Enantioselective N-acetylation of 2-phenylglycine by an unusual N-acetyltransferase from Chryseobacterium sp.

The demand for d-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the l-form of racemic d,l-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50 % and an enantiomer excess of >99.5 % under optimal culture conditions, consequently resulting in 99 % pure d-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by l-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates l-2-phenylglycine, d-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of d,l-2-phenylglycine.     

Stability of immobilized D-hydantoinase from Vigna angularis and repeated cycles of highly enantioenriched production of N-carbamoyl-D-phenylglycines

Summary. D-hydantoinase from Vigna angularis was immobilized by covalent linkage to aminopropyl glass beads. Thermal stability, resistance to storage at different pH values and temperatures of this biocatalyst were studied. This enzyme preparation was used as a catalyst to prepare enantioenriched N-carbamoyl-D-phenylglycine, N-carbamyl-D-p-fluorophenylglycine and N-carbamoyl-D-p-trifluoromethylphenylglycine, using a stirred batch reactor. Reactions were conducted during eight repeated reaction cycles, without loss of enzymatic activity or variation of the enantiomeric excess of the respective product (>98%).     

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.     

Effect of water activity on enzymatic synthesis of cephalexin

Summary In enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D--phenylglycine methyl ester (PGM) using an acylase fromXanthomonas citri, it was found that the synthetic activity and conversion yield were enhanced markedly by depressing the water activity (a _w ) of reaction system. This enhancement was probably resulted from the change of thermodynamic equilibrium and maximized at a range ofa _w from 0.96 to 0.97.     

Identification of Nucleotide Residues Essential for RNase P Activity from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5′ leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg²⁺ ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.     

Specificity of the action of lysoamidase on Staphylococcus aureus 209P cell walls

Specificity of Staphylococcus aureus 209P cell wall hydrolysis by the L1 and L2-bacteriolytic enzymes from lysoamidase lytic complex was studied. L1-peptidase was shown to display both glycyl-glycine endopeptidase and N-acetylmuramyl-L-alanine amidase enzymatic activities on the S. aureus peptidoglycan molecule, whereas L2-peptidase acts as N-acetylmuramyl-L-alanine amidase.     

Structure of arabinogalactan oligosaccharides derived from arabinogalactan-protein of Coffea arabica instant coffee powder

Arabinogalactan-protein, previously isolated from instant coffee powder of Coffea arabica, has been subjected to partial mild acidic and enzymatic hydrolyses. Separation of obtained mixtures by size exclusion and HPLC chromatographies afforded series of oligosaccharides, structure of which were studied by NMR spectroscopy. Mild acidic hydrolysis afforded oligosaccharides without any αAraf substituent while after enzymatic hydrolysis αAraf was found in di-, tri-, and tetrasaccharides. In all cases αAraf was a terminal substituent linked separately to O3, O6, and to both, O3 and O6, of βGal residues. Identification of di-, tri-, and tetrasaccharides containing α-Araf enabled to distinguish in the ¹H NMR spectra αAraf signals linked to O6 and O3 of neighboring βGal unit. Composition of polymeric residues after enzymatic and mild acidic hydrolyses was also analyzed.     

A two-step, one-pot enzymatic synthesis of ampicillin from penicillin G potassium salt

A two-step, one-pot synthesis of ampicillin from penicillin G potassium salt (PGK) in aqueous buffer/organic co-solvent has been achieved. Ethylene glycol (EG) was chosen as the organic co-solvent. Factors including co-solvent content, enzyme loading, reaction temperature and substrate concentration were investigated. The optimum conditions were as follow: pH 8.0 phosphate buffer solution, 50% EG (v/v), 25 °C, 100 mM PGK and 300 mM d-phenylglycine methyl ester (D-PGM), 43.2 IU/ml IPA-750. The maximum yield was 57.3% after a reaction time of 17 h. It is the first report about the synthesis of ampicillin from penicillin G potassium salt in one-pot combining the enzymatic hydrolysis and the subsequent enzymatic condensation, and the novel methodology will have important application in the β-lactam antibiotics industry.     

ABE production from yellow poplar through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation

ABE (acetone-butanol-ethanol) was produced through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation using yellow poplar as a raw material. In alkaline pre-hydrolysis, 51.1% of the biomass remained as a residue. In the main woody components, the degrees of lignin and xylan removal were 94.3 and 62.0%, respectively. A yield of 80.9% for cellulose-to-glucose and 81.2% for xylan-to-xylose were obtained by enzymatic hydrolysis. The sugar composition of enzymatic hydrolysate was 95.1 g/L of glucose and 21.4 g/L of xylose. The enzymatic hydrolysate also contained 0.5 g/L of acetic acid and 0.5 g/L of total phenolics. Furfural and 5-hydroxymethylfurfural (5-HMF) were not detected in this hydrolysate. The yellow poplar hydrolysate (YPH) from enzymatic saccharification was used for the production of ABE using Clostridium acetobutylicum and C. beijerinckii. In YPH fermentation, C. acetobutylicum produced 18.1 g/L total ABE (productivity 0.38 g/L h, and yield 0.42), and C. beijerinckii produced 12.1 g/L (productivity 0.25 g/L h, and yield 0.37). Although the ABE productivity by C. beijerinckii was slightly low, the general performance of ABE fermentation in YPH was similar to or higher than those reported previously. Therefore, alkaline pre-hydrolysis could be a very effective pretreatment step prior to enzymatic hydrolysis.     

Bioethanol production from Gracilaria verrucosa using Saccharomyces cerevisiae adapted to NaCl or galactose

This study examined the pretreatment, enzymatic saccharification, and fermentation of the red macroalgae Gracilaria verrucosa using adapted saccharomyces cerevisiae to galactose or NaCl for the increase of bioethanol yield. Pretreatment with thermal acid hydrolysis to obtain galactose was carried out with 11.7% (w/v) seaweed slurry and 373 mM H₂SO₄ at 121 °C for 59 min. Glucose was obtained from enzymatic hydrolysis. Enzymatic saccharification was performed with a mixture of 16 U/mL Celluclast 1.5L and Viscozyme L at 45 °C for 48 h. Ethanol fermentation in 11.7% (w/v) seaweed hydrolysate was carried out using Saccharomyces cerevisiae KCTC 1126 adapted or non-adapted to high concentrations of galactose or NaCl. When non-adapted S. cerevisiae KCTC 1126 was used, the ethanol productivity was 0.09 g/(Lh) with an ethanol yield of 0.25. Ethanol productivity of 0.16 and 0.19 g/(Lh) with ethanol yields of 0.43 and 0.48 was obtained using S. cerevisiae KCTC 1126 adapted to high concentrations of galactose and NaCl, respectively. Adaptation of S. cerevisiae KCTC 1126 to galactose or NaCl increased the ethanol yield via adaptive evolution of the yeast.

     

Enzymatic synthesis of ATP from RNA and adenine

Summary The title compound was prepared by a three-stage enzymatic procedure consisting of (i) RNA hydrolysis to a mixture of ribonucleosides using intact mycelium of Spicaria violacea, (ii) transribosylation of exogenous adenine employing whole cells of Escherichia coli as a biocatalyst, and (iii) conversion of formed adenosine into ATP by the enzymes of alcohol fermentation and the kinases extracted from baker's yeast.     

Identification of 20-hydroxyecdysone and ecdysone from the pupa of the gypsy moth,Lymantria dispar

Normal and reverse-phase high-performance liquid chromatography in conjunction with radioimmunoassay and mass spectrometry were used to identify the free and conjugated ecdysteroids (after enzymatic hydrolysis) from day-4 pupae of the gypsy moth, Lymantria dispar L. Seven ecdysteroids were searched for, but only 20-hydroxyecdysone (964 ng/g fresh weight) and ecdysone (367 ng/g fresh weight) were detected. Analysis of conjugated ecdysteriods after liberation by hydrolysis also indicated the presence of 20-hydroxyecdysone (21.6 ng/g fresh weight) and ecdysone (2.4 ng/g fresh weight). Neither 26-hydroxyecdysone nor the 3α-epimers of 20-hydroxyecdysone or ecdysone were detected.     

High-Pressure Effects on Vacuolar H+-ATPase from Etiolated Mung Bean Seedlings

A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H⁺-ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H⁺-ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both V _max and K _m of solubilized vacuolar H⁺-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg²⁺-ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H⁺-ATPase by pressure may result from changes in protein–protein interaction. The conformational change of solubilized vacuolar H⁺-ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H⁺-ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit–subunit interaction is crucial for the integrity and the function of plant vacuolar H⁺-ATPase. It is also suggested that the assembly of the vacuolar H⁺-ATPase complex is probably not random, but follows a sequestered pathway.     

Enzymatic hydrolysis of soybean oil using lipase from different sources to yield concentrated of polyunsaturated fatty acids

The ability of three commercially available lipases to mediate the hydrolysis of the soybean oil to yield concentrated of essential fatty acids was evaluated. The tested lipases were from microbial (Candida rugosa and Thermomyces lanuginosa) and animal cells (Porcine pancreatic lipase). In terms of free fatty acids, microbial lipases were more effective to promote the enzymatic hydrolysis of the soybean oil (over 70%) than the porcine pancreatic lipase (24%). In spite of this, porcine pancreatic lipase (PPL) showed the most satisfactory specificity towards both essential fatty acids and was, therefore, chosen to carry out additional studies. An experimental design was performed taking into consideration the enzyme and NaCl amounts as independent variables. The main effects were fitted by multiple regression analysis to a linear model and maximum fatty acids concentration could be obtained using 3.0 wt% of lipase and 0.08 wt% of NaCl. The mathematical model representing the hydrolysis degree was found to describe adequately the experimental results. Under these conditions, concentrations of 29.5 g/L and 4.6 g/L for linoleic and linolenic acids, respectively, were obtained.     

Controlled-release biocatalysis for the synthesis of D-phenylglycine

An isolated and immobilised aminotransferase cloned from Pseudomonas stutzeri ST-201 into Escherichia coli was used to synthesise d-phenylglycine. The reaction was characterised by an unfavourable equilibrium constant and substrate inhibition. The use of a controlled-release system via the use of Amberlite (IRA 400)-adsorbed benzoylformate proved a useful technique to circumvent these issues. This resulted in a four-fold improvement in product concentration achievable to yield a final d-phenylglycine concentration of 10.25 mg/ml.     

Profiles of fatty acids and triacylglycerols and their influence on the anaerobic biodegradability of effluents from poultry slaughterhouse

The hydrolysis of effluent from a poultry slaughterhouse containing 800 mg oil and grease (O&G)/L was conducted with 1% (w/v) of an enzymatic pool obtained by solid-state fermentation with the fungus Penicillium restrictum. The chromatographic evaluation of the lipid profile during hydrolysis indicated a higher concentration of acids after 4 h of reaction (2954 mg/L), with a predominance of oleic, palmitic, and linoleic acids. Effluent aliquots were collected after 4, 8, and 24 h of hydrolysis and tested for anaerobic biodegradation in sequential batches. An adaptation of the biomass was observed, both in the control experiment (with non-hydrolyzed raw effluent) and in the experiments with enzymatically pre-treated effluent. The specific methane production in the control experiment was 0.248 L CH₄/g COD_consumed, and in the experiment with effluent pre-treated for 4 h, this production was 0.393 L CH₄/g COD_consumed, indicating a higher methane production after enzymatic hydrolysis.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

The enzymatic mobilization of bound precarthamine from the flower florets ofCarthamus tinctorius L.

Floret pieces prepared from the methanol-treated and air-dried flowers of safflower (Carthamus tinctorius L.) were subjected to an enzymatic hydrolysis in a buffered solution and the rate of precarthamine solubilization was assessed spectrophotometrically. The amount of precarthamine was risen by treating the floral pieces with a β-glucosidase (EC 3.2.1.21), supporting a previous assumption that precarthamine is in the floral tissues associated with cellular components. The enzyme activity was reduced by various inhibitors. On the basis of the experimental data, it is proposed that the precarthamine may be bound through 0-β-glucosyl linkage(s).     

New Protein Hydrolysates from Collagen Wastes Used as Peptone for Bacterial Growth

A simple and low-cost procedure was developed for the effective processing of native calf skin and blood wastes to produce protein hydrolysates. The method includes extraction of high–molecular-weight protein from the raw material, followed by enzymatic hydrolysis of the extracted residue. The enzymatic hydrolysis was performed by inexpensive commercial subtilisin DY, produced by Bacillus subtilis strain DY possessing high specific activity. The contents of protein, nitrogen, ash, and amino acids of the obtained hydrolysates were determined and compared with those of the commonly used commercial casein hydrolysate (Fluka Biochemica, Switzerland). The newly obtained calf skin hydrolysate, called Eladin, was found to be suitable as a low-cost alternative peptone in growth media of different microorganisms, such as Escherichia coli, Pseudomonas aeruginosa, Salmonella dublin, and Staphylococcus aureus. The method allows utilization of waste materials by converting them into valuable protein products that could find widespread application in microbiologic practice.     

Liberation of fermentable sugars from soybean hull biomass using ionic liquid 1‐butyl‐3‐methylimidazolium acetate and their bioconversion to ethanol

Optimized hydrolysis of lignocellulosic waste biomass is essential to achieve the liberation of sugars to be used in fermentation process. Ionic liquids (ILs), a new class of solvents, have been tested in the pretreatment of cellulosic materials to improve the subsequent enzymatic hydrolysis of the biomass. Optimized application of ILs on biomass is important to advance the use of this technology. In this research, we investigated the effects of using 1‐butyl‐3‐methylimidazolium acetate ([bmim][Ac]) on the decomposition of soybean hull, an abundant cellulosic industrial waste. Reaction aspects of temperature, incubation time, IL concentration, and solid load were optimized before carrying out the enzymatic hydrolysis of this residue to liberate fermentable glucose. Optimal conditions were found to be 75°C, 165 min incubation time, 57% (mass fraction) of [bmim][Ac], and 12.5% solid loading. Pretreated soybean hull lost its crystallinity, which eased enzymatic hydrolysis, confirmed by Fourier Transform Infrared analysis. The enzymatic hydrolysis of the biomass using an enzyme complex from Penicillium echinulatum liberated 92% of glucose from the cellulose matrix. The hydrolysate was free of any toxic compounds, such as hydroxymethylfurfural and furfural. The obtained hydrolysate was tested for fermentation using Candida shehatae HM 52.2, which was able to convert glucose to ethanol at yields of 0.31. These results suggest the possible use of ILs for the pretreatment of some lignocellulosic waste materials, avoiding the formation of toxic compounds, to be used in second‐generation ethanol production and other fermentation processes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:312–320, 2016