Chitinolytic Enzyme Activity Research Articles

Evaluation of the crude chitinases toxicity on the reproductive system of Callosobruchus maculatus (Coleoptera: Bruchidae)

Insecticides represent the most used control method against the insect pests of stored food products. But there are strains of insects which are resistant to these insecticides: it is the case of the beetle of the chickpea Callosobruchus maculatus. The present study has for objective the evaluation of the biological activity of chitinolytic enzymes extracted from the fish offals Scorpaena scrofa (scorpion fish) on this beetle at different doses (3%, 6%, 9%, 12%, 15% and 21%) prepared with the buffer solution. The results obtained from three treatments (T1, T2 and T3) realized according to the dose of the crude enzyme and the time of exposure were compared with those of the controls and have showed a very significant efficiency of our crude enzyme on the reduction of the fertility rate of 100% by treating the couple (T1) at the same time as to treat the male (T3) and the female (T2) separately with the strongest dose (21%) for 48 h of exposure. It would be thus desirable to demonstrate the activity of these enzymes in the real conditions of storage.

Dehydrated and live black soldier fly larvae as environmental enrichment in indigenous slow-growing chickens: performance, gut health, and chitinolytic enzyme activity

The demand for sustainable and ethically farmed animal products is on the rise as consumers become more environmentally and animal welfare conscious. The need to diminish the consumption of soybean meal is urgent, and companies are looking for ways to respond to this necessity by looking for alternatives to soybean meal. This study assessed the impact of introducing whole dehydrated and live black soldier fly larvae (BSFL) into the diet of an indigenous chicken breed as environmental enrichment. A total of 144 39-day-old male Bianca di Saluzzo chickens were distributed among 18 pens and assigned to three different experimental groups. The control group received a diet where soybean meal was entirely replaced by alternative ingredients. The two experimental groups were given the same diet supplemented with 5% of the expected daily feed intake of whole dehydrated BSFL or whole live BSFL. Throughout the trial period (from the bird age of 39–174 days of age), live weight was recorded every 21 days, and the average daily gain, daily feed intake, and feed conversion ratio were calculated. The time required for the birds to consume the larvae was recorded three times a week. At age 147 and 174 days, 12 birds per treatment were selected based on mean live weight and slaughtered. Measurements included hot and chilled carcass weights, organ weights (spleen, liver, heart, stomach), breast and thigh muscle weights, and the corresponding yields were calculated. Acid protease activity was measured in proventriculus extract, and chitinase and chitosanase activity was calculated based on the release of reducing sugars from chitin or chitosan. The results showed little improvement in final live weights and daily feed intakes of the animals fed the insect larvae compared with control birds. Larva supplementation had no negative impact on the overall well-being of the animals assessed by blood analysis and histopathological assessment of the intestinal tract, spleen, and liver. No differences were found between the dehydrated vs live insect larvae consumption times, with all larvae being eaten up very rapidly (< 3 min). The birds fed BSFL showed an increase in chitinase activity. These findings support the potential use of whole BSFL as a form of environmental enrichment, particularly in their dehydrated form, being more convenient to use and store, which would also encourage the uptake of this practice by farmers.

Internal Transcribed Spacers (ITS) Based Identification of Trichoderma Isolates and Biocontrol Activity against Fusarium oxysporum and Chitinase Activity

Twelve Trichoderma spp. isolated from different locations in Uttar Pradesh, India. Internal transcribed spacer (ITS) amplification and sequencing were used to identify isolates of Trichoderma that showed 99–100% identification with Trichoderma harzianum, Trichoderma asperellum, and Trichoderma longibrachiatum, the three species of Trichoderma. Tests were conducted in Vitro to evaluate the biocontrol potential of Trichoderma isolates against Fusarium species. After seven days of incubation, the isolate TBT6 (T. harzianum) exhibited the greatest antagonism against Fusarium oxysporum, with inhibition rates of 87.1%) by TBT7 (82.2%), and the least amount of inhibition (59.7%) by T. longibrachiatum isolate TBT10. Under field conditions, the antagonistic T. harzianum isolate TBT6 can be employed as a bio-control agent against Fusarium oxysporum. and for the development of formulations based on Trichoderma. Using the dual plate approach, twelve highly antagonistic Trichoderma isolates were selected for chitinolytic activity. Observing the breakdown of chitin substrates, Trichoderma isolates TBT6 was shown to have strong chitinolytic enzyme activity, making it a good candidate for endochitinase gene isolation. Using two particular primers, the genomic DNA of the Trichoderma isolate TBT6 was isolated and amplified.

Screening of indigenous Rhizobacteria isolates from Patchouli Rhizosphere producing HCN, Siderophores and Chitinolytic Enzymes

The consortium of bacteria that inhabit the rhizosphere, acting as plant growth promoting rhizobacteria is known as indigenous rhizobacteria. Rhizobacteria can produce chemical compounds that can protect plants from pathogens and various plant-disturbing organisms. Optimization of the growth and yield of patchouli can be done by utilizing the potential of rhizobacteria derived from patchouli rhizosphere. This study aims to obtain rhizobacteria capable of producing secondary metabolites of HCN compounds, siderophores and chitinolytic enzymes which are thought of as biocontrol agents in plant protection. The soil samples used were taken from patchouli rhizosphere areas in three locations, namely Alue Abed, Aceh Jaya and Krueng Itam and Purwosari, Nagan Raya. The exploration results obtained 4 isolates of rhizobacteria from Alue Abed, Calang, 8 isolates from Krueng Itam, Nagan Raya and 37 isolates from Purwosari, Nagan Raya. Based on the test results of qualitative and quantitative physiological properties, 34 isolates were able to produce HCN, 49 isolates produced siderophores and 42 isolates were able to carry out chitinolytic enzyme activity. Rhizobacteria isolate producing HCN, siderophores and chitinolytic enzymes are potential indigenous rhizobacteria candidates and potential for future testing as bio-protections of plants from pathogen attack.

Detection of hydrolases of different subclasses in cell-free fractions of Vibrio cholerae O1 and O139 serogroups using radial enzymatic diffusion in agarose gel

Cholera remains an actual infection worldwide, which dictates the need for a comprehensive study of its pathogens and, in particular, their hydrolytic enzymes that interact with the cells of the macroorganism. We determined the presence of hydrolases from different subclasses in preparations of cell-free fractions obtained from 58 strains of Vibrio cholerae O1 and O139 serogroups of different epidemic significance and origins using radial enzyme diffusion in agarose gels with various substrates. The analysis revealed statistically significant differences in enzyme activity depending on the origin and epidemiological significance of the original strain. We found that preparations obtained from non-toxigenic strains had increased activity of proteases and chitinolytic enzymes, while those from toxigenic strains showed high activity of mucinases, lipolytic enzymes, and nucleases. These data can be applied in the microbiological diagnosis of cholera as an additional biochemical characterization of Vibrio cholerae strains, as well as contribute to the understanding of the role of enzymes in the pathogenesis and adaptation of cholera pathogens.

Exploring the Probiotic and Plant Growth Promoting Potential of Endophytic Bacteria Isolated from the Vegetable Crop Bhendi Abelmoschus esculentus

Endophytes are microbes that reside within the plant tissues and facilitate the growth of the host plant. Inthe present study, occurrence of endophytic bacteria in the most common vegetable plant of India, Bhendi(Abelmoschus esculentus) was screened and analysed for its plant growth promoting (PGP) potential. Presenceof a rod shaped bacterium within the tissues of the host plant was observed. The isolate was identified asBacillus firmus by its biochemical profile. Phosphate solubilization and IAA production by the isolate at 60h of incubation was found to be 37.3 g ml–1 and 39.47 g ml–1 respectively. B. firmus was found to producetrihydroxamate type siderophore with hexadentate nature. The isolate exhibited extracellular pectinolytic,proteolytic, cellulolytic, chitinolytic enzyme activity. Further, the isolate exhibited probiotic properties suchas tolerance to acid (pH2), bile salt (2%), gastric juice, auto-aggregation potential, antibiotic resistance andthe absence of haemolytic activity. Seed biopriming and scanning electron microscope (SEM) imagingconfirmed the endophytic and plant growth promoting potential of B. firmus. The results of this studyillustrate the possible utilization of the endophytic bacterium B. firmus as a bioinoculant for plant growthpromotion.

Effects of Ocean Acidification on Molting, Oxidative Stress, and Gut Microbiota in Juvenile Horseshoe Crab Tachypleus tridentatus.

Anthropogenic elevation of atmospheric carbon dioxide (CO2) drives global-scale ocean acidification (OA), which has aroused widespread concern for marine ecosystem health. The tri-spine horseshoe crab (HSC) Tachypleus tridentatus has been facing the threat of population depletion for decades, and the effects of OA on the physiology and microbiology of its early life stage are unclear. In this study, the 1st instar HSC larvae were exposed to acidified seawater (pH 7.3, pH 8.1 as control) for 28 days to determine the effects of OA on their growth, molting, oxidative stress, and gut microbiota. Results showed that there were no significant differences in growth index and molting rate between OA group and control group, but the chitinase activity, β-NAGase activity, and ecdysone content in OA group were significantly lower than those of the control group. Compared to the control group, reactive oxygen species (ROS) and malondialdehyde (MDA) contents in OA group were significantly increased at the end of the experiment. Superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatase (AKP) activities increased first and then decreased, glutathione peroxidase (GPX) decreased first and then increased, and GST activity changed little during the experiment. According to the result of 16S rRNA sequencing of gut microbiota, microbial-mediated functions predicted by PICRUSt showed that “Hematopoietic cell lineage,” “Endocytosis,” “Staphylococcus aureus infection,” and “Shigellosis” pathways significantly increased in OA group. The above results indicate that OA had no significant effect on growth index and molting rate but interfered with the activity of chitinolytic enzymes and ecdysone expression of juvenile horseshoe crabs, and caused oxidative stress. In addition, OA had adverse effects on the immune defense function and intestinal health. The present study reveals the potential threat of OA to T. tridentatus population and lays a foundation for the further study of the physiological adaptation mechanism of juvenile horseshoe crabs to environmental change.

Roundup® disrupts chitinolytic enzyme activity and ecdysteroid concentration in Macrobrachium potiuna.

The endocrine system of crustaceans regulates the molt cycle with ecdysteroid hormones, mainly the 20-hydroxyecdysone (20-HE). Moreover, the molt process requires the action of chitinolytic enzymes (e.g., chitinase, chitobiase) to break down the old cuticle. However, endocrine disrupting compounds (EDC) are capable of altering their normal functioning. Glyphosate-based herbicides (GBH), such as Roundup®, the most widely used herbicides, are found in freshwater environments and have been considered EDC for many aquatic organisms. Therefore, this study examined the effects of environmentally relevant GBH concentrations (0.0065, 0.065, and 0.28mgL-1) on the 20-HE concentration and chitobiase activity in the decapod prawn Macrobrachium potiuna exposed for 14days. Additionally, lipid peroxidation, a biomarker of membrane lipid degradation, was evaluated in hepatopancreas to assess cellular damage. Results showed that GBH decreased the 20-HE concentration in females at the two highest concentrations tested, while an increase was observed in males exposed to the highest GBH concentration. In addition, GBH also decreased chitobiase activity in males (all concentrations) and females (the two highest concentrations). Finally, GBH caused increased lipid peroxidation in males, indicating cellular damage in the hepatopancreas. In conclusion, this work suggests that GBH is an EDC for crustaceans by disrupting molting, which could lead to altered reproduction and thus population dynamics. Graphical abstract Decrease in the 20-HE concentration and chitobiase activity in muscle of males and females of the freshwater prawn Macrobrachium potiuna exposed to the herbicide Roundup® for 14days.

Effects of chitin degradation products N-acetylglucosamine and N,Nʹ-diacetylchitobiose on chitinase activity and bacterial community structure in an incubated upland soil

ABSTRACT Chitin, which is the polymer of N-acetylglucosamine (GlcNAc) linked by β1,4 glycoside bonds, has been reported as a soil amendment to mitigate plant soil diseases, increasing the population of chitin-degrading bacteria, and chitinolytic enzymatic activity in the soil. In some chitin-degrading bacteria, whose chitinolytic systems have been intensively studied, the chitin degradation product N,Nʹ-diacetylchitobiose {(GlcNAc)2} induces expression of genes for chitinases whereas GlcNAc does not. To evaluate the effects of these mono- and disaccharides on the population and activity of chitinolytic bacteria in soil, we investigated the chitinolytic enzyme activity and bacterial community structure in an incubated upland soil supplemented with GlcNAc or (GlcNAc)2. The added GlcNAc and (GlcNAc)2 (2 mg g−1) were consumed within 1 d when incubated at 25°C. Chitinase activity was induced by (GlcNAc)2 and chitin after 1-d and 7-d incubation, respectively, but not by GlcNAc. N-acetylglucosaminidase (GlcNAcase) activity was induced by GlcNAc but was lower than those by (GlcNAc)2 and chitin. Amplicon sequencing analysis targeting 16S rRNA genes demonstrated that both GlcNAc and (GlcNAc)2 significantly increased the rate of the order Bacillales, but the compositions of Bacillales differed from each other: the family Planococcaceae significantly increased in either GlcNAc- or (GlcNAc)2-added soil, but the genera Bacillus and Paenibacillus were increased mainly by GlcNAc and (GlcNAc)2, respectively. The family Streptomycetaceae of the order Actinomycetales was significantly increased by (GlcNAc)2 and chitin, but GlcNAc did not. Thus, GlcNAc and (GlcNAc)2, which were promptly consumed in the incubated soil, indicated partly similar but distinctive effects on chitinolytic enzyme activity and bacterial communities. Both aminosugars increased GlcNAcase activity and the population size of Planococcaceae. GlcNAc increased Bacillus. Chitinase activity and the populations of Paenibacillus and Streptomycetaceae, a number of strains of which are known as potent chitin-degraders, were increased by (GlcNAc)2, but not by GlcNAc.

Carbamazepine disrupts molting hormone signaling and inhibits molting and growth of Eriocheir sinensis at environmentally relevant concentrations

Carbamazepine (CBZ), one of the most frequently detected pharmaceutical compounds in aquatic environments, has recently been shown to cause chronic toxicity and endocrine disruption in a variety of non-target aquatic organisms. However, neither the effects nor the specific mechanism of CBZ action on the molting of crustaceans is well understood. The aim of this study was to investigate the effects of CBZ on the molting and growth of the Chinese mitten crab Eriocheir sinensis, a native and economically important species in China, and to elucidate the specific mechanisms through which molt inhibition occurs. Juvenile E. sinensis were treated with four nominal environmentally relevant concentrations (0.01, 0.1, 1, or 10 μg/L) of CBZ for acute (4 days) and chronic (40 days) exposures. After acute exposure, chitinase activity in the epidermis and 20-hydroxyecdysone (20-HE) concentration in the hemolymph were significantly decreased (p < 0.05) following treatment with 10 μg/L CBZ, whereas epidermal chitobiase activity significantly decreased (p < 0.05) in response to both 1 and 10 μg/L CBZ treatments. Transcript levels of the genes encoding crustacean hyperglycemic hormone (chh) and molt-inhibiting hormone (mih) in the eyestalks were also markedly induced after 1 or 10 μg/L CBZ treatment (p < 0.05), whereas the expression of genes encoding ecdysone receptor (ecr) and crustacean retinoid X receptor (rxr) in the hepatopancreas were significantly suppressed (p < 0.05) after acute exposure. Moreover, under chronic exposure, CBZ inhibited the molting and growth of E. sinensis, resulting in a longer time before molting was completed (TBM), as well as a lower molting increment (MI) with 1 or 10 μg/L treatment (p < 0.05). Collectively, these data indicate that CBZ can inhibit the molting process of E. sinensis by interfering with the activity of chitinolytic enzymes and molting hormone signaling, suggestive that carbamazepine may have long-term effects on crab development. We also confirmed that CBZ may function as an endocrine disruptor in decapod crustaceans, a phenomenon that has previously been reported in vertebrates.

Aktivitas Bakteri Kitinolitik dari Cangkang Perna viridis sebagai Antifungi Phytophthora palmivora Penyebab Penyakit Busuk Buah Kakao (Theobroma cacao L.)

Abstract. The emphasis on the growth of Phytophthora palmivora was important in order to reduce Pod Rot Disease of cacao (Theobrorna cacao L.) which could harm agriculture sector. Some bacteria had chitinolytic enzyme activity that is potentially used as an antifungal against Phytophthora palmivora, because the cell wall of the fungi composed of chitin. The purpose of this research was to know chitinolytic bacteria from Perna viridis shell which had higher activity of chitinase enzyme, the amount of chitinase enzyme activity of each selected isolate, and to know the effect of chitinolytic bacterial isolates from Perna viridis shell to reduce the growth of Phytophthora palmivora. The bacteria were isolated from Perna viridis shell at Depok Beach, Kretek, Bantul, Yogyakarta. This research was an explorative research which include bacterial characterization and experimental research which include antagonistic test of chitinolytic bacteria against Phytophthora palmivora. The chitinolytic bacteria was isolated using selective chitin agar medium by pour plate method and then screening the isolates that had chitinase enzyme activity by measuring the enzyme activity of each bacterial isolates by spectrophotometric method. Selected bacterial isolates were characterized by macroscopic, microscopic and physiological characters. The bacteria that had been selected tested for their ability to reduce the growth of Phytophthora palmivora by Kirby Bauer modification method. The result showed that there were 10 isolates that had chitinase enzyme activity which two selected isolates had the higher chitinase enzyme activity. There were 7D and 6B isolates. The isolate 7D had 1,258 u/ml chitinase enzyme activity and isolate 6B had 1,212 u/ml chitinase enzyme activity. The result of chitinolytic bacterial antagonist test on Phytophthora palmivora growth showed that both bacterial isolates were potential to antifungal Phytophthora palmivora and showed a real effect in inhibiting the growth of Phytophthora palmivora with significance value &lt; 0,05.Keywords: Chitinolytic Bacteria, Perna viridis, Phytophthora palmivora

The effect of rumen ciliates on chitinolytic activity, chitin content and the number of fungal zoospores in the rumen fluid of sheep

ABSTRACTThe objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 104 (D. affine) to 42.2 · 104 cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 105 cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.

Response of Vitis vinifera cell cultures to Eutypa lata and Trichoderma atroviride culture filtrates: expression of defence-related genes and phenotypes.

Cell suspension cultures of Vitis vinifera cv. Dauphine berries were used to study the response to the vascular pathogen, Eutypa lata, in comparison with a biological control agent, Trichoderma atroviride, that was previously shown to be effective in pruning wound protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins was profiled over a 48-h period using quantitative reverse transcriptase PCR. The cell cultures responded to elicitors of both fungi with a hypersensitive-like response that lead to a decrease in cell viability. Similar genes were triggered by both the pathogen and biocontrol agent, but the timing patterns and magnitude of expression was dependent on the specific fungal elicitor. Culture filtrates of both fungi caused upregulation of phenylalanine ammonia-lyase (PAL), 4-coumaroyl Co-A ligase (CCo-A) and stilbene synthase (STS), and a downregulation of chalcone synthase (CHS) genes. The pathogen filtrate caused a biphasic pattern in the upregulation of PAL and STS genes which was not observed in cells treated with filtrates of the biocontrol agent. Analytical assays showed significantly higher total phenolic content and chitinolytic enzyme activity in the cell cultures treated with the T. atroviride filtrate compared to the pathogen filtrate. These results corresponded well to the higher expression of PAL and chitinase class IV genes. The response of the cell cultures to T. atroviride filtrate provides support for the notion that the wound protection by the biocontrol agent at least partially relies on the induction of grapevine resistance mechanisms.

Effect of Pentoxifylline on Chitinolytic Enzyme Activity in the Eastern Subterranean Termite (Isoptera: Rhinotermitidae)

Abstract A two-molecule chitinolytic enzyme system (endo- and exo-chitinase) hydrolyzes and degrades the chitin polymers. Therefore, it is imperative to discover novel compounds for inhibiting chitinolytic enzymes to prevent insect growth. This research examined the effect of pentoxifylline (a dimethylxanthine chitinase inhibitor) on inhibition of endo- and exo-chitinolytic enzyme activities in eastern subterranean termite, Reticulitermes flavipes (Kollar). Enzyme activities were compared with amounts of treated diet consumed by termites and percent mortality observed over time. Pentoxifylline affected in vitro endo-chitinase activity in a concentration-dependent manner, while having minimal to no effect on in vitro exo-chitinase enzyme activity. However, pentoxifylline treatment affected in vivo endo- and exo-chitinase enzyme activity and caused measurable termite mortality. Moreover, pentoxifylline concentrations did not deter the amount of diet consumed by termites, thereby suggesting that it is palat...

Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Stomach of Two Species of Fish, Chub Mackerel (&amp;lt;i&amp;gt;Scomber japonicus&amp;lt;/i&amp;gt;) and Silver Croaker (&amp;lt;i&amp;gt;Pennahia argentata&amp;lt;/i&amp;gt;)

Chitinolytic activities were measured in two fish species having different feeding habits, chub mackerel (Scomber japonicus) and silver croaker (Pennahia argentata). Chitinase (an endo-type chitinolytic enzyme) activity was measured using pNP-(GlcNAc)n (n = 2, 3) as substrates; its level was significantly high in the stomachs of both species, as well as in the gills, intestine, pyloric appendage, testis, and liver of chub mackerel and in the spleen, kidney, pyloric appendage, ovaries, heart, and liver of silver croaker. β-N-Acetylhexosaminidase (an exo-type chitinolytic enzyme) activity was measured using pNP-(GlcNAc) as a substrate; it was detected at high levels in many parts apart from the digestive tracts of both species. The optimum pH for chitinase activity was 3.0 - 5.0 in the stomachs of both species, 4.0 in the liver of chub mackerel, and 4.0 and 8.0 in the kidney of silver croaker. Full-length cDNAs encoding two chitinase isozymes were obtained from the stomachs of the two fish species: SjChi-1 (1604 bp) and SjChi-2 (1512 bp) from chub mackerel and PaChi-1 (1630 bp) and PaChi-2 (1606 bp) from silver croaker. Expression analysis of these genes in the organs of the two species revealed strong expression of SjChi-1 in the stomach of chub mackerel and that of PaChi-1 andPaChi-2 in the stomach of silver croaker. The difference in the expression pattern of these genes is likely attributed to the difference in the feeding habits of the two fish species. Our results suggested the presence of novel chitinases in the two species.

Bioproduction of chitooligosaccharides: present and perspectives.

Chitin and chitosan oligosaccharides (COS) have been traditionally obtained by chemical digestion with strong acids. In light of the difficulties associated with these traditional production processes, environmentally compatible and reproducible production alternatives are desirable. Unlike chemical digestion, biodegradation of chitin and chitosan by enzymes or microorganisms does not require the use of toxic chemicals or excessive amounts of wastewater. Enzyme preparations with chitinase, chitosanase, and lysozymeare primarily used to hydrolyze chitin and chitosan. Commercial preparations of cellulase, protease, lipase, and pepsin provide another opportunity for oligosaccharide production. In addition to their hydrolytic activities, the transglycosylation activity of chitinolytic enzymes might be exploited for the synthesis of desired chitin oligomers and their derivatives. Chitin deacetylase is also potentially useful for the preparation of oligosaccharides. Recently, direct production of oligosaccharides from chitin and crab shells by a combination of mechanochemical grinding and enzymatic hydrolysis has been reported. Together with these, other emerging technologies such as direct degradation of chitin from crustacean shells and microbial cell walls, enzymatic synthesis of COS from small building blocks, and protein engineering technology for chitin-related enzymes have been discussed as the most significant challenge for industrial application.

극지유래 저온활성 Chitinase 생산균주의 스크리닝과 Chitinase 유전자 클로닝

극지의 다양한 환경으로부터 분리되어 극지연구소 PAMC(Polar and Alpine Microbial Collection)에 보관중인 169개 균주들을 0.4% colloidal chitin이 첨가된 ZoBell 고체배지에서 배양하여 chitinase 활성을 균주 27개를 선별하였다. 그 중 PAMC 21693 균주는 저온에서 pNP-<TEX>$(GlcNAc)_1$</TEX>를 기질로 사용했을 때 가장 큰 활성을 보였고, <TEX>$4-37^{\circ}C$</TEX>의 온도 범위 중 <TEX>$4^{\circ}C$</TEX>에서 가장 높은 개체수 증가율을 보였다. PAMC 21693의 chitinase 유전자를 클로닝한 결과 2,619 bp의 ORF를 포함하는 총 2,857 bp의 염기서열을 확보하였다. 대장균에서 chitinase 유전자의 재조합 단백질을 발현한 결과 분자량 96 kDa의 재조합 단백질을 확인할 수 있었다. 본 논문에서는 극지 미생물 유래 저온활성 chitinase들의 생물공학 분야에서의 이용가능성을 제시하였다. Of the 169 strains of microorganisms stored in Polar and Alpine Microbial Collection of Korea Polar Research Institute, 27 strains were selected for their chitinase activity on ZoBell plates supplemented with 0.4% colloidal chitin. Among them, PAMC 21693 strain have shown the highest chitinolytic enzyme activity toward pNP-<TEX>$(GlcNAc)_1$</TEX> at low temperature and the highest growth rate at <TEX>$4^{\circ}C$</TEX>. We cloned a full-length chitinase gene of 2,857 bp which contains an open reading frame of 2,169 bp encoding 872-amino acid polypeptide. Recombinant chitinase protein was expressed in E. coli and its molecular weight was confirmed 96 kDa. In this paper, we suggest the potential use of cold-active chitinase from polar microorganisms in the field of biotechnology.

Screening of Argentine native fungal strains for biocontrol of the grasshopper Tropidacris collaris: relationship between fungal pathogenicity and chitinolytic enzyme activity

Tropidacris collaris (Orthoptera: Romaleidae) is a large and voracious grasshopper, which, in recent years, has become a recurrent pest in increasingly extensive areas of Argentina's northern provinces. In the present work chitinase activity was measured in 59 entomopathogenic fungal isolates native to Argentina, and the relationship between enzymatic activity and fungal virulence was assessed. Isolate LPSC 1067 caused the highest mortality on T. collaris nymphs (97.7 ± 1.22%). Nine isolates caused no mortality, while the remaining 49 caused mortalities ranging from 6.6 ± 0.3% (LPSC 770) to 91.06 ± 1.51% (LPSC 906). Several isolates revealed chitinolytic capabilities on test plates, although the activities differed with respect to the ratio of the chitin-decay-halo and fungal-colony diameters. A principal component analysis indicated that isolate LPSC 1067, obtained from a long-horned grasshopper (Orthoptera: Tettigoniidae), would be a potential candidate for T. collaris biocontrol because the strain exhibited the highest mortality, a shorter median lethal time, and a high enzymatic activity and growth rate.

In vitro antifungal activity of chitinolytic enzymes produced by bio-agents against root rot pathogenic fungi

A study was carried out using simple laboratory techniques to examine the influence of the antagonistic isolates of Trichoderma harzianum, T. viride, Bacillius subtilis and Pseudomons flourescence and their culture filtrates on selected soil-borne root rot pathogens Rhizoctonia solani and Fusarium solani. Testing procedures were standardised using two different methods. The experiments were based on the principle of dual culture and agar diffusion techniques. The experiment involved the recording of the percentage of reduction in growth and inhibition zones formed by various filtrates of antagonistic culture growth. The results showed that the antagonists tested had the ability to reduce the linear growth of fungal pathogens. Also, the cultures filtrates of antagonists had antifungal activities by forming inhibition zones. Culture filtrates have shown a strong clear inhibition zone which increases in diameter as the incubation period of antagonists increases. This observation was related to the increase in the activity of chitinolytic enzymes as secondary metabolic compounds produced in growth media by prolonging the period of incubation. The study has proved that such enzymes can be effectively used for suppression of soil-borne pathogens and that it can evolve as a potential biocide.

Substrate supply, fine roots, and temperature control proteolytic enzyme activity in temperate forest soils

Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolytic activity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.