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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.120, #2)

Relative effectiveness of pretreatments on performance of Rhizomucor miehei lipase in nonpolar reaction media by Rafael A. Garcia; Mark R. Riley (pp. 81-95).

Enzymes can be used in nonpolar reaction media to modify waterinsoluble substrates. A variety of pretreatments, applied to the enzyme prior to introduction to the nonpolar media, can improve enzyme activity. However, the various pretreatments have not been studied using directly comparable conditions, nor have they been applied simultaneously to test for interactive effects. This work evaluates pretreatment of lipase with various classes of additives. The pretreated lipase is used to catalyze esterification between citronellol and acetic acid in a medium of n-hexane. The effectiveness of a particular pretreatment is presented in terms of relative performance (RP), which is equal to the number of times faster the pretreated lipase catalyzes the reaction relative to untreated lipase. The individual and interactive effects of the pretreatment factors were studied and compared. Buffer salts had a much stronger performance-enhancing effect than nonbuffer salts; pretreatment with 90% (w/w) sodium phosphate yielded lipase with an RP of approx 64. A strong interaction was found between the treatments with sodium phosphate and pH adjustment. These treatments may mitigate the inhibitory effect of acetic acid. Activating effects of phase interfaces and active-site protectants are shown to be complementary to other treatments, demonstrating that they likely act by distinct mechanisms.

Keywords: Nonaqueous enzymology; lipase; pretreatment; lyophilization; fragrance

Comparative study of amidase production by free and immobilized Escherichia coli cells by K. Madhavan Nampoothiri; Krishnan Roopesh; Sonia Chacko; Ashok Pandey (pp. 97-108).

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.

Keywords: Amidase; acetamide; Escherichia coli ; immobilization; fermentation

Feasibility of treating swine manure in an anaerobic sequencing batch biofilm reactor with mechanical stirring by Samantha Cristina de Pinho; Bruna Soares Fernandes; José Alberto D. Rodrigues; Suzana Maria Ratusznei; Eugenio Foresti; Marcelo Zaiat (pp. 109-120).

Anaerobic sequencing batch reactors containing granular or flocculent biomass have been employed successfully in the treatment of piggery wastewater. However, the studies in which these reactors were employed did not focus specifically on accelerating the hydrolysis step, even though the degradation of this chemical oxygen demand (COD) fraction is likely to be the limiting step in many investigations of this type of wastewater. The mechanically stirred anaerobic sequencing batch biofilm reactor offers an alternative for hastening the hydrolysis step, because mechanical agitation can help to speed up the reduction of particle sizes in the fraction of particulate organic matter. In the present study, a 4.5-L reactor was operated at 30°C, with biomass immobilized on cubic polyurethane foam matrices (1 cm of side) and mechanical stirring provided by three flat-blade turbines (6 cm) at agitation rates varying from 0 to 500 rpm. The reactor was operated to treat diluted swine waste, and mechanical stirring efficiently improved degradation of the suspended COD. The operational data indicate that the reactor remained stable during the testing period. After 2 h of operation at 500 rpm, the suspended COD decreased by about 65% (from 1500 to 380 mg/L). Apparent kinetic constants were also calculated by modified first-order expressions.

Keywords: Anaerobic sequencing batch reactor; anaerobic sequencing batch biofilm reactor; mechanical stirring; piggery wastewater; solids removal; swine manure

Effect of dehydration on photoinduced transformation in gelatin films made with 14-fluoro bacteriorhodopsin derivatives by Anna B. Druzhko; Sergey K. Pirutin; Angel R. de Lera; Rosana Alvarez; Howard H. Weetall (pp. 121-132).

Photoinduced transformation in gelatin films made with 14-fluoro bacteriorhodopsin derivatives, both wild-type (WT) and D96N mutant, were studied. Spectral and kinetic peculiarities for these two types of samples were compared over a wide range of relative humidity (9–92%). Analysis of the results considered two existing photoinduced processes that occur in suspensions and films of corresponding pigments. It was demonstrated that there is a range of humidity in which the performance of fluorine WT bacteriorhodopsin gelatin films may offer a technological advantage compared with fluorine D96N bacteriorhodopsin.

Keywords: Photoinduced transformation; bacteriorhodopsin; all-trans retinal; relative humidity; gelatin films; red-shifted species

Optimization of alkaline protease production by Bacillus sp. using taguchi methodology by R. S. Prakasham; Ch. Subba Rao; R. Sreenivas Rao; S. Rajesham; P. N. Sarma (pp. 133-144).

Optimization of alkaline protease production parameters by Bacillus sp. was investigated using Taguchi methodology. The pH of the medium was observed to be the most significant factor among all selected optimization parameters at an individual level. The combinatorial influence of least significant factors, inoculum level and salt solution concentration (at the individual level), resulted in an interacting severity index of 76%, suggesting their interactive role in the regulation of protease production in this microbial species. Protease production could be improved more than 100% with Taguchi’s optimized conditions of the medium composition by this microorganism.

Keywords: Alkaline protease; Bacillus sp.; production; optimization; Taguchi methodology

Medium optimization for polysaccharide production of Cordyceps sinensis by Chienyan Hsieh; Ming-Jin Tsai; Tai-Hao Hsu; Der-Ming Chang; Chaur-Tsuen Lo (pp. 145-157).

As a potential anticarcinogenic agent, polysaccharides from Cordyceps sinensis have been demonstrated to possess strong antioxidation activity. The aim of the present research was to study the optimal medium to produce polysaccharides of C. sinensis by using response surface methodology (RSM). The composition of optimized medium for polysaccharide production calculated from the regression model of RSM was 6.17% sucrose, 0.53% corn steep powder, 0.5% (NH₄)₂HPO₄, and 0.15% KH₂PO₄ at pH 4.44, with a predicted maximum polysaccharide production of 3.17 g/L. When applying this optimal medium, the maximum polysaccharide production was 3.05 and 3.21 g/L in a shake flask and a 5-L jar fermentor, respectively. When the pH was controlled at a higher level such as pH 5.0, both cell growth and polysaccharide production were inhibited. A low pH of 2.85 was required for maximum production of polysaccharides.

Keywords: Cordyceps sinensis ; polysaccharides; medium optimization; response surface methodology; corn steep powder; second-order model

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.120, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.120, #2)