A Cold Active Bifunctional Lipase with Protease Activity from Antarctic Microorganism Pi 12

A psychrophilic microorganism (cold loving) named PI 12 was isolated from the Antarctic sea ice near Casey station, Antarctica. This psychrophilic microorganism produced extracellular lipase and the activity was determined by using both qualitative and quantitative methods. Clearing zones was formed when it was grown at 4°C on top of tributyrin agar plates, indicating an extracellular cold active lipase with activity at 0.051U/ml. 16rRNA genes revealed an apparent homology of 99 % to Janthibacterium sp. However, further identification of isolate PI 12 confirmed that it was a yeast, Leucosporodium antarcticum. The lipase gene of isolate PI 12 was isolated via shotgun cloning. Gene analysis showed an open reading frame of 783 bp was found to encode a lipase. The lipase was assayed at 4ºC with activity at 0.1 U/ml. The gene was also successfully expressed extracellularly by co-transformation of pJL3 plasmid which encode Bacteriocin Release Protein (BRP) The lipase gene did not show high similarity to other lipases as anticipated. Interestingly, the gene shared high homology to protease. Thus more investigations in trying to understand such novel finding were done. In order to visualize the enzyme, LipPI12 was modeled using the template of psychrophilic protease from Pseudomonas sp. TACII18. The putative 3D structure of the enzyme showed the typical properties of psychrophilic enzyme, which is increasing number of loops and a non compact structure to cater the lipase structural flexibility. Purification of recombinant intracellular and extracellular LipPI12 was done using Nickel Sepharose affinity chromatography. The purified intracellular LipPI12 was a monomer with the size of ~30kDa as judge native and SDS PAGE respectively. LipPI12 holds huge prospect of greater finding therefore characterization of LipPI12 lipase and protease were done. Temperature profile of the bifunctional LipPI12 showed that the lipase functions optimally at 20°C and reached half life after 30 min whereas the protease was more active at 40°C but reaches half life even faster after 15 mins of incubation. pH profile showed that both LipPI12 lipase and protease were active at near neutral condition. Medium chain length fatty acid (C12) seemed to be the best substrate for LipPI12 lipase. The presence of organic solvents did not affect both the lipase and protease activities. The lipase was more stable at solvents with higher log P value whereas the protease was slightly activated at low log P value particularly with dimethylsulfonyl. Activity of LipPI12 lipase and protease were also activated in the presence of CaCl2 but its protease counterpart seemed to be more active in the presence of other metal ions such as ZnCl2 and MgCl2. Effect of surfactants showed LipPI12 lipase was activated by Tween 80 and SLS and in contrast, LipPI12 protease was almost deactivated in all surfactants tested. Inhibitor studies revealed that LipPI12 lipase was partially inhibited with EDTA and PMSF whereby the LipPI12 protease was inhibited by pepstatin and was also partially inhibited by EDTA and PMSF. Amino acid comparison showed patterns of cold adaptation with increasing number of glycine and lesser proline. Circular dichroism and fluorescence spectroscopy analysis, strengthens the findings which entails protein psychrophilicity. The findings of unique LipPI12 has led to better understanding of the enzyme as shown from its bifunctional properties. The contrasting figure of LipPI12 lipase and protease reveals greater elucidation on protein structure and function. Thus it is concluded that LipPI12 lipase and protease is a remarkable enzyme which has highlighted way of surviving the cold and also promises potential application in the future

Evaluation of students' acceptance of the leap motion hand gesture application in teaching biochemistry

This paper presents an early stage of the Leap Motion controller regarding user acceptance in the teaching and learning process. The Leap Motion is a new device for a hand-gesture-controlled user interface. For appropriate evaluation, a novel experiment and questionnaire were created utilizing 35 Biochemistry undergraduate students in Enzymology from the Universiti Putra Malaysia. The subjects participated in the user experiment and performed several tasks, such as rotating, translating and zooming in and out on the molecules. The tasks were performed using the Molecules application on an Airspace platform. The research compared the performance of Leap Motion with mouse interaction. As a result, 79.2% of the respondents gave a positive opinion about the Leap Motion because of its ease of use, acceptance, effectiveness and accuracy. These students were excited and looked forward to implementing the Leap Motion in class. Thus, the Leap Motion controller can potentially be used as a teaching tool for a better learning experience of the biomolecule

Review on fatty acid desaturases and their roles in temperature acclimatisation

Carbohydrates and proteins are polymers of polysaccharides and polypeptide residues, respectively. Conversely, lipids are made up of a wide range of compounds with tremendous differences in structures and lack building blocks. Therefore, fatty acids constitute the major components of various lipid classes such as glycerides and sphingolipids. Fatty acids are organic compounds containing a carboxylic acid group mostly at the end of an aliphatic chain and are categorized into saturated and unsaturated. Most fatty acids especially the so-called polyunsaturated fatty acids (PUFAs) and their derivatives play key biological roles in inflammatory response, cell division, control of lipid metabolism, as signaling molecules, supply of energy and protecting the biological membranes structure and function. In this study, a general overview of fatty acids, their biosynthesis mechanisms as well as biological importance have been discussed. Fatty acid desaturase enzymes and their sources have also been reviewed. Recent studies on the functional expression of different types of desaturase enzymes have also been discussed

Evaluation of banana (Musa sp.) flowers of selected varieties for their antioxidative and anti-hyperglycemic potentials

Consumption of banana flower as a vegetable is popular among many countries in Southeast Asia. In this study, banana flowers of six different Malaysian cultivars namely, pisang Abu (Musa balbisiana cv P. Abu), pisang Berangan (Musa acuminata cv P. Berangan), pisang Nipah (Musa balbisiana cv P. Nipah), pisang Susu (Musa acuminata cv P. Susu), pisang Mas (Musa acuminate cv P. Mas) and pisang Rastali (Musa paradisiaca cv P. Rastali) were investigated for their antioxidant and anti-hyperglyemic properties. The total poly phenolic content and antioxidant activities, the α-amylase and α-glucosidase inhibitory potentials of the banana flower extracts were studied in vitro using relevant assays. Among the six cultivars, cultivar Susu was found to have the highest phenolic content (80.13 ± 4.64 mg of GAE/g of extract) and displayed the highest ABTS+ and DPPH radical scavenging activities (24.73 ± 0.04 and 25.10 ± 0.15 μmole of Trolox equivalent/g of extract). The anti-amylase and antiglucosidase activity of the banana flowers extracts were in the range of 47.31-62.58% and 74.98-91.62%, respectively. All banana flower extracts inhibited the activity of α-glucosidase better than α-amylase at the concentration of 200 μg/ml. This study concluded that the extracts of Malaysian banana flowers were potent sources of natural antioxidants, which can be used as postprandial hyperglycemia regulators

CsoR metalloregulatory protein: function, mechanism and relevance

Transition metals are required constituent in bacterial metabolism to assist in some enzymatic reactions. However, intracellular accumulations of these metal ions are harmful to the bacteria as it can trigger unnecessary redox reactions. To overcome this condition, metalloregulatory proteins assist organisms to adapt to sudden elevated and deprived metal ion concentration in the environment via metal homeostasis. CsoR protein is a copper(I) [Cu(I)] sensing operon repressor that is found to be present in all major classes of eubacteria. This metalloregulatory protein binds to the operator region in its apo state under Cu(I) limiting condition and detaches off from the regulatory region when it binds to the excess cytosolic Cu(I) ion, thus derepressing the expression of genes involved in Cu(I) homeostasis. CsoR proteins exist in dimeric and tetrameric states and form certain coordination geometries upon attachment with Cu(I). Certain CsoR proteins have also been found to possess the ability to bind to other types of metals with various binding affinities in some Gram positive bacteria. The role of this metalloregulatory protein in host pathogen interaction and its relation to bacterial virulence are also discussed

Structural adaptation of cold-active RTX lipase from Pseudomonas sp. strain AMS8 revealed via homology and molecular dynamics simulation approaches

The psychrophilic enzyme is an interesting subject to study due to its special ability to adapt to extreme temperatures, unlike typical enzymes. Utilizing computer-aided software, the predicted structure and function of the enzyme lipase AMS8 (LipAMS8) (isolated from the psychrophilic Pseudomonas sp., obtained from the Antarctic soil) are studied. The enzyme shows significant sequence similarities with lipases from Pseudomonas sp. MIS38 and Serratia marcescens. These similarities aid in the prediction of the 3D molecular structure of the enzyme. In this study, 12 ns MD simulation is performed at different temperatures for structural flexibility and stability analysis. The results show that the enzyme is most stable at 0°C and 5°C. In terms of stability and flexibility, the catalytic domain (N-terminus) maintained its stability more than the noncatalytic domain (C-terminus), but the non-catalytic domain showed higher flexibility than the catalytic domain. The analysis of the structure and function of LipAMS8 provides new insights into the structural adaptation of this protein at low temperatures. The information obtained could be a useful tool for low temperature industrial applications and molecular engineering purposes, in the near future

Adaptational properties and applications of cold-active lipase from psychrophilic bacteria

Psychrophilic microorganisms are cold-adapted with distinct properties from other thermal classes thriving in cold conditions in large areas of the earth's cold environment. Maintenance of functional membranes, evolving cold-adapted enzymes and synthesizing a range of structural features are basic adaptive strategies of psychrophiles. Among the cold-evolved enzymes are the cold-active lipases, a group of microbial lipases with inherent stability-activity-flexibility property that have engaged the interest of researchers over the years. Current knowledge regarding these cold-evolved enzymes in psychrophilic bacteria proves a display of high catalytic efficiency with low thermal stability, which is a differentiating feature with that of their mesophilic and thermophilic counterparts. Improvement strategies of their adaptive structural features have significantly benefited the enzyme industry. Based on their homogeneity and purity, molecular characterizations of these enzymes have been successful and their properties make them unique biocatalysts for various industrial and biotechnological applications. Although, strong association of lipopolysaccharides from Antarctic microorganisms with lipid hydrolases pose a challenge in their purification, heterologous expression of the cold-adapted lipases with affinity tags simplifies purification with higher yield. The review discusses these cold-evolved lipases from bacteria and their peculiar properties, in addition to their potential biotechnological and industrial applications

Optimization and maximization of hexavalent molybdenum reduction to Mo-blue by Serratia sp. strain MIE2 using response surface methodology

Molybdenum has long been known to be toxic to ruminants, but not to humans. However, more recently it has been increasingly reported that molybdenum shows toxic effects to reproductive organs of fish, mouse and even humans. Hence, its removal from the environment is highly sought after. In this study, response surface methodology (RSM) was successfully applied in the optimization and maximization of Mo6+ reduction to Mo-blue by Serratia sp. MIE2 for future bioremediation application. The optimal conditions predicted by RSM were 20 mM molybdate, 3.95 mM phosphate, pH 6.25 and 25 g l−1 sucrose with absorbance of 19.53 for Mo-blue production measured at 865 nm. The validation experimental run of the predicted optimal conditions showed that the maximum Mo-blue production occurred at absorbance of 20.87, with a 6.75 % deviation from the predicted value obtained from RSM. Molybdate reduction was successfully maximized using RSM with molybdate reduction before and after optimization using RSM showing Mo-blue production starting at the absorbance value of 10.0 at 865 nm going up to an absorbance value above 20.87. The modelling kinetics of Mo6+ reduction showed that Teissier was the best model, with calculated Pmax, Ks and Ki values of 1.97 Mo-blue per hour, 5.79 mM and 31.48 mM, respectively

Modelling the kinetics of hexavalent molybdenum (Mo6+) reduction by the Serratia sp. strain MIE2 in batch culture

In the present work, the kinetics of hexavalent molybdenum reduction by the Serratia sp. strain MIE2 were investigated using several kinetic models, such as Monod, Haldane, Teissier, Aiba, Yano, Han and Levenspiel and Luong. The statistical analysis showed that the best model was Teissier, which had the lowest RMSE and AICc values, the highest adjusted R2 values, and an F test and with a bias factor and an accuracy factor nearest to unity (1.0). The calculated value for the Teissier constants, such as pmax, Ks and Ki, was 0.506 µmol Mo-blue h−1, 6.53 mM and 29.41 mM, respectively. The effect of heavy metals showed that hexavalent molybdenum reduction by the strain MIE2 was inhibited by silver, mercury and copper with a total inhibition of 96, 97, and 45 %, respectively, at a concentration of 1 ppm. Otherwise, the Mo-reducing enzyme was inhibited by mercury and zinc with an inhibition of 88 and 65 %, respectively. Most of the respiratory inhibitors did not inhibit the Mo-reducing enzyme activity, indicating that the respiratory system in this bacterium is not the site of the hexavalent molybdenum reduction. The results obtained from this study could be useful for estimating the relationship between molybdenum-blue production and the molybdate concentration, which may be important during the up scaling of the molybdenum bioremediation process