Seaweed Hydrocolloid Production: An Update on Enzyme Assisted Extraction and Modification Technologies

Agar, alginate, and carrageenans are high-value seaweed hydrocolloids, which are used as gelation and thickening agents in different food, pharmaceutical, and biotechnological applications. The annual global production of these hydrocolloids has recently reached 100,000 tons with a gross market value just above US$ 1.1 billion. The techno-functional properties of the seaweed polysaccharides depend strictly on their unique structural make-up, notably degree and position of sulfation and presence of anhydro-bridges. Classical extraction techniques include hot alkali treatments, but recent research has shown promising results with enzymes. Current methods mainly involve use of commercially available enzyme mixtures developed for terrestrial plant material processing. Application of seaweed polysaccharide targeted enzymes allows for selective extraction at mild conditions as well as tailor-made modifications of the hydrocolloids to obtain specific functionalities. This review provides an update of the detailed structural features of κ-, ι-, λ-carrageenans, agars, and alginate, and a thorough discussion of enzyme assisted extraction and processing techniques for these hydrocolloids

The Effects of Agaro-Oligosaccharides Produced by Marine Bacteria (Rheinheimera sp. (HY)) Possessing Agarose-Degrading Enzymes on Myotube Function

Agarase and its metabolites are reported to have applications in a variety of fields, but there have been few studies of the effects of agaro-oligosaccharide hydrolysate on muscle function. In this study, we analyzed the functionality of agarase and its metabolites in bacteria isolated from seawater. A bacterium with agar-degrading activity was isolated from Shimabara, Nagasaki, Japan. Through 16S rRNA sequence alignment, it was identified as being closely related to Rheinheimera sp. WMF-1 and was provisionally named Rheinheimera sp. (HY). Crude enzymes derived from this bacterium demonstrated an ability to hydrolyze various polysaccharides, including agar, agarose, and starch, with the highest specificity observed for agarose. The optimum pH and temperature were pH 10 and 50 °C. A glycoside bond specificity analysis of enzymatic activity indicated the cleavage of the α-linkage. Next, we investigated the functional effects of agaro-oligosaccharides on C2C12 myotubes. Treatment with 10–30 kDa oligosaccharides significantly increased the hypertrophy rate, diameter, and expression of myosin heavy-chain genes in C2C12 myotubes. These results indicate that the agaro-oligosaccharides produced by the enzymes identified in this study improve muscle mass, suggesting their potential contribution to muscle function.Marine Drugs, 22(11), art. no. 515; 2024journal articl

Microrganismos marinhos: um reservatório de hidrolases biotecnologicamente interessantes

This review aims to provide an updated narrative review about hydrolytic enzymes (agarases, amylases, cellulases, esterases, lipases, peptidases, and chitinases) isolated from the marine microbiome. These biocatalysts have unique biochemical properties, such as halotolerance, extremes of pH, temperature, and barophilicity, which make them attractive for use in several industrial sectors and stimulating future biotechnological applications. Considering the vast enzymatic repertoire of the various microbial communities living in the marine environment, it is highlighted the importance of harnessing this habitat, specifically these biocatalytic units. Consequently, with proper attention to the industrial perspective, new biocatalysts will be successfully discovered in the marine microbial communities, confirming their practical and valuable activity application to the attractive sectors and generating intellectual property.Esta revisão tem por objetivo fornecer uma revisão narrativa atualizada acerca de enzimas hidrolíticas (agarases, amilases, celulases, esterases, lipases, peptidases e quitinases) isoladas do microbioma marinho. Esses biocatalisadores apresentam propriedades bioquímicas únicas, como halotolerância, extremos de pH, temperatura e barofilicidade, que os tornam atraentes para uso em diversos setores industriais, estimulando futuras aplicações biotecnológicas. Considerando o vasto repertório enzimático dos diversos membros das comunidades microbianas vivendo no ambiente marinho, salienta-se a imprescindibilidade de acessar esse habitat, mais especificamente essas unidades biocatalíticas. Com a devida atenção voltada ao viés industrial, novos biocatalisadores serão descobertos com sucesso nas comunidades microbianas marinhas, desde a confirmação de sua atividade até a comprovação de seu emprego prático nos setores de interesse e geração de propriedade intelectual

The agarase system of saccharophagus degradans strain 2-40 : analysis of the agarase system and protein localization

Saccharophagus degradans (formerly "Microbulbifer degradans") strain 2-40 is a Gram-negative marine bacterium isolated from the Chesapeake Bay. Analysis of 16s rDNA sequence indicated that S. degradans is related to a group of marine proteobacteria adept at degrading complex polysaccharides (CPs). S. degradans can depolymerize at least ten CPs including agarose. Agarose, an algal galactan, is degraded by few organisms. The agarase system of S. degradans was shown to be composed of five enzymes AgaA, AgaB, AgaC, AgaD and AgaE. These proteins contain glycoside hydrolase domains GH16, GH50 and GH86. S. degradans is the only organism known to collectively encode agarases with at least one of these domains. Unusual for agarases, AgaB and AgaE also contain multiple type-six carbohydrate binding modules. Furthermore, AgaE contains four thrombospondin type-three repeats whose function in prokaryotic proteins were unknown. The predicted agarases were characterized using a variety of methods including genomics, biochemical assays, proteomics and a newly described mutagenic technique. Agar degradation by S. degradans includes two depolymerases, AgaB and AgaC, a B-agarase II (AgaE) and a possible a-neoagarobiose hydrolase (AgaA). AgaB was found to be freely secreted while AgaC and AgaE were surface associated. AgaC is a predicted lipoprotein while AgaE did not have domains characteristic of surface localization. The Tsp-3 repeats, which are similar to repeats found on other cell surface enzymes, are the proposed cell surface anchoring sequences of AgaE

Enzimas produzidas por bactérias associadas às esponjas Oscarella (Porifera, Homoscleromorpha)

As enzimas produzidas pelos microrganismos associados às esponjas constituem uma fonte promissora de biocatalisadores de relevância industrial, provavelmente devido às suas características adaptativas comuns ao ambiente marinho, como resistência a variações de salinidade, temperatura e pH. Considerando a potencial aplicação das enzimas desses simbiontes microbianos em diversas indústrias, como a de biocombustíveis, detergentes e farmacêutica, o microbioma de esponjas pode se revelar em um profícuo reservatório de biocatalisadores inéditos. O presente estudo focou em rastrear a produção de enzimas por bactérias isoladas de esponjas Oscarella coletadas em diferentes locais: Cabo Frio (RJ), cavernas submarinas de Marselha (França) e do Arquipélago de Fernando de Noronha (RJ). As bactérias isoladas das esponjas em Cabo Frio (n = 43) e Marselha (n = 27) já faziam parte da coleção de bactérias do laboratório e foram reativadas em meio BHI para as próximas etapas experimentais. Por sua vez, o isolamento bacteriano das esponjas coletadas em Fernando de Noronha foi conduzido no presente trabalho. Os extratos das onze amostras de esponjas coletadas em Fernando de Noronha foram diluídos (10-3 a 10-5) e semeados em seis meios de cultura: BHI, BHI 1:10, Marine, Marine 1:10, Meio Mineral Mínimo adicionado de ágar e de alginato de sódio. Todas as bactérias isoladas foram armazenadas no mesmo meio de isolamento adicionado de 20% de glicerol (v/v). As bactérias foram mantidas na bacterioteca do laboratório a -20 ºC e posteriormente foram identificadas por MALDI-TOF MS. Todas 314 estirpes foram testadas quanto à produção de diversas enzimas através de meios adicionados de 1,5% de ágar e substratos específicos: LB contendo amido (amilase); carboximetilcelulose (CMC, holocelulase); Sierra acrescido de Tween 80 (lipase) ou de Tween 20 (esterase); Leite Desnatado (peptidase), Mineral Mínimo e Hu (agarase), Mineral Mínimo suplementado de alginato de sódio e Nakamura (alginato-liase). A análise da produção enzimática foi feita através da observação de halos das zonas de degradação dos substratos. O cálculo do Índice Enzimático (IE) foi realizado através da razão entre o diâmetro de hidrólise e o diâmetro do crescimento bacteriano em milímetros (mm), sendo considerada potencial produtora da enzima àquela estirpe que na presença de determinado substrato apresentou um IE ≥ 2,0. Todos os experimentos foram realizados em triplicata. Das 43 bactérias isoladas de esponjas Oscarella em Cabo Frio, 11,6% foram potenciais produtoras de lipase, 16,2% de agarase e 25,5% de alginato-liase. Das 27 bactérias isoladas de Oscarella de Marselha, 7,4% com IE ≥ 2 para celulase, 18,5% para lipase, 3,7% para agarase e 66,6% alginato-liase. Das 244 bactérias isoladas de Oscarella de Fernando de Noronha, foram detectadas 13,5% para amilase, 48,7% para celulase, 9% lipase, 8,1% esterase, 3,6% peptidase, 55,7% agarase e 12,7% alginato-liase. A diversidade enzimática detectada nas bactérias isoladas das esponjas evidencia a importância de estudos sobre as potencialidades biotecnológicas desse microbioma

Análise a produção de hidrolases por bactérias associadas à esponja marinha Plakina cyanorosea do litoral de Cabo Frio (RJ)

Os microrganismos associados às esponjas deparam-se com uma grande quantidade de matéria orgânica na água filtrada, liberando exoenzimas para conversão da mesma em nutrientes mais simples. Esses simbiontes constituem uma fonte para busca desses biocatalisadores com propriedades únicas, como halotolerância e resistência a pH e temperatura, sendo de interesse para aplicação em diversos setores industriais O estudo objetivou o rastreio da produção de hidrolases por bactérias isoladas da esponja recém-descrita para a costa brasileira Plakina cyanorosea (Porifera, Homoscleromorpha). 683 bactérias foram isoladas nos meios BHI, BHI 1:10, Marine, Marine 1:10 e Gauze I a partir de cinco espécimes de P. cyanorosea. Após reativação do estoque e cultivo em seu respectivo meio de isolamento a 25ºC por 24-96 h, 305 bactérias isoladas foram semeadas em meios sólidos para a verificação da produção das exoenzimas: agarase, amilase, celulase total, lipase, esterase e peptidase. Seguidamente a 96-120 h de incubação a 25ºC, a leitura dos resultados foi efetuada, com o cálculo do Índice Enzimático (IE), considerando um IE ≥ 2,0 característico de uma estirpe potencialmente produtora da exoenzima. A termoestabilidade e a estabilidade a acidez ou alcalinidade foi verificada em meio sólido para as potenciais estirpes agarolíticas. Os extratos brutos de agarase e celulase total foram obtidos por fermentação submersa, com a determinação da atividade pela dosagem de açúcares redutores. A coleção bacteriana se destacou quanto à produção de celulases, em que 87,8% das estirpes tiveram IE ≥ 2,0 e de agarases, em que de 22 bactérias pré-selecionadas, 19 foram capazes de gerar agarases em meio sólido, com 94,2% demonstrando IE ≥ 2,0. Das bactérias testadas, 62,6% para peptidases, 61,8% para amilases, 50,4% para esterases e 32,7% para lipases, exibiram IE ≥ 2,0, respectivamente. Um conjunto de 207 estirpes foram positivas para dois ou mais desses cinco grupos de enzimas analisados, com 73 (35,3%) exibindo atividade para duas, 65 (31,4%) para três, e 55 (26,6%) para quatro hidrolases, respectivamente, e 14 bactérias isoladas (6,7%) apresentaram resultado positivo para os cinco grupos pesquisados. Duas estirpes agarolíticas, Bacillus sp. PA2MD8 (IE = 3,7) e Pseudoalteromonas sp. PA2MD11 (IE = 3,9), e duas celulolíticas, Vibrio sp. PL1G11 (IE = 8,3) e Vibrio sp. PA3G1 (IE = 7,1), foram selecionadas como potenciais produtoras. A temperatura de 28°C e o pH 5,0 foram as condições com maiores valores de IE de agarase para Pseudoalteromonas sp. PA2MD11. Após 72 h de crescimento em meio de produção mínimo otimizado, tal estirpe exibiu atividade de 0,345 U/mL. As seis hidrolases investigadas tiveram sua produção confirmada nas bactérias isoladas de P. cyanorosea, notoriamente as polissacaridases. Com base nesses resultados preliminares, o bacterioma cultivável dessa esponja marinha se notabiliza como uma promissora fonte de enzimas com aplicações relevantes nos âmbitos biotecnológico e industrial

Macroalgas como matéria-prima para a cultura de bactérias marinhas

Mestrado em Biotecnologia - Biotecnologia Industrial e AmbientalAlginate, laminarin and mannitol amount up to 60% of dry weight in brown macroalgae. The presence of alginate and laminarin-degrading enzymes and mannitol metabolic machinery have been confirmed by Matís, a partner in European BlueGenics project. Thus, in a biorefinery perspective, R. marinus can potentially perform the saccharification and fermentation of brown macroalgae carbohydrates to yield commercial valuable biocompounds, as thermostable enzymes and glycosidic carotenoids. Rhodothermus marinus is a moderate thermophilic (65ºC) and slight halophilic (1.0% NaCl) marine bacterium. Therefore, one of the objectives of this project was to decrease the NaCl concentration in the fermentation medium, since chloride leads to a lower equipment lifetime due to stainless steel corrosion of bioreactors. The main objective of this work was the study of the bacterium R. marinus pattern of growth when cultivated in the main brown macroalgal carbohydrates. This work was performed with five R. marinus strains, two of which were successfully acclimatized to cultivation in Medium 166, cryopreserved in glycerol and recultivated in liquid media, being subject of study in the assays with different carbon and sodium sources in shake flask. The growth studies with different carbon sources suggested that (i) strain 5 presented higher glucose consumption and growth, even though none of the strains consumed all the glucose available in the media; (ii) although none of strains consumed mannitol, strain 5 seemed to be more robust to its presence; and (iii) the growth differences between the controls and the assays with alginate and pretreated alginate were not significant enough to infer if any alginate consumption occurred. It was tested a partial and total substitution of NaCl by Na2SO4. The process was not successful, since Na2SO4 seem to represent a stress factor to both R. marinus strains. Interestingly, the strain 5, when cultivated in Medium 166 containing only a half of NaCl standard concentration, presented a similar growth pattern to control. In the operational conditions imposed in shake flask cultivations containing two tested brown macroalgae (orginial and pretreated) as feedstock for growth, mannitol was not consumed. It was not possible to monitor the alginate and laminarin saccharification and fermentation. Although, the results showed that brown macroalgae are a potential feedstock under the biorefinery concept, since some R. marinus growth was observed. The more promising result to BlueGenics project was obtained from shake flask cultivations of strain 5 in Medium 166 with 0.500% NaCl and 10.0 g.L-1 glucose, since the growth with low chloride content determinates the feasibility of the scale-up of the process to bioreactor . Because of that, the assay was validated in 3L controlled bioreactor. The process presented a μmax of 0.208 h-1, a maximum biomass concentration of 8.75 gX.L-1, a volumetric biomass production rate of 0.295 g.L-1.h-1 and a volumetric glucose uptake rate of 0.293 g.L-1.h-1. Some feeding strategies were tested but further assays have to be performed in order to optimize the bioprocess

Transcriptional regulation by sigma factor phosphorylation controls polymyxin resistance and swarming behavior in Vibrio parahaemolyticus

A major form of bacterial transcriptional regulation occurs by the exchange of the primary σ factor of the RNA polymerase with alternative ECF σ factors, which generally are retained in an inactive state by sequestration into σ/anti-σ factor complexes (until they are needed). Using Vibrio parahaemolyticus as a model organism, we report a novel mechanism of transcriptional regulation, which instead relies on intrinsically inactive ECF σ factors that in turn rely on σ factor phosphorylation for interaction with the RNA polymerase. Particularly, we show that upon polymyxin stress, the threonine kinase PknT phosphorylates the σ factor EcfP, resulting in EcfP activation and expression of an essential polymyxin resistance regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. EcfP is intrinsically inactive and unable to bind RNA polymerase due to the absence of a negatively charged DAED motif in this region. Our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP for interaction with RNA polymerase and activation of target gene expression. Regulation of gene expression by phosphorylation of ECF σ factor is likely a widespread mechanism in bacteria, presenting a new paradigm in transcriptional regulation. One of the unique features of V. parahaemolyticus is that it exhibits a dual lifestyle. In liquid environments, where the bacteria are free-living, they exist as short swimmer cells with a single polar flagellum. However, upon encountering solid surfaces, the bacteria differentiate into highly elongated swarmer cells that are characterized by the presence of numerous peritrichous lateral flagella. Here, we report the involvement of the aforementioned threonine kinase/ECF σ factor system, namely PknT/EcfP, in regulation of swarming behavior. Strikingly, our findings indicate that this regulatory role depends on a phosphorylation-driven mechanism. We also provide evidence for the role of two other proteins encoded by genes present within the same operon as that of pknT and ecfP, namely VP0054 and VP0056, in regulation of swarming behavior. Our findings also reveal several key targets such as transporters and proteins involved in certain biosynthetic processes that are regulated by PknT. This is the first time that STKs have been implicated in swarming behavior in bacteria