LAURA NAVIKA YAMANI (2011) KONSTRUKSI SEKRESI EKSTRASELULER DAN PENINGKATAN pH OPTIMUM a-L-ARABINOFURANOSIDASE dari Geobacillus thermoleovorans IT 08. Thesis thesis, UNIVERSITAS AIRLANGGA.

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Abstract

This study aims to obtain a-L-arabinofuranosidase extracellular expressed by M5 plasmid system in E. coli BL21 (DE3) and increased activity of a-L-arabinofuranosidase at alkaline pH through directed evolution. a-L-arabinofuranosidase from E. coli DH5a was successfully expressed and secreted in the M5 plasmid system (excretory expression system) with E. coli Bl21 (DE3) as host. Obtained optimum conditions for the expression of a-L-arabnofuranosidase extracellular from E. coli BL21 (DE3) / pBM5ABF done with the addition of 2,5 mM inducer IPTG and incubation time of 36 hours. In addition, the a-L-arabinofuranosidase extracellular enzyme has also been successfully purified by affinity chromatography system with Ni-NTA column. From the results of purification, the specific activity of crude enzyme was 8.191 U / mg, whereas a-L-arabinofuranosidase enzyme of purification with Ni-NTA has a specific activity of 11.427 U / mg. Pure a-L-arabinofuranosidase enzyme has optimum activity at 70 ° C and pH 8, while for the temperature stability ranging from 50 to 70 ° C and pH stability ranging from 4 to 7. a-L-arabinofuranosidase extracellular enzyme of E. coli BL21 (DE3) / pBM5ABF) that have been generated in this study had optimum activity at pH 8 and the activity decreased drastically at pH 9. For that would be an increase in the activity of a-L-arabinofuranosidase extracellular enzyme pH 9 by the method of directed evolution. Directed evolution process of gene a-L-arabinofuranosidase from mold pBM5ABF performed by PCR-errorprone. a-L-arabinofuranosidase extracellular enzyme variant of 10 colonies that have been selected from the first stage of screening results with MUA substrate with 96 well microtiter plate produces positive variants that emit high-intensity blue fluorescence at pH 9, the second stage of screening was then performed with pNPA substrate in buffer NaOH-glycine at pH 9. The highest activity in mutant A12 that is equal to 2.874223 U / ml, while for the activity of the control (wildtype) of 0.754 U / ml. This means that the activity of a-L-arabinofuranosidase enzyme varian with pNPA substrate in buffer at pH 9 increased 4-fold of the enzyme activity of a-L-arabinofuranosidase wildtype.

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