Dehalogenases are of high interest due to their potential applications in bioremediation and in synthesis of various industrial products. DehL is an L-2-haloacid dehalogenase (EC 3.8.1.2) that catalyses the cleavage of halide ion from L-2-halocarboxylic acid to produce D-2-hydroxycarboxylic acid. Although DehL utilises the same substrates as the other L-2-haloacid dehalogenases, its deduced amino acid sequence is substantially different (<25%) from those of the rest L-2-haloacid dehalogenases. To date, the 3D structure of DehL is not available. This limits the detailed understanding of the enzyme's reaction mechanism. The present work predicted the first homology-based model of DehL and defined its active site. The monomeric unit of the DehL constitutes α/β structure that is organised into two distinct structural domains: main and subdomains. Despite the sequence disparity between the DehL and other L-2-haloacid dehalogenases, its structural model share similar fold as the experimentally solved L-DEX and DehlB structures. The findings of the present work will play a crucial role in elucidating the molecular details of the DehL functional mechanism.
Persicobacter sp. CCB-QB2 belonging to the family Flammeovirga is an agarolytic bacterium and exhibits a diauxic growth in the presence of tryptone and agarose. A glycoside hydrolase (GH) 16 β-agarase, PdAgaC, was identified in the genome of the bacterium and was highly expressed during the second growth phase, indicating the agarase may play an important role in the diauxic growth. In this study, the catalytic domain of PdAgaC (PdAgaCgh) was cloned and characterized. PdAgaCgh showed thermostability at 50 °C and tolerance towards several detergents. In addition, the activity of PdAgaCgh after incubation with 0.1% of SDS and Triton X-100 increased approximately 1.2-fold. On the other hand, PdAgaCgh was sensitive to Fe2+, Ni2+, and Cu2+. The Km and Vmax of PdAgaCgh were 5.15 mg/ml and 2.9 × 103 U/mg, respectively. Interestingly, although the major hydrolytic product was neoagarobiose (NA2), monomeric sugar was also detected by thin-layer chromatographic analysis.
Enzymatic synthesis of maltooligosaccharides is hampered due to lack of stability of soluble enzyme. This limitation can be tackled by cross linked enzyme aggregates (CLEAs) immobilization approach. However, substrate diffusion is a major bottleneck in cross linking technology. Herein, CLEAs of maltogenic amylase from Bacillus lehensis G1 (Mag1) was developed with addition of porous agent (Mag1-p-CLEAs). Comparison of thermal, pH and kinetic analysis with CLEAs without porous agent (Mag1-CLEAs) and free Mag1 was performed. Mag1-p-CLEAs with porous structure prepared at 0.8% (w/v) of citrus pectin (porous agent), 0.25% (w/v) of chitosan (cross linker) and cross linked for 1.5 h yielded 91.20% activity. 80% of activity is retained after 30 min of incubation at 40 °C and showed longer half-life than free Mag1 and Mag1-CLEAs. Mag1-p-CLEAs also showed pH stability at acidic and alkaline pH. The 1.68-fold increase in Vmax value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin accessibility. The increase in high catalytic efficiency (Kcat/Km) value, 1.90-fold and 1.05-fold showed that it also has better catalytic efficiency than free Mag1 and Mag1-CLEAs, respectively. Mag1-p-CLEAs not only improved substrate diffusibility of CLEAs, but also leads to higher thermal and pH stability of Mag1.
Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (β-CD) (Km = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.
The l-2-haloacid dehalogenases (EC 3.8.1.2) specifically cleave carbon-halogen bonds in the L-isomers of halogenated organic acids. These enzymes have potential applications for the bioremediation and synthesis of various industrial products. One such enzyme is DehL, the l-2-haloacid dehalogenase from Rhizobium sp. RC1, which converts the L-isomers of 2-halocarboxylic acids into the corresponding D-hydroxycarboxylic acids. However, its catalytic mechanism has not been delineated, and to enhance its efficiency and utility for environmental and industrial applications, knowledge of its catalytic mechanism, which includes identification of its catalytic residues, is required. Using ab initio fragment molecular orbital calculations, molecular mechanics Poisson-Boltzmann surface area calculations, and classical molecular dynamic simulation of a three-dimensional model of DehL-l-2-chloropropionic acid complex, we predicted the catalytic residues of DehL and propose its catalytic mechanism. We found that when Asp13, Thr17, Met48, Arg51, and His184 were individually replaced with an alanine in silico, a significant decrease in the free energy of binding for the DehL-l-2-chloropropionic acid model complex was seen, indicating the involvement of these residues in catalysis and/or structural integrity of the active site. Furthermore, strong inter-fragment interaction energies calculated for Asp13 and L-2-chloropropionic acid, and for a water molecule and His184, and maintenance of the distances between atoms in the aforementioned pairs during the molecular dynamics run suggest that Asp13 acts as the nucleophile and His184 activates the water involved in DehL catalysis. The results of this study should be important for the rational design of a DehL mutant with improved catalytic efficiency.
Immobilization of cross-linked tannase on pristine multiwalled carbon nanotubes (MWCNT) was successfully performed. Cross-linking of tannase molecules was made through glutaraldehyde. The immobilized tannase exhibited significantly improved pH, thermal, and recycling stability. The optimal pH for both free and immobilized tannase was observed at pH 5.0 with optimal operating temperature at 30°C. Moreover, immobilized enzyme retained greater biocatalytic activities upon 10 repeated uses compared to free enzyme in solution. Immobilization of tannase was accomplished by strong hydrophobic interaction most likely between hydrophobic amino acid moieties of the glutaraldehyde-cross-linked tannase to the MWCNT.
New thermostable β-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 μmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.
The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus) waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent K m and V max of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0). The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe(2+) and Zn(2+), while protease activity was increased in the presence of Ca(2+) and Mg(2+) and Cu(2+) by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.
An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.
The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.
Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
Bacterial proteases are an important group of enzymes that have very diverse biochemical and cellular functions. Proteases from prokaryotic sources also have a wide range of uses, either in medicine as pathogenic factors or in industry and therapeutics. ProLysED (Prokaryotic Lysis Enzymes Database), our meta-server integrated database of bacterial proteases, is a useful, albeit very niche, resource. The features include protease classification browsing and searching, organism-specific protease browsing, molecular information and visualisation of protease structures from the Protein Data Bank (PDB) as well as predicted protease structures.
The ability of thermophilic microorganisms and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability, and decreased risk of contamination. Exploitation of efficient thermostable glycoside hydrolases (GHs) could accelerate the industrialization of biofuels and biochemicals. However, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y-shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y-shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus, Desulfurobacterium, Fervidobacterium, Geobacillus, Meiothermus, Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes.
Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.
Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease.
Penicillium candidum (PCA 1/TT031) synthesizes different types of extracellular proteases. The objective of this study is to optimize polyethylene glycol (PEG)/citrate based on an aqueous two-phase system (ATPS) and Response Surface Methodology (RSM) to purify protease from Penicillium candidum (PCA 1/TT031). The effects of different PEG molecular weights (1500-10,000 g/mol), PEG concentration (9%-20%), concentrations of NaCl (0%-10%) and the citrate buffer (8%-16%) on protease were also studied. The best protease purification could be achieved under the conditions of 9.0% (w/w) PEG 8000, 5.2% NaCl, and 15.9% sodium citrate concentration, which resulted in a one-sided protease partitioning for the bottom phase with a partition coefficient of 0.2, a 6.8-fold protease purification factor, and a yield of 93%. The response surface models displayed a significant (p ≤ 0.05) response which was fit for the variables that were studied as well as a high coefficient of determination (R²). Similarly, the predicted and observed values displayed no significant (p > 0.05) differences. In addition, our enzyme characterization study revealed that Penicillium candidum (PCA 1/TT031) produced a slight neutral protease with a molecular weight between 100 and 140 kDa. The optimal activity of the purified enzyme occurred at a pH of 6.0 and at a temperature of 50 °C. The stability between different pH and temperature ranges along with the effect of chemical metal ions and inhibitors were also studied. Our results reveal that the purified enzyme could be used in the dairy industry such as in accelerated cheese ripening.
The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases—key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.
A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates.
Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.