Background Acetyl xylan esterase plays an important role in the complete enzymatic hydrolysis of lignocellulosic materials. It hydrolyzes the ester linkages of acetic acid in xylan and supports and enhances the activity of xylanase. This study was conducted to identify and overexpress the acetyl xylan esterase (AXE) gene revealed by the genomic sequencing of the marine bacterium Ochrovirga pacifica . Results The AXE gene has an 864-bp open reading frame that encodes 287 aa and consists of an AXE domain from aa 60 to 274. Gene was cloned to pET-16b vector and expressed the recombinant AXE (rAXE) in Escherichia coli BL21 (DE3). The predicted molecular mass was 31.75 kDa. The maximum specific activity (40.08 U/mg) was recorded at the optimal temperature and pH which were 50 °C and pH 8.0, respectively. The thermal stability assay showed that AXE maintains its residual activity almost constantly throughout and after incubation at 45 °C for 120 min. The synergism of AXE with xylanase on beechwood xylan, increased the relative activity 1.41-fold. Conclusion Resulted higher relative activity of rAXE with commercially available xylanase on beechwood xylan showed its potential for the use of rAXE in industrial purposes as a de-esterification enzyme to hydrolyze xylan and hemicellulose-like complex substrates. Electronic supplementary material The online version of this article (10.1186/s12934-019-1169-y) contains supplementary material, which is available to authorized users.
Cellulase and xylanase are main hydrolysis enzymes for the degradation of cellulosic and hemicellulosic biomass, respectively. In this study, our aim was to develop and test the efficacy of a rapid, high-throughput method to screen hydrolytic-enzyme-producing microbes. To accomplish this, we modified the 3,5-dinitrosalicylic acid (DNS) method for microwell plate-based screening. Targeted microbial samples were initially cultured on agar plates with both cellulose and xylan as substrates. Then, isolated colonies were subcultured in broth media containing yeast extract and either cellulose or xylan. The supernatants of the culture broth were tested with our modified DNS screening method in a 96-microwell plate, with a 200 μl total reaction volume. In addition, the stability and reliability of glucose and xylose standards, which were used to determine the enzymatic activity, were studied at 100°C for different time intervals in a dry oven. It was concluded that the minimum incubation time required for stable color development of the standard solution is 20 min. With this technique, we successfully screened 21 and 31 cellulase- and xylanase-producing strains, respectively, in a single experimental trial. Among the identified strains, 19 showed both cellulose and xylan hydrolyzing activities. These microbes can be applied to bioethanol production from cellulosic and hemicellulosic biomass.
We report here the annotated genome sequence of the marine bacterium Alteromonas sp. S89 and the identification of six genes coding for agar-degrading enzymes. The sequenced Alteromonas sp. S89 genome is composed of a 3,864,871-bp circular chromosome that includes 3,236 complete open reading frames.The genus Alteromonas contains Gram-negative, aerobic marine bacteria, including both nonpigmented and pigmented species (1, 2). Agar-degrading marine bacteria such as Alteromonas spp. that produce different types of agarases have been reported previously (4). Recently, new Alteromonas species have been reported in different parts of the world (3, 5, 6). However, the genome sequence of Alteromonas sp. S89 has not yet been reported. Therefore, the purpose of this study was to sequence the Alteromonas sp. S89 genome and screen it for identification of agarase-producing genes.We isolated and identified Alteromonas sp. S89 from a seawater sample collected from Chuuk State in Micronesia and analyzed its 16S rRNA sequence. Analysis of the 16S rRNA revealed 95.7% identity to the 16S rRNA sequence of Alteromonas alvinellae; hence, our organism was named Alteromonas sp. S89. The Roche GSFLX 454 sequencing approach (Macrogen, Korea) was used for determining the Alteromonas sp. S89 genome. The sequenced genome of Alteromonas sp. S89 was determined from 384,131 total reads and represents 161,008,227 bases with an average read length of 903 bp. The whole genome was determined from 376,240 assembled reads and 3,023 partial assembled reads. Whole assembled reads could be divided into two groups of contigs, including 57 large contigs (length of Ͼ500 bp) and 65 small contigs (length of Ͼ100 bp). The average contig size was 67,767 bp. The Alteromonas sp. S89 genome was shown to have 3,236 complete open reading frames (ORFs) which represent different enzymatic categories. The Alteromonas sp. S89 whole genome consists of a 3,864,871-bp circular chromosome with an average GC content of 56.7%. Moreover, it contains 22 rRNA operons.We analyzed the identified genes for ontology (GO tools) and found that the percentages of genes related to biological processes, molecular functions, and cellular components were 35%, 36%, and 9%, respectively. From the genes required for biological process, a large number of genes (40%) were classified under metabolic processes. The cellular components encoded by most of the genes (53%) were unknown. Based on their molecular function, 45% of the genes were identified as encoding catalytic activities. More importantly, we identified six complete coding sequences for agarases from the Alteromonas sp. S89 genome, and these could be used for production of recombinant agar-degrading agarases for various commercial applications. Finally, genome sequencing of Alteromonas sp. S89 provides a foundation for functional characterization of various agarase enzymes from marine bacteria. Nucleotide sequence accession number. The genome sequence of Alteromonas sp. S89 has been deposited in GenBank under accession number AF...
A strain designated as S85(T) was isolated from a seaweed collected from coastal area of Chuuk State in Micronesia. The strain was gram-negative, rod-shaped, and non-motile and formed yellow colonies on the SWY agar (0.2 % yeast extract and 1.5 % agar in seawater) and Marine agar 2216. The strain grew at pH 5-9 (optimum, pH 8), at 15-40 °C (optimum, 25-28 °C), and with 1-9 % (w/v) NaCl (optimum, 3 %). The phylogenetic analysis based on 16S rRNA gene sequence showed that strain S85(T) was related to Lutibacter litoralis CL-TF09(T) and Maritimimonas rapanae A31(T) with 91.4 % and with 90.5 % similarity, respectively. The dominant fatty acids were iso-C15:0, iso-C15:0 3-OH and iso-C17:0 3-OH, C16:0 3-OH and summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH). The major isoprenoid quinone was MK-6. The DNA G+C content of the type strain was 34.6 mol %. The major polar lipids were phosphatidylethanolamine, an unknown glycolipid and two unknown polar lipids. Based on this polyphasic taxonomic data, strain S85(T) stands for a novel species of a new genus, and we propose the name Ochrovirga pacifica gen. nov., sp. nov. The type strain of O. pacifica is S85(T) (=KCCM 90106 =JCM 18327(T)).
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