determine the OGTs of these 39 new strains of Colwelliaceae. We found that growth rates among all isolates were comparable at −1℃, but varied widely above 10 ℃, indicating higher variability in the ability to tolerate warmer temperatures. To analyze the phenotypic differences on a genomic level, we examined indices of amino acid substitutions that have previously been linked with cold adaptation via an increase in protein flexibility. We found that these indices were significantly correlated with OGT at the whole genome level, although the sign of some correlations were opposite of the predicted positive correlation between temperature and the indices. Using these data, we fit a multiple linear regression model for OGT within the Colwelliaceae family that incorporates the three most informative amino acid indices: GRAVY, Aliphatic Index, and Acidic Residue Proportion. Additionally, a putative cold-adaptive gene cassette was identified that was likely introduced by horizontal gene transfer between two closely related clades with different OGTs. These contributions offer key insights into OGT variability and its underlying genomic foundation in the family Colwelliaceae.The National Ocean Partnership Program (NOPP grant NA14NOS0120158), The National Oceanographic and Atmospheric Administration (NOAA grant NA14OAR0110266 and NA15OAR0110208), the Robert and Kathleen Byrd Award, the Frances & Alfred Baker Scholarship, and the UAF Thesis Completion Fellowship, Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 2P20GM10339

Marine Proteins and Peptides

Marine proteins and peptides have great potential application in developing pharmaceuticals, nutraceuticals, and cosmeceuticals. Proteins and peptides from marine sources are considered to be safe and inexpensive. Protein- and peptide-based drugs have been increasing in recent days to cure various diseases by serving multiple roles, such as antioxidants, anticancer drugs, antimicrobials, and anticoagulants. There are different marine sources (macroalgae, fish, shellfish, and bivalves), which possibly contain specific protein and peptides