Phosphorylase-catalyzed N-formyl-α-glucosaminylation of maltooligosaccharides

This paper describes the phosphorylase-catalyzed enzymatic N-formyl-α-glucosaminylation of maltooligosaccharides for direct incorporation of 2-deoxy-2-formamido-α-d-glucopyranose units into maltooligosaccharides. When the reaction of 2-deoxy-2-formamido-α-d-glucopyranose-1-phosphate (GlcNF-1-P) as the glycosyl donor and maltotetraose as a glycosyl acceptor was performed in the presence of phosphorylase, the N-formyl-α-d-glucosaminylated pentasaccharide was produced, as confirmed by MALDI-TOF MS. Furthermore, the glucoamylase-catalyzed reaction of the crude products supported that the 2-deoxy-2-formamido-α-d-glucopyranoside unit was positioned at the non-reducing end of the pentasaccharide. The pentasaccharide was isolated from the crude products and its structure was further determined by the ¹H NMR analysis. On the other hand, when the phosphorylase-catalyzed reactions of maltotriose and maltopentaose using GlcNF-1-P were conducted, no N-formyl-α-glucosaminylation took place in the former system, whereas the latter system gave N-formyl-α-d-glucosaminylated oligosaccharides with various degrees of polymerization. These results could be explained by the recognition behavior of phosphorylase toward maltooligosaccharides.     

Homogeneous,heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: A review

In the last few years, biodiesel has emerged as one of the most potential renewable energy to replace current petrol-derived diesel. It is a renewable, biodegradable and non-toxic fuel which can be easily produced through transesterification reaction. However, current commercial usage of refined vegetable oils for biodiesel production is impractical and uneconomical due to high feedstock cost and priority as food resources. Low-grade oil, typically waste cooking oil can be a better alternative; however, the high free fatty acids (FFA) content in waste cooking oil has become the main drawback for this potential feedstock. Therefore, this review paper is aimed to give an overview on the current status of biodiesel production and the potential of waste cooking oil as an alternative feedstock. Advantages and limitations of using homogeneous, heterogeneous and enzymatic transesterification on oil with high FFA (mostly waste cooking oil) are discussed in detail. It was found that using heterogeneous acid catalyst and enzyme are the best option to produce biodiesel from oil with high FFA as compared to the current commercial homogeneous base-catalyzed process. However, these heterogeneous acid and enzyme catalyze system still suffers from serious mass transfer limitation problems and therefore are not favorable for industrial application. Nevertheless, towards the end of this review paper, a few latest technological developments that have the potential to overcome the mass transfer limitation problem such as oscillatory flow reactor (OFR), ultrasonication, microwave reactor and co-solvent are reviewed. With proper research focus and development, waste cooking oil can indeed become the next ideal feedstock for biodiesel.     

Insights into the fatty acid chain length specificity of the carboxylesterase PA3859 from Pseudomonas aeruginosa: A combined structural,biochemical and computational study

The open reading frame PA3859 of Pseudomonas aeruginosa encodes an intracellular carboxylesterase belonging to a group of microbial enzymes (EC 3.1.1.1) that catalyze the hydrolysis of aliphatic and aromatic esters with a broad substrate specificity. With few exceptions, for this class of enzymes, belonging to the α/β-hydrolase fold superfamily, very little information is available regarding their biochemical activity and in vivo function. The X-ray crystal structure of recombinant PA3859 has been determined for two crystal forms (space groups P2₁ and P2₁2₁2). The kinetic properties of the enzyme were studied using p-nitrophenyl esters as substrates and data fitted to a surface dilution mixed micelle kinetic model. Enzymatic assays and computational docking simulations, pinpointed the enzyme’s preference for esters of palmitic and/or stearic acids and provided insights into the enzyme–substrate favorable binding modes.     

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.     

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81 kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35 °C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35 °C for 1 h. Higher activity was observed in the presence of surfactants, Na⁺, NH₄⁺ ions, NaN₃ and ethylenediaminetetraacetic acid (EDTA), while Co²⁺ and Cu²⁺ ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10 days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone.     

Immobilized lipase on porous silica particles: Preparation and application for biodegradable polymer syntheses in ionic liquid at higher temperature

Porous silica particles (PSP) modified with different surface active groups were prepared for covalent immobilization of porcine pancreas lipase (PPL). Organosilanes combined with reactive end amino-group or epoxy-group were employed for the modification through silanization process. Polyethylenimine and long chain alkyl silane coupling agent were also used in the modification process. Several modification-immobilization strategies were performed, while good coupling yield could be achieved within the range of 86.2–158.2 mg of native PPL per gram of the carrier. Furthermore, at higher temperature, the resulting immobilized PPL (IPPL) could successfully perform the syntheses of polycaprolactone (PCL) and poly(5,5-dimethyl-1,3-dioxan-2-one) (PDTC) in ionic liquid medium. No polymers could be obtained catalyzed by native PPL, suggesting that IPPL showed much higher catalytic activity than native PPL. Effect of different treatments on the activity of IPPL also showed the long time high temperature stability in ionic liquid medium, contributing to a good combination of immobilization and ionic liquids effect. The catalytic activity of IPPL for polymerization was closely related to both the properties of immobilized enzyme and cyclic monomer. This work would be expected to highlight further careful design of immobilized enzyme for a wide range of application, especially in biodegradable polymers syntheses.     

Enzymatic rearrangement of chitine hydrolysates with β-N-acetylhexosaminidase from Aspergillus oryzae

The role of higher chitooligomers in medical applications is increasing due to their interesting biological activities. Transglycosylation activity of β-N-acetylhexosaminidase from Aspergillus oryzae was employed to produce higher chitooligosaccharides (chitohexaose–chitooctaose) from a mixture of lower chitooligomers prepared by acid hydrolysis of chitin. Enzymatic rearrangement of the chitooligomer mixture was optimized in respect of substrate concentration, presence of inorganic salts, enzyme activity, and reaction time to achieve the highest production of longer chitooligomers.     

An improved solid-phase synthesis of resonance energy transfer fluorescent peptides and depsipeptides employing the EDANS/DABCYL donor-acceptor pair

Summary Fluorescent peptide substrates of the resonance energy transfer type, employing the donor-acceptor pair 5-[(2′-aminoethyl)amino]naphthalene sulphonic acid (EDANS) and 4-[[4′-(dimethylamino)phenyl]azo]benzoic acid (DABCYL), are widely used for continuous monitoring of the activity of various proteases. We describe here a flexible synthetic scheme which allows the synthesis of peptides having EDANS and DABCYL in any position of the sequence; the synthesis is entirely performed on solid phase with standard methods and makes use only of EDANS, DABCYL and commercially available amino acid derivatives. Moreover, we show that our scheme can be equally applied to the synthesis of EDANS/DABCYL-derivatised depsipeptides.     

Effect of proteolytic and glycolytic enzymes on a factor inSorghum bicolor that induces mycelial growth in the smut fungus,Sporisorium reilianum

Proteins obtained from seedling shoots and floral meristems ofSorghum bicolor (L.) Moench cv. NK 1210 induced mycelial growth in the smut fungus,Sporisorium reilianum in vitro. Proteins precipitated with trichloroacetic acid and ammonium sulfate were equally effective as inducers, although there were minor variations in the pattern of mycelial growth. Hydrolysis of the protein fraction with the proteolytic enzyme pronase E resulted in considerable reduction in the proteins' ability to induce mycelial growth. Digestion of the protein fraction with driselase, resulted in a slight enhancement of biological activity. The results suggest that amino sugar moieties in glycoproteins may act as inducers of mycelial growth inSporisorium reilianum.     

Kinetic and Thermodynamic Approach to Design of Processes for Enzymatic Synthesis of Betalactams

An optimal way to design an enzymatic process for the production of betalactam antibiotics based on thermodynamic and kinetic studies is described. The study was performed on model reactions involving synthesis of cephalosporin-acids (cephalotin, cefazolin, cefoxitin) using immobilised cephalosporin-acid synthetase from Escherichia coli as biocatalyst, and aminocephalosporins (cephalexin) using immobilised cells of Xanthomonas rubrilineans containing the aminocephalosporin synthetase. The possibility of direct synthesis of cephalotin and cefoxitin was shown, the main equilibrium parameters were determined and the operation conditions were evaluated. The maximum key amino acid conversion to product of approximately 90% for cefoxitin and cephalotin was achieved using initial concentrations of the corresponding key amino acids of 0.05 λM and, respectively, 2-fold and 4-fold molar excess of the carboxylic acids. Cefazolin and cephalexin production by enzymatic synthesis with using of corresponding biocatalyst with a mechanism of action involving the acylenzyme intermediate was shown possible. The kinetic parameters of the process were estimated and the relationship between the maximum antibiotic yield and the initial concentrations of the substrate and nucleophile in the kinetically controlled synthesis was determined. The technologies for cefazolin and cephalexin enzymatic synthesis were designed and the cefazolin technology was optimised. Maximum yields of cefazolin and cephalexin of more than 90% were predicted by the kinetic model using 4-6-fold molar excess of the acylating agents and maximum yields of approximately 85% were achieved in experiments.