Functional expression of the chitinase to examine the virtually screened inhibitor candidates.
Zhang L,Guan Z,Pan Z,Ge H,Zhou D,Xu J,Zhang W
Bulletin of entomological research
Chitinase is responsible for insect chitin hydrolyzation, which is a key process in insect molting and pupation. However, little is known about the chitinase of Spodoptera exigua (SeChi). In this study, based on the SeChi gene (ADI24346) identified in our laboratory, we constructed the recombinant baculovirus P-Chi for the expression of recombinant SeChi (rSeChi) in Hi5 cells. The rSeChi was purified by chelate affinity chromatography, and the purified protein showed activity comparable with that of a commercial SgChi, suggesting that we harvested active SeChi for the first time. The purified protein was subsequently tested for enzymatic properties and revealed to exhibit its highest activity at pH 8 and 40 C. Using homology modeling and molecular docking techniques, the three-dimensional model of SeChi was constructed and screened for inhibitors. In two rounds of screening, twenty compounds were selected. With the purified rSeChi, we tested each of the twenty compounds for inhibitor activity against rSeChi, and seven compounds showed obvious activity. This study provided new information for the chitinase of beet armyworm and for chitinase inhibitor development.
10.1017/S0007485319000191
Structure-Function Insights into the Fungal -Chitinase Chit33 Depict its Mechanism on Chitinous Material.
International journal of molecular sciences
Chitin is the most widespread amino renewable carbohydrate polymer in nature and the second most abundant polysaccharide. Therefore, chitin and chitinolytic enzymes are becoming more importance for biotechnological applications in food, health and agricultural fields, the design of effective enzymes being a paramount issue. We report the crystal structure of the plant-type -chitinase Chit33 from and its D165A/E167A-Chit33-(NAG) complex, which showed an extended catalytic cleft with six binding subsites lined with many polar interactions. The major trait of Chit33 is the location of the non-conserved Asp117 and Arg274 acting as a clamp, fixing the distorted conformation of the sugar at subsite -1 and the bent shape of the substrate, which occupies the full catalytic groove. Relevant residues were selected for mutagenesis experiments, the variants being biochemically characterized through their hydrolytic activity against colloidal chitin and other polymeric substrates with different molecular weights and deacetylation percentages. The mutant S118Y stands out, showing a superior performance in all the substrates tested, as well as detectable transglycosylation capacity, with this variant providing a promising platform for generation of novel Chit33 variants with adjusted performance by further design of rational mutants'. The putative role of Tyr in binding was extrapolated from molecular dynamics simulation.
10.3390/ijms23147599
Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.
Biotechnology journal
Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.
10.1002/biot.202300219
Suppression of Plant Immunity by Fungal Chitinase-like Effectors.
Fiorin Gabriel Lorencini,Sanchéz-Vallet Andrea,Thomazella Daniela Paula de Toledo,do Prado Paula Favoretti Vital,do Nascimento Leandro Costa,Figueira Antonio Vargas de Oliveira,Thomma Bart P H J,Pereira Gonçalo Amarante Guimarães,Teixeira Paulo José Pereira Lima
Current biology : CB
Crop diseases caused by fungi constitute one of the most important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M. roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens.
10.1016/j.cub.2018.07.055
A marine chitinase from with antifungal activity and broad specificity toward crystalline chitin degradation.
Preparative biochemistry & biotechnology
Chitinases convert chitin into chitin oligomers and are also known antifungal agents. Chitin oligomers have numerous industrial applications. However, chitin's crystalline nature requires pretreatment before breakdown into oligomers. In the study, a novel marine bacterium is isolated from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, chitin flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin with the fractional release of oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains which help enzyme action on crystalline chitin. Statistical optimization of media and Process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulfate as significant media components along with colloidal chitin. The optimum process parameters such as pH 7, temperature 40 °C, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific enzyme activity to ±146.2 U/mL, ±114.9 U/mL and ±175.4 U/mL against chitin powder, chitin flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. The antifungal activity of chitinase against plant pathogenic fungi like and revealed a zone of inhibition with 14 mm diameter.
10.1080/10826068.2022.2033994
From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts.
ACS synthetic biology
Genetic parts and hosts can be sourced from nature to realize new functions for synthetic biology or to improve performance in a particular application environment. Here, we proceed from the discovery and characterization of new parts to stable expression in new hosts with a particular focus on achieving sustained chitinase activity. Chitinase is a key enzyme for various industrial applications that require the breakdown of chitin, the second most abundant biopolymer on the earth. Diverse microbes exhibit chitinase activity, but for applications, the environmental conditions for optimal enzyme activity and microbe fitness must align with the application context. Achieving sustained chitinase activity under broad conditions in heterologous hosts has also proven difficult due to toxic side effects. Toward addressing these challenges, we first screen ocean water samples to identify microbes with chitinase activity. Next, we perform whole genome sequencing and analysis and select a gene for heterologous expression. Then, we optimize transformation methods for target hosts and introduce chitinase. Finally, to achieve robust function, we optimize ribosome binding sites and discover a beneficial promoter that upregulates chitinase expression in the presence of colloidal chitin in a sense-and-respond fashion. We demonstrate chitinase activity for >21 days in standard () and nonstandard () hosts. Besides enhancing chitinase applications, our pipeline is extendable to other functions, identifies natural microbes that can be used directly in non-GMO contexts, generates new parts for synthetic biology, and achieves weeks of stable activity in heterologous hosts.
10.1021/acssynbio.3c00637
Chitinase activation in patients with fungus-associated cystic fibrosis lung disease.
Hector Andreas,Chotirmall Sanjay H,Lavelle Gillian M,Mirković Bojana,Horan Deirdre,Eichler Laura,Mezger Markus,Singh Anurag,Ralhan Anjai,Berenbrinker Sina,Mack Ines,Ensenauer Regina,Riethmüller Joachim,Graepler-Mainka Ute,Murray Michelle A,Griese Matthias,McElvaney N Gerry,Hartl Dominik
The Journal of allergy and clinical immunology
BACKGROUND:Chitinases have recently gained attention in the field of pulmonary diseases, particularly in asthma and chronic obstructive pulmonary disease, but their potential role in patients with cystic fibrosis (CF)-associated lung disease remains unclear. OBJECTIVE:The aim of this study was to assess chitinase activity systemically and in the airways of patients with CF and asthma compared with healthy subjects. Additionally, we assessed factors that regulate chitinase activity within the lungs of patients with CF. METHODS:Chitinase activities were quantified in serum and bronchoalveolar lavage fluid from patients with CF, asthmatic patients, and healthy control subjects. Mechanistically, the role of CF airway proteases and genetic chitinase deficiency was assessed. RESULTS:Chitinase activity was systemically increased in patients with CF compared with that in healthy control subjects and asthmatic patients. Further stratification showed that chitinase activity was enhanced in patients with CF colonized with Candida albicans compared with that in noncolonized patients. CF proteases degraded chitinases in the airway microenvironment of patients with CF. Genetic chitinase deficiency was associated with C albicans colonization in patients with CF. CONCLUSION:Patients with CF have enhanced chitinase activation associated with C albicans colonization. Therefore chitinases might represent a novel biomarker and therapeutic target for CF-associated fungal disease.
10.1016/j.jaci.2016.01.031
Chitinase: diversity, limitations, and trends in engineering for suitable applications.
Bioscience reports
Chitinases catalyze the degradation of chitin, a ubiquitous polymer generated from the cell walls of fungi, shells of crustaceans, and cuticles of insects. They are gaining increasing attention in medicine, agriculture, food and drug industries, and environmental management. Their roles in the degradation of chitin for the production of industrially useful products and in the control of fungal pathogens and insect pests render them attractive for such purposes. However, chitinases have diverse sources, characteristics, and mechanisms of action that seem to restrain optimization procedures and render standardization techniques for enhanced practical applications complex. Hence, results of laboratory trials are not usually consistent with real-life applications. With the growing field of protein engineering, these complexities can be overcome by modifying or redesigning chitinases to enhance specific features required for specific applications. In this review, the variations in features and mechanisms of chitinases that limit their exploitation in biotechnological applications are compiled. Recent attempts to engineer chitinases for improved efficiency are also highlighted.
10.1042/BSR20180323
Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications.
Khan Faez Iqbal,Bisetty Krishna,Singh Suren,Permaul Kugen,Hassan Md Imtaiyaz
Extremophiles : life under extreme conditions
Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of chitinases, of which chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanuginosus and its industrial application.
10.1007/s00792-015-0792-8
Chitin and chitinase: Role in pathogenicity, allergenicity and health.
Patel Seema,Goyal Arun
International journal of biological macromolecules
Chitin, a polysaccharide with particular abundance in fungi, nematodes and arthropods is immunogenic. It acts as a threat to other organisms, to tackle which they have been endowed with chitinase enzyme. Even if this enzyme is not present in all organisms, they possess proteins having chitin-binding domain(s) (ChtBD). Many lethal viruses like Ebola, and HCV (Hepatitis C virus) have these domains to manipulate their carriers and target organisms. In keeping with the basic rule of survival, the self-origin (own body component) chitins and chitinases are protective, but that of non-self origin (from other organisms) are detrimental to health. The exogenous chitins and chitinases provoke human innate immunity to generate a deluge of inflammatory cytokines, which injure organs (leading to asthma, atopic dermatitis etc.), and in persistent situations lead to death (multiple sclerosis, systemic lupus erythromatosus (SLE), cancer, etc.). Unfortunately, chitin-chitinase-stimulated hypersensitivity is a common cause of occupational allergy. On the other hand, chitin, and its deacetylated derivative chitosan are increasingly proving useful in pharmaceutical, agriculture, and biocontrol applications. This critical review discusses the complex nexus of chitin and chitinase and assesses both their pathogenic as well as utilitarian aspects.
10.1016/j.ijbiomac.2017.01.042
Chitin and chitin-related compounds in plant-fungal interactions.
Mycology
Chitin is the second abundant polysaccharide in the world after cellulose. It is a vital structural component of the fungal cell wall but not for plants. In plants, fungi are recognised through the perception of conserved microbe-associated molecular patterns (MAMPs) to induce MAMP-triggered immunity (MTI). Chitin polymers and their modified form, chitosan, induce host defence responses in both monocotyledons and dicotyledons. The plants' response to chitin, chitosan, and derived oligosaccharides depends on the acetylation degree of these compounds which indicates possible biocontrol regulation of plant immune system. There has also been a considerable amount of recent research aimed at elucidating the roles of chitin hydrolases in fungi and plants as chitinase production in plants is not considered solely as an antifungal resistance mechanism. We discuss the importance of chitin forms and chitinases in the plant-fungal interactions and their role in persistent and possible biocontrol. ET, ethylene; GAP, GTPase-activating protein; GEF, GDP/GTP exchange factor; JA, jasmonic acid; LysM, lysin motif; MAMP, microbe-associated molecular pattern; MTI, MAMP-triggered immunity; NBS, nucleotide-binding site; NBS-LRR, nucleotide-binding site leucine-rich repeats; PM, powdery mildew; PR, pathogenesis-related; RBOH, respiratory burst oxidase homolog; RLK, receptor-like kinase; RLP, receptor-like protein; SA, salicylic acid; TF, transcription factor.
10.1080/21501203.2018.1473299
An overview of fungal chitinases and their potential applications.
Protoplasma
Chitin, the world's second most abundant biopolymer after cellulose, is composed of β-1,4-N-acetylglucosamine (GlcNAc) residues. It is the key structural component of many organisms, including crustaceans, mollusks, marine invertebrates, algae, fungi, insects, and nematodes. There has been a significant increase in the generation of chitinous waste from seafood businesses, resulting in a big amount of scrap. Although several organisms, such as plants, crustaceans, insects, nematodes, and animals, produce chitinases, microorganisms are promising candidates and a sustainable option that mediates chitin degradation. Fungi are the dominant group of chitinase producers among microorganisms. In fungi, chitinases are involved in morphogenesis, cell division, autolysis, chitin acquisition for nutritional purposes, and mycoparasitism. Many efficient chitinolytic fungi with potential applications have been identified in a variety of environments, including soil, water, marine wastes, and plants. The current review highlights the key sources of chitinolytic fungi and the characterization of fungal chitinases. It also discusses the applications of fungal chitinases and the cloning of fungal chitinase genes.
10.1007/s00709-023-01839-5
Microbial chitinases: properties, enhancement and potential applications.
Gomaa Eman Zakaria
Protoplasma
Chitinases are a category of hydrolytic enzymes that catalyze chitin and are formed by a wide variety of microorganisms. In nature, microbial chitinases are primarily responsible for chitin decomposition and play a vital role in the balance of carbon and nitrogen ratio in the ecosystem. The physicochemical attributes and the source of chitinase are the main bases that determine their functional characteristics and hydrolyzed products. Several chitinases have been reported and characterized, and they obtain a wider consideration for their utilization in a large number of uses such as in agriculture, food, environment, medicine and pharmaceutical companies. The antifungal and insecticidal impacts of several chitinases have been extensively studied, aiming to protect crops from phytopathogenic fungi and insects. Chitooligosaccharides synthesized by chitin degradation have been shown to improve human health through their antimicrobial, antioxidant, anti-inflammatory and antitumor properties. This review aims at investigating chitinase production, properties and their potential applications in various biotechnological fields.
10.1007/s00709-021-01612-6
Chitinases: Therapeutic Scaffolds for Allergy and Inflammation.
Madan Kirtika,Madan Mansi,Sharma Swapnil,Paliwal Sarvesh
Recent patents on inflammation & allergy drug discovery
BACKGROUND:Chitinases are the evolutionary conserved glycosidic enzymes that are characterized by their ability to cleave the naturally abundant polysaccharide chitin. The potential role of chitinases has been identified in the manifestation of various allergies and inflammatory diseases. In recent years, chitinases inhibitors are emerging as an alluring area of interest for the researchers and scientists and there is a dire need for the development of potential and safe chitinase antagonists for the prophylaxis and treatment of several diseases. OBJECTIVE:The present review expedites the role of chitinases and their inhibitors in inflammation and related disorders. METHODS:At first, an exhaustive survey of literature and various patents available related to chitinases were carried out. Useful information on chitinases and their inhibitor was gathered from the authentic scientific databases namely SCOPUS, EMBASE, PUBMED, GOOGLE SCHOLAR, MEDLINE, EMBASE, EBSCO, WEB OF SCIENCE, etc. This information was further analyzed and compiled up to prepare the framework of the review article. The search strategy was conducted by using queries with key terms " chitin", "chitinase", "chitotrisidase", "acidic mammalian chitinase", "chitinase inhibitors", "asthma" and "chitinases associated inflammatory disorders", etc. The patents were searched using the key terms "chitinases and uses thereof", "chitinase inhibitors", "chitin-chitinase associated pathological disorders" etc. from www.google.com/patents, www.freepatentsonline.com, and www.scopus.com. RESULTS:The present review provides a vision for apprehending human chitinases and their participation in several diseases. The patents available also signify the extended role and effectiveness of chitinase inhibitors in the prevention and treatment of various diseases viz. asthma, acute and chronic inflammatory diseases, autoimmune diseases, dental diseases, neurologic diseases, metabolic diseases, liver diseases, polycystic ovary syndrome, endometriosis, and cancer. In this regard, extensive pre-clinical and clinical investigations are required to develop some novel, potent and selective drug molecules for the treatment of various inflammatory diseases, allergies and cancers in the foreseeable future. CONCLUSION:In conclusion, chitinases can be used as potential biomarkers in prognosis and diagnosis of several inflammatory diseases and allergies and the design of novel chitinase inhibitors may act as key and rational scaffolds in designing some novel therapeutic agents in the treatment of variety of inflammatory diseases.
10.2174/1872213X14666200114184054
Microbial chitinases: properties, current state and biotechnological applications.
Le Bao,Yang Seung Hwan
World journal of microbiology & biotechnology
Chitinases are a group of hydrolytic enzymes that catalyze chitin, nd are synthesized by a wide variety of organisms. In nature, microbial chitinases are primarily responsible for chitin decomposition. Several chitinases have been reported and characterized, and they are garnering increasing attention for their uses in a wide range of applications. In the food industry, the direct fermentation of seafood, such as crab and shrimp shells, using chitinolytic microorganisms has contributed to increased nutritional benefits through the enhancement of chitin degradation into chitooligosaccharides. These compounds have been demonstrated to improve human health through their antitumor, antimicrobial, immunomodulatory, antioxidant, and anti-inflammatory properties. Moreover, chitinase and chitinous materials are used in the food industry for other purposes, such as the production of single-cell proteins, chitooligosaccharides, N-acetyl D-glucosamines, biocontrol, functional foods, and various medicines. The functional properties and hydrolyzed products of chitinase, however, depend upon its source and physicochemical characteristics. The present review strives to clarify these perspectives and critically discusses the advances and limitations of microbial chitinase in the further production of functional foods.
10.1007/s11274-019-2721-y
Microbial chitinases and their relevance in various industries.
Folia microbiologica
Chitin, the second most abundant biopolymer on earth after cellulose, is composed of β-1,4-N-acetylglucosamine (GlcNAc) units. It is widely distributed in nature, especially as a structural polysaccharide in the cell walls of fungi, the exoskeletons of crustaceans, insects, and nematodes. However, the principal commercial source of chitin is the shells of marine or freshwater invertebrates. Microbial chitinases are largely responsible for chitin breakdown in nature, and they play an important role in the ecosystem's carbon and nitrogen balance. Several microbial chitinases have been characterized and are gaining prominence for their applications in various sectors. The current review focuses on chitinases of microbial origin, their diversity, and their characteristics. The applications of chitinases in several industries such as agriculture, food, the environment, and pharmaceutical sectors are also highlighted.
10.1007/s12223-022-00999-w
Enzymatic properties of β-N-acetylglucosaminidases.
Zhang Rui,Zhou Junpei,Song Zhifeng,Huang Zunxi
Applied microbiology and biotechnology
β-N-Acetylglucosaminidases (GlcNAcases) hydrolyse N-acetylglucosamine-containing oligosaccharides and proteins. These enzymes produce N-acetylglucosamine (GlcNAc) and have a wide range of promising applications in the food, energy, and pharmaceutical industries, such as synergistic degradation of chitin with endo-chitinases and using GlcNAc to produce sialic acid, bioethanol, single-cell proteins, and pharmaceutical therapeutics. GlcNAcases also play an important role in the dynamic balance of cellular O-linked GlcNAc levels, catabolism of ganglioside storage in Tay-Sachs disease, and bacterial cell wall recycling and flagellar assembly. In view of these important biological functions and the wide range of industrial applications of GlcNAcases, this review aims to provide a better understanding of various advances for these enzymes. It focuses on enzymatic properties of GlcNAcases, including substrate specificity, catalytic activity, pH optimum, temperature optimum, thermostability, the effects of various metal ions and organic reagents, and transglycosylation.
10.1007/s00253-017-8624-7
Chitin, Characteristic, Sources, and Biomedical Application.
Shahbaz Umar
Current pharmaceutical biotechnology
BACKGROUND:Chitin stands at second, after cellulose, as the most abundant polysaccharide in the world. Chitin is found naturally in marine environments as it is a crucial structural component of various marine organisms. METHODS:Different amounts of waste chitin and chitosan can be discovered in the environment. Chitinase producing microbes help to hydrolyze chitin waste to play an essential function for the removal of chitin pollution in the Marine Atmosphere. Chitin can be converted by using chemical and biological methods into prominent derivate chitosan. Numerous bacteria naturally have chitin degrading ability. RESULTS:Chitin shows promise in terms of biocompatibility, low toxicity, complete biodegradability, nontoxicity, and film-forming capability. The application of these polymers in the different sectors of biomedical, food, agriculture, cosmetics, pharmaceuticals could be lucrative. Moreover, the most recent achievement in nanotechnology is based on chitin and chitosan-based materials. CONCLUSION:In this review, we examine chitin in terms of its natural sources and different extraction methods, chitinase producing microbes and chitin, chitosan together with its derivatives for use in biomedical and agricultural applications.
10.2174/1389201021666200605104939