No abstract

Error-prone PCR was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant of the α6 helix (lid domain), with an 189E to V substitution at residue 189, lost its thermostability but exhibited higher activity than that of its wild-type predecessor (r03Lip). Site-saturation mutagenesis was used to explore the sequence-function relationship. Five other mutants also lost thermostability (20-40%) but exhibited enhanced thermoactivity (6.3-79-fold). The mutant E189I showed the highest activity retaining 50% activity after maintaining it at 65 °C for 24 h. In comparison to r03Lip, the mutant E189I had a higher affinity for p-nitrophenyl palmitate and p-nitrophenyl stearate (61 and 56% decreased Km) and catalytic efficiency (42-fold and 18-fold increased kcat/Km). The mutant lipase retained its tolerance to n-hexane, but had an improved transesterification activity. The results suggest that residue Glu189 plays a significant role in the thermostability and activity of this thermoalkalophilic lipase.

A batch culture of Geobacillus sp. NTU 03 was subjected to a rapid temperature shift for investigating the stress response. Several known heat-shock responses for protein, DNA, and cell membrane recurring were observed on two-dimensional (2D) gels. Heat caused protein and cell membrane disruption greatly affected the electron transport chain. Further, heat caused lower dissolved oxygen (DO) solubility resulting in insufficient oxygen to be electron acceptor, and the NADH could not be reoxidized. Hence, we observed seven dehydrogenase that used NADH as electron donor were downregulated on the 2D gels. In contrast, succinate dehydrogenase that used FADH(2) as electron donor was upregulated. However, this induction may simultaneously increase generation of superoxide; therefore the cellular redox state was imbalanced. We observed that superoxide dismutase (2D gel) and zinc ion ABC transporter (mRNA quantification) were upregulated, whereas ferric ion ABC transporter (2D gel and mRNA quantification) was downregulated. Increase in the reactive oxygen or nitrogen species scavenging activities were also observed. For responding the lower DO solubility, a transient activation of nitrate respiration was observed at transcriptional level. Our results support the view that both heat stress and heat-induced stress should be considered together when investigating the stress responses of thermophiles.

No abstract

In this study, we used polymerase chain reaction (PCR) to detect genetically modified Roundup Ready soybean (RRS) in miso.Several different DNA extraction methods had been tested. The CTAB method published by Lipp et al. and a commercial kit, Nucleospin Food, were used, because they had the most appropriate performance for miso sample. Four pairs of primers specific for the inserted genes and crop endogenous genes in RRS were applied in PCR. Using these primers, the method showed a false-negative result for the miso sample during a later period. When another more sensitive method, nested-PCR had been used, we obtained false-negative result for the sample in later fermentative stage. Since PCR and nested-PCR can not yield to positive results, using these two methods to detect transgenic components in miso is not efficient.