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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.80, #3)

Rapid and high-frequency in vitro plant regeneration from leaflet and petiole explants of groundnut (Arachis hypogaea L.) by P. Venkatachalam; N. Geetha; K. Sankara Rao; N. Jayabalan (pp. 193-203).

An efficient protocol for regeneration of groundnut plantlets from immature and mature leaflet and petiole explants excised from axenic seedlings has been developed. The highest frequency of callus induction obtained from leaflet explants was on Murashige and Skoog (MS) medium containing 2.0 mg/L of α-naphthalene acetic acid (NAA) and 0.5 mg/L of kinetin combination. MS medium containing different auxins in combination with 6-benzylaminopurine (BAP) induced shoot buds. A BAP (2.0 mg/L) and NAA (0.5mg/L) combination resulted in the highest frequency of shoot-bud regeneration. Subsequent shoot multiplication was obtained on MS medium supplemented with either BAP or kinetin (5.0 mg/L) in combination with NAA (1.0 mg/L). Immature leaflet explants were found to be more responsive to shoot induction than mature leaflet explants. Direct shoot-bud regeneration was also observed from petiole explants on the same regeneration medium used for leaflet callus. Regenerated shoots were rooted on MS medium containing indole-3-butyric acid (2.0 mg/L) and kinetin (0.5 mg/L). Plantlets obtained were successfully established in the field, where they grew to maturity and set viable seeds.

Keywords: Arachis hypogaea ; organogenesis; shoot regeneration

Lipase-catalyzed synthesis of (S)-naproxen ester prodrug by transesterification in organic solvents by Shau-Wei Tsai; Chin-Shain Tsai; Chun-Sheng Chang (pp. 205-219).

A lipase-catalyzed enantioselective transesterification process was developed for the synthesis of (S)-naproxen 2-N-morpholinoethyl ester prodrug from racemic 2,2,2-trifluoroethyl naproxen ester in organic solvents. By selecting isooctane and 37°C as the best solvent and temperature, the apparent fits of the initial conversion rates for transesterification and hydrolysis side reaction suggest a ping-pong Bi-Bi enzymatic mechanism with the alcohol as a competitive enzyme inhibitor. Improvements in the initial conversion rate and the productivity for the desired (S)-ester product were obtained after comparing with the result of an enantioselective esterification process. Studies of water content in isooctane and alcohol containing various N,N-dialkylamino groups on the enzyme activity and enantioselectivity, as well as the recovery of (S)-ester product by using extraction, were also reported.

Keywords: Lipase; enantioselective transesterification; (S)-naproxen ester prodrugs

Oxidation of polycyclic aromatic hydrocarbons catalyzed by soybean peroxidase by Jennifer J. Kraus; Inmar Z. Munir; James P. McEldoon; Douglas S. Clark; Jonathan S. Dordick (pp. 221-230).

Soybean peroxidase (SBP) catalyzes the oxidation of a variety of polycyclic aromatic hydrocarbons (PAHs) in the presence of water-miscible organic cosolvents, including acetonitrile, tetrahydrofuran, and dimethylformamide (DMF). Oxidation was optimal at pH 2.0–2.5, with substantially lower reactivity at pH 1.5 as well as at pH > 3.0. Despite the low pH activity optimum, SBP had an observed half-life of 120 h at pH 2.5. Conversions of greater than 90% were observed with anthracene and 9-methylanthracene in the presence of 50% (v/v) DMF. Anthracene oxidation yielded exclusively anthraquinone, thereby demonstrating that SBP catalyzes a formal six-electron oxidation of the unactivated aromatic substrate to the quinone. A mechanism is proposed to account for this reaction that includes the initial one-electron oxidation of the PAH followed by addition of water to the oxidized PAH. 9-Methylanthracene was more reactive than anthracene, and its enzymatic oxidation yielded two products: anthraquinone and 9-methanol-9,10-dihydroanthracene. The former product indicates that loss of the methyl group occurs during enzymatic oxidation. These results suggest that SBP could be useful in the conversion of PAHs into more environmentally benign materials.

Keywords: Peroxidase; oxidation of polycyclic aromatic hydrocarbons; water-miscible organic cosolvents; anthracene

Alteration of glucose consumption kinetics with progression of baculovirus infection in Spodoptera frugiperda cells by Natarajan Raghunand; Bruce E. Dale (pp. 231-242).

We have used the initial-rate approach to characterize changes in the glucose consumption kinetics of baculovirus-infected Spodoptera frugiperda clone 9 (Sf9) cells with the progression of the infection process. The specific glucose consumption rate (q _G) of cultured baculovirus-infected Sf9 cells was measured at 4, 8, 12, 16, and 24 h postinfection (h.p.i.) in media containing 4–35 mM glucose. Higher medium glucose concentrations resulted in higher final extracellular virus and recombinant β-galactosidase yields. q _G was related to the extracellular glucose concentration by means of a Michaelis-Menten relationship. The apparent Michaelis-Menten constant (K _m) for glucose consumption was found not to change significantly during the progression of the infection process, and remained between 6.2 and 7.2 mM. However, the maximal specific glucose consumption rate (q _Gmax) was found to rapidly increase after infection, peaking at 16 h.p.i. at a value four times that for uninfected Sf9 cells. The kinetic analysis of glucose consumption rates in baculovirus-infected Sf9 cells presented here will aid in the optimal design and operation of bioreactor systems for the large-scale production of recombinant products from the baculovirus/insect cell system.

Keywords: Spodoptera frugiperda ; insect cells; glucose consumption; kinetics; baculovirus; AcMNPV; Sf9

Phylogenetic characterization of a mixed microbial community capable of degrading carbon tetrachloride by Jizhong Zhou; Anthony V. Palumbo; Janet M. Strong (pp. 243-253).

Two bacterial communities (NO92 and GBS) capable of degrading carbon tetrachloride (CT) were enriched from in-house CT-contaminated water. These communities are able to degrade CT in the presence of toluene. To characterize the community structure and diversity, one enrichment (NO92) was subjected to 16S ribosomal RNA (rRNA) gene-based molecular analysis. The 16S rRNA genes were amplified from the bulk genomic community DNA and cloned into plasmid vectors. Unique 16S rRNA gene clones, i.e., phylotypes, were detected by four tetrameric restriction enzymes. Together, 123 16S rRNA gene clones were obtained; thirty-one showed different restriction fragment length polymorphism (RFLP) patterns. About 73% of the clones belong to two dominant RFLP patterns. Phylogenetic analysis based on the partial 16S rRNA gene sequences of 10 major phylotypes showed that all the phylotypes that were sequenced were affiliated with the high G+C Gram-positive bacteria. Whereas seven of the phylotypes (∼80% of the clones) were closely related to Rhodococcus, the other three (∼5% of the clones) were related to Curtobacterium. These results suggest that this CT-degrading community is diverse but is predominated by closely related bacterial groups.

Keywords: Carbon tetrachloride; bioremediation; phylogeny; microbial community

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.80, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.80, #3)