Stress responses at elevated temperature in thermophile Geobacillus thermocatenulatus NTU 03 and the property and application of its thermostable lipase
Date Issued
2011
Date
2011
Author(s)
Shih, Tsung-Wei
Abstract
Thermophiles have been reported to be a good thermostable enzyme source due to their living environment. Thermostable enzymes not only exhibit potential for industrial applications but are also used as protein structure models in studies on protein thermostability. In addition, because of the high temperature of these environments, many of their physical and chemical properties differ from those of lower-temperature environments. Hence, to deal with elevated temperature-induced changes in high temperature environmental conditions, the stress responses of thermophiles should differ from mesophiles.
In this study, a thermophile (isolate NTU 03) was isolated in Taiwan on the basis of its lipolytic activity. This isolate was classified as Geobacillus thermocatenulatus NTU 03 on the basis of analysis pertaining to biochemical characteristics and 16S rRNA, recA, and rpoB sequence homology.
The stress response of G. thermocatenulatus NTU 03 at elevated temperatures was investigated by proteomic analysis. Two-dimensional (2-D) gel analysis showed that heat stress induced modulation of protein expression, imbalance in the redox state, and a sudden decrease in the oxygen supply. The elevated temperatures adversely affected cellular energy metabolism, including respiration and electron transport, and subsequently elevated the reactive oxygen species level. However, the induced reactive oxygen species level was not high enough to cause oxidative stress. Transient activation of nitrate respiration due to heat-induced insufficiency in oxygen supply modulated ammonium metabolism. Our results support the view that both heat stress and heat-induced stress should be considered together when investigating the stress responses of thermophiles.
A thermoalkalophilic lipase was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant NTU 03 lipase (r03Lip) showed optimal reaction activity at 55°C and pH 9. r03Lip exhibited increased activity and stability in the presence of sodium and calcium ions; this effect was caused by the structure stabilization effect of these ions. r03Lip exhibited good tolerance to various organic solvents under experimental conditions, and the non-polar organic solvent used enhanced r03Lip activity.
Error-prone polymerase chain reaction was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant of the α6 helix (lid domain) with a Glu to Ile substitution at residue 189 (E189I) lost its thermostability but exhibited 79-fold higher activity than its wild-type predecessor (r03Lip) did. Glu189 could form a salt bridge interaction with K185, and the salt bridge interaction could interact with the AXXXA motif at the C-terminal of the α6 helix to stabilize the helical structure. Furthermore, E189I increased the local hydrophobicity of the α6 helix and then increased its activity by improving its substrate affinity with fatty acids.
To develop a cost-efficient process for production biodiesel, we developed a whole-cell biocatalyst harboring mutated lipase E189I. The process developed could reduce the cost of producing fatty acid isopropyl ester; however, the recycling and reaction rates of the biocatalyst need to be improved for industrial application of this kind of biocatalyst.
Subjects
thermophile
stress responses
proteomic
thermostable lipase
direct evolution
biodiesel
fatty acid isopropyl ester
whole-cell biocatalyst
SDGs
Type
thesis
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