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. 2020 Mar;51(1):217-228.
doi: 10.1007/s42770-019-00162-7. Epub 2019 Nov 18.

Mangrove soil as a source for novel xylanase and amylase as determined by cultivation-dependent and cultivation-independent methods

Kelly Jaqueline Alves  1 , Mylenne Calciolari Pinheiro da Silva  2 , Simone Raposo Cotta  3 , Júlia Ronzella Ottoni  4 , Jan Dirk van Elsas  5 , Valeria Maia de Oliveira  6 , Fernando Dini Andreote  2
Affiliations

Mangrove soil as a source for novel xylanase and amylase as determined by cultivation-dependent and cultivation-independent methods

Kelly Jaqueline Alves et al. Braz J Microbiol. 2020 Mar.

Abstract

Xylanase and α-amylase enzymes participate in the degradation of organic matter, acting in hemicellulose and starch mineralization, respectively, and are in high demand for industrial use. Mangroves represent a promising source for bioprospecting enzymes due to their unique characteristics, such as fluctuations in oxic/anoxic conditions and salinity. In this context, the present work aimed to bioprospect xylanases from mangrove soil using cultivation-dependent and cultivation-independent methods. Through screening from a metagenomic library, three potentially xylanolytic clones were obtained and sequenced, and reads were assembled into contigs and annotated. The contig MgrBr135 was affiliated with the Planctomycetaceae family and was one of 30 ORFs selected for subcloning that demonstrated only amylase activity. Through the cultivation method, 38 bacterial isolates with xylanolytic activity were isolated. Isolate 11 showed an enzymatic index of 10.9 using the plate assay method. Isolate 39 achieved an enzyme activity of 0.43 U/mL using the colorimetric method with 3,5-dinitrosalicylic acid. Isolate 39 produced xylanase on culture medium with salinity ranging from 1.25 to 5%. Partial 16S rRNA gene sequencing identified isolates in the Bacillus and Paenibacillus genera. The results of this study highlight the importance of mangroves as an enzyme source and show that bacterial groups can be used for starch and hemicellulose degradation.

Keywords: Biotechnology; Culture-dependent method; Enzyme; Metagenomic library.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Map of the contig MgrBr135. The highlighted ORF (red) represents the ORF used in subcloning
Fig. 2
Fig. 2
Representation of the taxonomic affiliation of the 30 ORFs generated through the RAST server. Sequences were compared with those present in the GenBank database through the BLASTx program. a Phylum. b Class. c Order. d Family
Fig. 3
Fig. 3
Phylogenetic reconstruction demonstrating the relationships between the metagenomic insert (amino acids) and sequences of the most similar organisms present in the BLASTp database. Phylogeny was determined by distance matrix–based model JTT. Values on the branches indicate the frequency of grouping, as determined by the bootstrap test with 1000 replicates. The obtained α-amylase is indicated with bold letters
Fig. 4
Fig. 4
Enzymatic index (EI) of the 38 bacterial isolates from a composite sample of mangrove sediment, an oil spill area, located in the city of Bertioga, São Paulo. The EI was calculated by the ratio of the mean diameter of the degradation halo and the mean diameter of the colony (cm). Error bars indicate the standard deviation above the mean (n = 3)
Fig. 5
Fig. 5
The quantitative xylanolytic activity of 38 bacterial isolates from a composite sample of mangrove sediment located in the city of Bertioga, São Paulo. The activity was measured by the proportion of reducing sugars released by the enzymatic hydrolysis of xylan. The error bars indicate the standard deviation above the mean (n = 3)
Fig. 6
Fig. 6
Enzymatic reaction of isolate 39 with different concentrations of NaCl. Growth of isolate 39 at different concentrations of NaCl added to the culture medium and enzymatic activity under addition of the same NaCl concentration range in the reaction buffer
Fig. 7
Fig. 7
Phylogenetic reconstruction demonstrating relationships between isolates analyzed with sequences of the most similar organisms present in the database. Phylogeny was determined by the maximum likelihood method using the distance matrix Kimura 2-parameter model. The Escherichia coli sequence was used as an external group for tree rooting. Values on the branches indicate the frequency of isolate grouping, determined by the bootstrap test with 1000 replicates
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