TfxA is a thermostable xylanase produced by the thermophilic soil bacterium Thermomonospora fusca. The enzyme was purified to homogeneity from the culture supernatant of Streptomyces lividans transformed by plasmid pGG92, which carries the gene for TfxA, xynA. The molecular mass of TfxA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 32 kDa. TfxA is extremely stable, retaining 96% of its activity after 18 h at 75°C. It has a broad pH optimum around pH 7 and retains 80% of its maximum activity between pH 5 and 9. The native enzyme binds strongly to both cellulose and insoluble xylan even though it has no activity on cellulose. Treatment of TfxA with a T. fusca protease produced a 24-kDa catalytically active fragment that had the same N-terminal sequence as TfxA. The fragment does not bind to cellulose and binds weakly to xylan. The Vmax values for TfxA and the fragment are 600 and 540 ,umol/min/mg, respectively, while the Kms are 1.1 and 2.3 mg of xylan per ml, respectively. The DNA sequence of the xynA gene was determined, and it contains an open reading frame that codes for a 42-amino-acid (42-aa) actinomycete signal peptide followed by the 32-kDa mature protein. There is a 21-aa Gly-Pro-rich region that separates the catalytic domain from an 86-aa C-terminal binding domain. The amino acid sequence of the catalytic domain of TfxA has from 40 to 72% identity with the sequences of 12 other xylanases from seven different organisms and belongs to family G. Two glutamic acid residues, previously identified as essential for catalytic activity in BaciUlus pumilus XynA, are conserved in all 13 proteins. TfxA is the only thermophilic xylanase in family G as well as one of only two family-G xylanases to contain a binding domain. Xylanases cleave the 3-1,4 linkage of xylan, a polymer with a linear backbone of P-1,4-D-xylopyranoside residues, which are commonly substituted with acetyl, arabinosyl, and glucuronosyl residues. Xylan is a major component of hemicellulose from monocots and is usually associated with the cellulose and lignin components of plant cell walls. Numerous bacteria and fungi grow on xylan as a carbon source by using a variety of enzymes, including exoxylanases, endoxylanases, ,B-xylosidases, c-glucuronidases, a-arabinofuranosidases, and esterases (3, 5, 30, 33). Zymogram analysis of isoelectric focusing gels has revealed six protein bands with xylanase activity from Thermomonospora fusca BD21 (1) and four from T. fusca YX (9). The gene that codes for one of these, xynA, has been cloned and

The DNA sequences of the Thermomonosporafusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonasfimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.An important step toward understanding the mechanism of action of an enzyme is the determination of its amino acid sequence. In recent years, this usually has been done by determining the DNA sequence of the structural gene that encodes the protein, as DNA sequencing is simpler and more precise than protein sequencing. The sequences of a number of cellulase genes have been determined, and this work has been reviewed by Beguin et al. (1).We have been studying the cellulases of a thermophilic, filamentous soil bacterium, Thermomonospora fusca, and have purified five antigenically distinct cellulases, designated E1 to E5, from the culture supernatant of an extracellularprotease-negative mutant of T. fusca (34). All five enzymes are P-1,4-endoglucanases, but they show considerable variation in their specific activities on several substrates and in their physical properties. The enzymes from T. fusca are heat stable and active over a broad pH range with an optimum centered at pH 6.5. While no complex formation between the cellulases has been seen, enzyme E3 acts synergistically with E2 and E5. Evidence for coordinate regulation (20,22) As part of our study of enzymatic mechanisms of cellulose degradation, we determined the DNA sequences of the structural genes encoding three (E2, E4, E5) of the five purified T. fusca cellulases. Comparisons of the amino acid sequences of these cellulases with each other and with other cellulases yielded information about the similarities and differences among cellulases. Such comparisons may provide insight into the catalytic and regulatory mechanisms of these enzymes. MATERIALS AND METHODSBacterial strains and plasmids. The host strain for all transformations and transfections was Escherichia coli JM101 (rK' mK' supE thi A(/ac-proAB) [F' traD36 proAB lacl"ZAM15]) (36), except for the subcloning of the E4 gene, for which E. coli HB101 (F-hsdS20 [rB-mB-] supE44 * Corresponding author. ara-14 ga/K2 lacYl proA2 rspL20 xyl-5 mtl-l recA13) was used. The cellulase genes were cloned from T. fiusca YX, acquired from Dexter Bellamy, Cornell University (3). T...

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Two genes encoding cellulases El and E4 from Thermomonosporafusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. El and E4 were expressed 18and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that El digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. El shows 59% identity to Cellulomonas fimi CenC in a 689-amino-acid overlap, and E4 shows 80%o identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, El, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of El and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding

Low-temperature growth of InO films was demonstrated at 70-250 °C by plasma-enhanced atomic layer deposition (PEALD) using a newly synthesized liquid indium precursor, dimethyl(N-ethoxy-2,2-dimethylcarboxylicpropanamide)indium (MeIn(EDPA)), and O plasma for application to high-mobility thin film transistors. Self-limiting InO PEALD growth was observed with a saturated growth rate of approximately 0.053 nm/cycle in an ALD temperature window of 90-180 °C. As-deposited InO films showed negligible residual impurity, film densities as high as 6.64-7.16 g/cm, smooth surface morphology with a root-mean-square (RMS) roughness of approximately 0.2 nm, and semiconducting level carrier concentrations of 10-10 cm. Ultrathin InO channel-based thin film transistors (TFTs) were fabricated in a coplanar bottom gate structure, and their electrical performances were evaluated. Because of the excellent quality of InO films, superior electronic switching performances were achieved with high field effect mobilities of 28-30 and 16-19 cm/V·s in the linear and saturation regimes, respectively. Furthermore, the fabricated TFTs showed excellent gate control characteristics in terms of subthreshold swing, hysteresis, and on/off current ratio. The low-temperature PEALD process for high-quality InO films using the developed novel In precursor can be widely used in a variety of applications such as microelectronics, displays, energy devices, and sensors, especially at temperatures compatible with organic substrates.