GDSL esterase is designated as a member of Family II of lipolytic enzymes known to catalyse the synthesis and hydrolysis of ester bonds. The enzyme possesses a highly conserved motif Ser-Gly-Asn-His in the four conserved blocks I, II, III and V respectively. The enzyme characteristics, such as region-, chemo-, and enantioselectivity, help in resolving the racemic mixture of single-isomer chiral drugs. Recently, crystal structure of GDSL esterase from Photobacterium J15 has been reported (PDB ID: 5XTU) but not in complex with substrate. Therefore, GDSL in complex with substrate could provide insights into the binding mode of substrate towards inactive form of GDSL esterase (S12A) and identify the hot spot residues for the designing of a better binding pocket. Insight into molecular mechanisms is limited due to the lack of crystal structure of GDSL esterase-substrate complex. In this paper, the crystallization of mutant GDSL esterase (S12A) (PDB ID: 8HWO) and its complex with butyric acid (PDB ID: 8HWP) are reported. The optimized structure would be vital in determining hot spot residue for GDSL esterase. This preliminary study provides an understanding of the interactions between enzymes and hydrolysed p-nitro-phenyl butyrate. The information could guide in the rational design of GDSL esterase in overcoming the medical limitations associated with racemic mixture.
In eukaryotes, serine proteases are cellular localized hydrolases reported to regulate essential biological reactions. Improved industrial applications of proteins are aided by prediction and analysis of their 3-dimensional structures (3D). A serine protease was identified from CTG-clade yeast Meyerozyma guilliermondii strain SO and its 3D structure as well as its catalytic attributes have not been fully understood yet, thus we seek to report on the catalytic mechanism of M. guilliermondii strain SO MgPRB1 using substrate PMSF via in silico docking as well as its stability by way of disulfide bonds formation. Herein, bioinformatics tools and techniques were used to predict, validate and analyze the possible changes of CUG ambiguity (if any) in strain SO using template PDB ID: 3F7O. Structural assessments confirmed the classic catalytic triad Asp305, His337, and Ser499. Superimposition of MgPRB1 and template 3F7O structures revealed the unlinked cysteine residues between Cys341, Cys440, Cys471 and Cys506 of MgPRB1 compared to template 3F7O with two disulfide bonds formation, which confers structural stability. In conclusion, serine protease structure from strain SO was successfully predicted and studies towards understanding at the molecular level may be undertaken for its potential applications in the degradation of peptide bonds.
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.
Photobacterium species are Gram-negative coccobacilli which are distributed in marine habitats worldwide. Some species are unique because of their capability to produce luminescence. Taxonomically, about 23 species and 2 subspecies are validated to date. Genomes from a few Photobacterium spp. have been sequenced and studied. They are considered a special group of bacteria because some species are capable of producing essential polyunsaturated fatty acids, antibacterial compounds, lipases, esterases and asparaginases. They are also used as biosensors in food and environmental monitoring and detectors of drown victim, as well as an important symbiont.
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.
Hyperthermostable enzymes are very attractive biocatalysts due to their exceptional stability at extreme temperatures. Recently, a hyperthermostable carboxylesterase EstD9 from Anoxybacillus geothermalis D9 was biochemically characterized. The enzyme displayed remarkable stability at high temperature. In this study, we attempted to probe the conformational adaptability of EstD9 under extreme conditions via in silico approaches. Circular dichroism revealed that EstD9 generated new β-sheets at 80 °C that make up the core of the hydrolase fold. Interestingly, the profiles of molecular dynamics simulation showed the lowest scores of radius of gyration and solvent accessible surface area (SASA) at 80 °C. Three loops were responsible for protecting the catalytic site, residing at the interface between the two domains. To further investigate molecular adaptation in extreme conditions, the intramolecular interactions of native structure were investigated. 18 hydrogen bond networks, 7 salt bridges, and 9 hydrophobic clusters were revealed within EstD9, which is higher than the reported thermostable carboxylesterase Est30. Collectively, the analysis indicates that intramolecular interactions and structural dynamics play distinct roles in preserving the overall EstD9 structure at elevated temperatures. This work is relevant to fundamental and applied research involving protein engineering of industrial thermostable enzymes.
Waxy crude oil is a problem to the oil and gas industry because wax deposition in pipelines reduces the quality of the crude oil. Currently, the industry uses chemicals to solve the problem but it is not environmentally friendly. As an alternative, the biodegradation approach is one of the options. Previously eleven thermophilic bacteria were isolated and exhibited high ability to degrade hydrocarbon up to 70% of waxy crude oil. However, despite the successful study on these single bacteria strains, it is believed that biodegradation of paraffin wax requires more than a single species. Five consortia were developed based on the biodegradation efficiency of 11 bacterial strains. Consortium 3 showed the highest biodegradation (77.77%) with more long-chain alkane degraded throughout the incubation compared to other consortia. Enhancement of hydrocarbon degradation was observed for all consortia especially in long chain alkane (C18-C40). Consortium 3 exhibited higher alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase activities. Moreover, the dominant bacteria in the consortia were determined by denaturing gradient gel electrophoresis (DGGE), which showed the domination of genera Geobacillus, Parageobacillus, and Anoxybacillus. It can be concluded that the bacterial consortia showed higher biodegradation and improved degrading more long-chain hydrocarbon compared to a single isolate.
Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268-275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29-residues signal peptide, 77-residues propeptide, and 275-residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.
Carboxylesterases (CEs) are members of prominent esterase, and as their name imply, they catalyze the cleavage of ester linkages. By far, a considerable number of novel CEs have been identified to investigate their exquisite physiological and biochemical properties. They are abundant enzymes in nature, widely distributed in relatively broad temperature range and in various sources; both macroorganisms and microorganisms. Given the importance of these enzymes in broad industries, interest in the study of their mechanisms and structural-based engineering are greatly increasing. This review presents the current state of knowledge and understanding about the structure and functions of this ester-metabolizing enzyme, primarily from bacterial sources. In addition, the potential biotechnological applications of bacterial CEs are also encompassed. This review will be useful in understanding the molecular basis and structural protein of bacterial CEs that are significant for the advancement of enzymology field in industries.
Pseudomonas fluorescens AMS8 lipase lid 1 structure is rigid and holds unclear roles due to the absence of solvent-interactions. Lid 1 region was stabilized by 17 hydrogen bond linkages and displayed lower mean hydrophobicity (0.596) compared to MIS38 lipase. Mutating lid 1 residues, Thr-52 and Gly-55 to aromatic hydrophobic-polar tyrosine would churned more side-chain interactions between lid 1 and water or toluene. This study revealed that T52Y leads G55Y and its recombinant towards achieving higher solvent-accessible surface area and longer half-life at 25 to 37 °C in 0.5% (v/v) toluene. T52Y also exhibited better substrate affinity with long-chain carbon substrate in aqueous media. The affinity for pNP palmitate, laurate and caprylate increased in 0.5% (v/v) toluene in recombinant AMS8, but the affinity in similar substrates was substantially declined in lid 1 mutated lipases. Regarding enzyme efficiency, the recombinant AMS8 lipase displayed highest value of kcat/Km in 0.5% (v/v) toluene, mainly with pNPC. In both hydrolysis reactions with 0% and 0.5% (v/v) toluene, the enzyme efficiency of G55Y was found higher than T52Y for pNPL and pNPP. At 0.5% (v/v) toluene, both mutants showed reductions in activation energy and enthalpy values as temperature increased from 25 to 35 °C, displaying better catalytic functions. Only T52Y exhibited increase in entropy values at 0.5% (v/v) toluene indicating structure stability. As a conclusion, Thr-52 and Gly-55 are important residues for lid 1 stability as their existence helps to retain the geometrical structure of alpha-helix and connecting hinge.
The deposition of paraffin wax in crude oil is a problem faced by the oil and gas industry during extraction, transportation, and refining of crude oil. Most of the commercialized chemical additives to prevent wax are expensive and toxic. As an environmentally friendly alternative, this study aims to find a novel thermophilic bacterial strain capable of degrading paraffin wax in crude oil to control wax deposition. To achieve this, the biodegradation of crude oil paraffin wax by 11 bacteria isolated from seawater and oil-contaminated soil samples was investigated at 70°C. The bacteria were identified as Geobacillus kaustophilus N3A7, NFA23, DFY1, Geobacillus jurassicus MK7, Geobacillus thermocatenulatus T7, Parageobacillus caldoxylosilyticus DFY3 and AZ72, Anoxybacillus geothermalis D9, Geobacillus stearothermophilus SA36, AD11, and AD24. The GCMS analysis showed that strains N3A7, MK7, DFY1, AD11, and AD24 achieved more than 70% biodegradation efficiency of crude oil in a short period (3 days). Notably, most of the strains could completely degrade C37-C40 and increase the ratio of C14-C18, especially during the initial 2 days incubation. In addition, the degradation of crude oil also resulted in changes in the pH of the medium. The degradation of crude oil is associated with the production of degradative enzymes such as alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase. Among the 11 strains, the highest activities of alkane monooxygenase were recorded in strain AD24. A comparatively higher overall alcohol dehydrogenase, lipase, and esterase activities were observed in strains N3A7, MK7, DFY1, AD11, and AD24. Thus, there is a potential to use these strains in oil reservoirs, crude oil processing, and recovery to control wax deposition. Their ability to withstand high temperature and produce degradative enzymes for long-chain hydrocarbon degradation led to an increase in the short-chain hydrocarbon ratio, and subsequently, improving the quality of the oil.
The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- β-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.
The conversion of aldehydes to valuable alkanes via cyanobacterial aldehyde deformylating oxygenase is of great interest. The availability of fossil reserves that keep on decreasing due to human exploitation is worrying, and even more troubling is the combustion emission from the fuel, which contributes to the environmental crisis and health issues. Hence, it is crucial to use a renewable and eco-friendly alternative that yields compound with the closest features as conventional petroleum-based fuel, and that can be used in biofuels production. Cyanobacterial aldehyde deformylating oxygenase (ADO) is a metal-dependent enzyme with an α-helical structure that contains di‑iron at the active site. The substrate enters the active site of every ADO through a hydrophobic channel. This enzyme exhibits catalytic activity toward converting Cn aldehyde to Cn-1 alkane and formate as a co-product. These cyanobacterial enzymes are small and easy to manipulate. Currently, ADOs are broadly studied and engineered for improving their enzymatic activity and substrate specificity for better alkane production. This review provides a summary of recent progress in the study of the structure and function of ADO, structural-based engineering of the enzyme, and highlight its potential in producing biofuels.
Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C.
GDSL esterase J15 (EstJ15) is a member of Family II of lipolytic enzyme. The enzyme was further classified in subgroup SGNH hydrolase due to the presence of highly conserve motif, Ser-Gly-Asn-His in four conserved blocks I, II, III, and V, respectively. X-ray quality crystal of EstJ15 was obtained from optimized formulation containing 0.10 M ammonium sulphate, 0.15 M sodium cacodylate trihydrate pH 6.5, and 20% PEG 8000. The crystal structure of EstJ15 was solved at 1.38 Å with one molecule per asymmetric unit. The structure exhibits α/β hydrolase fold and shared low amino acid sequence identity of 23% with the passenger domain of the autotransporter EstA of Pseudomonas aeruginosa. The active site is located at the centre of the structure, formed a narrow tunnel that hinder long substrates to be catalysed which was proven by the protein-ligand docking analysis. This study facilitates the understanding of high substrate specificity of EstJ15 and provide insights on its catalytic mechanism.
Engineered thermostable microbial enzymes are widely employed to catalyze chemical reactions in numerous industrial sectors. Although high thermostability is a prerequisite of industrial applications, enzyme activity is usually sacrificed during thermostability improvement. Therefore, it is vital to select the common and compatible strategies between thermostability and activity improvement to reduce mutants̕ libraries and screening time. Three functional protein engineering approaches, including directed evolution, rational design, and semi-rational design, are employed to manipulate protein structure on a genetic basis. From a structural standpoint, integrative strategies such as increasing substrate affinity; introducing electrostatic interaction; removing steric hindrance; increasing flexibility of the active site; N- and C-terminal engineering; and increasing intramolecular and intermolecular hydrophobic interactions are well-known to improve simultaneous activity and thermostability. The current review aims to analyze relevant strategies to improve thermostability and activity simultaneously to circumvent the thermostability and activity trade-off of industrial enzymes.
Biocatalysts have been gaining extra attention in recent decades due to their industrial-relevance properties, which may hasten the transition to a cleaner environment. Carboxylic acid reductases (CARs) are large, multi-domain proteins that can catalyze the reduction of carboxylic acids to corresponding aldehydes, with the presence of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). This biocatalytic reaction is of great interest due to the abundance of carboxylic acids in nature and the ability of CAR to convert carboxylic acids to a wide range of aldehydes essentially needed as end products such as vanillin or reaction intermediates for several compounds production such as alcohols, alkanes, and amines. This modular enzyme, found in bacteria and fungi, demands an activation via post-translational modification by the phosphopantetheinyl transferase (PPTase). Recent advances in the characterization and structural studies of CARs revealed valuable information about the dynamics, mechanisms, and unique features of the enzymes. In this comprehensive review, we summarize the previous findings on the phylogeny, structural and mechanistic insight of the domains, post-translational modification requirement, strategies for the cofactors regeneration, the extensively broad aldehyde-related industrial application properties of CARs, as well as their recent immobilization approaches.
Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
The stability and activity of lipase in organic media are important parameters in determining how quickly biocatalysis proceeds. This study aimed to examine the effects of two commonly used alcohols in industrial applications, methanol (MtOH) and ethanol (EtOH) on the conformational stability and catalytic activity of G210C lipase, a laboratory-evolved mutant of Staphylococcus epidermidis AT2 lipase. Simulation studies were performed using an open-form predicted structure under 30, 40 and 50% of MtOH and EtOH at 25 °C and 45 °C. The overall enzyme structure becomes more flexible with increasing concentration of MtOH and exhibited the highest flexibility in 40% EtOH. In EtOH, the movement of the lid was found to be temperature-dependent with a noticeable shift in the lid position at 45 °C. Lid opening was evidenced at 50% of MtOH and EtOH which was supported by the increase in SASA of hydrophobic residues of the lid and catalytic triad. The active site remained mostly intact. An open-closed lid transition was observed when the structure was re-simulated in water. Experimental evaluation of the lipase stability showed that the half-life reduced when the enzyme was treated with 40% (v/v) and 50% (v/v) of EtOH and MtOH respectively. The finding implies that a high concentration of alcohol and elevated temperature can induce the lid opening of lipase which could be essential for the activation of the enzyme, provided that the catalytic performance in the active site is not compromised.Communicated by Ramaswamy H. Sarma.
Aquaporin is a water channel protein that facilitates the movement of water across the cell membrane. Aquaporin from the Antarctic region has been noted for its psychrophilic properties and its ability to perform at a lower temperature but there remains limited understanding of the water mechanism of Antarctic Pseudomonas sp. strain AMS3 However, studies regarding aquaporin isolated from psychrophilic Pseudomonas sp. are still scattered. Recently, the genome sequence of an Antarctic Pseudomonas sp. strain AMS3 revealed a gene sequence encoding for a putative aquaporin designated as AqpZ1 AMS3. In this study, structure analysis and a molecular dynamics (MD) simulation of a predicted model of a fully hydrated aquaporin tetramer embedded in a lipid bilayer was performed at different temperatures for structural flexibility and stability analysis. The MD simulation results revealed that the structures were able to remain stable at low to medium temperatures. The protein was observed to have high flexibility in the loop region as compared to the helices region throughout the simulated temperatures. The selectivity filter and NPA motifs play a major role in solute selectivity and the pore radius of the protein. The structural and functional characterization of this psychrophilic aquaporin provides new insights for the future applications of this protein.Communicated by Ramaswamy H. Sarma.