Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S407

acid) and as unwanted by-products deriving from lignocellulosespre-treatments.

Therefore, the development of “robust cell factories” withimproved production rates and resistance is of crucial importance.

We previously demonstrated that yeast strains engineered toendogenously produce vitamin C become more tolerant to variousstress conditions (Branduardi et al., 2007) including oxidative andlow pH ones. In this work we extended the panel of stresses coun-teracted through ascorbic acid production, focusing our attentionon several acids and toxic compounds commonly deriving frombiomass pre-treatments. To this purpose wild type and vitamin Cproducing strains were grown in presence of increasing concen-trations of said limiting agents. The obtained results, supportedby flow cytometric data, demonstrated the beneficial effect of theendogenously produced antioxidant agent to overcome organicacids stress.

To better characterise this aspect, the behaviour of said strainsunder acetic acid treatment was analysed more in detail. Viabilityfor both strains was determined on plates, in presence of increasingconcentrations of acetic acid. Moreover, activities of the main ROSdetoxifying enzymes (superoxide dismutase and catalase) as wellas glutathione content were measured and compared between thetwo strains.

doi:10.1016/j.jbiotec.2010.09.541

[P-I.176]

Development of a novel bidirectional deletion system for mini-mizing the genome of Escherichia coli

B.H. Sung ∗, O.J. Yoo, S.C. Kim

Korea Advanced Institute of Science and Technology, Republic of Korea

Numerous genome information obtained by genomics studiesafter complete genome sequencing of diverse species have beenused to define and understand the cellular life at the molecularlevel. To facilitate the understanding of the life, we have developeda powerful bidirectional deletion system for identifying essentialgenes and minimizing bacterial genomes. The technique, whichproduces nested sets of deletions efficiently, involves a hybridtransposable element that includes components of two transpos-able element, mariner and �� as well as two contraselectable tetRand sacB genes for phenotypic selection. We made a large pool ofindependent transposon insertion mutants in Escherichia coli usingmariner and precisely mapped the chromosomal location of 600of these transposons. By the action of �� transposase, bidirectionaldeletion was performed from the �� ends to the adjacent chromo-somal DNA with various distances and selected on the mediumcontaining kanamycin or sucrose. We obtained E. coli strains inwhich large genomic segments were deleted by serial or simulta-neous bidirectional deletion. This provides a robust technology foreliminating dispensable genes and constructing a minimal genome.

doi:10.1016/j.jbiotec.2010.09.542

[P-I.177]

Production and Characterization of Thermostable Alpha Amy-lase Produced by Local Thermophilic Isolate Geobacillusstearothermophilus HP GU984043

Samy Selim

Suez canal University, EgyptKeywords: Thermostable; Alpha Amylase; Geobacillus stearother-mophilus

This study reported the production and characterization of anovel highly thermostable alkaline amylase from a newly iso-lated Geobacillus stearothermophilus HP GU984043. ThermophilicGeobacillus stearothermophilus HP GU984043 was isolated from soilcollected from the hot spring in Sinai geothermal site, Egypt. Max-imum production of enzyme was obtained in minimal mediumsupplemented with 1% sucrose. The enzyme was found to be pro-duced constitutively even in the absence of starch. The optimumtemperature and pH for the enzyme production was 65 ◦C and 9.0,respectively. The amylase powder obtained from the culture filtrateby prechilled acetone treatment was stable over a wide pH rangeand liquefied thick starch slurries at 75 ◦C. The crude amylase, after(NH4)2SO4 fractionation, had an activity of 285 U mg−1. The enzymewas stable for 2 h at 65 ◦C, while at 70 ◦C and 90 ◦C, 5% and 17% of theoriginal activities were lost, respectively. The optimum pH of theenzyme was 8.5. After incubation of crude enzyme solution for 24 hat pH 9, a decrease of about 5% of its original activity was observed.The enzyme was strongly inhibited by Co2+, Cu2+ and Ba2+, but lessaffected by Ca2+, Mg2+, Ni2+, Sr2+ and Mn2+. The enzyme in 1 Mand 3 M NaCl solutions the enzyme retained 70% and 47% of theoriginal activity after 24 h of incubation at 4 ◦C, respectively. Ca2+

was required for the thermostability of the enzyme preparation.The present purified amylase therefore could be defined as a ther-mostable and alkalitolerant with new properties make the presentenzyme applicable for many starch processing and food industries.

doi:10.1016/j.jbiotec.2010.09.543

[P-I.178]

Optimization for lacticin Production by Bacillus vallismortis BK6Isolated from Jeot-gal

Ju-Sang Kim 1,∗, Ramasamy Harikrishnan 1, Gi-Young Kim 2,Moon-Soo Heo 1

1 Department of Aquatic Life Medicine, College of Ocean Science, JejuNational University, Jeju, Republic of Korea2 Faculty of Applied Marine Science, Jeju National University, Jeju,Republic of KoreaKeywords: Jeot-kal; Antibacterial activities; Bacillus valismortisBK6; Cultural Characteristics

The aim of the present study was isolate BK6 from Koreanfermented Joet-gal food to investigate their antibacterial andantioxidant activity of solvents (ethyl acetate and butanol) extractsfrom the supernatant and culture characteristics of the bacteriocinantibacterial substance. BK6 was identified as Bacillus Vallismortisbased on the biochemical properties and 16S rRNA gene sequence.In the experiment to test the stability of various enzyme treat-ment, such as trypsin, protease, proteinase K, lipase, lysozyme,�-amylase, �-chemotrypsin, and pepsin. The hydroxyl, alkyl, andDPPH radical scavenging activity were investigated using a ESRspectrophotometer and compared with the ESR signal intensity.The antibacterial activity was found to be stable below 50 ◦C and