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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.76, #3)

Production of laccase by immobilized cells of Agaricus sp. by V. M. Kaluskar; B. P. Kapadnis; C. Jaspers; M. J. Pennickx (pp. 161-170).

Laccase was produced in the supernatant of culture of a local isolate of Agaricus sp. obtained from decaying Ficus religiosa wood. The enzyme was produced at a constitutive level when growing the fungus in a nitrogenlimited medium supplemented with either glycerol, glucose, fructose, mannitol, arabinose, maltose, sacch arose, cellulose, or cellobiose. Atwo-to sixfold increase in enzyme specific activity was observed when growing the strain in the presence of straw, xylan, xylose, lignosulfonate, veratryl alcohol, and ferulic and veratric acid. Experimentsare consistent with the existence of an induction control on laccase and the absence of a form of carbon catabolite repression mediated by noninducing carbon sources. Immobilization of the Agaricus sp. on several supports, including polyurethane foam, textilestrips, and straw, resulted in an increase of enzyme production as compared to cultivation in liquid medium.

Keywords: Agaricus ; white-rot fungi; laccase; polyphenol oxidase; cell immobilization; paper industry application

Improved activity in acidic media of immobilized lysozyme by Marina Zacchigna; Gabriella Di Luca; Lucia Lassiani; Antonio Varnavas; Anna Pitotti; Enrico Boccù (pp. 171-181).

Polyethylene glycols (PEGs) of various chain length were used tocrosslink lysozyme onto an insoluble support such as oxirane. A very high degree of modification and noinactivation of lysozyme were obtained with PEG 20000, but enzymatic activity increased up to 20 times at pH 3.0, at which point the activity of the native enzyme was lower when using Leuconos tok oenus as a macromolecular substrate.

Keywords: Lysozyme; enzymeimmobilization; Eupergit C; polyethylene glycol; covalent attachment

Preparation and properties of immobilized pig kidney aminoa cylase and optical resolution of N-acyl-dl-alanine by Hao-Jing Wang; Ji-Hong Bai; De-Shan Liu; Tong Zhang; Hai-Meng Zhou (pp. 183-191).

Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-dl-alanine. The effects of pH, temperature, and Co²⁺ concentration on the activity of free and immobilized enzymes were in vestigated along with the operational and the thermal stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature for the hydrolysis of N-acyl-l-alanine in the dl-isomer mixture were 8.0 and 65°C, respectively. Co²⁺ was an activator for the immobilized enzyme in a similarroleas for the free enzyme. Nosignificant loss of activity was observed for at least 300 h of continuous operation. The yield of l-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation, but could be completely reactivated by regeneration.

Keywords: Aminoacylase; pig kidney; immobilization; optical resolution

Anaerobic upflow fixed-film bioreactor for biomethanation of salty cheese whey by Priti Patel; Chirag Patel; Datta Madamwar (pp. 193-201).

In order to develop a suitable reactor for the biomethanation of high-strength salty cheese whey, the performance of anaerobic upflow fixed-film reactors packed with different support materials, such as charcoal, gravel, brick pieces, pumicestones, and PVC pieces, has been studied. The charcoal-bedded reactor gave the best performance, with the maximum gas production (3.3 L/L digester/d) and an enriched methane content (69% CH₄). Temperature and hydraulic retention time were optimized, with the ultimate aim of improving biomethanation. Maximum gas production (3.3 L/L digester/d) was achieved at a hydraulic retention time of 2 d at 40°C.

Keywords: Salty cheese whey; biomethanation; anaerobic digestion; methane; energy; fixed-film bioreactor; charcoal

Laccase from Coriolus hirsutus as alternate label for enzyme immunoassay by Anatoliy V. Zherdev; Nadezhda A. Bizova; Alexander I. Yaropolov; Natalija V. Lyubimova; Olga V. Morozava; Boris B. Dzantiev (pp. 203-215).

A new label—laccase from the fungus Coriolus hirsutus—was applied for solid-phaseenzyme-linked immunosorbentassays of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). Two proposed assays are based on (1) competitive binding of antibody-laccase conjugate with immobilized 2,4-D-protein conjugate and 2,4-D in tested sample, and (2) competition of 2,4-D and 2,4-D-laccase conjugate for binding with immobilized antibodies. Kinetic and concentration dependencies for these reactions were studied, and the ELISAs were optimized in accordance with the data obtained. The elaborated systems perm it the detection of 2,4-D in concentrations down to 10–20ng/mL; time of the assays is 1.5–2 h. The main advantage of the laccase label, in comparison with the widely used peroxidase one, lies in the lack of hydrogen peroxide from substrate mixture, because dissolved oxygen plays the role of oxidizer.

Keywords: Enzyme immunoassay; laccase; pesticide; 2,4-dichlorophen-oxyacetic acid

Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system by Eiry Kobatake; Akira Ebisawa; Orie Asaka; Yasuko Yanagida; Yoshihito Ikariyama; Masuo Aizawa (pp. 217-227).

The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly (A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3′-terminus. The mRNA was stabilized against three types of nucleases (3′-OH exonuclease, 5′-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.

Keywords: Stabilization of mRNA; immobilized mRNA; cell-free protein synthesis; continuous flow system; Saccharomyces cerevisiae ; protein A

Partial purification and properties of putrescine oxidase from Candida guilliermondii by Muthukumaran Gunasekaran; Uma Gunasekaran (pp. 229-236).

Putrescine oxidase ([PO]; E.C. 1.4.3.4), which catalyzes the oxidative deamination of putrescine into γ-aminobutyraldehyde, has been partially purified from Candida guilliermondii. Among the substrates tested, putrescine has the highest reaction rate, followed by spermidine and cadaverine. The K _IN values for putrescine, spermidine, and cadaverine were 20, 200, and 1.1 mM, respectively. The optimum pH and the temperature for PO were 8.0 and 37°C, respectively. Growth of Candida species on putrescine as the solenitrogen source induced the synthesis of PO that converts putrescine into Δ¹-pyrroline and γ-aminobutyric acid. These two products were detected and identified from the culture medium. The enzyme was not activated by divalent cations. Among the species of Candida tested, the highest enzyme activity was found in cell-free extracts of C. guilliermondii. The pathway of putrescine degradation was identified by substrate analysis to be along the nonacetylated pathway in C. guilliermondii.

Keywords: Candida sp.; enzyme purification; spermidine; polyamines

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.76, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.76, #3)