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A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 degrees C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 degrees C for 6 h. TtcGA can hydrolyze both alpha-1, 4- and alpha-1, 6-glycosidic linkages in various alpha-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca2+, Mn2+, Co2+, Mg2+, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn2+, Pb2+, Cu2+, and EDTA.

Heat shock protein 60 (HSP60) is one of the important chaperonins, which can assist proper protein folding and configuration,and can prevent denaturation and degradation of proteins as well. It was found that HSP60 in Thermoanaerobacter tengcongensis (T. tengcongensis) was typically temperature-dependent with the highest protein expression at 80 degrees C. To understand the molecular mechanisms of thermophiles in their responding to heat stress, further investigation was conducted to examine the dynamic expression of HSP60 gene induced by higher temperature. The T. tengcongensis cells cultured at optimal temperature (75 degrees C) were transferred to 80 degrees C followed by taking the aliquots at different time intervals. To monitor the expression levels of HSP60 mRNA and protein, these bacterial gene products were analyzed by two-dimensional electrophoresis, MALDI-TOF, HPLC-MS/MS, Western blot and quantitative PCR. The experimental data were calculated statistically and quantitatively, which indicate that the HSP60 protein increases steadily and significantly within 4h period after temperature raised, but its mRNA level is shown as a peak shape change within 1h. Obviously, the expression response of HSP60 protein to heat stress is significantly slower than that of its mRNA expression. Additionally, the expression extent of HSP60 mRNA and protein induced by raised-temperature is very different. In E. coli strains, the expression of HSP60 is usually regulated by the sigma32 factor, which disassociates with DnaK immediately when the environmental temperature elevated. Since the genome of T. tengcongensis contains two sigma32 genes, thus, it is likely that both bacteria share the similar mechanisms which regulate HSP60 transcription responding to the changes of temperature. Interestingly, the mRNA of HSP60 drops down about 70% within 30m responding to heat shock, but goes to the increased expression after then. Although this phenomenon is hard to explain based upon the current theory, it is plausible that the transcription in T. tengcongensis is temporally inhibited due to the culture temperature sharply raised.

According to analysis of proteomic profiling for Thermoanaerobacter tencongensis, TTE0090 could be a novel gene of glucokianse (GLK), though no GLK gene was annotated in the genomic data. With the methods of cloning and expression in vitro, the recombinant TTE0090 was successfully expressed and purified. The recombinant TTE0090 exhibited the catalysis of GLK, even at high temperatures. Detection of expression levels and catalysis of TTE0090 in vivo was furthermore carried out at different temperatures. The expression of TTE0090 was attenuated during the culture temperature elevated; however, the specific activity was positively correlated to temperature raised. This leads a possibility that the metabolic capacity of glycolysis in T. tencongensis is relatively constant at different temperatures. All the results herein demonstrate that TTE0090 is a novel gene of GLK. The studies on TTE0090 and its protein product, thus, may deepen our understanding of the adaptation mechanism of thermophilic bacteria living in harsh environment.

The enzyme of 6-phosphofructokinase (PFK) is a key element in glycolysis, widely distributed in most eukaryote and prokaryote. Although the gene TTE1816 from Thermoanaerobacter tengcongenisis was annotated as a PFK based upon genomic analysis, its catalytic properties have to be examined experimentally. The cells of T. tengcongenisis were cultured at optimal temperature followed by the separation of the bacterial proteins with two-dimensional electrophoresis (2-DE). These 2-DE spots located around the theoretical values of pI and MW for TTE1816 were picked up and identified by mass spectrometry, suggesting that TTE1816 indeed expressed at such high temperature. Furthermore, TTE1816 was cloned into an expression vector and expressed soluble protein in E. coli BL-21 strain. The kinetic data revealed that the recombinant TTE1816 exhibited the catalysis to phosphorylate fructose-6-phosphate (F-6-P). Not only converting F-6-P to F-1,6-BP, does TTE1816 also catalyze the phosphorylation of glucose, fructose, mannose and glucose-6-phosphate(G-6-P) with optimal temperature at 60 degrees C . Interestingly, TTE1816 is capable to catalyze the reverse reaction as a bisphosphatase for dephosphorylation of F-1,6-BP under the reaction conditions with high concentrations of enzyme as well as substrates. The data reported herein demonstrate that a new member of PFK family has been identified in T. tengcongenisis.