Eduardo Bárzana

Effect of pH in the synthesis of ampicillin by penicillin acylase

Recombinant Escherichia coli cells with high penicillin acylase (PA) activity were immobilized by gel entrapment with agar. This biocatalyst was used to study the effect of pH on the synthesis of ampicillin from phenylglycine methylester (PGME) and 6-aminopenicillanic acid (6-APA). The parallel hydrolysis reactions of PGME and ampicillin were also studied. A selective inhibition of the hydrolysis of the ester was possible by controlling the pH at 6.0. At such conditions, and using 6-APA solutions ranging from 50–200 mm, a 75% conversion to ampicillin was obtained. This yield was higher than obtained with other strategies. The reaction kinetics was described by a second-order model for ampicillin synthesis with experimentally determined Michaelis-Menten constants of 27 and 25 mm for 6-APA and PGME, respectively. In addition, ampicillin and PGME were hydrolyzed by the enzyme following Michaelis-Menten kinetics with Km values of 40.5 and 30 mm, respectively. A good correlation was found between experimental results of synthesis reactions and the kinetic model derived from initial rate experiments with only slight deviations at high substrate concentrations. This is the first report where the specific effect of pH on the synthesis of ampicillin was studied in detail. It is shown that, by controlling the pH, it is possible to inhibit the lateral undesirable reactions increasing the yield of the main reaction.

Structural Characterization of Chitin and Chitosan Obtained by Biological and Chemical Methods

Chitin production was biologically achieved by lactic acid fermentation (LAF) of shrimp waste (Litopenaeus vannameii) in a packed bed column reactor with maximal percentages of demineralization (D(MIN)) and deproteinization (D(PROT)) after 96 h of 92 and 94%, respectively. This procedure also afforded high free astaxanthin recovery with up to 2400 μg per gram of silage. Chitin product was also obtained from the shrimp waste by a chemical method using acid and alkali for comparison. The biologically obtained chitin (BIO-C) showed higher M(w) (1200 kDa) and crystallinity index (I(CR)) (86%) than the chemically extracted chitin (CH-C). A multistep freeze-pump-thaw (FPT) methodology was applied to obtain medium M(w) chitosan (400 kDa) with degree of acetylation (DA) ca. 10% from BIO-C, which was higher than that from CH-C. Additionally, I(CR) values showed the preservation of crystalline chitin structure in BIO-C derivatives at low DA (40-25%). Moreover, the FPT deacetylation of the attained BIO-C produced chitosans with bloc copolymer structure inherited from a coarse chitin crystalline morphology. Therefore, our LAF method combined with FPT proved to be an affective biological method to avoid excessive depolymerization and loss of crystallinity during chitosan production, which offers new perspective applications for this material.

Effect of temperature on chitin and astaxanthin recoveries from shrimp waste using lactic acid bacteria.

The chitin and astaxanthin recoveries by lactic acid fermentation of shrimp wastes (Litopenaeus sp) were conducted in bed-column reactors at 15, 20, 25, 30, 35, 40 and 45 degrees C. The response surface methodology showed that the fermentations carried out in the 27-36 degrees C temperature range with lactic acid above 0.319 mmol/g resulted in the highest demineralization. The maximal deproteinizations were attained from 30 to 40 degrees C. The extraction of free-astaxanthin did not present significant differences between 20 and 35 degrees C and the proportion of cis-stereoisomer forms increased with temperature. The growth rates of Lactobacillus plantarum were estimated in the 15-45 degrees C range and analyzed by Arrhenius and square root models. The cardinal values were 3.94 and 51.7 degrees C for minimum and maximum temperatures, respectively, with activation energy of 43.38 Jmol(-1).

Kluyveromyces marxianus CDBB-L-278: A wild inulinase hyperproducing strain

Abstract Kluyveromyces marxianus CDBB-L-278 is an inulinase hyperproducing strain. It was able to grow in a medium containing inulin as the unique carbon source in the presence of 2-deoxyglucose. It produced up to 3.3 times the activity of the control strain K. marxianus NCYC-1429 in an inulin medium, and 3.6 times in a medium with glycerol as the sole carbon source. Although the strain CDBB-L-278 was able to produce inulinase in the presence of 2-deoxyglucose, it was demonstrated that it is not a de-repressed strain since enzyme production was reduced when the concentration of glucose or fructose was increased in the medium. Since inulinase was produced in a glycerol medium without an inducer, it can be considered that the enzyme production was partially constitutive in K. marxianus CDBB-L-278 as well as strain NCYC-1429. The inulinase from K. marxianus CDBB-L-278 was characterized. It had a higher affinity for inulin than for sucrose. Temperature and pH profiles were different for both of these two substrates. The enzyme was stable to high temperatures, with a half-life of 180 min at 50°C.

Ultrasonication and steam-explosion as chitin pretreatments for chitin oligosaccharide production by chitinases of Lecanicillium lecanii

In this study, chitin oligosaccharides have been successfully produced using chitinases from submerged fermentation of Lecanicillium lecanii. The highest Hex, Chit and Prot production was 0.14, 0.26 and 2.05 U/mg of protein, respectively, which were attained varying pH from 5 to 8 after 96 h. Culture conditions conducted at constant pH of 6 resulted in significantly lower enzyme production. The crude enzyme was partially purified by salting out with (NH4)2SO4 followed by size exclusion chromatography to isolate the chitinase mixture for further chitin hydrolysis assays. In this regard, chitin substrates were pretreated with sonication and steam explosion prior to enzymatic reaction. Structural changes were observed with steam explosion with 11.28% reduction of the crystallinity index attained with the lowest chitin/water ratio (0.1g/mL). Pretreated chitins reached the highest production of reducing sugars (0.37 mg/mL) and GlcNAc (0.59 mg/mL) in 23.6% yield.

Fungal Removal of Gaseous Hexane in Biofilters Packed With Poly(Ethylene Carbonate) Pine Sawdust or Peat Composites

The removal of volatile organic compounds (VOC) in biofilters packed with organic filter beds, such as peat moss (PM) and pine sawdust (PS), frequently presents drawbacks associated to the collapse of internal structures affecting the long‐term operation. Poly(ethylene ether carbonate) (PEEC) groups grafted to these organic carriers cross linked with 4,4′‐methylenebis(phenylisocyanate) (MDI) permitted fiber aggregation into specific shapes and with excellent hexane sorption performance. Modified peat moss (IPM) showed very favorable characteristics for rapid microbial development. Water‐holding capacity in addition to hexane adsorption almost equal to the dry samples was obtained. Pilot scale hexane biofiltration experiments were performed with the composites after inoculation with the filamentous fungus Fusarium solani. During the operation of the biofilter under non‐aseptic conditions, the addition of bacterial antibiotics did not have a relevant effect on hexane removal, confirming the role of fungi in the uptake of hexane and that bacterial growth was intrinsically limited by an adequate performance of the composites. IPM biofilter had a start‐up period of 8–13 days with concurrent CO2 production of ∼90 g m−3 h−1 at day 11. The final pressure drop after 70 days of operation was 5.3 mmH2O m−1 reactor. For modified pine sawdust (IPS) packed biofilter, 5 days were required to develop an EC of about 100 g m−3 h−1 with an inlet hexane load of ∼190 g m−3 h−1. Under similar conditions, 12–17 days were required to observe a significant start‐up in the reference perlite biofilter to reach gradually an EC of ∼100 g m−3 h−1 at day 32. Under typical biofiltration conditions, the physical–chemical properties of the modified supports maintained a minimum water activity (aw) of 0.925 and a pH between 4 and 5.5, which allowed the preferential fungal development and limited bacterial growth. Biotechnol. Bioeng. 2008;100: 864–871.

Studies on the simultaneous production of single cell protein and polygalacturonase fromKluyveromyces fragilis

SummaryKluyveromyces fragilis produces polygalacturonase (PG) on a lactose medium. Although the enzyme is normally repressed at high aeration levels, significant amounts of PG can be produced under such conditions when pectin is added as inducer. The productivity and yield of cell mass were not significantly affected by the presence of inducer, suggesting potential applications to current single cell protein processes from whey.

Thermodynamic hydrophobicity of aqueous mixtures of water-miscible organic solvents predicts peroxidase activity

The effect of different water-miscible organic solvents on biocatalytic activities of chloroperoxidase from Caldariomyces fumago and horseradish peroxidase was determined. A new hydrophobicity parameter for water-organic solvent mixtures was used to predict the enzyme behavior. This thermodynamic concept of hydrophobicity also describes the catalytic behavior of three other biocatalysts with peroxidase activity. So far, all reported data of peroxidase activity in increasing concentrations of water-miscible organic solvents are effectively predicted by the thermodynamic model presented in this work. q 1998 Elsevier Science B.V.

Lipase-catalyzed synthesis of hyperbranched poly-L-lactide in an ionic liquid

Hyperbranched poly-l-lactides have been synthesized by eROP in [C4MIM][PF6] media. The bis(hydroxymethyl)butyric acid molecule was used as the AB2 core co-monomer and immobilized lipase B from Candida antarctica as biocatalyst. The degree of branching could be controlled by the reaction conditions, with the maximum achieved being 0.21. The successful achievement of the hyperbranched structure is attributed to the high solvent power of substrates and products in the ionic liquid besides sustained lipase activity.

Alcoholysis and reverse hydrolysis reactions in organic one‐phase system with a hyperthermophilic β‐glycosidase

Alcoholysis and reverse hydrolysis reactions were performed enzymatically in one-phase water-saturated 1-heptanol systems. Lactose or glucose was used as substrate to produce heptyl-beta-galactoside and/or heptyl-beta-glucoside, respectively. When alcoholysis of lactose was performed at 37 degrees C with beta-galactosidase from Escherichia coli, the initial rate was 14 nmol/mL min, and the limiting factors were the poor solubility of the substrate in 1-heptanol and low thermal stability of the enzyme. When a hyperthermophilic beta-glycosidase was used at 90 degrees C, the rate was 3.14-fold higher; in this case a higher concentration of soluble lactose in the water-saturated heptanol was available to the enzyme due to the higher temperature. The hyperthermophilic beta-glycosidase was also able to use glucose and galactose as substrates to achieve the reverse hydrolysis reaction. As a consequence, when lactose was used as substrate, heptyl-beta-galactoside was formed by alcoholysis, while the released glucose moiety was used in a secondary reverse hydrolysis reaction to produce heptyl-beta-glucoside. Both reactions followed Michaelis-Menten kinetics behavior. Neither lactose nor heptyl glycosides were hydrolyzed by this enzyme in water-saturated heptanol. However, the conversion was limited by a strong product inhibition and the formation of oligosaccharides, especially at high substrate concentrations, reducing the final glycoside yield.