Application of immobilized aminoacylase from Micrococcus agilis for isolation of D-phenylglycine from its racemic mixture

The procedure for isolation of D -phenylglycine from its racemic mixture by enzymatic hydrolysis of L -enantiomer of N-acetyl-D ,L -phenylglycine is described. For this hydrolysis. aminoacylase from Micrococcus agilis immobilized by sorption of DEAE-cellulose was applied. As is also shown, the course of enzymatic reaction can be directly controlled by spectrophotometric method.     

Poly(3-hydroxybutyrate) biosynthesis in the biofilm of Alcaligenes eutrophus, using glucose enzymatically released from pulp fiber sludge

Glucose, enzymatically released from pulp fiber sludge, was combined with inorganic salts and used as a growth medium for Alcaligenes eutrophus, a gram-negative strain producing poly(3-hydroxybutyrate) (PHB). By controlling the concentrations of the inorganic salts in the growth medium, almost 78% of the cell mass was converted to pure PHB. Efforts were made to find conditions for bacterial growth in the form of a biofilm on a cheap and reusable carrier. A number of positively charged carriers were tested, and the anion exchanger DEAE-Sephadex A-25 was chosen as a microcarrier for packed-bed biofilm cultures of A. eutrophus. Conditions for attachment, growth, and detachment were established. Biofilm formation on the microcarrier is strongly dependent on the ionic strength of the attachment medium. In order to achieve formation of the biofilm and its recovery from the microcarrier, the ionic strengths of the attachment and the detachment media were varied. Low ionic strength was tested for attachment, and high ionic strength was tested for detachment. Although biofilm formation in the packed-bed reactor is limited, the volumetric yield of cells based on the void volume of the packed bed is comparable with the batch culture yield.     

The potential use of silicon compounds as oxygen carriers for free and immobilized cells containing l-amino acid oxidase

Summary The relatively low solubility of oxygen in water presents a problem in particular when immobilized cells are used or enzymes are applied for oxygen dependent reactions. The other main purpose is the requirement of oxygen for the increase of biomass. In this investigation the usefulness of silicon emulsions as oxygen carriers is demonstrated.In case of l-amino acid oxidase activity of immobilized cells, an increase by a factor of four was found in the presence of silicon emulsions. Likewise, growth medium enriched with silicon emulsions showed a significantly increased growth of cells inside alginate beads compared to normal growth medium.     

A novel procedure for the isolation of glycolipids from Bifidobacterium adolescentis 94 BIM using supercritical carbon dioxide

This study describes a novel isolation procedure for major glycolipids from Bifidobacterium adolescentis 94 BIM. The procedure consists of the use of supercritical carbon dioxide (scCO(2)) with hydro-methanolic solution as co-solvent. The major glycolipids were isolated using the following operating conditions: pressure, 30 MPa, co-solvent concentration, 10% (9:1, methanol/water, v/v), CO(2) flow rate, 5 g/min, extraction time and temperature, 2h and 55 degrees C, respectively. The reference glycolipids sample was prepared by classical organic solvent extraction followed by chromatographic purification. All isolates were characterized by TLC and the major glycolipids additionally by enzyme linked immunosorbent (ELISA). Sixty milligrams of glycolipids with similar immunoreactivity as the reference glycolipids were isolated from 1g of freeze-dried biomass (6% of yield).     

Improvement of NaNO2-Oxidizing Activity in Nitrobacter vulgaris by Coentrapment in Polyacrylamide Containing Polydimethylsiloxane Copolymer and DEAE-Sephadex

Removal of nitrite and nitrate from drinking water has attracted great attention in recent years because of the human health risk induced by the exposure to contaminated groundwater and surface water. We have therefore tested a model nitrite oxidation system by coentrapping the NaNO₂ oxidizer Nitrobacter vulgaris with polydimethylsiloxane (PDMS) copolymer and DEAE-Sephadex in a polyacrylamide gel. The copolymer and the anion exchanger facilitate the diffusion of oxygen and NaNO₂, respectively, into the gel matrix. To test the nitrite-oxidizing activity, the entrapped cells were coupled to a thermal sensor. Coentrapment of 5% (wt/vol) DEAE-Sephadex with Nitrobacter vulgaris increased the nitrite-oxidizing activity by a factor of 3.7 compared to entrapped cells alone, and by the addition of 0.86% (wt/vol) artificial oxygen carrier PDMS copolymer increased the activity further to 4.3 times higher. Operational and storage stability of the coentrapped N.vulgaris also improved. This suggests that this enhanced immobilized cell system can also be used for nitrite oxidation to nitrate in drinking water as an on-line thermally monitored bioreactor.     

Production of α-keto acids: 2. Immobilized whole cells of Providencia sp. PCM 1298 containing -amino acid oxidase

A number of bacteria belonging to the genera Proteus, Providencia, Pseudomonas and Erwinia have been tested for their capacity to oxidize -amino acids to their corresponding α-keto acids. Members of the Proteus and the Providencia genera were active towards various -amino acids. Immobilized cell preparations of Providencia sp. PCM 1298 were shown to form up to 80 mg α-keto-γ-methiol butyric acid from -methionine per g of gel preparation (containing 4% w/w cells) per day. The productivity was highly dependent on the size of the beads. Oxygen appeared to be the rate-limiting substrate and oxygen transfer rates of 3–4 μmol cm⁻² h⁻¹ were calculated. The entrapment of activated charcoal to remove H₂O₂ formed during the oxidation extended the half-life of the immobilized biocatalyst considerably. A decrease in -amino acid oxidase [ -amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.2] activity during operation could be compensated for by reinoculation of the alginate-entrapped cells in fresh growth medium, allowing use of these preparations of immobilized bacterial cells for more than one month.     

Characterization of antimicrobial compounds produced by Pseudomonas aurantiaca S-1.

Pseudomonas aurantiaca S-1 can serve as a natural source of pesticides towards phytopathogens like Fusarium oxysporum P1 and Pseudomonas syringae pv. glycinea BIM B-280. The largest pool of produced antimicrobial compounds was found in four days-old spent culture supernatant. At least two groups of bioactive substances were identified, one responsible for the antibacterial activity and the other for the antifungal activity. The fraction with strong antibacterial activity had the molecular mass 282.8 and formula C18H36NO, and the fraction with strong antifungal activity had molecular mass 319.3 and molecular formula C20H31O3 which could be a new fungicide. Additionally, P. aurantiaca S-1 was able to produce indoleacetic acid and siderophores.     

Noladin ether, a putative endocannabinoid, inhibits mu-opioid receptor activation via CB2 cannabinoid receptors

We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.     

Novel serine penicillocarboxypeptidase CPD-S3 from Penicillium janthinellum IBT 3991: Purification, characterization, and uses in peptide synthesis and modification

A novel carboxypeptidase (CPD-S3) from Penicillium janthinellum IBT 3991 has been isolated in a two-step purification procedure by cation exchange and affinity chromatography. The enzyme is a serine carboxypeptidase with a denatured molecular mass determined by SDS of 62 kDa of which 32% is carbohydrate. The isoelectric point is 5.1. CPD-S3 exhibits a high stability towards organic solvents and elevated temperatures. Besides the carboxypeptidase activity, CPD-S3 exhibits esterase, amidase, and carboxamidohydrolase activities. CPD-S3 favors substrates of -configuration with basic amino acid residues in either P₁ or P_1', and particularly dibasic substrates and medium-sized straight-chain alkyl esters for hydrolysis. In aminolysis of esters, amino acid amides and hydrazines coupled in good yield, but methyl esters poorly, and unlike other carboxypeptidases, free amino acids could not be coupled or transpeptidation effected to form amides. In ester semisynthesis, peptides with neutral, but not basic, residues in P₁ could be esterified. The scope of applicability for enzymatic peptide synthesis is limited.