Papers by Adam Thean Chor Leow
The dynamics and conformational landscape of proteins in organic solvents are events of potential... more The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H 2 O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA) for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent systems, which could lead to weaknesses in the catalytic H-bond network and most likely a drop in catalytic activity. The conformational variation of the lid domain caused by the How to cite this article Maiangwa et al. (2017), Lid opening and conformational stability of T1 Lipase is mediated by increasing chain length polar solvents. PeerJ 5:e3341; DOI 10.7717/peerj.3341
The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role o... more The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.
Advances in Bioscience and Biotechnology, 2012
In-silico and experimental investigations were conducted to explore the effects of substituting h... more In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues; Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyanion Q114, involved in substrate binding is also associated with modulation of conformational stability and in conferring specific enzyme attributes. The in-silico investigations accurately predicted the quality of the protein packing in some of the variants. Our study found by altering the hydrophobicity of the oxyanion 114, remarkably altered enzyme conformational stability and catalytic attributes. Substitution with Leu resulted improvements in four out of the six tested characteristics. The hydrophobic Leu might have improved local structure folding and increased hydrophobic interactions with other residues in the vicinity of the mutation. The Met variant showed higher activity over the wild-type in hydrolyzing a wider range of natural oils. The bulky amino acids, Phe and Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability and inferior enzyme characteristics. We have successfully illustrated that a single point residue changes at oxyanion 114 could result in a myriad of enzyme attributes, which implied there was some interplay between hydrophobicity and conformation for lipase catalytic functions.
In-silico and experimental investigations were conducted to explore the effects of substituting h... more In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues; Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyanion Q114, involved in substrate binding is also associated with modulation of conformational stability and in conferring specific enzyme attributes. The insilico investigations accurately predicted the quality of the protein packing in some of the variants. Our study found by altering the hydrophobicity of the oxyanion 114, remarkably altered enzyme conformational stability and catalytic attributes. Substitution with Leu resulted improvements in four out of the six tested characteristics. The hydrophobic Leu might have improved local structure folding and increased hydrophobic interactions with other residues in the vicinity of the mutation. The Met variant showed higher activity over the wild-type in hydrolyzing a wider range of natural oils. The bulky amino acids, Phe and Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability and inferior enzyme characteristics. We have successfully illustrated that a single point residue changes at oxyanion 114 could result in a myriad of enzyme attributes, which implied there was some interplay between hydrophobicity and conformation for lipase catalytic functions.
Most substrate for esterification has the inherent problem of low miscibility which requires addi... more Most substrate for esterification has the inherent problem of low miscibility which requires addition of solvents into the reaction media. In this contribution we would like to present an alternative and feasible option for an efficient solvent-free synthesis of menthyl butyrate using a novel thermostable crude T1 lipase. We investigated the effects of incubation time, temperature, enzyme loading and substrate molar ratio and determine the optimum conditions. The high conversion of menthyl butyrate catalyzed by crude T1 lipase in a solvent-free system is greatly affected by temperature and time of the reaction media. The highest yield of menthyl butyrate was 99.3% with the preferred low incubation time and minimal enzyme loading at optimized reaction conditions of 60°C, incubation time 13.15 h, 2.53 mg, 0.43% (w/w) enzyme to substrate ratio and at molar ratio of butyric anhydride/menthol 2.7:1. Hence, the investigation revealed the thermostable crude T1 lipase successfully catalyzed the high yield production of menthyl butyrate in a solvent free system. The finding suggests crude T1 lipase was a promising alternative to overcome shortcomings associated with solvent-assisted enzymatic reactions.
In silico and experimental investigations were conducted to explore the effects of substituting h... more In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and ×Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with ×Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with ×Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the ×Phe and Trp mutation.
BMC Structural Biology, 2014
Background: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) whi... more Background: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) which are largely non-similar to other known, well-characterized proteins. Predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. The present study highlights the primary effort to classify and cluster 1202 HPs of Bacillus lehensis G1 alkaliphile to serve as a platform to mine and select specific HP(s) to be studied further in greater detail. Results: All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the βαβαββ core structure of Trx-like proteins as well as three flanking β-sheets, a 3 10 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes. Conclusions: We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.
Cellular and Molecular Biology Letters, 2015
soy broth; YNB -yeast nitrogen base; YPD -yeast extract peptone dextrose; YPDS -yeast peptone dex... more soy broth; YNB -yeast nitrogen base; YPD -yeast extract peptone dextrose; YPDS -yeast peptone dextrose sorbitol; YPTG -yeast extract peptone tryptic soy broth glycerol; YPTM -yeast extract peptone tryptic soy broth methanol
4th Annual International Conference on Advances in Biotechnology (BioTech 2014), 2014
Protein & Peptide Letters, 2010
An all-atom level MD simulation in explicit solvent at high temperature is a powerful technique t... more An all-atom level MD simulation in explicit solvent at high temperature is a powerful technique to increase our knowledge about the structurally important regions modulating thermal stability in thermenzymes. In this respect, two large-sized thermoalkalophilic enzymes from Bacillus stearothermophilus L1 (L1 lipase) and Geobacillus zalihae strain T1 (T1 lipase) are well-established representatives. In this paper, comparative results from temperature-induced MD simulations of both model systems at 300 K, 400 K and 500 K are presented and discussed with respect to identification of highly flexible regions critical to thermostability. From our MD simulation results, specific regions along the L1 lipase and T1 lipase polypeptide chain including the small domain and the main catalytic domain or core domain of both enzymes show a marked increase in fluctuations and dynamics followed by clear structural changes. Overall, the N-terminal moiety of both enzymes and their small domains exhibit hyper-sensitivity to thermal stress. The results appear to propose that these regions are critical in determining of the overall thermal stability of both organisms.
Journal of Applied Phycology, 2014
ABSTRACT Short-chain dehydrogenases (SDRs) which catalyze the dehydrogenation/reduction reaction ... more ABSTRACT Short-chain dehydrogenases (SDRs) which catalyze the dehydrogenation/reduction reaction have a wide range of substrate specificities. In this study, we report the molecular characterization of a transcript encoding SDR from the red seaweed, Gracilaria changii (Rhodophyta) as part of our effort to elucidate the functions of novel transcripts from this marine alga. The transcript, denoted as GcSDR, encodes a protein of 282 amino acids with a predicted size of approximately 31 kDa. The GxxxGxG coenzyme binding motif and YxxxK active-site motif of SDRs are well conserved in GcSDR. The coding sequence of GcSDR was cloned and expressed as recombinant protein in Escherichia coli BL21 (DE3) pLysS. Kinetic analysis of recombinant GcSDR using pyruvaldehyde dimethyl acetal as a substrate has a Km value of 116.52 mM and a Vmax value of 720 nmol product formed per minute per milligram. It has a higher affinity towards NADPH compared to NADP+ as a cofactor. Homology modeling showed that the three-dimensional structure of GcSDR has 34.5 % sequence identity to the SDR from a soil bacterium. Virtual screening of the possible substrates for GcSDR revealed that CMP-N-acetyl-beta-neuraminate (2-) which belongs to a group of amino sugars has the lowest binding energy among the compounds examined. The predicted cis-acting regulatory elements (CREs) at the 5′-flanking genomic sequence of GcSDR include CREs associated with abscisic acid, methyl jasmonic acid, light, anoxia, endosperm, low-temperature stress, and heat stress. The transcripts of GcSDR were found to accumulate in seaweed samples treated under low-salinity stress, thus suggesting its involvement during saline deprivation.
BMC Structural Biology, 2014
Background: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) whi... more Background: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) which are largely non-similar to other known, well-characterized proteins. Predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. The present study highlights the primary effort to classify and cluster 1202 HPs of Bacillus lehensis G1 alkaliphile to serve as a platform to mine and select specific HP(s) to be studied further in greater detail. Results: All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the βαβαββ core structure of Trx-like proteins as well as three flanking β-sheets, a 3 10 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes. Conclusions: We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.
Psychrophilic microorganisms are cold-adapted
with distinct properties from other thermal classes... more 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 coldactive
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 characterizationsof 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.
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Papers by Adam Thean Chor Leow
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 coldactive
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 characterizationsof 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.
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 coldactive
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 characterizationsof 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.