Isolation, characterisation, and genome sequencing of Rhodococcus equi: a novel strain producing chitin deacetylase

Abstract

chitin deacetylase (cDA) can hydrolyse the acetamido group of chitin polymers to produce chitosans, which are used in various fields including the biomedical and pharmaceutical industries, food production, agriculture, and water treatment. cDA represents a more environmentally-friendly and easier to control alternative to the chemical methods currently utilised to produce chitosans from chitin; however, the majority of identified CDAs display activity toward low-molecular-weight oligomers and are essentially inactive toward polymeric chitin or chitosans. therefore, it is important to identify novel cDAs with activity toward polymeric chitin and chitosans. in this study, we isolated the bacterium Rhodococcus equi F6 from a soil sample and showed that it expresses a novel CDA (ReCDA), whose activity toward 4-nitroacetanilide reached 19.20 U/mL/h during fermentation and was able to deacetylate polymeric chitin, colloidal chitin, glycol-chitin, and chitosan. Whole genome sequencing revealed that RecDA is unique to the R. equi F6 genome, while phylogenetic analysis indicated that RecDA is evolutionarily distant from other cDAs. in conclusion, RecDA isolated from the R. equi F6 strain expands the known repertoire of CDAs and could be used to deacetylate polymeric chitosans and chitin in industrial applications.Chitin is the second most abundant biopolymer after cellulose and is mainly obtained as a waste product of the seafood industry at a relatively low cost 1 . The chitin derivative, chitosan, is a linear polysaccharide comprised of β-(1→4)-linked glucosamine and N-acetyl glucosamine units which are randomly arranged within the chitosan polysaccharide chain 2 . Due to its ability to dissolve in dilute acids, chitosan is more useful than its crystalline precursor chitin in various industrial applications 3 , including the biomedical and pharmaceutical industries, food production, agriculture, and water treatment 4 .Chitosan occurs naturally and is mainly found in the cell walls of certain fungi, the exoskeletons of certain insects (such as the abdominal wall of termite queens), and in some yeasts 5,6 . Although chitosan can be extracted from fungal sources 7 , the method is commercially inapplicable as it provides too low a yield at too high a cost. Therefore, chitosan is still obtained by treating marine-derived chitin with thermo-alkaline 8 , a method that is inexpensive and results in high yields but is environmentally unsafe and difficult to control, producing a heterogeneous range of products depending on the degree of deacetylation 9 . The enzymatic production of chitosan using microbial chitin deacetylase (CDA; EC 3.5.1.41), which can hydrolyse the acetamido group in chitin polymers to produce chitosans 10 , is an environmentally-friendly process that is easy to control and results in a high yield of homogeneous end products 11 .CDAs have been identified in marine and soil bacteria, several fungi, a few insects, and at least one virus 12,13 . Fungal CDAs exist mostly as N-glycosylated (20-70%) glycoproteins, w...

Enhanced Chitin Deacetylase Production Ability of Rhodococcus equi CGMCC14861 by Co-culture Fermentation With Staphylococcus sp. MC7

“…MC7 was found accidentally during the screening process of R. equi CGMCC14861. Its colony size was smaller than R. equi CGMCC14861, but the colony morphology was identical except for the colony color ( Ma et al, 2020b ). After incubation for 1–two days at 37°C, the colonies of strain MC7 appeared to be milky-white, round, smooth, opaque, glistening, gram-positive, non-motile, aerobic, and did not produce spores.…”

“…In addition, the CDA gene from Rhodococcus genus was amplified for the first time ( Ma et al, 2020a ). Recently, we have shown the enzymatic properties of this ReCDA by phylogenetic analysis, biochemical characterization, and substrate spectrum determination ( Ma et al, 2020b ). The gene encoding ReCDA was 882 bp and located at 3,400 kb in the genomic map ( Ma et al, 2020b ).…”

“…Recently, we have shown the enzymatic properties of this ReCDA by phylogenetic analysis, biochemical characterization, and substrate spectrum determination ( Ma et al, 2020b ). The gene encoding ReCDA was 882 bp and located at 3,400 kb in the genomic map ( Ma et al, 2020b ). Phylogenetic analysis, biochemical characterization, and substrate spectrum determination have indicated the novelty of this newly identified ReCDA ( Ma et al, 2020b ).…”

“…The gene encoding ReCDA was 882 bp and located at 3,400 kb in the genomic map ( Ma et al, 2020b ). Phylogenetic analysis, biochemical characterization, and substrate spectrum determination have indicated the novelty of this newly identified ReCDA ( Ma et al, 2020b ). The rapid production of CDA by R. equi CGMCC14861 has advantages for industrial applications such as the recycling of the fermenters, bulky production, and cost-cutting ( Ma et al, 2020a ).…”

“…The rapid production of CDA by R. equi CGMCC14861 has advantages for industrial applications such as the recycling of the fermenters, bulky production, and cost-cutting ( Ma et al, 2020a ). Our recent reports have shown the enzyme proficiency of ReCDA using diverse chitin substrates ( Ma et al, 2020b ). However, further improvements in the fermentation performance based on R. equi CGMCC14861 should be conducted to obtain higher CDA yields.…”