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Microbiol Spectr
2022 Aug 31;104:e0107822. doi: 10.1128/spectrum.01078-22.
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Carbohydrate-Active Enzymes of a Novel Halotolerant Alkalihalobacillus Species for Hydrolysis of Starch and Other Algal Polysaccharides.
Masasa M
,
Kushmaro A
,
Chernova H
,
Shashar N
,
Guttman L
.
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Halotolerant bacteria capable of starch hydrolysis by their amylases will benefit various industries, specifically since the hydrolytic activity of current industrial amylases is inhibited or even absent in salt-rich or alkaline environments. Seeking novel enzymes, we analyzed the entire genome content of a marine bacterium isolated from the gut of sea urchins to compare it against other bacterial genomes. Conditions underlying α-amylase activity were examined in vitro at various salinities (0 to 4%) and temperatures (25°C to 37°C). Genomic analyses revealed the isolated bacterium as a new species of Alkalihalobacillus. Comparative analysis of the contents of carbohydrate-active enzymes revealed various α-amylases, each with its respective carbohydrate-binding module for starch hydrolysis. Functional analysis identified the hydrolysis of starch and the maltooligosaccharides maltose and dextrin into d- and UDP-glucose. The fastest growth and α-amylase production occurred at 3% salinity at a temperature of 30°C. The Alkalihalobacillus sp. consists of exclusive contents of α-amylases and other enzymes that may be valuable in the hydrolysis of the algal polysaccharides cellulose and laminarin. IMPORTANCE Toward the discovery of novel carbohydrate-active enzymes that may be useful in the hydrolysis of starch, we examined a halotolerant bacterial isolate of Alkalihalobacillus sp. regarding its genomic content and conditions underlying the production of active α-amylases. The production of α-amylases was measured in bacterial cultures at relatively high temperature (37°C) and salinity (4%). The Alkalihalobacillus sp. revealed an exclusive content of amylases and other carbohydrate-active enzymes compared to other relevant bacteria. These enzymes may be valuable for the hydrolysis of algal polysaccharides. The enzymatic cascade of the Alkalihalobacillus sp. for starch metabolism allows polysaccharide degradation into monosugars while preventing the accumulation of intermediate inhibitors of maltose or dextrin.
FIG 1. Circular map of the genome of the isolated Alkalihalobacillus sp. bacterium generated by the CGView comparison tool. Colored circles (from outside to inside) identify the (i) forward sequence feature, (ii) reverse sequence feature, (iii) GC content, and (iv) GC skew. Colors identify the genome contents as CDSs (blue), tRNAs (black), rRNAs (red), tmRNAs (pink), and positive (+) (green) or negative (−) (purple) GC skews.
FIG 2. Phylogenetic closeness tree based on comparative analysis of the 16S rRNA gene sequences of the isolated Alkalihalobacillus sp. bacterium and phylogenetically closely related bacteria in the database. The evolutionary distances were computed using the maximum composite likelihood method. The tree was generated using the MEGAX tool according to the neighbor-joining method. A bootstrap test was performed in 1,000 replicates; the number next to each branch of the tree identifies the percentage of replicate trees in which the associated taxa were clustered together by the bootstrap test.
FIG 3. In vitro growth performance and α-amylase activity of the isolated Alkalihalobacillus sp. bacterium under different culture conditions. Bacterial growth (a and c) and α-amylase activity (b and d) were measured in cultures of different salinities (a and b) and temperatures (c and d) in a sampling regime of once every 4 h during the first 48 h and once every 12 h during the following 48-h period. One unit of α-amylase activity represents a rate of disappearance of 1 mg/min of the iodine binding starch in the assay reaction mixture. Values are means ± standard deviations (SD) (n = 3).
FIG 4. Pie chart of the contents of functional categories in the genome of the isolated Alkalihalobacillus sp. bacterium. Genes were annotated and categorized into Clusters of Orthologous Groups (COG), while the number of orthologue genes in each category is displayed.
FIG 5. Comparative heat map diagrams of the entire contents of CAZymes (a) and GH13 family enzymes (b) in the genomes of the isolated Alkalihalobacillus sp. bacterium and other bacteria for which a complete genome is available in the database. The maps highlight the presence and the cumulative number of copies in the genome (dark blue [0] to dark red [5 or above]) of CAZymes of different families (a) or those of GH13 family only (b). A comparison of the isolated Alkalihalobacillus sp. was made against several species of Bacillus currently used for α-amylase production in various industries as well as against halophilic bacteria that contain hydrolases or polylyases in their genomes. Bacterial species that were reported for their capability of starch hydrolysis in in vitro experiments are marked with an asterisk. Similarity analysis based on the entire contents and number of copies of the CAZymes in the different bacteria was performed and is demonstrated by a similarity tree on the left side of the heat map in panel a.
FIG 6. Hypothetical pathway for the metabolism of starch by the isolated Alkalihalobacillus sp. bacterium. Metabolic mapping was performed according to the Kyoto Encyclopedia of Genes and Genomes (KEGG). Green boxes represent the genes (CAZymes) that have been identified in the genome of the isolated bacterium, while clear boxes represent other known genes in the referred pathway between two given compounds (marked with ○). Arrows present the direction of the metabolic function. The highlighted red lines identify pathways for the complete metabolism of starch as suggested by KEGG Mapper.
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