Hydrolytic enzymes such as chitinases and glucanases are implicated in plant defense responses against fungal pathogens. These enzymes are responsible for the breakdown of chitin and glucan, two major components of the fungal cell walls. Genes encoding these enzymes have been used to genetically engineer plants to enhance their protection against fungal pathogens. Western Australia has over 4000 endemic plant species and a largely unknown fungal biota. Given that fungi possessing chitinases and glucanases with novel activities have been isolated in other parts of the world, we propose that fungi from Western Australian soils may possess novel biochemical/enzymatic activities. The aims of this research project were to isolate chitinolytic and glucanolytic fungi from soil and to clone the genes encoding for chitinase and glucanase enzymes. To achieve these aims, fungi with activity against chitin and glucan were isolated, the activity quantified by colorimetric and inhibition assays and gene fragments with homology to known chitinase and glucanase genes were isolated and their sequences determined. Soil fungi were isolated from five locations in and around the Perth Metropolitan area of Western Australia with the use of a medium containing Rose Bengal that eliminates all actinomycetes and most bacteria and reduces the growth of fast growing mold colonies. Forty-one isolates were obtained by this method. Twenty four chitinolytic and glucanolytic fungal isolates were identified by growing them on chitin-containing media to select for those species that utilised chitin/glucan as a carbon source. These were assayed for production of exo- and endochitinolytic and glucanolytic enzymes. Enzyme activity was compared between crude and dialysed supernatants. Exochitinase activity was determined in the supernatants of 4-day old fungal cultures by the release of p-nitrophenol from p-nitrophenyl-N-acetyl-â-D glucosaminide. The supernatants were measured for endochitinase activity determined by the reduction of turbidity of suspensions of colloidal chitin. Glucanase activity was determined by release of reducing sugar (glucose) from laminarin. Supernatants from eleven of the twenty four isolates showed significant levels of enzyme activity. Eleven isolates were assayed for activity against purified cell walls of phytopathogenic fungi. Activity was determined by measuring reducing sugars in the fungal supernatants against cell wall preparations of six economically important plant pathogens. Chitinolytic activity was detected in seven isolates against cell wall preparations of Botrytis cinerea and Rhizoctonia solani, in four isolates against Fusarium solani and Sclerotinia sclerotium; in five isolates against Ascochyta faba and in six isolates against Leptosphaeria maculans. Similarly glucanolytic activity was detected in eight isolates against B. cinerea, in seven against R. solani, in two against F. solani, in three against S. sclerotium and A. faba and in one against L. maculans. The supernatants derived from the isolates were used in a bioassay to determine growth inhibition against live B. cinerea spores by measuring turbidity reduction. Growth inhibition was measured against a control (B. cinerea, grown in medium with no added supernatant). Boiled supernatant did not inhibit the growth of B. cinerea spores but there was 100% inhibition by the crude supernatant from ten of the twenty four isolates. Similarly, supernatants were used to assess growth inhibition against live mycelia cultures of F. solani and S. sclerotium. Growth inhibition of F solani ranged from 9- 59%, boiled and crude supernatants respectively whilst growth inhibition of S. sclerotium ranged from 46-75%, boiled and crude supernatants respectively. Two partial chitinase genes from the soil filamentous ungus Trichoderma asperellum,(ChiA and ChiB) and a novel glucanase gene from the filamentous fungus Aspergillus (Glu1) were cloned. ChiA, was 639 bp long, encoding 191 amino acids with identity to other chitinase genes. Two highly conserved regions, characteristic of glycosyl hydrolases from family 18, were present. ChiB, was 887 bp long and encoded a 293 amino acid sequence that was closely related to an endochitinase gene from the filamentous fungus Trichoderma asperellum. The two highly conserved regions corresponding to the substrate binding and active sites that characterise the glycosyl hydrolases from family 18, also found in ChiA, were found in this gene. Glu1 was 2844 bp long and encoded a 948 amino acid sequence that shared high identity with a â-1, 3-glucanase from the filamentous fungus Aspergillus oryzae. The sequence contained conserved regions found in glycosyl hydrolases from family 17 that encode for substrate binding, N-terminal sequences and putative asparagine linked glycosylation sites. The partial putative sequence ChiA is probably a pseudogene because it has two inframe stop codons. However, once the entire sequence of ChiB is known, both ChiB and the novel glucanase gene Glu1 could be useful contenders for engineering resistance in crop plants.

There are six known secretion systems in Gram negative bacteria, referred to as Type 1 to Type 6 respectively, which are dedicated to moving substrate across the outer membrane. Secretion systems are broadly separated into those that move their substrate across the cell envelope in a single translocation event (one-step systems), and those that are dependent on the Sec or Tat machineries for export to the periplasm (two-step systems). Serratia marcescens is an important opportunistic human pathogen and has gathered a lot of interest due to its repertoire of secreted proteins. These include the haem-scavenging protein HasA, which is secreted by a Type 1 secretion system, and the cytotoxic haemolysin ShlA, which is secreted as part of a two-partner Type 5 secretion system. Serratia marcescens also encodes a Type 6 secretion system, which is known to translocate at least six effector molecules directly into other bacterial target cells. Serratia marcescens is a model organism in terms of its ability to degrade the quite intractable polymer chitin, for which it produces three chitinase enzymes ChiA, ChiB, ChiC and a chitin-binding protein Cbp21, which hydrolyse the ß-1,4 link in the chitin chain and promote binding of chitinase to the chitin substrate respectively. These chitinolytic enzymes are utilised by S. marcescens for both basic physiology and also in pathogenesis. In this work, genetic, biochemical and proteomic approaches identified, for the first time, genes that are essential for the secretion of all three chitinases as well as Cbp21. A genetic screen identified genes encoding a holin-like membrane protein (ChiW) and a putative L-alanyl-D-glutamate endopeptidase (ChiX). Subsequent quantitative proteomics experiments and biochemical analyses established that ChiW and ChiX were required for secretion of the entire chitinolytic machinery. Chitinase secretion was observed to be blocked at a late stage in the mutant strains as normally secreted enzymes were found to accumulate in the periplasm, thus implicating ChiW and ChiX in a novel outer membrane protein translocation process. It is proposed that the bacterial genome-encoded holin-like protein and endopeptidase identified represent a putative secretion system utilised by Gram-negative bacteria. In addition to this, genes encoding the chitinolytic machinery and the putative secretion apparatus were shown to be bimodally regulated and co-ordinately expressed.

N-acetil-glucosamina é um composto de importância biotecnológica com grande potencial de aplicação nas áreas de farmácia, medicina e dermatologia. É atualmente produzido pela hidrólise química da quitina, o polissacarideo mais abundante no ambiente marinho e o principal constituinte do exoesqueleto dos artrópodes. No entanto, os processos geralmente utilizados são prejudiciais ao meio ambiente, têm baixo rendimento e alto custo. Este estudo demonstra um dos mais eficientes processos de produção enzimática de N-acetil-glucosamina a partir de -quitina utilizando quitinases bacterianas como uma alternativa sustentável aos processos atuais e isolados de A. caviae com grande potencial para produção de quitinases e derivados de quitina. / N-acetyl-glucosamine (GlcNAc) is a compound of biotechnological importance with great application potential in the areas of pharmacy, medicine and dermatology. GlcNAc is currently produced by the chemical hydrolysis of chitin, the polysaccharide most abundant in the marine environment and the principal constituent of the exoskeleton of arthropods. However, the processes generally used are environmentally unfriendly, have low yield and high cost. This study demonstrates the most efficient processes for enzymatic production of N-acetyl-glucosamine from -chitin using bacterial chitinases as a sustainable alternative to the current processes and A. caviae isolates with great potential for chitinases and chitin derivatives production.

Submitted by MARIA LAURA VIOLA AUGUSTO (marialauraa@gmail.com) on 2018-06-15T17:16:40Z No. of bitstreams: 1 augusto_mlv_dr_jabo.pdf: 1777889 bytes, checksum: 32d8b34f05ca40dec4ae93cca2a67527 (MD5) / Approved for entry into archive by Neli Silvia Pereira null (nelisps@fcav.unesp.br) on 2018-06-15T18:34:35Z (GMT) No. of bitstreams: 1 augusto_mlv_dr_jabo.pdf: 1777889 bytes, checksum: 32d8b34f05ca40dec4ae93cca2a67527 (MD5) / Made available in DSpace on 2018-06-15T18:34:35Z (GMT). No. of bitstreams: 1 augusto_mlv_dr_jabo.pdf: 1777889 bytes, checksum: 32d8b34f05ca40dec4ae93cca2a67527 (MD5) Previous issue date: 2018-05-14 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O controle da broca da cana, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), pode ser feito pela aplicação de inseticidas químicos, mas o uso dessa tática de manejo pode resultar em danos ambientais e ao homem. De forma alternativa e com efeitos negativos reduzidos, o manejo de D. saccharalis e outros insetos-praga pode ser feito pela utilização do entomopatógeno Bacillus thuringiensis. Além do uso de formulações de B. thuringiensis para o controle de pragas, um ou mais genes que codificam proteínas inseticidas Cry e Vip de B. thuringiensis estão presentes nas plantas transgênicas com resistência a insetos comercialmente disponíveis no Brasil. Apesar de alta adoção, efeitos colaterais reduzidos e eficiência das plantas Bt, o uso desta tecnologia pode resultar na seleção de populações de insetos resistentes às plantas transgênicas. Por isso, a busca por novas proteínas e combinações de proteínas com diferentes modos de ação para retardar a evolução da resistência deve ser constante. Nesse cenário, uma quitinase de B. thuringiensis apresenta potencial para ser incorporada em uma nova geração de plantas transgênicas. Quitinases são enzimas que apresentam domínios catalíticos para hidrólise de quitina e domínios ligantes em quitina (CBD) que podem estar envolvidos com a atividade inseticida. A atividade inseticida das quitinases é relacionada com a desestruturação da membrana peritrófica dos insetos e consequente perda de suas propriedades e funções. Além da descoberta de proteínas inseticidas com diferentes modos de ação, é de interesse que essas apresentem interações positivas com outras proteínas inseticidas. No presente trabalho, duas formas de uma quitinase de B. thuringiensis - ChiΔSP (sem o peptídio sinal) e ChiΔSP.CBD (sem o peptídio sinal e sem o domínio ligante em quitina) - foram produzidas de forma heteróloga em Escherichia coli para avaliações bioquímicas e biológicas sobre D. saccharalis. Ensaios bioquímicos das enzimas ChiΔSP e ChiΔSP.CBD indicam que a atividade quitinolítica sobre quitina coloidal de ChiΔSP (3,07±0,15 U/nmol) foi maior que de ChiΔSP.CBD (1,83±0,06 U/nmol), a ligação de ChiΔSP à quitina coloidal também foi superior à ligação de ChiΔSP.CBD e as maiores atividades (60-100% da atividade total) de ChiΔSP e ChiΔSP.CBD foram detectadas a 30 e 40°C em uma ampla faixa de pH (4,0-9,0). Após estimar a CL50 da proteína Vip3Aa42 sobre lagartas neonatas de D. saccharalis em 352,132 ng/cm2 de dieta, foram avaliadas interações entre Vip3Aa42 e ChiΔSP ou ChiΔSP.CBD. Os resultados indicam que a adição isolada de 2,41 nmol/cm2 de ChiΔSP ou ChiΔSP.CBD não resultou na mortalidade de lagartas de D. saccharalis. A combinação da CL50 de Vip3Aa42 com 2,41 nmol/cm2 de ChiΔSP apresentou efeito sinérgico para mortalidade das lagartas (mortalidade de 90%), mas nenhum efeito (aditivo, sinérgico ou antagônico) foi observado para a combinação da CL50 de Vip3Aa42 com 2,41 nmol/cm2 de ChiΔSP.CBD. A ausência do CBD em ChiΔSP.CBD e consequentes alterações bioquímicas impediu sinergismo de ChiΔSP.CBD com Vip3Aa42 sobre lagartas de D. saccharalis. / Chemical control is applied to manage the sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae). However, this control tactic may result in great damages to the environment and non-target species, including men. Alternativelly, the use of the entomopathogen Bacillus thuringiensis can be made to manage D. saccharalis and other insect pests with reduced undisarable side effects. Beyond the use of formulations of B. thuringiensis for pest control, one or more genes of insecticidal proteins from B. thuringiensis such as Cry and Vip are incorporated into commercially available transgenic plants with insect resistance in Brazil. Despite of the high adoption rate, reduced side effects, and high efficency, the intensive use of this technology may result in the selection of insect-resistant populations to transgenic plants. For this reason, the search for new genes with different modes of action to delay the evolution of resistance must be constant. In this scenario, a chitinase from B. thuringiensis has potential to be incorporated into a new generation of transgenic plants. Chitinases are enzymes of the family of glycosyl hydrolases which catalyze the hydrolysis of chitin. Chitinases present catalytic domains responsible for the hydrolysis of chitin and chitin-binding domains (CBD), both associated to insecticidal activity. Insecticidal activity of chitinases is related to the disruption of the peritrophic membrane of insects, which results in the loss of properties and functions of this structure. In addition to the discovery of insecticidal proteins with different modes of action, positive interactions with other insecticidal proteins is desirable for pest management. In the present work, two forms of a chitinase of B. thuringiensis - ChiΔSP (without signal peptide) and ChiΔSP.CBD (without signal peptide and without the chitin-binding domain) - were produced by heterologous expression in Escherichia coli for biochemical and biological analyses. Chitinolytic activity of ChiΔSP (3.07 ± 0.15 U/nmol) was higher than that observed for ChiΔSP.CBD (1.83 ± 0.06 U/nmol) towards colloidal chitin. The binding of ChiΔSP to colloidal chitin was also higher than that observed for ChiΔSP.CBD, and the ChiΔSP or ChiΔSP.CBD chitinolytic activity (60-100% of total chitinolytic activity) was detected at 30 and 40°C in a broad pH range (4.0-9.0). After estimating the LC50 of the Vip3Aa42 protein at 352,132 ng/cm2 of diet, interactions between combinations of Vip3Aa42 and ChiΔSP or ChiΔSP.CBD were evaluated. Results indicate that the adition of 2,41 nmol/cm2 of ChiΔSP or ChiΔSP.CBD do not result in larval mortality of D. saccharalis. The combination of the LC50 of Vip3Aa42 with 2,41 nmol/cm2 nmol of ChiΔSP resulted in a synergic effect (mortality of 90%), but no effect (aditive, sinergic or antagonic) was observed when the LC50 of Vip3Aa42 was combined to 2,41 nmol/cm2 nmol of ChiΔSP.CBD. Lack of CBD in ChiΔSP.CBD resulted in biochemical changes in this form of B. thuringiensis chitinase which impacted on the sinergic effect of this protein with Vip3Aa42 against D. saccharalis. / CNPq: 140093/2015-0

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-10-05T10:38:29Z No. of bitstreams: 2 Dissertação - Enio Saraiva Soares - 2016.pdf: 2392510 bytes, checksum: 37b84e9c9bc76eaf406e92f41d3828bb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-10-05T10:58:52Z (GMT) No. of bitstreams: 2 Dissertação - Enio Saraiva Soares - 2016.pdf: 2392510 bytes, checksum: 37b84e9c9bc76eaf406e92f41d3828bb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-05T10:58:52Z (GMT). No. of bitstreams: 2 Dissertação - Enio Saraiva Soares - 2016.pdf: 2392510 bytes, checksum: 37b84e9c9bc76eaf406e92f41d3828bb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-29 / Currently there are different approaches to synthesize and discover new compounds, but the pursuit of these products on biodiversity is still advantageous. In bioprospecting microorganisms, which often are seeking their properties that can be exploited in biotechnology products. This is the case of chitinases, enzymes that degrade chitin. Chitinases (EC 3.2.1.29) are glycosyl hydrolases type enzymes that specifically cleave β-1,4 bonds between N-acetylglucosamines units of chitin with sizes ranging from 20 kDa to 90 kDa. The main producers of chitinase are the bodies that have chitin in their cell wall or exoskeleton, such as insects, crustaceans, fungi, algae, among others. This study aimed to select and identify producing bacteria chitinase in soil samples from different coastal regions of southern Brazil. Seventeen soil samples, collected close to fishing for shellfish waste disposal sites, were prepared and seeded in four minimum culture medium containing colloidal chitin as the sole source of carbon and energy, incubated and the colonies were isolated and purified. After yielded a total of thirteen isolates that were submitted to enzymatic index test, stressed that four isolates. The four isolated genomic DNA was extracted, amplified and purified, and sequenced region encoding 16S rRNA of these organisms. Bacteria were then pooled and identified by construction of a phylogenetic tree. The results showed the presence of the species Paenibacillus illinoisensis and Paenibacillus chitinolyticus and two members of the genus Bacillus. Future studies may indicate the possibility of its use as a source of genes for biotechnological applications such as the production of new biopesticides. / Existem atualmente diferentes abordagens para se sintetizar e descobrir novos compostos, mas a busca desses produtos na biodiversidade ainda é vantajosa. Na bioprospecção de microrganismos, o que muitas vezes se busca são as suas propriedades que possam ser aproveitadas em produtos biotecnológicos. Esse é o caso das quitinases, enzimas capazes de degradar a quitina. As quitinases (EC 3.2.1.29) são enzimas do tipo glicosilhidrolases, com tamanhos que variam de 20 kDa até 90 kDa, que clivam especificamente as ligações β-1,4 entre unidades de N-acetilglicosaminas da quitina. Os principais produtores de quitinases são os organismos que possuem quitina no seu exoesqueleto ou parede celular, como insetos, crustáceos, fungos, algas, bactérias entre outros. O presente estudo teve como objetivo selecionar e identificar bactérias produtoras de quitinases em amostras de solos de diferentes locais litorâneos da região Sul do Brasil. Dezessete amostras de solo, coletadas próximo a locais de descarte de resíduos de crustáceos por pescadores, foram preparadas e semeadas em meio de cultura mínimo contendo quitina coloidal como única fonte de carbono e energia, incubadas e as colônias foram isoladas e purificadas. Ao fim obteve-se um total de treze isolados de bactérias, que foram submetidas ao teste de índice enzimático, que destacou desses quatro isolados. O DNA genômico de quatro isolados foi extraído, amplificado e purificado, sendo sequenciada a região codificadora do gene 16S rRNA destes microrganismos. As bactérias foram então agrupadas e identificadas pela construção de uma árvore filogenética. Os resultados mostraram a presença das espécies Paenibacillus illinoisensis e Paenibacillus chitinolyticus além de dois membros do gênero Bacillus. Estudos futuros poderão indicar a possibilidade de seu uso como fonte de genes para aplicação biotecnológica, como a produção de novos bioinseticidas.

Bactérias quitinolíticas são autóctones do ecossistema marinho e tem um importante papel no processo de degradação de quitina. Relativamente pouco é conhecido sobre a diversidade e potencial enzimático de bactérias quitinolíticas isoladas de ambientes tropicais costeiros. Amostras de água do mar e de plâncton foram coletadas no Canal de São Sebastião, Baixada Santista e Ubatuba. As bactérias quitinolíticas foram enumeradas e isoladas em meio mínimo contendo quitina coloidal e caracterizadas através de métodos fenotípicos e genotípicos. As maiores contagens de bactérias quitinolíticas foram observadas em amostras de água do mar e plâncton coletadas na Baixada Santista. A diversidade de bactérias quitinolíticas e o potencial de produção de quitinases foram influenciados pelo nível de contaminação fecal presente no ecossistema marinho. Uma maior diversidade foi encontrada em ambiente com médio e baixo impacto antropogênico, mas bactérias quitinolíticas isoladas de ambiente com alta atividade antropogênica mostraram os maiores valores de produção de quitinases. / Chitinolytic bacteria are autochthonous in marine ecosystems and have an important role in chitin degradation process. A very little is know about the diversity and enzymatic potential of chitinolytic bacteria isolated from coastal tropical environments. Seawater and plankton samples were collected at Canal de São Sebastião, Baixada Santista and Ubatuba. Chitinolytic bacteria were counted and isolated in minimal media containing colloidal chitin and characterized using phenotypic and genotypic methods. Highest counts of chitinolytic bacteria were observed in seawater and plankton samples collected at Baixada Santista. The diversity of chitinolytic bacteria and the potential of chitinases production were influenced by the level of fecal contamination present in the marine ecosystem. Highest diversity was found in environment with medium and low anthropogenic impact, but chitinolytic bacteria isolated from environment with high anthropogenic influences showed highest chitinases production.

Bactérias quitinolíticas são autóctones do ecossistema marinho e tem um importante papel no processo de degradação de quitina. Relativamente pouco é conhecido sobre a diversidade e potencial enzimático de bactérias quitinolíticas isoladas de ambientes tropicais costeiros. Amostras de água do mar e de plâncton foram coletadas no Canal de São Sebastião, Baixada Santista e Ubatuba. As bactérias quitinolíticas foram enumeradas e isoladas em meio mínimo contendo quitina coloidal e caracterizadas através de métodos fenotípicos e genotípicos. As maiores contagens de bactérias quitinolíticas foram observadas em amostras de água do mar e plâncton coletadas na Baixada Santista. A diversidade de bactérias quitinolíticas e o potencial de produção de quitinases foram influenciados pelo nível de contaminação fecal presente no ecossistema marinho. Uma maior diversidade foi encontrada em ambiente com médio e baixo impacto antropogênico, mas bactérias quitinolíticas isoladas de ambiente com alta atividade antropogênica mostraram os maiores valores de produção de quitinases. / Chitinolytic bacteria are autochthonous in marine ecosystems and have an important role in chitin degradation process. A very little is know about the diversity and enzymatic potential of chitinolytic bacteria isolated from coastal tropical environments. Seawater and plankton samples were collected at Canal de São Sebastião, Baixada Santista and Ubatuba. Chitinolytic bacteria were counted and isolated in minimal media containing colloidal chitin and characterized using phenotypic and genotypic methods. Highest counts of chitinolytic bacteria were observed in seawater and plankton samples collected at Baixada Santista. The diversity of chitinolytic bacteria and the potential of chitinases production were influenced by the level of fecal contamination present in the marine ecosystem. Highest diversity was found in environment with medium and low anthropogenic impact, but chitinolytic bacteria isolated from environment with high anthropogenic influences showed highest chitinases production.