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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.

In industrial application, immobilized lipase are typically not reused and served as industrial waste after a certain process is completed. The capacity on the reusability of the spent lipase is not well studied. This current study embarks on reusing the remaining lipase from the spent immobilized enzyme. Active lipases were recovered using a simple reverse micellar extraction (RME). RME is the extraction process of targeted biomolecules using an organic solvent and a surfactant. This method was the first attempt reported on the recovery of the lipase from the used immobilized lipase. RME of the spent lipase was done using the nonionic Triton X-100 surfactant and toluene. Various parameters were optimized to maximize the lipase recovery from the used immobilized lipase. The optimum forward extraction condition was 0.075 M KCl, and backward conditions were at 0.15 M Triton X-100/toluene (pH 6, 2 M KCl) with recovery of 66%. The extracted lipase was immobilized via simple adsorption into the ethanol pretreated carrier. The optimum conditions of immobilization resulted in 96% of the extracted lipase was reimmobilized. The reimmobilized lipase was incubated for 20 h in pH 6 buffer at 50 °C of water bath shaker. The reimmobilized lipase still had 27% residual activity after 18 h of incubation, which higher thermal stability compared to the free lipase. In conclusion, the free lipase was successfully extracted from the spent immobilized lipase and reimmobilized into the new support. It exhibited high thermal stability, and the reusability of the spent lipase will promote continued use of industrial lipase and reduce the cost of the manufacturing process.

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.

Aminoacylase (EC 3.5.1.14) hydrolyzes N–acetylated amino acids to produce amino acids. Although thermostable aminoacylase has been commercially produced since 2004, there was a knowledge gap in the field of understanding aminoacylase thermostability from a structural point of view. This study investigated the physical and structural properties of the purified thermostable aminoacylase SZN. The spectropolarimetry data for structural determination has indicated a gradual decrease of α-helix from 36 to 27.6%, followed by tremendous disorientation of the structure at the transition of temperatures from 60 to 70°C (27.6 to 19.5%). In contrast, the percentage of β-sheet has increased steadily over the tested temperatures. The α-helix, where notable metal binding and catalytic residues are located, was totally weakened at temperatures above 70C, thus resulted in loss of activity. The loss of the α-helical structure could further explain drastic deterioration of activity at temperatures beyond 70C. The activity of aminoacylase SZN was enhanced by divalent metal ions, such as Mn2+ and Cu2+, and inhibited by detergent Triton-X-100. As a conclusion, the isolated aminoacylase SZN was characterized as a thermostable enzyme based on the α-helical structure integrity and functional stability in high temperatures. This enzyme could be used as an alternative enzyme for bioindustries in view of its activity enhancement in high temperatures and stability in various tested inhibitors.

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.

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.

Riboflavin synthase is an important enzyme catalyzing the last step of riboflavin biosynthesis in microorganisms. Due to the absolute dependency of the microbes to this biosynthetic pathway coupled with its nonexistence in humans, riboflavin synthase (RiS) is considered as a prospective drug target. The riboflavin synthase for this study was derived from Leptospira kmetyi, a pathogenic bacterium locally isolated in Malaysia. Leptospirosis, an infectious disease caused by pathogenic Leptospira, is a serious growing public health issue. Treatment of leptospirosis with antibiotics over time resulted in the evolution of antibiotic resistance strains, thus requiring the development of newer but safe antimicrobial agents. In this study, a computational approach involving virtual screening followed by molecular dynamics (MD) simulation was implemented in order to identify possible inhibitors against riboflavin synthase. The model of Leptospira kmetyi riboflavin synthase predicted from E. coli riboflavin synthase (1I8D) was used as the drug target to screen for potential compounds from the ZINC database through virtual screening. The potential compound with the highest Glide score (−10.987 Kcal/mol) was identified to be ZINC21883831. Chemically it is 2-[(2-chloro-4-fluoro-phenyl)methylsulfanyl]-7-phenyl-3,5-dihydropyrrolo[2,3-e]pyrimidin-4-one. The top three docked complexes from the virtual screening and apo-structures were subjected to molecular dynamics simulation to predict the stabilities of the Leptospira kmetyi riboflavin synthase-ligand complexes. Stability parameters including RMSD, RMSF, SASA and Rg of the complexes were evaluated from 60 ns of the MD simulation trajectories. Insights from this study provide promising starting points for the rational designs of new effective and safe anti-leptospirosis drugs.

In order to determine the potential of biochemical and structural features of Elaeis guineensis Jacq. oil palm mesocarp lipases, the LIP2 gene was isolated, expressed, purified and characterized through the Escherichia coli microbial recombinant system. Gene analysis of LIP2 revealed that it is composed of 1584 base pairs which are encoded in 528 amino acid residues with a molecular weight of around 57 kDa. LIP2 has distinctive lipolytic properties in terms of α/β fold and the catalytic triad for lipase. The LIP2 lipase was successfully expressed and purified from E. coli Rosetta (DE3) via affinity chromatography. The optimal temperature and pH for the lipase activity was 30 °C and a pH of 9, respectively. Stability was profoundly increased with the addition of metal ions (Ca2+, Mg2+, Mn+, and Ni+), along with organic solvents (ethanol and octanol). pNP myristate was the most suitable among all pNP esters. In biophysical characterization analysis, LIP2 has a thermal denaturing point at 66 °C, which mostly consists of random patterns (39.8%) followed by α-helix (30.3%), turns (23.8%) and β-sheet (6.2%). From the successful purification and characterization, the potential of oil palm mesocarp lipase was able to be further explored.

5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 Å with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl–esters.

Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter &lsquo, ragi&rsquo, from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 &deg, C and 70 &deg, C) and Hygromycin B (50 &mu, g/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDh&alpha, ). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDh&alpha, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.

Calcium-binding plays a decisive role in the folding and stabilization of many RTX proteins, especially for the RTX domain. Although many studies have been conducted to prove the contribution of Ca2+ ion toward the folding and stabilization of RTX proteins, its functional dynamics and conformational structural changes remain elusive. Here, molecular docking and molecular dynamics (MD) simulations were performed to analyze the contribution of Ca2+ ion toward the folding and stabilization of the RTX lipase (AMS8 lipase) structure. AMS8 lipase contains six Ca2+ ions (Ca1&ndash, Ca6). Three Ca2+ ions (Ca3, Ca4, and Ca5) were bound to the RTX parallel &beta, roll motif repeat structure (RTX domain). The metal ion (Ca2+) docking analysis gives a high binding energy, especially for Ca4 and Ca5 which are tightly bound to the RTX domain. The function of each Ca2+ ion is further analyzed using the MD simulation. The removal of Ca3, Ca4, and Ca5 caused the AMS8 lipase structure to become unstable and unfolded. The results suggested that Ca3, Ca4, and Ca5 stabilized the RTX domain. In conclusion, Ca3, Ca4, and Ca5 play a crucial role in the folding and stabilization of the RTX domain, which sustain the integrity of the overall AMS8 lipase structure.

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 two 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.

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 &deg, C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

Background Metal polluted environments have been found to harbor acid and metal tolerant bacterial communities. Metal oxidizing bacteria in particular are industrially important microorganisms that can be utilized for potential applications in biomining and bioremediation. However, some well-characterized strains are not readily culturable as they are obligate and fastidious chemolithotrophs requiring special techniques for their cultivation. Hence, this study was aimed at isolating, identifying, and characterizing indigenous metal tolerant heterotroph(s) from abandoned mines that can potentially be used for biomining or bioremediation processes in the future. Results Seventeen bacteria from former mining lakes were isolated and identified using 16S rRNA. Minimal inhibition concentration (MIC) and growth study of isolated bacteria carried out in Luria-Bertani media containing three different metals ions, zinc (II), copper (II), and iron (II), showed that a particular isolate termed Enterobacter cloacae NZS was found to exhibit better growth and tolerance for copper (up to 90 mM), zinc (up to 200 mM), and iron (up to 170 mM). Growth of the strain was notably well in the presence of iron (II). Compared to all the isolates, only E. cloacae NZS was able to be enumerated at pH lower than 5 while other strains were culturable only at pH 7. Its capability in iron (II) oxidation was preliminary assessed based on the pH, cell count, glucose consumption, and amount of iron oxidized throughout incubation in 9K media. E. cloacae NZS strain was found to be capable of oxidizing iron (II) supplied in 9K media to iron (III). Conclusion As preliminary investigation showed that E. cloacae NZS was able to oxidize iron (II) in 9K media at pH2, further optimization on the strain, medium, and culture conditions in future may be able to provide a better insight on this strain to be possibly used as an iron oxidizer for various applications.

It is hypothesized that the Ca2+ ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three RTX motifs. So, this research aimed to examine the influence of Ca2+ ion towards the activity and folding of AMS8 lipase through various biophysical characterizations. The results showed that CaCl2 increased lipase activity. The far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) analysis suggested that the secondary structure content was improved with the addition of CaCl2. Fluorescence spectroscopy analysis showed that the presence of CaCl2 increased protein folding and compactness. Dynamic light scattering (DLS) analysis suggested that AMS8 lipase became aggregated at a high concentration of CaCl2.The binding constant (Kd) value from the isothermal titration calorimetry (ITC) analysis proved that the Ca2+ ion was tightly bound to the AMS8 lipase. In conclusion, Ca2+ ions play crucial roles in the activity and folding of the AMS8 lipase. Calcium binding to RTX nonapeptide repeats sequences will induced the formation and folding of the RTX parallel &beta, roll motif repeat structure.

A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.

Aminopeptidase P (APPro, E.C 3.4.11.9) cleaves N-terminal amino acids from peptides and proteins where the penultimate residue is proline. This metal-ion-dependent enzyme shares a similar fold, catalytic mechanism, and substrate specificity with methionine aminopeptidase and prolidase. It adopts a canonical pita bread fold that serves as a structural basis for the metal-dependent catalysis and assembles as a tetramer in crystals. Similar to other metalloaminopeptidase, APPro requires metal ions for its maximal enzymatic activity, with manganese being the most preferred cation. Microbial aminopeptidase possesses unique characteristics compared with aminopeptidase from other sources, making it a great industrial enzyme for various applications. This review provides a summary of recent progress in the study of the structure and function of aminopeptidase P and describes its various applications in different industries as well as its significance in the environment.

Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The km and kcat values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.

The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris–HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris–HCl pH 8, while for L2 lipase it was at 70 °C in Glycine–NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.

Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 &deg, C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability.

Sago starch is traditionally used as food especially in Southeast Asia. Generally, sago is safe for consumption, biodegradable, easily available and inexpensive. Therefore, this research was done to expand the potential of sago by using it as a support for enzyme immobilization. In this study, ARM lipase, which was isolated fromGeobacillussp. strain ARM, was overexpressed inEscherichia colisystem and then purified using affinity chromatography. The specific activity of the pure enzyme was 650 U/mg, increased 7 folds from the cell lysate. The purified enzyme was immobilized in gelatinized sago and spray-dried by entrapment technique in order to enhance the enzyme operational stability for handling at high temperature and also for storage. The morphology of the gelatinized sago and immobilized enzyme was studied by scanning electron microscopy. The results showed that the spray-dried gelatinized sago was shrunken and became irregular in structure as compared to untreated sago powder. The surface areas and porosities of spray-dried gelatinized sago with and without the enzyme were analyzed using BET and BJH method and have shown an increase in surface area and decrease in pore size. The immobilized ARM lipase showed good performance at 60–80 °C, with a half-life of 4 h and in a pH range 6–9. The immobilized enzyme could be stored at 10 °C with the half-life for 9 months. Collectively, the spray-dried immobilized lipase shows promising capability for industrial uses, especially in food processing.

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 &deg, C and at pH 6. Stable lipolytic activity was reported between temperatures of 5&ndash, 30 &deg, C and at pH 6&ndash, 8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.

T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.

The immobilization of Rhizopus oryzae lipase (RoL) by hydrophobic adsorption on polypropylene supports with additives was investigated. Additives such as hen egg albumin, sodium caseinate and CAVAMAX® W6 were used to coat the support during immobilization where the immobilized RoL on coated support was compared to those of noncoated support. Following the immobilization, the catalytic activity of immobilized RoL was characterized based on different temperatures and pH. The immobilized RoL without additives showed optimal lipase activity at an optimum temperature of 50 °C and pH 6. However, RoL lipase that was immobilized on support treated with CAVAMAX® W6 had better performance in terms of hydrolytic activity and stability as compared to other additives. In addition, by having a support treated with hen egg albumin, the immobilized RoL was capable of yielding higher ester during esterification reactions.

The PT7(A1/O4/O3) is a promoter resulted from construction of O3 and O4 operators into PA1, a promoter derived from coliphage T7, that evidenced lower the occupancy of the promoter by RNA polymerase and thereby increases the repression factor. A new expression system, pTEL, was successfully constructed via shuttle vector pUCP19 as the backbone as the former carries pre-existing stabilizing fragment (SF) that enables replication of the plasmid in both E. coli and Pseudomonas sp. The leaky lac operon-based promoter found in pUCP19 was subsequently replaced by the PT7 (A1/O4/O3). Meanwhile, structural gene of the organic solvent tolerant elastase strain K was used as DNA insert (passenger enzyme) for repression and overexpression studies. The success of pTEL was evidenced by detection of non-significant protein expression level in the absence of IPTG as the inducer, indicating tight regulation possessed by the modified promoter. The addition of IPTG, however, relieved repression and demonstrated overexpression of the elastase strain K in various strains of E. coli following several optimization studies.

Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 &deg, C. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 &deg, C change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 &deg, C (T1/2 = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.

The molecular structure of DNA polymerase I or family A polymerases is made up of three major domains that consist of a single polymerase domain with two extra exonuclease domains. When the N-terminal was deleted, the enzyme was still able to perform basic polymerase activity with additional traits that used isothermal amplification. However, the 3&prime, 5&prime, exonuclease domain that carries a proofreading activity was disabled. Yet, the structure remained attached to the 5&prime, 3&prime, polymerization domain without affecting its ability. The purpose of this non-functional domain still remains scarce. It either gives negative effects or provides structural support to the DNA polymerase. Here, we compared the effect of deleting each domain against the polymerase activity. The recombinant wild type and its variants were successfully purified and characterized. Interestingly, SK72-Exo (a large fragment excluding the 5&prime, exonuclease domain) exhibited better catalytic activity than the native SK72 (with all three domains) at similar optimum temperature and pH profile, and it showed longer stability at 70 &deg, C. Meanwhile, SK72-Exo2 (polymerization domain without both the 5&prime, and 3&prime, exonuclease domain) displayed the lowest activity with an optimum at 40 &deg, C and favored a more neutral environment. It was also the least stable among the variants, with almost no activity at 50 &deg, C for the first 10 min. In conclusion, cutting both exonuclease domains in DNA polymerase I has a detrimental effect on the polymerization activity and structural stability.

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 &deg, C and 76.0 &deg, C in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in &beta, Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.

Resistance to high temperature, acidic pH and proteolytic degradation during the pelleting process and in the digestive tract are important features of phytases as animal feed. The integration of insights from structural and in silico analyses into factors affecting thermostability, acid stability, proteolytic stability, catalytic efficiency and specific activity, as well as N-glycosylation, could improve the limitations of marginal stable biocatalysts with trade-offs between stability and activity. Synergistic mutations give additional benefits to single substitutions. Rigidifying the flexible loops or inter-molecular interactions by reinforcing non-bonded interactions or disulfide bonds, based on structural and roof mean square fluctuation (RMSF) analyses, are contributing factors to thermostability. Acid stability is normally achieved by targeting the vicinity residue at the active site or at the neighboring active site loop or the pocket edge adjacent to the active site. Extending the positively charged surface, altering protease cleavage sites and reducing the affinity of protease towards phytase are among the reported contributing factors to improving proteolytic stability. Remodeling the active site and removing steric hindrance could enhance phytase activity. N-glycosylation conferred improved thermostability, proteases degradation and pH activity. Hence, the integration of structural and computational biology paves the way to phytase tailoring to overcome the limitations of marginally stable phytases to be used in animal feeds.

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

Four major enzymes commonly used in the market are lipases, proteases, amylases, and cellulases. For instance, in both academic and industrial levels, microbial lipases have been well studied for industrial and biotechnological applications compared to others. Immobilization is done to minimize the cost. The improvement of enzyme properties enables the reusability of enzymes and facilitates enzymes used in a continuous process. Immobilized enzymes are enzymes physically confined in a particularly defined region with retention to their catalytic activities. Immobilized enzymes can be used repeatedly compared to free enzymes, which are unable to catalyze reactions continuously in the system. Immobilization also provides a higher pH value and thermal stability for enzymes toward synthesis. The main parameter influencing the immobilization is the support used to immobilize the enzyme. The support should have a large surface area, high rigidity, suitable shape and particle size, reusability, and resistance to microbial attachment, which will enhance the stability of the enzyme. The diffusion of the substrate in the carrier is more favorable on hydrophobic supports instead of hydrophilic supports. The methods used for enzyme immobilization also play a crucial role in immobilization performance. The combination of immobilization methods will increase the binding force between enzymes and the support, thus reducing the leakage of the enzymes from the support. The adsorption of lipase on a hydrophobic support causes the interfacial activation of lipase during immobilization. The adsorption method also causes less or no change in enzyme conformation, especially on the active site of the enzyme. Thus, this method is the most used in the immobilization process for industrial applications.

Microorganisms, especially those that survive in extremely cold places such as Antarctica, have gained research attention since they produce a unique feature of the protein, such as being able to withstand at extreme temperature, salinity, and pressure, that make them desired for biotechnological application. Here, we report the first hormone-sensitive lipase (HSL)-like esterase from a Glaciozyma species, a psychrophilic yeast designated as GlaEst12-like esterase. In this study, the putative lipolytic enzyme was cloned, expressed in E. coli, purified, and characterised for its biochemical properties. Protein sequences analysis showed that GlaEst12 shared about 30% sequence identity with chain A of the bacterial hormone-sensitive lipase of E40. It belongs to the H group since it has the conserved motifs of Histidine-Glycine-Glycine-Glycine (HGGG)and Glycine-Aspartate-Serine-Alanine-Glycine (GDSAG) at the amino acid sequences. The recombinant GlaEst12 was successfully purified via one-step Ni-Sepharose affinity chromatography. Interestingly, GlaEst12 showed unusual properties with other enzymes from psychrophilic origin since it showed an optimal temperature ranged between 50&ndash, 60 &deg, C and was stable at alkaline pH conditions. Unlike other HSL-like esterase, this esterase showed higher activity towards medium-chain ester substrates rather than shorter chain ester. The 3D structure of GlaEst12, predicted by homology modelling using Robetta software, showed a secondary structure composed of mainly &alpha, /&beta, hydrolase fold, with the catalytic residues being found at Ser232, Glu341, and His371.

Large-scale production of T1 lipase using conventional culture media is costly. To reduce the cost of production, an alternative growth medium using local resources has been developed. In this study, the growth of recombinant Escherichia coli and expression of T1 lipase were tested using different agroindustrial wastes as carbon and nitrogen sources by conventional method. Subsequently, by using central composite rotatable design (CCRD), a set of 30 experiments was generated to evaluate the effect of different parameters, including the amount of molasses (as carbon source), fish waste (as nitrogen source), NaCl, and inducer concentration on production of T1 lipase. Response surface methodology (RSM) analysis indicated that all factors had significant effects on T1 lipase production. This statistical analysis was utilised to develop a quadratic model to correlate various important variables for the growth of the recombinant strain and regulation of gene expression to the response (T1 lipase activity). Optimum conditions for T1 lipase production were observed to be 1.0 g/L of molasses, 2.29 g/L of fish waste, 3.46 g/L of NaCl, and 0.03 mM of IPTG (Isopropyl &beta, d-1-thiogalactopyranoside). Based on these conditions, the actual lipase activity was found to be 164.37 U/mL, which fitted well with the maximum predicted value of 172.89 U/mL. Therefore, the results demonstrated that, the statistical analysis, performed using RSM, was efficient in optimising T1 lipase production. Moreover, the optimum conditions obtained can be applied to scale up the process and minimise the cost of enzyme production.

A moderate yield of a purified enzyme can be achieved by using the simple technique of reverse micellar extraction (RME). RME is a liquid&ndash, liquid extraction method that uses a surfactant and an organic solvent to extract biomolecules. Instead of traditional chromatographic purification methods, which are tedious and expensive, RME using the nonionic surfactant Triton X-100 and toluene is used as an alternative purification technique to purify a recombinant cold-adapted lipase, AMS8. Various process parameters were optimized to maximize the activity recovery of the AMS8 lipase. The optimal conditions were found to be 50 mM sodium phosphate buffer, pH 7, 0.125 M NaCl, and 0.07 M Triton X-100 in toluene at 10 &deg, C. Approximately 56% of the lipase activity was successfully recovered. Structural analysis of the lipase in a reverse micelle (RM) was performed using an in silico approach. The predicted model of AMS8 lipase was simulated in the Triton X-100/toluene reverse micelles from 5 to 40 &deg, C. The lid 2 was slightly opened at 10 &deg, C. However, the secondary structure of AMS8 was most affected in the non-catalytic domain compared to the catalytic domain, with an increased coil conformation. These results suggest that an AMS8 lipase can be extracted using Triton X-100/water/toluene micelles at low temperature. This RME approach will be an important tool for the downstream processing of recombinant cold-adapted lipases.