Identification of a novel tailor-made chitinase from white shrimp fenneropenaeus merguiensis

Fenneropenaeus merguiensis (commonly named banana shrimp) is one of the most important farmed crustacean worldwide species for the fisheries and aquaculture industry. Besides its nutritional value, it is a good source of chitinase, an enzyme with excellent biological and catalytic properties for many industrial applications. In the present study, a putative chitinase-encoding cDNA was synthesized from mRNA from F. merguiensis hepatopancreas tissue. Subsequently, the corresponding cDNA was cloned, sequenced and functionally expressed in Escherichia coli, and the recombinant F. merguiensis chitinase (rFmCHI) was purified by His-tag affinity chromatography. The bioinformatics analysis of aminoacid sequence of rFmCHI displayed a cannonical multidomain architecture in chitinases which belongs to glycoside hydrolase family 18 (GH18 chitinase). Biochemical characterization revealed rFmCHI as a monomeric enzyme of molecular weight 52 kDa with maximum activity at 40 °C and pH 6.0 Moreover, the recombinant enzyme is also stable up to 60 °C, and in the pH range 5.0-8.0. Steady-state kinetic studies for colloidal chitin revealed KM, Vmax and kcat values of 78.18 μM, 0.07261 μM. min−1 and 43.37 s−1, respectively. Overall, our results aim to demonstrate the potential of rFmCHI as suitable catalyst for bioconversion of chitin waste

Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis

Due to the importance of chitinolytic enzymes for insect, nematode and fungal growth, they are receiving attention concerning their development as biopesticides or chemical defense proteins in transgenic plants and as microbial biocontrol agents. Targeting chitin associated with the extracellular matrices or cell wall by insect chitinases may be an effective approach for controlling pest insects and pathogenic fungi. The ability of chitinases to attack and digest chitin in the peritrophic matrix or exoskeleton raises the possibility to use them as insect control method. In this study, an insect chitinase cDNA from cotton leaf worm (Spodoptera littoralis) has been synthesized. Transgenic maize plant system was used to improve its tolerance against insects. Insect chitinase transcripts and proteins were expressed in transgenic maize plants. The functional integrity and expression of chitinase in progenies of the transgenic plants were confirmed by insect bioassays. The bioassays using transgenic corn plants against corn borer (Sesamia cretica) revealed that ~50% of the insects reared on transgenic corn plants died, suggesting that transgenic maize plants have enhanced resistance against S. cretica

Comparative Genomic Analysis of Chitinase and Chitinase-Like Genes in the African Malaria Mosquito (Anopheles gambiae)

Chitinase is an important enzyme responsible for chitin metabolism in a wide range of organisms including bacteria, yeasts and other fungi, nematodes and arthropods. However, current knowledge on chitinolytic enzymes, especially their structures, functions and regulation is very limited. In this study we have identified 20 chitinase and chitinase-like genes in the African malaria mosquito, Anopheles gambiae, through genome-wide searching and transcript profiling. We assigned these genes into eight different chitinase groupings (groups I–VIII). Domain analysis of their predicted proteins showed that all contained at least one catalytic domain. However, only seven (AgCht4, AgCht5-1, AgCht6, AgCht7, AgCht8, AgCht10 and AgCht23) displayed one or more chitin-binding domains. Analyses of stage- and tissue-specific gene expression revealed that most of these genes were expressed in larval stages. However, AgCht8 was mainly expressed in the pupal and adult stages. AgCht2 and AgCht12 were specifically expressed in the foregut, whereas AgCht13 was only expressed in the midgut. The high diversity and complexity of An. gambiae chitinase and chitinase-like genes suggest their diverse functions during different developmental stages and in different tissues of the insect. A comparative genomic analysis of these genes along with those present in Drosophila melanogaster, Tribolium castaneum and several other insect species led to a uniform classification and nomenclature of these genes. Our investigation also provided important information for conducting future studies on the functions of chitinase and chitinase-like genes in this important malaria vector and other species of arthropods

Molecular mechanisms of extracellular matrix dynamics at barrier tissues and control of molting in <em>Drosophila</em>

The insect epidermal cuticle acts as the exoskeleton and has crucial barrier functions, protecting against wounding, desiccation, invading pathogens and other environmental dangers. During the life of insects, the epidermal cuticle is periodically shed in a process called molting in order to adapt to increasing body growth. This requires coordinated cuticle rearrangement to reestablish the barrier functions. However, the molecular mechanisms of cuticle reorganization during molting and upon wounding are incompletely understood. Two proteins were identified which have key roles in epidermal cuticle assembly in Drosophila larvae. First, the chitin-binding protein Obstructor-A (Obst-A) was shown to act as a central scaffold for cuticle organization. Obst-A builds a core complex of enzymes and proteins involved in the formation and protection of chitinous structures, which form a major part of the cuticle. Thereby Obst-A mediates the build-up of an organized, stable cuticle. Second, the Chitinase 2 is essential for lamellar cuticle organization and was shown to be required for Obst-A function. Loss of both obst-A and Cht2 leads to similar defects in cuticle integrity and to death in larval stages. A structural analysis of the mutant larvae revealed that a prominent part of the cuticle, the chitin-matrix, is defective, leading to fragile cuticles at epithelial organs. On the molecular level, both Obst-A and Cht2 share an essential function in the so called cuticle assembly zone at the apical cell surface of epidermal cells. There, they contribute to proper chitin-matrix assembly and localization of enzymes and proteins that organize maturation and protection of newly synthesized cuticles. In addition, Obst-A was shown to contribute to the hormonal control of molting in early larvae by influencing the production of the steroid molting hormone 20-hydroxyecdysone (20E). Obst-A is expressed in the hormone-producing cells of the ring gland. Loss of obst-A results in reduced size and severe malformation of this gland and impairment of upregulation of genes involved in 20E biosynthesis and signaling. Altogether, new molecular factors in cuticle assembly and regulation of molting were characterized