Isolation, identification, and whole genome sequence analysis of the alginate-degrading bacterium Cobetia sp. cqz5-12

Abstract

Alginate-degrading bacteria or alginate lyases can be used to oligomerize alginate. In this study, an alginate-degrading bacterium with high alginolytic activity was successfully screened by using Sargassum fusiforme sludge. When the strain was grown on a plate containing sodium alginate, the transparent ring diameter (D) was 2.2 cm and the ratio (D/d) of transparent ring diameter to colony diameter (d) was 8.8. After 36 h in culture at a temperature of 28 °C shaken at 150 r/min, the enzymatic activity of the fermentation supernatant reached 160 U/mL, and the enzymatic activity of the bacterial precipitate harvested was 2,645 U/mL. The strain was named Cobetia sp. cqz5-12. Its genome is circular in shape, 4,209,007 bp in size, with a 62.36% GC content. It contains 3,498 predicted coding genes, 72 tRNA genes, and 21 rRNA genes. The functional annotations for the coding genes demonstrated that there were 181 coding genes in the genome related to carbohydrate transport and metabolism and 699 coding genes with unknown functions. Three putative coding genes, alg2107, alg2108 and alg2112, related to alginate degradation were identified by analyzing the carbohydrate active enzyme (CAZy) database. Moreover, proteins Alg2107 and Alg2112 were successfully expressed and exhibited alginate lyase activity. Alginate is a kind of water-soluble acidic polysaccharide found on the cell walls of algae, such as Gigantophtha, Charcot algae, Laminaria japonica, and Sargassum fusiformis. It is formed by the random combination of beta-d-1,4-mannosuronic acid and alpha-l-1,4-gulosuronic acid 1, 2. Because of its high degree of polymerization, high molecular weight, and high viscosity, it not easily absorbed and utilized by the human body, which limits its potential applications 3. The oligomerization of alginate can widen the scope of its potential applications and results in more biological activities. The oligomeric alginate is called alginate oligosaccharide (AOS). AOS, a kind of functional oligosaccharide, exhibits a variety of properties such as increasing fruit quality 4 , and antioxidative 5 , anticoagulant 6 , and antineoplastic 7 potential. Moreover, AOS has broad application prospects in the fields of pharmaceuticals, functional food, and green agriculture. Nowadays, there are many ways to achieve alginate oligomerization. At present, it is recognized that the environmentally friendly and mildly efficient method of alginate oligomerization is to use alginate-degrading microorganisms or alginate lyase 8 extracted from microorganisms. Until now, scientists have isolated several strains of alginate-degrading microorganisms, including Cobetia sp. NAP1 9 , Microbulbifer mangrovi DD-13 T10 , Vibrio weizhoudaoensis M010 11 , Isoptericola halotolerans NJ-05 12 , V. splendidus 12B01 13 , Sphingomonas sp. A1 14 , Pseudoalteromonas sp. CY24 15 , etc. Some alginate lyase enzymes have also been isolated and purified from these strains 11-13, 15. With the improvement of sequencing technology and the dramatic reduction in sequencing costs, th...

Cloning, expression, and characterization of a novel endo‐type alginate lyase from Microbulbifer sp. BY17

“…[3][4][5][6] Researchers have attached increasing importance to alginate oligosaccharides (AOS). 7 Alginate oligosaccharides, a mixture of carbohydrates composed of 2-20 monosaccharides, connected by glycoside bonds, can be obtained by physical, chemical, and biological enzymatic methods. 8,9 The chemical and physical methods have some disadvantages, such as severe reaction conditions, difficulty controlling the reaction process, environmental pollution, and destruction of the alginate oligosaccharide structure.…”

“…It has therefore been used widely in the pharmaceutical industry, the food industry, the chemical industry, agriculture, and other fields 3‐6 . Researchers have attached increasing importance to alginate oligosaccharides (AOS) 7 . Alginate oligosaccharides, a mixture of carbohydrates composed of 2–20 monosaccharides, connected by glycoside bonds, can be obtained by physical, chemical, and biological enzymatic methods 8,9 .…”

Are the Closely Related Cobetia Strains of Different Species?

“…The whole genome sequence analysis is presented only for three strains of the genus Cobetia , including a type strain C. marina JCM 21022 T [ 8 ], and the non-type strains C. amphilecti KMM 296 (formerly C. marina KMM 296) [ 9 ] and Cobetia sp. cqz5-12 [ 10 ]. At the same time, more and more information appears about Cobetia isolates, which are promising sources of unique enzymes and secondary metabolites degrading oil [ 11 ], bacterial biofilms [ 12 ], alginate [ 10 , 13 ], and phenol [ 14 ].…”

“…cqz5-12 [ 10 ]. At the same time, more and more information appears about Cobetia isolates, which are promising sources of unique enzymes and secondary metabolites degrading oil [ 11 ], bacterial biofilms [ 12 ], alginate [ 10 , 13 ], and phenol [ 14 ]. However, their species identification are complicated due to the high level of identity for their 16S rRNA genes and the absence of whole genome sequences for the type strains of known species, excluding from C. marina JCM 21022 T [ 8 ].…”

Decolorization of azo dyes with Klebsiella Pneumoniae under high saline conditions

“…The cell cultures were maintained in M9 minimal media and transferred to 96 well plate with the initial cell concentrations adjusted to 0.01%. [165], [166] and the same was repeated with the media spiked with salts as mentioned in section 3. replicate runs, keeping the other default settings in the [168], [169] MEGAX software version 10.1.8-1 [170]. The results for minimum evolution and maximum likelihood algorithms were found to be similar.…”