| Acidophilus, widely distributed in extremely acidic environments, are important microorganisms; the knowledge of this group of microorganisms is very limited however. Alicyclobacillus sp., as the typical thermalacidophilus, has attracted much attention in the recent years. Herein a thermalacidophilus, denoted as A4, was isolated from the soil close to the outflow of a hot spring in Baoshan, Yunnan, China, and was identified as Alicyclobacillus sp. by comparison of the 16s rDNA sequence with known ones. The strain showed optimal growth at 55–60°C and pH 3.0–3.5. Activities of several glycosyl hydrolyses were detected when induced by various carbon sources, suggesting that stain A4 is a producer of multiple glycosyl hydrolyses.During the double-enzymatic sugar-making process, thermophilicα-amylase and mesophilic saccharifying enzyme are widely applied. Because both enzymes have different optimum temperature and pH, the pH values and temperatures must be adjusted repeatedly. Moreover, thermophilicα-amylase has no ability to degrade raw starch, which must be gelatinized before treatment, thus causing problems such as high energy consumption and environmental contamination. Using soluble starch as carbon source, an acidα-amylase (AmyA4) was isolated and purified. AmyA4 had the optimum pH of 4.2 and optimal temperature at 75°C, showed high activity at 40?65°C, and possessed strong capacity to degrade raw starch. Under simulated double-enzymatic sugar-making conditions, AmyA4 combined with commercialized saccharifying enzyme could catalyze the hydrolysis of 96.7% substrate, and thus has great potential for application.Using barleyβ-glucan as carbon source, an extremely acidβ-1,4-glucanase (CelA4) was isolated and purified from the culture supernatant of Alicyclobacillus sp. A4. CelA4 showed optimal activity at pH 2.6, was stable in acidic conditions, showed catalytic activity towards various substrates including barleyβ-glucan, cellulose, oat spelt xylan and mannan, and had high resistance to pepsin. Under simulated gastric conditions, CelA4 can effectively reduce the viscosity of soybean-barley meal and has potential to be a candidate of pig feed additives. The gene sequence of CelA4 was determined by the analysis of MALDI-TOF-MS and whole genome sequence. CelA4 coded 715 AAs, and its deduced amino acid sequence shared the highest identity (44%) with an endo-β-1,4-glucansae from Alicyclobacillus acidocaldarius that belongs to family 51 of the glycoside hydrolases. CelA4 has three domains, namely the signal peptide (0–39 AAs), catalytic doamin (40–498 AAs), and threonine-rich region (499-715 AAs). Furthermore, the genes coding for full-length CelA4F (40–715 AAs) and truncated CelA4T (40–498 AAs) were also expressed in Pichia pastries. Biochemical characteristics of CelA4T and CelA4F were similar with native CelA4, but CelA4T achieved higher throughout than CelA4F did in 3.7 L fermentor. Based on the analysis of deglycosylation and SDS-PAGE, CelA4F was determined to be truncated by itself as native CelA4 did, and the threonine-rich region might have a significant effect on enzyme expression. The gene encoding a GH 9 glucanase (Agl9A) from Alicyclobacillus sp. A4 was cloned and expressed in P. pastries. The deduced amino acid sequence of Agl9A shared the highest identity (48%) with an endo-β-1,4-glucansae from A. acidocaldarius that belongs to family 9 of the glycoside hydrolases. The purified recombinant Agl9A had optimal pH at 5.8 and temperature optimumat 65°C, was Ca2+-dependent, and showed strong resistance to various neutral proteases including Neutrase 0.8L (Novozymes), a protease widely added to the mash. Under simulated mashing conditions, addition of Agl9A alone or in combination with a commercial xylanase can both effectively reduce the filtration rate and viscosity. These characteristics indicate that Agl9A is a good candidate to improve glucan degradation in the malting and brewing industry.The gene coding for a GH 10 xylanase (XynA4) from Alicyclobacillus sp. A4 was cloned and expressed in Escherichia coli. The deduced amino acid sequence of XynA4 shared the highest identity (53%) with an endo-β-1,4-xylanase from Geobacillus stearohermophilus that belongs to family 10 of the glycoside hydrolases. Purified recombinant XynA4 exhibited maximum activity at 55°C and pH 7.0, had broad pH adaptability (>40% activity at pH 3.8?9.4) and stability (retaining >80% activity at pH 2.6?12.0). The superior properties, such as good thermostability and pH stability and adapatability over a broad pH range, make XynA4 promising for application in the paper industry.Considering the harm to beverage caused by Alicyclobacillus sp., its special demand for extreme environment, and low 16S rDNA sequence identity (only 95%) with A. acidocaldarius DSM 446 (whole genome sequenced), the whole genome sequencing of Alicyclobacillus sp. A4 and elementary analysis was carried out. Results showed that the genome of Alicyclobacillus sp. A4 consisted of a chromosome genome and a plasmid genome, and shared near genomic synteny with A. acidocaldarius DSM 446. The chromosome genome was 2,815,170 bp in length, coded 2,884 genes, and the GC content was 46.5%. Among the 2,884 coding genes, 2,359 (81.8%) genes were identified to be homologous with A. acidocaldarius DSM 446, and the average identity was 65.4%. The plasmid genome length was 80,481 bp, coding 88 genes, with 52.1% GC content. Fifty-eight genes (65.9%) were identified to be the homologues of pAACI01 from A. acidocaldarius DSM 446 with average identity of 83.3%. The secretion of histone proteins was also analyzed, and 10.9% of the sequences were identified with signal peptides. In addition, the genome of Alicyclobacillus sp. A4 contained 31 glycosyl hydrolyse-encoding sequences, and 6 of them had signal peptides.
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