Chitinase Production Research Articles

Growth promotion and biocontrol activity of Nocardiopsis dassonvillei strain YM12: an isolate from coastal agricultural land of Khambhat

Ecological niche of coastal agricultural land of Khambhat, Gujarat (India) has an impact of marine and terrestrial process. Present study explored soil microbial biomass from a unique niche, which can contribute to the sustainable agricultural approach. Total 68 isolates with percent ratio of 42:58 of Actinobacteria:bacteria were isolated from the alkaline soil with pH 8.1. Twenty-eight isolates were identified as cultivable Actinobacteria associated with the order Streptomycetales, Streptosporengiales, micrococcales, Pseudonocardials and it belonged to 14 different genera. All the strains were screened primarily for Plant growth promoting traits like IAA production, ammonia production, phosphate as well as potassium solubilisation, glucanase and chitinase production. Strain YM12 having maximum traits positive for promoting growth and biocontrol activity was further used and characterized. Strain YM12 was identified as Nocardiopsis dassonvillei which showed 262.4 µg ml−1 IAA production, 7.2 µmol ml−1 Ammonia production along with mineral solubilisation capacity (Phosphate—288 ppm, Potassium—48.82 ppm). Apart from having plant growth promoting activity, strain showed inhibition of Fusarium growth which might be due to production of 0.004 μM ml−1 Chitinase and 0.0093 µM ml−1 Glucanase enzyme. Further it was tested for growth promotion of Pearl Millet (Pennisetum glaucum) and showed 20–30% increased growth as compared to control plants.

Lactobacillus crustorum ZHG 2-1 as novel quorum-quenching bacteria reducing virulence factors and biofilms formation of Pseudomonas aeruginosa

Quorum quenching (QQ) activity of crude extract (CE) from Lactobacillus crustorum ZHG 2-1 against Pseudomonas aeruginosa was evaluated at three sub-minimum inhibitory concentrations (MIC) 1.0, 2.0 and 3.0 mg/mL. Agar diffusion method results showed that CE can degrade N-homoserine lactone (AHLs) by 37.1–87.6% at three sub-MICs. Moreover, the degradation activity was further confirmed by gas chromatography-mass spectrometer (GC-MS). The inhibition rate of biofilms and the removal of preformed biofilms of the CE alone and in combination with azithromycin were determined by 96-well plate method. The results demonstrated that CE can significantly enhance the sensitivity of azithromycin to biofilms, and the inhibition rate were 58.4–90.3% at three sub-MICs. Scanning electron microscopy (SEM) images demonstrated that the CE combined with azithromycin can inhibit biofilms formation and reduce the thickness of preformed biofilms. Quantitative real-time polymerase chain reaction analysis indicated that the expressions of quorum sensing (QS)-regulated genes lasI/R and rhlI/R were down-regulated. Additionally, the production of virulence factors, including protease, chitinase, pyocyanin, rhamnolipid, alinate and exopolysaccharide, were also suppressed by CE at three sub-MICs. And all the above effects showed a dose dependent. These results demonstrated that ZHG 2-1 can consider as a novel potential QQ bacteria to against foodborne pathogenic bacteria.

Chitin Heterodisaccharide, Released from Chitin by Chitinase and Chitin Oligosaccharide Deacetylase, Enhances the Chitin-Metabolizing Ability of Vibrio parahaemolyticus.

Vibrio parahaemolyticus RIMD2210633 secretes both chitinase and chitin oligosaccharide deacetylase and produces β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) from chitin. Previously, we reported that GlcNAc-GlcN induces chitinase production by several strains of Vibrio harboring chitin oligosaccharide deacetylase genes (T. Hirano, K. Kadokura, T. Ikegami, Y. Shigeta, et al., Glycobiology 19:1046-1053, 2009). The metabolism of chitin by Vibrio was speculated on the basis of the findings of previous studies, and the role of chitin oligosaccharide produced from chitin has been well studied. However, the role of GlcNAc-GlcN in the Vibrio chitin degradation system, with the exception of the above-mentioned function as an inducer of chitinase production, remains unclear. N,N'-Diacetylchitobiose, a homodisaccharide produced from chitin, is known to induce the expression of genes encoding several proteins involved in chitin metabolism in Vibrio strains (K. L. Meibom, X. B. Li, A. Nielsen, C. Wu, et al., Proc Natl Acad Sci U S A 101:2524-2529, 2004). We therefore hypothesized that GlcNAc-GlcN also affects the expression of enzymes involved in chitin metabolism in the same manner. In this study, we examined the induction of protein expression by several sugars released from chitin using peptide mass fingerprinting and confirmed the expression of genes encoding enzymes involved in chitin metabolism using real-time quantitative PCR analysis. We then confirmed that GlcNAc-GlcN induces the expression of genes encoding many soluble enzymes involved in chitin degradation in Vibrio parahaemolyticus Here, we demonstrate that GlcNAc-GlcN enhances the chitin-metabolizing ability of V. parahaemolyticusIMPORTANCE We demonstrate that β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) enhances the chitin-metabolizing ability of V. parahaemolyticus Members of the genus Vibrio are chitin-degrading bacteria, and some species of this genus are associated with diseases affecting fish and animals, including humans (F. L. Thompson, T. Iida, and J. Swings, Microbiol Mol Biol Rev 68:403-431, 2004; M. Y. Ina-Salwany, N. Al-Saari, A. Mohamad, F.-A. Mursidi, et al., J Aquat Anim Health 31:3-22, 2019). Studies on Vibrio are considered important, as they may facilitate the development of solutions related to health, food, and aquaculture problems attributed to this genus. This report enhances the current understanding of chitin degradation by Vibrio bacteria.

Immobilization, Thermodynamic studies and Application of Chitinase enzyme from Penicillium chrysogenum

This study was conducted to investigate the production of an efficient chitinase from a marine isolate. Accordingly, a marine fungus was isolated from the soft coral; Heteroxenia fuscescens and identified by molecular 18S rRNA technique as Penicillium chrysogenum. The isolate was chitinase producer (331, 8 U/mL) and the result was confirmed by TLC and HPLC. The enzyme was partially purified and immobilized completely in a modified bentonite. The free and immobilized enzymes recorded a maximum relative activity (100%) at 50 and 55°C, respectively, the activation energy of the immobilized enzyme was 10.60 Kjmol -1 with 40% reduction than that of the free enzyme. Thermal and pH stability studies showed that the enzyme maintained its complete activity at (50 and 55) °C for 120 min and at pH 5 and 6. The deactivation rate constant (Kd), deactivation energy (Ed), half-life (t1/2) , D, ∆ G, ∆ H, ∆ S values were calculated for both enzyme forms and insured the success of the immobilization process in enzyme protection. The immobilized enzyme was reused for three cycles without loss in activity. Toxicological studies of chitinase against 3rd instar larvae of Cx. pipiens L revealed high mortality rates.

Screening and characterization of actinomycetes isolated from soybean rhizosphere for promoting plant growth

Abstract. Fatmawati U, Meryandini A, Nawangsih AA, Wahyudi AT. 2019. Screening and characterization of actinomycetes isolated from soybean rhizosphere for promoting plant growth. Biodiversitas 20: 2970-2977. Actinomycetes which colonized plant rhizosphere has a vital role in improving plant growth by producing Indole-3-Acetic Acid (IAA). The aim of this study was to evaluate the potency of actinomycetes isolated from soybean rhizosphere as promoter agents for plant growth in vitro. Fifty actinomycetes isolates were successfully isolated from soybean rhizosphere. Based on the colorimetric methods, 35 isolates can produce IAA in various concentration, in the range of 0.46-30.6 mg/L. Seed germination assay using Ragdoll methods revealed that 26 isolates significantly promoted germination parameters, including the hypocotyl and the radicular length, the number of the lateral roots, and dry weight of the plant. Also, 14 from 26 isolates showed phosphate solubilizing activity in different phosphate-solubilizing index ranging from 1.25-2.62. Eight isolates were able to grow in N-free medium, indicating that these isolates have the ability in fixing nitrogen. Out of 23 from 26 isolates were detected to produce siderophore. All the tested isolates show chitinase production except ASR 55. Based on the observed parameters, it showed that there are four potential isolates (ASR 46, ASR 58, ASR 75 and ASR 76) as promising plant-growth promoters, phosphate solubilizer, nitrogen fixer, siderophore and chitinase producer. Based on the result of 16S rRNA sequence analysis, four potential isolates were identified as Streptomyces spp. in different taxa of strains and species.

Potential biocontrol and superlative plant growth promoting activity of indigenous Bacillus mojavensis PB-35(R11) of soybean (Glycine max) rhizosphere

Disease control using microbes that exhibit beneficial effects on plants to strengthen the host is a foremost requirement in agriculture. The aim of the present study is to search for an effective biocontrol agent against Rhizoctonia solani endowed with high plant growth potential. A total of 95 bacterial strains were isolated from the soybean plant rhizosphere and screened in vitro against R. solani by dual culture technique, revealing that strain PB-35(R11) was the most efficient for controlling R. solani with 54.835% inhibition. Isolate PB-35 was identified by 16S ribosomal RNA (rRNA) gene sequencing as Bacillus mojavensis. It was observed that, in order to effectively control R. solani in vitro, B. mojavensis produced volatile metabolites. Fourier-transform infrared (FTIR) analysis revealed the presence of aldehyde (CHO) group (1739.79 cm−1), acetyl group (1896.03 cm−1), and cyanide group (2360.87 cm−1) in the crude extract of isolate PB-35(R11). Furthermore, the siderophore, indole acetic acid (IAA), catalase, oxidase, and chitinase production ability as well as phosphate solubilization potential of PB-35(R11) make it beneficial for crop growth and soil biofortification.

Optimization of Chitinase Production by <i>Trichoderma</i> <i>virens</i> in Solid State Fermentation Using Response Surface Methodology

Physical factor for chitinase production by Trichodermavirens was first carried out using screening factor of 2-level factorial. The design was employed by selecting incubation time, temperature, moisture substrate, pH, inoculums size and concentration ammonium sulphate as a model factors. The result of 2-level factorial design experiment showed thal all three independent variable have significant effect on chitinase production. The physical factor was further optimized using Central Composite Design in which response surface was generated later from the derived model. An experimental design of three variables including various incubation time, temperature and moisture substrate were created using Design Expert® Software, Version 6.0.4 The design consist of 20 experiments, which include 6 replicate at center points. The optimal value for each variable are incubation time sixth days, temperature 27.83°C and moisture substrate 54% with predicted chitinase activity of 0.48738 U/g of dry substrate. These predicted parameters were tested in the laboratory and the final chitinase activity obtained was 0.48864 U/g of dry substrate, which is similar to the predicted value. The obtained value of the chitinase production was 0.48738 U/g IDS, which was 1.2 fold higher than that of the 2-level factorial design (0.261 U/gds)

Bioproduction of N-acetyl-glucosamine from colloidal α-chitin using an enzyme cocktail produced by Aeromonas caviae CHZ306.

N-acetyl-D-glucosamine (GlcNAc) is an important amino-monosaccharide with great potential for biotechnological applications. It has traditionally been produced by the chemical hydrolysis of chitin, despite certain industrial and environmental drawbacks, including acidic wastes, low yields and high costs. Therefore, enzymatic production has gained attention as a promising environmentally-friendly alternative to the chemical processes. In this study we demonstrate the GlcNAc bioproduction from colloidal α-chitin using an enzyme cocktail containing endochitinases and exochitinases (chitobiosidases and N-acetyl-glucosaminidases). The enzyme cocktail was extracted after fermentation in a bioreactor by Aeromonas caviae CHZ306, a chitinolytic marine bacterium with great potential for chitinase production. Hydrolysis parameters were studied in terms of temperature, pH, enzyme and substrate concentration, and reaction time, achieving over 90% GlcNAc yield within 6h. The use of colloidal α-chitin as substrate showed a substantial improvement of GlcNAc yields, when compared with β-chitin and α-chitin polymorphs. Such result is directly related to a significant decrease in crystallinity and viscosity from natural α-chitin, providing the chitinase with greater accessibility to the depolymerized chains. This study provides valuable information on the GlcNAc bioproduction from chitin using an enzymatic approach, addressing the key points for its production, including the enzyme cocktail composition and the substrate structures.

Peat substrate amended with chitin modulates the N-cycle, siderophore and chitinase responses in the lettuce rhizobiome

Chitin is a valuable peat substrate amendment by increasing lettuce growth and reducing the survival of the zoonotic pathogen Salmonella enterica on lettuce leaves. The production of chitin-catabolic enzymes (chitinases) play a crucial role and are mediated through the microbial community. A higher abundance of plant-growth promoting microorganisms and genera involved in N and chitin metabolism are present in a chitin-enriched substrate. In this study, we hypothesize that chitin addition to peat substrate stimulates the microbial chitinase production. The degradation of chitin leads to nutrient release and the production of small chitin oligomers that are related to plant growth promotion and activation of the plant’s defense response. First a shotgun metagenomics approach was used to decipher the potential rhizosphere microbial functions then the nutritional content of the peat substrate was measured. Our results show that chitin addition increases chitin-catabolic enzymes, bacterial ammonium oxidizing and siderophore genes. Lettuce growth promotion can be explained by a cascade degradation of chitin to N-acetylglucosamine and eventually ammonium. The occurrence of increased ammonium oxidizing bacteria, Nitrosospira, and amoA genes results in an elevated concentration of plant-available nitrate. In addition, the increase in chitinase and siderophore genes may have stimulated the plant’s systemic resistance.

Bioconversion of Colloidal Chitin Using Novel Chitinase from Glutamicibacter uratoxydans Exhibiting Anti-fungal Potential by Hydrolyzing Chitin Within Fungal Cell Wall

Chitin is a unique structural exopolysaccharide abundantly found in nature. This exopolysaccharide has a unique chemical structure that acts as a protective outermost covering for most of the crustaceans in aquatic ecosystem. This fortification is because of the insoluble nature of this exopolysaccharide which consist of a linear chain of β-(1→4)-linked-N-acetylglucosamine units. Chitin is hydrolyzed with the help of a hydrolase known as chitinase. Variety of microbial species have been explored for chitinase production. Chitinolytic microbial species can be alternatively used for degradation of chitin instead of chemical treatment in agricultural sector. This biological approach has lesser environmental impact because of its apparently safe nature. In the current study, bioprospecting of chitinase producing species was conducted and different chitinolytic bacterial strains were screened for chitinase production which could have anti-fungal potential. Bacterial isolates were identified based on polyphasic approach and the enzyme production was optimized using one-variable-at-a-time technique. Hyphal extension method was used for determination of anti-fungal potential of chitinase.Glutamicibacter uratoxydans was indigenously isolated and identified for chitinase production. G. uratoxydans is a novel bacterial species which has not been previously explored to produce chitinase or other hydrolases. G. uratoxydans biosynthesized chitinase utilizing colloidal chitin as a sole source of carbon. The chitinase biosynthesized by G. uratoxydans is effectively potent against Aspergillus fumigatus thus, suggesting that this extracellular enzyme could be used for the treatment of fungal infection caused by filamentous fungi.

Draft genome of Streptomyces sp. strain 130 and functional analysis of extracellular enzyme producing genes.

Streptomyces sp. strain 130 possesses multiple uncharacterized extracellular enzyme producing genes. Enzymes from these genes may fulfil the intense demand of stable and effective extracellular enzymes in various industries. Taxonomy of Streptomyces sp. strain 130 was validated by FAME analysis. Strain 130 was screened for the presence of chitinase producing genes of family 18 and 19 using SC1F/SC2R and F19F2/F19R primer sets respectively. Whole genome sequencing was done using Illumina Next Seq500 system. In the analysis of draft genome of Streptomyces sp. strain 130, the genome size was found to be 7.1Mb. Blastn and NCBI-conserved domain search tool were used to find similarity percentage with genes in existing database and enzyme family respectively. Ten chitinase, six xylanase and one cellulase producing genes were present in draft genome. Among the ten chitinase producing genes, two were belonging to GH19 family and other eight to GH18 family chitinase. Six out of ten chitinase producing genes were uncharacterized and one belonged to family GH18_PF-ChiA (PF-ChiA is a chitinase found in the hyperthermophilic archaea, prokaryotes). In case of xylanase, four out of six (GH9, 43, 10 and 11 enzyme family) were not showing nucleotide based similarity with any characterized gene. The study of reported genome sequence will help us to identify gene sequence of characterized and uncharacterized extracellular enzyme producing genes. Cloning of each gene and enzyme activity assay of their products will reveal the activity and stability at different variables; and resulting products may have huge applications at industrial scale.

Modulation of hydrolytic profiles of cell-bound and cell-free exoenzymes in Antarctic marine bacterial isolates

Microbial degradation of high molecular weight organic matter in the marine environment is dependent on the synthesis and activity of hydrolytic exoenzymes. These can be found both associated to the cell (i.e., attached to cell-wall or in the periplasmic space) and cell-free, dissolved in the surrounding waters. Recent evidences suggest that exoenzymes location is linked to different foraging strategies. ‘Selfish’ bacteria use cell-bound enzymes to obtain large oligomers which are then further degraded in the confined periplasmic space, whereas cell-free enzymes are thought to be advantageous for particle-attached prokaryotes, consequently ‘sharing’ the hydrolysis product with other members of the degrading consortium. We examined whether different bacterial isolates exhibit different exoenzymatic activity profiles when exposed to different growth condition. Seven bacterial strains, isolated from the Ross Sea, were screened for the production of β-glucosidase, alkaline-phosphatase, lipase, chitinase and leucine aminopeptidase, assaying the hydrolysis rates of both cell-bound and cell-free exoenzymatic fractions. Furthermore, to test whether bacteria growing on particles are more prone to produce cell-free exoenzymes, we set up a controlled experiment, amending one of the bacterial isolates with phytodetritus, in order to promote the colonization of aggregates. Cell specific hydrolytic rates were highly variable and different isolates expressed different dominant hydrolytic activities. These results demonstrate a specialization for different substrates in different bacteria, further suggesting that some of the tested isolates may have an intrinsic potential to copiously produce cell-free exoenzymes. Moreover, our results suggest that this specialization is not strictly genetically determined but varies according to growth conditions. The experiment performed with phytodetrital particles highlighted an increasing contribution of the dissolved exoenzymatic activity in samples bearing aggregates and associated bacteria. Also, the tested isolate further modulated its hydrolytic machinery, expressing a different enzymatic profile when exposed to phytodetrital particles. The continuation of these experimental activities, testing the response of different bacterial isolates to different substrates, will help to determine the conditions under which different hydrolysis patterns develop, deepening the current knowledge on the organic matter cycling in the ocean.

Antagonistic potential of bacterial endophytes and induction of systemic resistance against collar rot pathogen Sclerotium rolfsii in tomato

A diverse pool of 310 bacterial endophytes isolated from tomato plants growing in the Indo-Gangetic plains of India was evaluated for their antagonistic potential against three soil-borne fungal pathogens causing root rot (Rhizoctonia solani), collar rot (Sclerotium rolfsii) and, fungal wilt (Fusarium oxysporum f. sp. lycopersici). Preliminary screening indicated that most of the isolates could inhibit R. solani and F. oxysporum f. sp. lycopersici to the greater extent but failed to reduce mycelia growth of S. rolfsii by more than 50 percent. The only exceptions were 1PR7a, 2P2, and 2PR9b which were further characterized and evaluated against S. rolfsii. Molecular identification of 15 isolates based on 16s rRNA gene sequence similarity indicated the presence of three genera viz. Bacillus, Lysinibacillus, and Stenotrophomonas. In planta trial in tomato challenged with pathogen S. rolfsii in presence and absence of endophyte inoculation revealed that Bacillus sp. 2P2 showed the highest protection against S. rolfsii. These strains elicited induced systemic resistance of plant and significantly higher activity (p ≤ 0.05) of phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and ascorbate oxidase indicating the further strengthening of cell wall barrier through lipid peroxidation, cross linking of cell walls, lignifications, suberization and other cell wall strengthening processes. It was further confirmed by confocal scanning laser micrographs of upper collar region. It was evident that the inoculation of endophyte inhibited the colonization and movement of the pathogen. In addition, endophytes upregulated the expression of three pathogenesis-related genes PR1a, PR2a, and PR3, which are responsible for production of glucanases and chitinases contributing to pathogen inhibition. Further, oxidative stress alleviation was evident from decreased superoxide accumulation and enhanced dry matter content. Results of the present study indicated suppressive potential of endophyte Bacillus sp. 2P2 against S. rolfsii which could be useful in collar rot management in the nursery as well as after transplantation.

Production of cell wall degrading enzymes and antibiotic by Pseudomonads for assessing their biocontrol potential

A comprehensive study on bio-control efficacy of different Pseudomonas species was made in terms of cell wall degrading enzymes, viz. chitinase, lipase, protease and antibiotic 2, 4- diacetylphloroglucinol (2,4- DAPG) production abilities in respect to changing temperatures and pH. Observations revealed that all the isolates of Pseudomonas spp. under study produced all the three cell wall degrading enzymes and 2,4- DAPG significantly. However, the isolates PS-11 and PS-30 exhibited maximum enzyme producing abilities. Moreover, PS-11 could be considered to be the maximum heat tolerant among the isolates as it maintain remarkable sustainability even at 55° C in the ability to produce enzymes with minimum losses in activities 0.1026 to 0.718 in chitinase, 3.1167 to 2.1794 in lipase and 0.5827 to 0.4075 in protease activities due to rise of temperature from 45° C to 55° C. In pH variation studied at 35°C, it was observed that chitinase, lipase and protease production abilities of most of the isolates of Pseudomonas spp. were maximum at pH value of 6.5 and decreased on further lowering to 5.5 or rising to pH 8.5. PS-30 recorded the highest production of 2,4-DAPG (767.37 μg/ml) at 30° C and pH 6.5.

Use of low cost natural resources for enhanced chitinase production and optimization using CCD and RSM: a new initiative for bio-control of plant pathogen

A total number of 160 native fluorescent Pseudomonads were isolated from rhizospheric soil of tomato cultivated in different agro-ecological regions of West Bengal. Out of them 25 (15.63%) were found to be chitinase positive by semi-quantitative spot culture assay on chitinase detection agar media. By quantifying the chitinase production based on spectrophotometric assay, best performing isolate Pseudomonas aeruginosa FPK22 (Gene bank accession number: KY575332) was selected for further study of media optimization. Ten different media compositions were designed with cheap and readily available nutrient and substrate sources viz. aqueous extract of cow dung manure, aqueous extract of vermi compost, aqueous extract of de-oiled neem cake, sugar, sugarcane molasses, Baker’s yeast powder and crab shell powder in their different combinations. The media I, comprising with aqueous extract of vermi compost (V), sugarcane molasses (M), Baker’s yeast (Y) and crab shell powder (C) exhibited maximum biomass and highest chitinase production of the strain FPK22. The media was named, based on its composition, as VMYC media. In VMYC media chitinase production of the strain was enhanced by 1.15 fold than from the standard chitinase detection media. The composition of the VMYC media was then optimized by a four factor (A, B, C, D) central composite design (CCD) and response surface methodology for the response (Y) chitinase activity. By using this design a total of 30 runs were tested and the data were fitted. The regression analysis showed good fit of the experimental data to the second order polynomial model with coefficient of determination (R2) value of 0.9093 and model F value 10.74. The optimum concentrations of vermi wash (250 g/l), sugarcane molasses (20.7 g/l), Baker’s yeast (15 g/l), crab shell powder (5.2 g/l) were recorded from desirability function with a predicted value of chitinase production of 1.125 EU/ml. Ten repetitive runs were tested under the optimized condition of the four variables and observed that they yielded 92.9–98.7% of the predicted chitinase production. Two soil borne fungal pathogens R. solani and S. rolfsii were co-inoculated with the strains P. aeruginosa FPK22 and P. monteilii FPK4 in the optimized VMYC media in dual culture study. The enhancement of inhibition of mycelial growth of the fungal pathogens was also observed in optimized VMYC media compared to PDA media. The mycelial percent inhibition of R. solani challenged with FPK22 and FPK4 were increased by 1.17 and 1.13 fold respectively, besides, the mycelial percent inhibition of S. rolfsii co-inoculated with FPK22 and FPK4 were observed to be enhanced by 1.04 and 1.12 fold, respectively. The field evaluation study with the strains FPK22 and FPK4 grown in optimized VMYC media exhibited that the consortia of the strains could efficiently check the damping off disease, 69.2% and 62%, over control in case of tomato and chilli respectively.

Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion

In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46) were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc)2 (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.

Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens

BackgroundThe use of chemical fungicides against fungal pathogens adversely affects soil and plant health thereby resulting in overall environmental hazards. Therefore, biological source for obtaining antifungal agents is considered as an environment-friendly alternative for controlling fungal pathogens.ResultsIn this study, seven endophytic bacteria were isolated from sugarcane leaves and screened for its antifungal activity against 10 fungal isolates belonging to the genera Alternaria, Cochliobolus, Curvularia, Fusarium, Neodeightonia, Phomopsis and Saccharicola isolated from diseased leaves of sugarcane. Among the seven bacterial isolates, SCB-1 showed potent antagonistic activity against the tested fungi. Based on the phenotypic data, Fatty Acid Methyl Esters (FAME) and 16S rRNA gene sequence analysis, the isolate SCB-1 was identified as Bacillus subtilis. The bacterial isolate was screened negative for chitinase production; however, chloroform and methanol extracts of the bacterial culture caused significant inhibition in the growth of the fungal isolates on semisolid media. Volatile component assay showed highest inhibitory activity against Saccharicola bicolor (SC1.4). A PCR based study detected the presence of the genes involved in biosynthesis of surfactin, bacillaene, difficidin, macrolactins and fengycin. Mass spectrometric analysis of the bacterial extract detected the presence of antifungal lipopeptide surfactin, but other metabolites were not detected. The biocontrol activity of the bacterial isolate was established when bacterial pretreated mung bean seeds were able to resist Fusarium infection, however, the untreated seeds failed to germinate.ConclusionThe antifungal potential of isolate Bacillus subtilis SCB-1 was established against taxonomically diverse fungal pathogens including the genera Saccharicola, Cochliobolus, Alternaria and Fusarium. The potent antifungal compound surfactin as well as volatiles produced by the bacterial isolate could be responsible for its bio-control activity against fungal infections.

Entomopathogenic efficacy of the chitinolytic bacteria: Aeromonas hydrophila isolated from Siwa Oasis, Egypt

Thirty-four bacterial isolates were isolated from soil samples collected from the North Western Coast and a water sample collected from brackish water at Siwa Oasis, Matrouh Governorate, Egypt. Only six isolates showed chitinase activity when screened on colloidal chitin agar medium. The highest chitinolytic activity was achieved by a bacterial isolate labeled as A.S. This isolate was identified as Aeromonas hydrophila based on analysis of 16S rRNA gene sequence and morphological, physiological, and biochemical characteristics. Optimization of the cultural conditions for maximum chitinase production by A. hydrophila revealed that the highest level of chitinase was recorded when the bacterium was grown in malt nitrogen-based medium containing 1% colloidal chitin at pH7 for 48 h incubation at 30 °C. Crude chitinase from isolate A. hydrophila was evaluated against first instar larvae of the greater wax moth; Galleria mellonella L. (Lepidoptera: Pyralidae) at different concentrations of 0, 185, 205, 235, 265, 295 U/mg protein. It increased larval and pupal mortality rates in a concentration-dependent manner. The tested crude chitinase significantly induced a decrease in adults’ emergence rate and their fecundity.

BIOLOGICAL CONTROL OF SOIL -BORNE FUNGAL PATHOGENS

Seventeen bacterial isolates have been successfully isolated and purified from soil rhizosphere samples collected from different agricultural areas Buhayra, Sharqia and Alqilyubia of Egypt, and screened for production of chitinase enzymes had the highest chitinolytic activities amongst those investigated. Isolate S3-C and S1-C were Identified by biochemical, physiological tests and 16s rRNA gene primer as Bacillus cereus S3C, Gene Bank NCBI accession MK185696 and Bacillus cereus S1C accession MK185697. The production of chitinase by B.cereus S3C and Bacillus cereus S1C was optimized using colloidal chitin medium amended with 1% colloidal chitin at 30°C after five days of incubation. B.cereus S3C had potential for cell wall lysis of many phytopathogenic fungi tested such as Fusarium oxysporum and Rhizoctonia solani Potato phytopathogenic fungi by in vitro antagonistic test. The addition of B. cereus S3C chitinase was more effective than that of B.cereus S1C in increasing the resistance of Potato plants infected with various Soil- borne phytopathogenic fungi.

Genome-wide association study in two populations to determine genetic variants associated with Toxoplasma gondii infection and relationship to schizophrenia risk

T. gondii (TOXO) infects over one billion people worldwide, yet the literature lacks a Genome Wide Association Study (GWAS) focused on genetic variants controlling the persistence of TOXO infection.To identify putative T. gondii susceptibility genes, we performed a GWAS using IgG seropositivity as the outcome variable in a discovery sample (n = 790) from an Ashkenazi dataset, and a second sample of predominately African Americans (The Grady Trauma Project, n = 285). We also performed a meta-analyses of the 2 cohorts.None of the SNPs in these analyses was statistically significant after Bonferroni correction for multiple comparisons. In the Ashkenazi population, the gene region of CHIA (chitinase) showed the most nominally significant association with TOXO. Prior studies have shown that the production of chitinase by macrophages in the brain responding to TOXO infection is crucial for controlling the burden of T. gondii cysts. We found a surprising number of genes involved in neurodevelopment and psychiatric disorders among our top hits even though our outcome variable was TOXO infection. In the meta-analysis combining the Ashkenazi and Grady Trauma Project samples, there was enrichment for genes implicated in schizophrenia spectrum disorders (p < .05). Upon limiting our sample to those without mental illness, two schizophrenia related genes (CNTNAP2, GABAR2) still had significant TOXO–associated variants at the p < .05 level, but did not pass the genome wide significance threshold after correction for multiple comparisons.Using Ingenuity Systems molecular network analysis, we identified molecular nodes suggesting that while different genetic variants associated with TOXO in the two population samples, the molecular pathways for TOXO susceptibility nevertheless converged on common pathways. Molecular nodes in these common pathways included NOTCH1, CD44, and RXRA. Prior studies show that CD44 participates in TOXO-induced immunopathology and that RXRA is instrumental in regulating T-helper immune responses. These data provide new insights into the pathophysiology of this common neurotropic parasite.