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

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

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

Value of chitinase-like protein YKL-40 in bronchoalveolar lavage fluid for predicting refractory / 中国当代儿科杂志

OBJECTIVE@#To evaluate the value of chitinase-like protein YKL-40 in bronchoalveolar lavage fluid (BALF) for predicting refractory @*METHODS@#A total of 50 children with common @*RESULTS@#Compared with the common MPP group, the RMPP group had significantly higher incidence rates of fever, shortness of breath, lung consolidation, and pleural effusion (@*CONCLUSIONS@#There is an increase in the level of YKL-40 in BALF in children with RMPP, and the level of YKL-40 in BALF has a certain value for predicting RMPP.

Expression and characterization of β-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-β-D glucosamine production / 生物工程学报

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Overexpression of Chitinase A Gene from Serratia marcescens in Bacillus subtilis and Characterization of Enhanced Chitinolytic Activity

Abstract Chitinase enzymes possess various usages in agriculture, biotechnology and medicine due to their chitin degrading property. Thus, efficient production of chitinase enzymes with desired properties has importance for its use. In this study, chitinase A (chiA) gene from Serratia marcescens Bn10 was cloned and heterologously overexpressed using pHT43 vector in Bacillus subtilis 168. The recombinant chitinase was characterized in terms of temperature, pH, and various effectors. The extracellular chitinase activity in recombinant B. subtilis was found 2.15-fold higher than the parental strain after 2 h of IPTG induction. Optimum temperature and pH for the extracellular chitinase activity in the recombinant B. subtilis were determined as 60 oC and pH 9.0, respectively. NaCl, Ca2+, Mn2+, Cu2+, Zn2+, sodium dodecyl sulfate (SDS), Tween-20, and ethanol increased the chitinase activity whereas Mg2+ caused an inhibition. The most notable increment on the chitinase activity was provided by Zn2+ (3.2 folds) and then by SDS (2.9 folds). The chitinase, overproduced by the recombinant B. subtilis 168 heterologously expressing chiA, was determined to have optimum activity at high temperature and alkaline conditions as well as various effectors increase its activity. The extracellular chitinase of recombinant B. subtilis might be a promising source for agricultural, biotechnological and medical applications.

The potential of plant and fungal proteins in the control of gastrointestinal nematodes from animals / Potencial de proteínas de plantas e fungos no controle de nematoides gastrintestinais de animais

Abstract Gastrointestinal nematode infection is an important cause of high economic losses in livestock production. Nematode control based on a synthetic chemical approach is considered unsustainable due to the increasing incidence of anthelmintic resistance. Control alternatives such as the use of natural products are therefore becoming relevant from an environmental and economic point of view. Proteins are macromolecules with various properties that can be obtained from a wide range of organisms, including plants and fungi. Proteins belonging to different classes have shown great potential for the control of nematodes. The action of proteins can occur at specific stages of the nematode life cycle, depending on the composition of the external layers of the nematode body and the active site of the protein. Advances in biotechnology have resulted in the emergence of numerous protein and peptide therapeutics; however, few have been discussed with a focus on the control of animal nematodes. Here, we discuss the use of exogenous proteins and peptides in the control of gastrointestinal.

Resumo A infecção por nematoides gastrintestinais é uma importante causa de grandes perdas econômicas na pecuária. O controle de nematoides com compostos químicos sintéticos é considerado insustentável devido ao aumento da resistência anti-helmíntica. Alternativas de controle, como o uso de produtos naturais, estão se tornando relevantes do ponto de vista ambiental e econômico. As proteínas são macromoléculas com várias propriedades que podem ser obtidas de uma ampla gama de organismos, incluindo plantas e fungos. Proteínas pertencentes a diferentes classes têm mostrado grande potencial para o controle de nematoides. A ação das proteínas pode ocorrer em estágios específicos do ciclo de vida do nematoide, dependendo da composição das camadas externas do parasito e do sítio ativo da proteína. Avanços na biotecnologia resultaram no surgimento de numerosas terapias de proteínas e peptídeos; no entanto, pouco foi discutido com foco no controle de nematoides parasitos de animais. Na presente revisão foi discutido o uso de proteínas exógenas e peptídeos no controle de nematoides gastrintestinais, os mecanismos sugeridos de ação, e os desafios e perspectivas para o uso dessas biomoléculas como uma classe de anti-helmínticos.

Transcriptome and proteome analyses of resistant preharvest peanut seed coat in response to Aspergillus flavus infection

BACKGROUND: The infection of peanut (Arachis hypogaea L.) seed coat by the pathogenic fungus Aspergillus flavus has highly negative economic and health impacts. However, the molecular mechanism underlying such defense response remains poorly understood. This study aims to address this issue by profiling the transcriptomic and proteomic changes that occur during the infection of the resistant peanut cultivar J11 by A. flavus. RESULTS: Transcriptomic study led to the detection of 13,539 genes, among which 663 exhibited differential expression. Further functional analysis found the differentially expressed genes to encode a wide range of pathogenesis- and/or defense-related proteins such as transcription factors, pathogenesis-related proteins, and chitinases. Changes in the expression patterns of these genes might contribute to peanut resistance to A. flavus. On the other hand, the proteomic profiling showed that 314 of the 1382 detected protein candidates were aberrantly expressed as a result of A. flavus invasion. However, the correlation between the transcriptomic and proteomic data was poor. We further demonstrated by in vitro fungistasis tests that hevamine-A, which was enriched at both transcript and protein levels, could directly inhibit the growth of A. flavus. Conclusions: The results demonstrate the power of complementary transcriptomic and proteomic analyses in the study of pathogen defense and resistance in plants and the chitinase could play an important role in the defense response of peanut to A. flavus. The current study also constitutes the first step toward building an integrated omics data platform for the development of Aspergillus-resistant peanut cultivars

Mesenchymal Stem Cells Ameliorate Renal Inflammation in Adriamycin-induced Nephropathy

Mesenchymal stem cells (MSCs) ameliorate the renal injury in Adriamycin (ADR)-induced nephropathy, but the mechanisms underlying their efficacy remain incompletely understood. In this study, we demonstrated that MSCs increased the survival, recovered body weight loss, and decreased proteinuria and serum creatinine levels in ADR-treated mice. MSCs also prevented podocyte damage and renal fibrosis by decreasing the expression of fibronectin, collagen 1α1, and α-smooth muscle actin. From a mechanistic perspective, MSCs inhibited renal inflammation by lowering the expression of CCL4, CCL7, CCL19, IFN-α/β, TGF-β, TNF-α, and chitinase 3-like 1. In summary, our data demonstrate that MSCs improve renal functions by inhibiting renal inflammation in ADR-induced nephropathy.

Directed evolution of chitinase Chisb and biosynthesis of chitooligosaccharides / 生物工程学报

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.

Identification of a novel strain, Streptomyces blastmyceticus JZB130180, and evaluation of its biocontrol efficacy against Monilinia fructicola / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Peach brown rot, caused by Monilinia fructicola, is one of the most serious peach diseases. A strain belonging to the Actinomycetales, named Streptomyces blastmyceticus JZB130180, was found to have a strong inhibitory effect on M. fructicola in confrontation culture. Following the inoculation of peaches in vitro, it was revealed that the fermentation broth of S. blastmyceticus JZB130180 had a significant inhibitory effect on disease development by M. fructicola. The fermentation broth of S. blastmyceticus JZB130180 had an EC50 (concentration for 50% of maximal effect) of 38.3 µg/mL against M. fructicola, as determined in an indoor toxicity test. Analysis of the physicochemical properties of the fermentation broth revealed that it was tolerant of acid and alkaline conditions, temperature, and ultraviolet radiation. In addition, chitinase, cellulase, and protease were also found to be secreted by the strain. The results of this study suggest that S. blastmyceticus JZB130180 may be used for the biocontrol of peach brown rot.

Bioinformatics analysis and expression of chitinase genes in Armillaria gallica / 中国中药杂志

Armillaria gallica is a facultative parasitic fungus which is the only nutrient source of Gastrodia elata during its cultivation.Chitinase,as a glycosidic hydrolytic enzyme,plays an important role in the growth,development,stress tolerance and symbiotic signal transduction of A. gallica. There were 22 chitinase genes in A. gallica. Bioinformatics analysis of amino acid sequence of these chitinase genes revealed that 12 chitinase genes contained glycosidase 18 family( GH18) domain. Chitinase amino acid sequences of A. gallica,A. ostoyae,G. elata,Saccharomyces cerevisiae and Trichoderma harzianum were analyzed byclustering trees,so as to further predict the gene function of chitinase in A. gallica. Induction of A. gallica branching with strigolactone analogue GR24,high-throughput sequencing technology based on the induction of branch group( MHJ1),uninduced branch group( MHJ2) and blank control group( MHJ3) is used to detect the expression quantity,the transcription level data of 22 chitinase genes were obtained and the heat map was generated for expression pattern analysis. It was found that 8 genes may be involved in physiological processes such as A. gallica branching,cell wall degradation and remodeling. In this paper,the function of chitinase gene in A. gallica was just preliminarily analyzed and predicted.

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Enzymatic comparison and mortality of Beauveria bassiana against cabbage caterpillar Pieris brassicae LINN

Abstract Beauveria bassiana, an entomopathogenic fungus, is the alternative biocontrol agent exploited against major economic crop pests. Pieris brassicae L. is an emerging pest of the Brassicaceae family. Therefore, in the present study, fungal isolates of Beauveria bassiana, viz. MTCC 2028, MTCC 4495, MTCC 6291, and NBAII-11, were evaluated for their virulence against third instar larvae of P. brassicae. Among all these fungal isolates, maximum mortality (86.66%) was recorded in B. bassiana MTCC 4495 at higher concentration of spores (109 conidia/ml), and the minimum mortality (30.00%) was recorded in B. bassiana MTCC 6291 at a lower concentration (107 conidia/ml) after ten days of treatment. The extracellular cuticle-degrading enzyme activities of fungal isolates were measured. Variability was observed both in the pattern of enzyme secretion and the level of enzyme activities among various fungal isolates. B. bassiana MTCC 4495 recorded the maximum mean chitinase (0.51 U/ml), protease (1.12 U/ml), and lipase activities (1.36 U/ml). The minimum mean chitinase and protease activities (0.37 and 0.91 U/ml, respectively) were recorded in B. bassiana MTCC 6291. The minimum mean lipase activity (1.04 U/ml) was recorded in B. bassiana NBAII-11. Our studies revealed B. bassiana MTCC 4495 as the most pathogenic isolate against P. brassicae, which also recorded maximum extracellular enzyme activities, suggesting the possible roles of extracellular enzymes in the pathogenicity of B. bassiana against P. brassicae.

Induction of Defense-Related Physiological and Antioxidant Enzyme Response against Powdery Mildew Disease in Okra (Abelmoschus esculentus L.) Plant by Using Chitosan and Potassium Salts

Foliar sprays of three plant resistance inducers, including chitosan (CH), potassium sorbate (PS) (C₆H₇kO₂), and potassium bicarbonates (PB) (KHCO₃), were used for resistance inducing against Erysiphe cichoracearum DC (powdery mildew) infecting okra plants. Experiments under green house and field conditions showed that, the powdery mildew disease severity was significantly reduced with all tested treatments of CH, PS, and PB in comparison with untreated control. CH at 0.5% and 0.75% (w/v) plus PS at 1.0% and 2.0% and/or PB at 2.0% or 3.0% recorded as the most effective treatments. Moreover, the highest values of vegetative studies and yield were observed with such treatments. CH and potassium salts treatments reflected many compounds of defense singles which leading to the activation power defense system in okra plant. The highest records of reduction in powdery mildew were accompanied with increasing in total phenolic, protein content and increased the activity of polyphenol oxidase, peroxidase, chitinase, and β-1,3-glucanase in okra plants. Meanwhile, single treatments of CH, PS, and PB at high concentration (0.75%, 2.0%, and/or 3.0%) caused considerable effects. Therefore, application of CH and potassium salts as natural and chemical inducers by foliar methods can be used to control of powdery mildew disease at early stages of growth and led to a maximum fruit yield in okra plants.

Antifungical Activity of Autochthonous Bacillus subtilis Isolated from Prosopis juliflora against Phytopathogenic Fungi

The ability of Bacillus subtilis, strain ALICA to produce three mycolytic enzymes (chitinase, β-1,3-glucanase, and protease), was carried out by the chemical standard methods. Bacillus subtilis ALICA was screened based on their antifungal activity in dual plate assay and cell-free culture filtrate (25%) against five different phytopathogenic fungi Alternaria alternata, Macrophomina sp., Colletotrichum gloeosporioides, Botrytis cinerea, and Sclerotium rolfesii. The B. subtilis ALICA detected positive for chitinase, β-1,3-glucanase and protease enzymes. Fungal growth inhibition by both strain ALICA and its cell-free culture filtrate ranged from 51.36% to 86.3% and 38.43% to 68.6%, respectively. Moreover, hyphal morphological changes like damage, broken, swelling, distortions abnormal morphology were observed. Genes expression of protease, β-1,3-glucanase, and lipopeptides (subtilosin and subtilisin) were confirmed their presence in the supernatant of strain ALICA. Our findings indicated that strain ALICA provided a broad spectrum of antifungal activities against various phytopathogenic fungi and may be a potential effective alternative to chemical fungicides.

Expression and secretion of cyan fluorescent protein [CFP] in B. subtilis using the chitinase promoter from bacillus pumilus SG2

Background: Improved cyan fluorescent protein [ICFP] is a monochromic, green fluorescent protein [GFP] derivative produced by Aequorea macrodactyla in a process similar to GFP. This protein has strong absorption spectra at wavelengths 426-446 nm. ICFP can be used in cell, organelle or intracellular protein labeling, investigating the protein-protein interactions as well as assessing the promoter activities

Methods: In our previous study, the promoters of two chitinases [ChiS and ChiL] from Bacillus pumilus SG2 were assessed in B. subtilis and their regulatory elements were characterized. In the present study, icfp was cloned downstream of several truncated promoters obtained in the former study, and ICFP expression was evaluated in B. subtilis

Results: Extracellular expression and secretion of ICFP were analyzed under the control of different truncated versions of ChiSL promoters grown on different media. Results from SDS-PAGE and fluorimetric analyses showed that there were different expression rates of CFP; however, the UPChi-ICFP3 construct exhibited a higher level of expression and secretion in the culture medium

Conclusion: Our presented results revealed that inserting this truncated form of Chi promoter upstream of the ICFP, as a reporter gene, in B. subtilis led to an approximately ten fold increase in ICFP expression

A broad pH range and processive chitinase from a metagenome library

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization

Abstract Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and V max values of chitinase to be 5.6 mg/mL and 0.87 µmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

Abstract Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

Isolation and Identification of Chitinolytic Bacteria as Biocontrol Agent of Pathogenic Fungi on Gold Silkworm Cocoon Cricula trifenestrata

Aims: Cricula trifenestrata is one of natural insects which has not been domesticated yet, thus called as the wild silkworm. C. trifenestrata is known as a silk producer which has high economic and market value. However, the fungi attack on C. trifenestrata cocoon decreased quality and quantity of silk yarn. Chitinolytic bacteria have a high potential as biological control against pathogenic fungi. This research aimed to isolate, select, characterize, and identify chitinolytic bacteria as pathogenic fungi growth inhibitors on C. trifenestrata cocoon. Methodology and results: Chitinolytic bacteria was isolated from the uninfected and infected cocoon while fungi was isolated from the uninfected cocoon. Inhibition test was conducted by Fokkema method and chitinase activity was measured by Spindler method. A total of 36 chitinolytic bacteria and 10 suspected pathogenic fungi isolates have been isolated. Fungal pathogenicity test showed that isolate CSAJ.2 was suspected as fungal pathogen. In vitro inhibition test indicated that chitinolytic bacteria isolate BSEP.3 could inhibit the growth of pathogenic fungi CSAJ.2 with percentage of inhibition 50%. Isolate BSEP.3 showed highest chitinase activity (5.11 U/mL) at the 15th h. It able to inhibit the growth of pathogenic fungi with percentage of inhibition of 47.5% and 46.25%, respectively. Conclusion, significance and impact of study: Identification of bacteria targeted on 16S rRNA gene showed that isolate BSEP.3 had 98% identity with Bacillus amyloliquefaciens B5 while identification of fungi using ITS region of the rDNA showed that isolate CSAJ.2 had 100% identity with Trichoderma virens TV242. Chitinase crude extract was effective to be used as a biological control agent of T. virens CSAJ.2.