Expression Of Chitinase Research Articles

CDNA cloning, expression, and antifungal activity of chitinase from Ficus microcarpa latex: difference in antifungal action of chitinase with and without chitin-binding domain.

A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.

Inclusion of Oat and Yeast Culture in Sow Gestational and Lactational Diets Alters Immune and Antimicrobial Associated Proteins in Milk.

Simple SummaryThis study investigated the impact that supplementing sow’s gestation and lactation feed with oat alone or together with brewer’s yeast has on milk proteins and piglet growth and health. Oat and yeast supplements increased abundance of several milk proteins involved in immune protection. Piglets born from either the oat- or yeast-supplemented sows had decreased incidence of diarrhea after weaning. The average birth weights for piglets born of dams that consumed Oat were significantly greater than those that did not. However, piglets born to sows that consumed yeast in combination with oat weighed less at weaning and gained the least amount of weight post-weaning. These data suggest that oat, and to a lesser extent, yeast, added to maternal diets during gestation and lactation can positively impact milk, growth, and health of offspring but given in combination can potentially negatively affect piglet weight gain.Maternal diet supplementation with pro- and prebiotics is associated with decreased incidence of diarrhea and greater piglet performance. This study investigated the impact adding whole ground oat as a prebiotic, alone or in combination with a probiotic, yeast culture (YC) (Saccharomyces cerevisiae), to sow gestation and lactation rations had on milk protein composition, piglet growth, and incidence of post-weaning diarrhea (PWD). Diets: control (CON), CON + yeast culture (YC) [5 g/kg], CON + oat (15% inclusion rate) (Oat) or CON+ YC [5 g/kg] + Oat (15%) were fed the last 30 days of gestation and throughout lactation (18–21 days). Shotgun proteome analysis of day 4 and 7 postpartum milk found 36 differentially abundant proteins (P-adj < 0.1) in both Oat and YC supplemented sows relative to CON. Notable was the increased expression of antimicrobial proteins, lactoferrin and chitinase in milk of Oat and YC sows compared to CON. The levels of IgA, IgM (within colostrum and milk) and IgG (within milk) were similar across treatments. However, colostral IgG levels in Oat-supplemented sows were significantly lower (p < 0.05) than that of the control sows, IgG from Oat-supplemented sows displayed greater reactivity to E. coli-antigens compared with CON and YC. Piglets from sows that consumed Oat alone or in combination weighed significantly more (p < 0.05) at birth compared to CON and YC. However, piglets in the Oat + YC group weighed less at weaning and had the lowest weight gain (p < 0.05) postweaning, compared with CON. Taken together with the observation that piglets of either YC- or Oat-fed sows had less PWD compared to CON and YC+ Oat suggests that Oat or YC supplementation positively impacts piglets through expression of certain milk-associated immune and antimicrobial proteins.

Effect of Extracts of Cupressus macrocarpa and Alpinia officinarum on Expression of Chitinase and FGF Receptor Genes in Spodoptera frugiperda Sf9 Cell Line

Effect of Extracts of Cupressus macrocarpa and Alpinia officinarum on Expression of Chitinase and FGF Receptor Genes in Spodoptera frugiperda Sf9 Cell Line

Combined strategies to improve the expression of acidic mammalian chitinase in Pichia pastoris for the production of N, N'-diacetylchitobiose

Acidic mammalian chitinase (AMCase) could hydrolyze chitin into (GlcNAc)2 at pH 2.0. Therefore, it was essential to develop an engineered strain with a high-yield AMCase. Herein, multiple strategies including codon optimization, multiple expression cassettes insertion, co-expression of helper proteins, were developed to construct a recombinant Pichia pastoris with the high-level secretion of AMCase. The recombinant strain harboring four copies of codon-optimized AMCase-encoding gene ChiD and co-expression of HAC1 presented the highest chitinase activity of 570 U/L in high density fermentation, which was 934 folds higher than that in E. coli reported before. Then, the recombinant AMCase was applied to hydrolyze colloidal chitin with a conversion rate of 90 % at pH 2.0, which could save half of the water consumption during the preparation of colloidal chitin. Finally, we also demonstrated that the hydrolysate of AMCase could promote seed germination of wheat and rice, improving the growth of roots and seedling.

Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC50 value of 0.81μM.

Rice transcription factor WRKY114 directly regulates the expression of OsPR1a and Chitinase to enhance resistance against Xanthomonas oryzae pv. oryzae

Rice (Oryza sativa L.) is a global staple crop, but its yield is severely threatened by bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo). The OsWRKY transcription factor family play a central role in innate plant immunity against Xoo, but the various biological functions of a large number of OsWRKYs remain to be understood. We characterized the role of OsWRKY114 against Xoo. OsWRKY114 has transcriptional activity in yeast and localizes in the nucleus. When OsWRKY114 is overexpressed in transgenic plants they show enhanced disease resistance against Xoo compared with wild types. By using genetic- and cell-based functional analyses, we showed OsWRKY114 directly associates with the promoters of OsPR1a and Chitinase and increases the promoter activities. These results suggest that OsWRKY114 enhances the innate immunity of Asian rice against Xoo through direct activation of defense genes that include OsPR1a and chtinase. This is the first report to functionally characterize OsWRKY114 in Xoo infection.

Transcriptional Changes of Plant Defense-Related Genes in Response to Clavibacter Infection in Pepper and Tomato.

Pepper and tomato plants infected with two Clavibacter species, C. capsici and C. michiganensis have shown different patterns of disease development depending on their virulence. Here, we investigated how pepper and tomato plants respond to infection by the high-virulent or low-virulent Clavibacter strains. For this, we chose two strains of each Clavibacter species to show different virulence level in the host plants. Although low-virulent strains showed less disease symptoms, they grew almost the same level as the high-virulent strains in both plants. To further examine the response of host plants to Clavibacter infection, we analyzed the expression patterns of plant defense-related genes in the leaves inoculated with different strains of C. capsici and C. michiganensis. Pepper plants infected with high-virulent C. capsici strain highly induced the expression of CaPR1, CaDEF, CaPR4b, CaPR10, and CaLOX1 at 5 days after inoculation (dai), but their expression was much less in low-virulent Clavibacter infection. Expression of CaSAR8.2 was induced at 2 dai, regardless of virulence level. Expression of GluA, Pin2, and PR2 in tomato plants infected with high-virulent C. michiganensis were much higher at 5 dai, compared with mock or low-virulent strain. Expression of PR1a, Osmotin-like, Chitinase, and Chitinase class 2 was increased, regardless of virulence level. Expression of LoxA gene was not affected by Clavibacter inoculation. These results suggested that Clavibacter infection promotes induction of certain defense-related genes in host plants and that differential expression of those genes by low-virulent Clavibacter infection might be affected by their endophytic lifestyle in plants.

DNA methylation suppresses chitin degradation and promotes the wing development by inhibiting Bmara-mediated chitinase expression in the silkworm, Bombyx mori

BackgroundDNA methylation, as an essential epigenetic modification found in mammals and plants, has been implicated to play an important role in insect reproduction. However, the functional role and the regulatory mechanism of DNA methylation during insect organ or tissue development are far from being clear.ResultsHere, we found that DNA methylation inhibitor (5-aza-dC) treatment in newly molted pupae decreased the chitin content of pupal wing discs and adult wings and resulted in wing deformity of Bombyx mori. Transcriptome analysis revealed that the up-regulation of chitinase 10 (BmCHT10) gene might be related to the decrease of chitin content induced by 5-aza-dC treatment. Further, the luciferase activity assays demonstrated that DNA methylation suppressed the promoter activity of BmCHT10 by down-regulating the transcription factor, homeobox protein araucan (Bmara). Electrophoretic mobility shift assay, DNA pull-down and chromatin immunoprecipitation demonstrated that Bmara directly bound to the BmCHT10 promoter. Therefore, DNA methylation is involved in keeping the structural integrity of the silkworm wings from unwanted chitin degradation, as a consequence, it promotes the wing development of B. mori.ConclusionsThis study reveals that DNA methylation plays an important role in the wing development of B. mori. Our results support that the indirect transcriptional repression of a chitin degradation-related gene BmCHT10 by DNA methylation is necessary to keep the proper wing development in B. mori.

Dataset on recombinant expression of an ancient chitinase gene from different species of Leishmania parasites in bacteria and in Spodoptera frugiperda cells using baculovirus.

The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.

Cloning and Expression of a New Chitinase from Carnivorous Plants Drosera capensis

A novel chi19 gene encoding the chitinase, belonging to the glycosyl hydrolase family 19 from the predatory plant Drosera capensis is cloned and expressed in the E. coli bacterial system. The amino acid sequence of the translated enzyme consists of 325 a.a. divided into four functional parts: the N-terminal signal sequence and of the catalytic and chitin-binding domains that were linked by the –S–P- linker. The chi19 gene is expressed in two forms: with and without a chitin-binding domain. Refolding is carried out and homogeneous soluble forms are obtained for both variants of the enzyme. The chitinases obtained exhibit predominantly specific activity towards crystalline chitin, with the optimal pH level ranging from 5.0 to 5.5 and the optimum temperature in the range of 52 to 55°C for both forms of the enzyme. The catalytic activity of the full-sized form of chitinase was 360 U/g with a 64% decrease of the catalytic activity for the form with the chitin-binding domain removed.

Expression of Specific Class I Chitinase mRNA Levels in Different Grape Varieties and Their Antimicrobial Activity

The aim of this study was to compare the natural variability of the expression of class I chitinase gene in grapes of Vitis vinifera L. varieties as well as investigate their antimicrobial activity. Further, the effect of antimicrobial activity in chitinase expression was analyzed. Matured berries of thirteen grape varieties were used in the study—Alibernet, Dornfelder, Cabernet Sauvignon, Blaufränkisch, Sauvignon Blanc, Welschriesling, Weisser Riesling, Irsai Oliver, Pinot Blanc, Pálava, Müller-Thurgau, Grűner Veltliner, and Feteasca Regala, grown in the Small Carpathians wine region of Slovakia, Vrbové. Chitinase I gene expression was found to be similar in most of the analyzed varieties with the except of two varieties, where the expression of chitinase I was significantly higher—Pinot Blanc and Savignon Blanc. By comparing the varieties between the lowest and highest expression fold change, they were statistically different in the level of its chitinase I gene expression. Grape pomace extracts of analyzed Vitis vinifera L. were effective against Gram-positive, Gram-negative bacteria and yeasts, but a weak correlation tendency was found between chitinase I gene expression and antimicrobial activity, only against Staphylococcus aureus subsp. aureus, Candida albicans, and Candida glabrata.

The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

Wheat (Triticum aestivum L.) is an important staple crop for global human. The necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot, a devastating disease of wheat. Herein, we identified RcMEP1, a zinc metalloproteaseencoding gene from R. cerealis genomic sequences, and characterized its pathogenesis function. RcMEP1 expressed at markedly-high levels during R. cerealis infection process to wheat. The predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif (HEVGHWLGLYH). The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death, and that the predicted signal peptide functions and is required for secretion and cell death-induction. The purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2O2 accumulation, and to inhibit expression of host chitinases when infiltrated into wheat and N. benthamiana leaves, while the M43 domain-deleting peptide and negative control lacked the capacity. Moreover, compared with the control pretreatment, the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat, whereas the M43 domain-deleting peptide failed. These results suggest that RcMEP1 acted as an important pathogenicity factor during R. cerealis infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of RcMEP1. This study provides insights into pathogenesis role of M43 domain-containing metalloproteases during R. cerealis infection to wheat.

Identification of key genes and modules in response to Cadmium stress in different rice varieties and stem nodes by weighted gene co-expression network analysis

Soil cadmium (Cd) pollution threatens food safety. This study aimed to identify genes related to Cd accumulation in rice. Low- (Shennong 315, short for S315) and high- (Shendao 47, short for S47) Cd-accumulative rice cultivars were incubated with CdCl2·2.5H2O. RNA-seq and weighted gene co-expression network analysis (WGCNA) were performed to identify the modules and genes associated with Cd-accumulative traits of rice. After Cd stress treatment, the Cd content in various tissues of S315 was significantly higher than that of S47. In the stem nodes, the Cd distribution results of the two varieties indicated that the unelongated nodes near the root (short for node A) had a stronger ability to block Cd transfer upwards than the panicle node (short for node B). Cd stress induced huge changes in gene expression profiles. After analyzing the differentially expressed genes (DEGs) in significantly correlated WGCNA modules, we found that genes related to heavy metal transportation had higher expression levels in node A than that in node B, such as Copper transporter 6 (OS04G0415600), Zinc transporter 10 (OS06G0566300), and some heavy-metal associated proteins (OS11G0147500, OS03G0861400, and OS10G0506100). In the comparison results between S315 and S47, the expression of chitinase (OS03G0679700 and OS06G0726200) was increased by Cd treatment in S315. In addition, OsHSPs (OS05G0460000, OS08G0500700), OsHSFC2A (OS02G0232000), and OsDJA5 (OS03G0787300) were found differentially expressed after Cd treatment in S315, but changed less in S47. In summary, different rice varieties have different processes and intensities in response to Cd stress. The node A might function as the key tissue for blocking Cd upward transport into the panicle via vigorous processes, including of heavy metal transportation, response to stress, and cell wall.

Ocean acidification inhibits initial shell formation of oyster larvae by suppressing the biosynthesis of serotonin and dopamine

Ocean acidification has severely affected the initial shell formation of marine bivalves during their larval stages. In the present study, it was found that dopamine (DA) content in early D-shape larvae was significantly higher than that in trochophore and D-shape larvae, while the serotonin (5-HT) content in early D-shape larvae and D-shape larvae was obviously higher than that in trochophore. Incubation of trochophore with 5-HT or DA could accelerate the formation of calcified shell, and the treatments with selective antagonists of receptors for 5-HT and DA (Cg5-HTR-1 and CgD1DR-1) obviously inhibited the formation of calcified shells. When oyster larvae were subjected to an experimental acidified treatment (pH 7.4), the biosynthesis of 5-HT and DA was inhibited, while the mRNA expression levels of the components in TGF-β pathway were significantly up-regulated in D-shape larvae. Moreover, the phosphorylation of TIR and the translocation of smad4 were hindered upon acidification treatments, and the expression patterns of chitinase and tyrosinase were completely reverted. These results collectively suggested that monoamine neurotransmitters 5-HT and DA could modulate the initial shell formation in oyster larvae through TGF-β smad pathway by regulating the expression of tyrosinase and chitinase to guarantee the chitin synthesis for shell formation. CO2-induced seawater acidification could suppress the biosynthesis of 5-HT and DA, as well as the activation of TGF-β smad pathway, which would subvert the expression patterns of chitinase and tyrosinase and cause the failure of initial shell formation in oyster early D-shape larvae.

Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002

BackgroundChitinases are enzymes which degrade β-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance.MethodsPrimers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out.ResultsThe exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg−1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0–10.0 and 25–45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and β-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products.ConclusionsThis is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.

Expression Activity of Allergen Coding Genes in Grape Varieties Used for Tokaj Wine Production

Abstract Vitis vinifera L. is adapted to a very variable range of climates but it mostly grows in the temperate regions of continental Europe. In the Slovak Republic, the Tokaj wine region is one of the territories, where wine production is concentrated. Grape is a popular fruit and when processed, it is used as musts, juices or wine. Some people may suffer from allergic reactions to grapes. Up to now, endochitinases, lipid-transfer protein, and thaumatin were identified as grape allergens. In this study, expression of chitinase and thaumatin allergen was analysed in the grapes of Tokaj region varieties – Hachat Lovelin, Tokay and Muskat Blanc. Expression changes were calculated by the delta delta Ct method. Expression differences of chitinase were found to be similar in these varieties. Thaumatin was found to be variable in its transcription.

Genome-Wide Identification of M35 Family Metalloproteases in Rhizoctonia cerealis and Functional Analysis of RcMEP2 as a Virulence Factor during the Fungal Infection to Wheat.

Rhizoctonia cerealis is the causal pathogen of the devastating disease, sharp eyespot, of the important crop wheat (Triticum aestivum L.). In phytopathogenic fungi, several M36 metalloproteases have been implicated in virulence, but pathogenesis roles of M35 family metalloproteases are largely unknown. Here, we identified four M35 family metalloproteases from R. cerealis genome, designated RcMEP2–RcMEP5, measured their transcriptional profiles, and investigated RcMEP2 function. RcMEP2-RcMEP5 are predicted as secreted metalloproteases since each protein sequence contains a signal peptide and an M35 domain that includes two characteristic motifs HEXXE and GTXDXXYG. Transcription levels of RcMEP2-RcMEP5 markedly elevated during the fungus infection to wheat, among which RcMEP2 expressed with the highest level. Functional dissection indicated that RcMEP2 and its M35 domain could trigger H2O2 rapidly-excessive accumulation, induce cell death, and inhibit expression of host chitinases. This consequently enhanced the susceptibility of wheat to R. cerealis and the predicated signal peptide of RcMEP2 functions required for secretion and cell death-induction. These results demonstrate that RcMEP2 is a virulence factor and that its M35 domain and signal peptide are necessary for the virulence role of RcMEP2. This study facilitates a better understanding of the pathogenesis mechanism of metalloproteases in phytopathogens including R. cerealis.

Effects of ammonia on shrimp physiology and immunity: a review

AbstractThe rapid expansion in shrimp farming comes with problems such as inadequate culture technology, disease outbreak, water pollution and other environmental degradation‐related problems. Prominent among these is the level of ammonia, which has been rising steadily in the last couple of years, mainly due to farming practices and other anthropogenic activities, and therefore has had consequent effects on marine life, especially shrimp. While optimal ammonia levels are a good source of nitrogen for marine phytoplankton that increases the level of dissolved oxygen in water as well serves as food for shrimp, high levels of ammonia are harmful to shrimp. When shrimp pond ammonia levels go beyond the tolerance limits, it inhibits chitinase expression, moulting, growth, phenoloxidase and haemolymph antimicrobial activity, thereby attenuating shrimp innate immune response. In this review, we bring together recent information on the effects of ammonia stress on the growth, physiology, biochemistry, ammonia‐metabolizing enzymes and immunity of shrimp. We also propose areas of research that can be explored in the breeding of new ammonia‐tolerant and disease‐resistant shrimp with robust immune and/or physiological systems that could withstand environmental stress and pathogens.

Functional expression of recombinant hybrid enzymes composed of bacterial and insect’s chitinase domains in E. coli

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect’s domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one’s leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other’s chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi’s LCL connected with BtChi’s ChBD (MbLCL-BtChBD) and BtChi’s LCL connected with MbChi’s ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

Correlation of acidic mammalian chitinase expression with disease severity in patients with moderate/severe persistent allergic rhinitis

Aim of the studyTo assess the level of acidic mammalian chitinase (AMCase) expression and IL-8 in nasal inferior turbinate mucosa in patients with mild and moderate to severe allergic rhinitis (AR).Material and methodsParticipants in this case-control study were divided into three groups, including patients with moderate and severe persistent allergic rhinitis, cases with mild forms of persistent AR, and control or healthy group. We obtained biopsies of nasal inferior turbinate mucosa from all participants. Expression of AMCase and IL-8 mRNAs were evaluated by real-time polymerase chain reaction (PCR). The serum levels of AMCase and IL-8 were determined by ELISA. The number of eosinophils per field, blood eosinophils, total serum IgE levels, and specific serum IgE levels were measured. Patients’ clinical manifestations were assessed by total nasal syndrome score (TNSS).ResultsExpression of AMCase and IL-8 in patients with moderate and severe perineal allergic rhinitis were significantly elevated compared to the control group and patients with mild persistent allergic rhinitis. Serum levels of AMCase and IL-8 were associated with specific IgE, nasal eosinophil count, and TNSS.ConclusionsAccording to the results of this study, there might be a relationship between the expression of AMCase and IL-8 in nasal turbinate mucosa and the severity of allergic rhinitis.