Extraction of chitin from edible crab shells of Callinectes sapidus and comparison with market purchased chitin

Abstract Chitin and its derived products have immense economic value due to their vital role in various biological activities as well as biomedical and industrial application. Insects, microorganism and crustaceans are the main supply of chitin but the crustaceans shell like shrimp, krill, lobsters and crabs are the main commercial sources. Chitin content of an individual varies depending on the structures possessing the polymer and the species. In this study edible crabs shells (Callinectes sapidus) were demineralized and deproteinized resulting in 13.8% (dry weight) chitin recovery from chitin wastes. FTIR and XRD analyses of the experimental crude as well as purified chitins revealed that both were much comparable to the commercially purchased controls. The acid pretreatment ceded 54g of colloidal chitin that resulted in 1080% of the crude chitin. The colloidal chitin was exploited for isolation of eighty five chitinolytic bacterial isolates from different sources. Zone of clearance was displayed by the thirty five isolates (41.17%) succeeding their growth at pH 7 on colloidal chitin agar medium. Maximum chitinolytic activity i.e. 301.55 U/ml was exhibited by isolate JF70 when cultivated in extracted chitin containing both carbon and nitrogen. The study showed wastes of blue crabs can be utilized for extraction of chitin and isolation of chitinolytic bacteria that can be used to degrade chitin waste, resolve environmental pollution as well as industrial purpose.

Resumo A quitina e seus produtos derivados têm imenso valor econômico devido ao seu papel vital em várias atividades biológicas, bem como em aplicações biomédicas e industriais. Insetos, microrganismos e crustáceos são o principal suprimento de quitina, mas a casca dos crustáceos como camarão, krill, lagosta e caranguejo são as principais fontes comerciais. O conteúdo de quitina de um indivíduo varia dependendo das estruturas que possuem o polímero e da espécie. Neste estudo, as cascas de caranguejos comestíveis (Callinectes sapidus) foram desmineralizadas e desproteinizadas, resultando em 13,8% (peso seco) de recuperação de quitina a partir de resíduos de quitina. As análises de FTIR e XRD do bruto experimental, bem como das quitinas purificadas, revelaram que ambas eram muito comparáveis aos controles adquiridos comercialmente. O pré-tratamento com ácido cedeu 54 g de quitina coloidal que resultou em 1.080% da quitina bruta. A quitina coloidal foi analisada para isolamento de 85 isolados bacterianos quitinolíticos de diferentes fontes. A zona de eliminação foi exibida pelos 35 isolados (41,17%) que sucederam seu crescimento a pH 7 em meio de ágar de quitina coloidal. A atividade quitinolítica máxima, ou seja, 301,55 U / ml, foi exibida pelo isolado JF70 quando cultivado em quitina extraída contendo carbono e nitrogênio. O estudo mostrou que resíduos de caranguejos azuis podem ser utilizados para extração de quitina e isolamento de bactérias quitinolíticas que podem ser usadas para degradar resíduos de quitina, resolver a poluição ambiental e também para fins industriais.

Extraction of chitin from edible crab shells of Callinectes sapidus and comparison with market purchased chitin / Extração de quitina de cascas de caranguejo comestíveis de Callinectes sapidus e comparação com quitina adquirida no mercado

Chitin and its derived products have immense economic value due to their vital role in various biological activities as well as biomedical and industrial application. Insects, microorganism and crustaceans are the main supply of chitin but the crustaceans shell like shrimp, krill, lobsters and crabs are the main commercial sources. Chitin content of an individual varies depending on the structures possessing the polymer and the species. In this study edible crabs’ shells (Callinectes sapidus) were demineralized and deproteinized resulting in 13.8% (dry weight) chitin recovery from chitin wastes. FTIR and XRD analyses of the experimental crude as well as purified chitins revealed that both were much comparable to the commercially purchased controls. The acid pretreatment ceded 54g of colloidal chitin that resulted in 1080% of the crude chitin. The colloidal chitin was exploited for isolation of eighty five chitinolytic bacterial isolates from different sources. Zone of clearance was displayed by the thirty five isolates (41.17%) succeeding their growth at pH 7 on colloidal chitin agar medium. Maximum chitinolytic activity i.e. 301.55 U/ml was exhibited by isolate JF70 when cultivated in extracted chitin containing both carbon and nitrogen. The study showed wastes of blue crabs can be utilized for extraction of chitin and isolation of chitinolytic bacteria that can be used to degrade chitin waste, resolve environmental pollution as well as industrial purpose.

A quitina e seus produtos derivados têm imenso valor econômico devido ao seu papel vital em várias atividades biológicas, bem como em aplicações biomédicas e industriais. Insetos, microrganismos e crustáceos são o principal suprimento de quitina, mas a casca dos crustáceos como camarão, krill, lagosta e caranguejo são as principais fontes comerciais. O conteúdo de quitina de um indivíduo varia dependendo das estruturas que possuem o polímero e da espécie. Neste estudo, as cascas de caranguejos comestíveis (Callinectes sapidus) foram desmineralizadas e desproteinizadas, resultando em 13,8% (peso seco) de recuperação de quitina a partir de resíduos de quitina. As análises de FTIR e XRD do bruto experimental, bem como das quitinas purificadas, revelaram que ambas eram muito comparáveis aos controles adquiridos comercialmente. O pré-tratamento com ácido cedeu 54 g de quitina coloidal que resultou em 1.080% da quitina bruta. A quitina coloidal foi analisada para isolamento de 85 isolados bacterianos quitinolíticos de diferentes fontes. A zona de eliminação foi exibida pelos 35 isolados (41,17%) que sucederam seu crescimento a pH 7 em meio de ágar de quitina coloidal. A atividade quitinolítica máxima, ou seja, 301,55 U / ml, foi exibida pelo isolado JF70 quando cultivado em quitina extraída contendo carbono e nitrogênio. O estudo mostrou que resíduos de caranguejos azuis podem ser utilizados para extração de quitina e isolamento de bactérias quitinolíticas que podem ser usadas para degradar resíduos de quitina, resolver a poluição ambiental e também para fins industriais.

Isolation and identification of proteolytic, amylolytic, lipolytic, and chitinolytic bacteria from shrimp waste / Aislamiento e identificación de bacterias proteolíticas, amilolíticas, lipolíticas y quitinolíticas de residuos de langostinos

Abstract Bacteria and microbial enzymes are biocatalysts and can be used as an alternative to industrial chemical processes. The present study focused on isolating and identifying bacterial strains from shrimp waste, that produce amylases, lipases, proteases and chitinases with potential use on shrimp waste treatment. Thirtytwo bacterial strains were isolated, phenotypically characterized, and identified by the API system and the molecular analysis of the 16S rDNA. It was found that 28.13% of the isolated bacterial strains had amylolytic capacity, 87.50% lipolytic, 96.88% proteolytic and 28.13% chitinolytic capacity on agar plates with specific substrates. The genera Bacillus, Burkholderia, Ochrobactrum, Vibrio, Pseudomonas and Shewanella were identified. Bacteria with enzymatic capacities isolated in the present study, could be used to obtain by-products from shrimp waste as well as other industrial applications.

Resumen Las bacterias y enzimas microbianas son biocatalizadores y pueden ser usadas como alternativa en los procesos químicos industriales. El presente estudio se centró en aislar e identificar cepas bacterianas a partir de desechos de langostinos, capaces de producir amilasas, lipasas, proteasas y quitinasas, que tuvieran potencial aplicación en el tratamiento de residuos de langostinos. Se aisló treinta y dos cepas bacterianas, caracterizadas fenotípicamente e identificadas mediante el sistema API 20 y mediante análisis molecular basado en el ADNr 16S. Se encontró que el 28.13% de las cepas bacterianas aisladas tenían capacidad amilolítica, 87.50% lipolítica, 96.88% proteolítica y 28.13% capacidad quitinolítica en placas de agar con sustratos específicos. Los géneros identificados fueron Bacillus, Burkholderia, Ochrobactrum, Vibrio, Pseudomonas y Shewanella. Las bacterias con capacidades enzimáticas aisladas en el presente estudio, podrían ser usadas para obtener subproductos de los desechos de langostinos, así como en otras aplicaciones industriales.

Cloning and expression analysis of a chitinase gene PnCHI1 from Panax notoginseng / 中国中药杂志

Chitinases(EC3.2.1.14), which are present in various organisms, catalyze the hydrolytic cleavage of chitin and play a vital role in plant defense mechanisms against fungal pathogens.In addition, the chitinases are well known to regulate plant growth and development and are involved in programmed cell death(PCD).A chitinase expressed sequence tag(EST) was isolated from Panax notoginseng, and the full-length cDNA of this EST was cloned with the method of rapid amplification of cDNA ends and named as PnCHI1. PnCHI1 was 1 022 bp in length and contained an intact open reading frame(ORF) of 822 bp, a 26 bp 5'-untranslated region(UTR), and a 174 bp 3'-UTR.The predicted protein of PnCHI1 with 273 amino acid residues belongs to glycoside hydrolase family 19 and fell into the class IV of chitinases through phylogenetic analysis.QRT-PCR analysis showed that the expression of PnCHI1 was induced by methyl jasmonate, ethylene, H2O2, and salicylic acid.PnCHI1 was quickly induced after inoculation with Alternaria panax.Moreover, the expression level of PnCHI1 was increased after pretreatment with methyl jasmonate, and then the transcription level of PnCHI1was sharp increased after inoculation with Fusarium solani,and the highest transcription level was achieved at 4 h post inoculation.But the expression level of PnCHI1 in the sterile water pretreated P.notoginseng was increased gradually after inoculation with F.solani, and the highest expression level was achieved at 48 h post inoculation.All the results of present study indicated that PnCHI1 was involved in defense response of P.notoginseng against the F.solani and A.panax.

Evaluación de microorganismos con potencial de promoción de crecimiento vegetal y biocontrol de Spongospora subterranea / Evaluation of microorganisms with potential for plant growth promotion and biological control of Spongospora subterranea

La sarna polvosa de la papa es causada por el patógeno Spongospora subterranea, que disminuye la calidad y producción de tubérculos y facilita la entrada de otros patógenos. Esta enfermedad afecta las principales zonas productoras del mundo, debido a la falta de tratamientos efectivos contra el patógeno y al comercio de tubérculos-semilla infectados. Algunas investigaciones indican que los agentes biocontroladores podrían contribuir a reducir la actividad de S. subterranea a través de efectos sobre la viabilidad de sus quistosoros o mediante efectos estimulantes del crecimiento de la planta. En este estudio se emplearon bacterias aisladas del interior de raí­z, rizósfera y superficie de tubérculos de un cultivo de papa ( Solanum tuberosum variedad Diacol Capiro) y seleccionadas por su capacidad para producir indoles totales y quitinasas. En estudios paralelos se determinó su capacidad para promover la velocidad de germinación en brotes de tubérculos o para controlar S. subterranea en raíces y promover el crecimiento vegetal de plántulas en invernadero. La mayoria de los aislamientos evaluados incrementraron la longitud de brotes de tubérculos en el laboratorio. En el invernadero, en suelo no estéril y en presencia del patógeno, se encontró que 2 de los 10 aislamientos seleccionados por su capacidad para producir indoles totales y quitinasas, presentaron promoción de crecimiento vegetal y posible biocontrol del patógeno. Estos resultados sugieren un gran potencial para la selección de microorganismos biocontroladores y desarrollo de bioproductos a partir de recursos microbiológicos locales.

The powdery scab of potato is caused by the pathogen Spongospora subterranea which reduces the quality and yield of tubers and facilitates the establishment of other pathogens. This disease affects main potato production zones in the world, because there is not a completely effective and available control method against the disease, and due to the trading of infested seed tubers. Some research suggests that biological control agents could reduce the activity of S. subterranea through effects on the viability of their cystosoris or zoospores or by stimulating plant growth. In this research, bacteria previously isolated, from inside the roots, the rhizosphere and tuber peel of potato plants (Solanum tuberosum var. Diacol Capiro), were used, and then selected according to their capacity for producing total indoles and chitinases. Here was tested in parallel studies the capacity of nine bacterial isolates differing in total indole and chitinase production, for its capacity at increasing tuber sprout length and in promoting plant growth and biocontrol of S. subterranea . Inoculated in excised minitubers in laboratory most indole producing isolates tested resulted in increased tuber sprout length. In the greenhouse assay, in non-sterile soil and under a high pathogen pressure, two of the ten isolates selected because for their ability to produce total indoles and chitinases, showed plant growth promotion and possible biocontrol of the pathogen. These results suggest a great potential for the selection of biocontrol microorganisms and the development of new bioproducts from local microbial resources.

Selección de cepas nativas con actividad quitino-proteolítica de bacillus sp aisladas de suelos tropicales / Selecting native Bacillus sp strains having chitinolytic and proteolytic activity which have been isolated from tropical soils

El objetivo de este trabajo fue la selección de cepas nativas del género Bacillus con actividad quitinolíticay proteolítica, en suelo tropical en la costa de Oaxaca, México. Se aislaron 150 cepas, de las cuales 22fueron seleccionadas por presentar actividad quitinolítica y proteolítica. Dicha actividad se evaluó porla formación de halo de hidrólisis alrededor de la colonia en medios de cultivo suplementados con quitinacoloidal al 5% y leche descremada al 1% respectivamente. Las cepas LUM B001, B003, B013, B015y B065 presentaron mayor actividad quitinolítica y proteolítica, por lo que tienen el potencial para serevaluadas en control biológico de hongos fitopatógenos. Se encontró al género Bacillus distribuido ensuelos cultivados y no cultivados, no se encontraron diferencias estadísticas según el cultivo establecido(P<0,05), sin embargo se encontraron diferencias significativas (P<0,05) entre las zonas estudiadas, correspondiendolas menores recuperaciones de cepas a los terrenos del municipio de Tututepec, Oaxaca.

This work was aimed at selecting native strains from the Bacillus genus having chitinolytic and proteolytic activityfrom soil from the tropical coast of Oaxaca, Mexico. 150 strains were isolated, 22 of which were selectedas they presented chitinolytic and proteolytic activity. Such activity was assessed by the formation of a hydrolysishalo around the colony in culture media supplemented with 5% colloidal chitin and 1% skimmed milk.The LUM B001, B003, B013, B015 and B065-chitin strains presented higher quitinolytic and proteolytic activity,thereby having the potential for being evaluated in the biological control of phytopathogenic fungi. TheBacillus genus was found in cultivated and uncultivated soils; no statistical differences were found accordingto established crop (p <0.05); however, significant differences (p <0.05) were found between the areas beingstudied regarding the smaller amount of strains collected from land in the municipality of Tututepec, Oaxaca.

Chitin, Chitinases and Chitinase-like Proteins in Allergic Inflammation and Tissue Remodeling

Chitin, the second most abundant polysaccharide in nature after cellulose, consist exoskeleton of lower organisms such as fungi, crustaceans and insects except mammals. Recently, several studies evaluated immunologic effects of chitin in vivo and in vitro and revealed new aspects of chitin regulation of innate and adaptive immune responses. It has been shown that exogenous chitin activates macrophages and other innate immune cells and also modulates adaptive type 2 allergic inflammation. These studies further demonstrate that chitin stimulate macrophages by interacting with different cell surface receptors such as macrophage mannose receptor, toll-like receptor 2 (TLR-2), C-type lectin receptor Dectin-1, and leukotriene B4 recepptor (BLT1). On the other hand, a number of chitinase or chitinase-like proteins (C/CLP) are ubiquitously expressed in the airways and intestinal tracts from insects to mammals. In general, these chitinase family proteins confer protective functions to the host against exogenous chitin-containing pathogens. However, substantial body of recent studies also set light on new roles of C/CLP in the development and progression of allergic inflammation and tissue remodeling. In this review, recent findings on the role of chitin and C/CLP in allergic inflammation and tissue remodeling will be highlighted and controversial and unsolved issues in this field of studies will be discussed.

Beta-1,3 Glucanases e quitinases: aplicação na lise de leveduras e inibição de fungos / Beta-1,3 glucanases and chitinases: application in the yeast cell lysis and fungi inhibition

Objetivou-se, no presente trabalho, a aplicação de β-1,3 glucanases e quitinases da linhagem Cellulosimicrobium cellulans 191 na lise de leveduras e inibição de fungos, respectivamente. O delineamento experimental mostrou que as melhores condições para a lise de Saccharomyces cerevisiae KL-88 pela β-1,3 glucanase foi pH 6,5 e 35ºC. As células de leveduras incubadas por 10 h em frascos sem agitação mostraram-se mais susceptíveis à lise pela ação da enzima. Foi obtido maior lise da levedura quando a suspensão de células foi submetida ao tratamento com β-1,3 glucanase e cisteína 1mM. A enzima invertase intracelular ou ligada à célula de S. cerevisiae KL-88 e K. marxianus NCYC 587 foi extraída após tratamento da suspensão celular com β-1,3 glucanase, sendo que o tratamento prévio das leveduras com a enzima aumentou a susceptibilidade das células à lise com ultra-som. A preparação de quitinase foi capaz de formar halos de inibição de alguns fungos.

The aim of this work was the application of β-1,3 glucanases and chitinases by Cellulosimicrobium cellulans 191 strain on yeast cell lysis and fungi inhibition, respectively. The experimental design study showed that the best conditions to Saccharomyces cerevisiae KL-88 lysis by β-1,3 glucanase extract were pH 6,5 and 35ºC. This study also demonstrated that the yeast cells were more susceptible to lysis after 10 h of cultivation in flasks without agitation. Lysis activity was increased when S. cerevisiae KL-88 cell suspension was treated with β-1,3 glucanase and cystein 1mM. The enzyme invertase of S. cerevisiae KL-88 and Kluyveromyces marxianus NCYC 587 was extracted after treatment of cell suspension with β-1,3 glucanase and the previous treatment of yeasts with the enzyme, increased the susceptibility to lysis when ultrasonic treatment was used. The chitinase presented growth inhibition halos for some of the fungi.