Chitinolytic enzymes: their contribution to basic and applied research

After cellulose, chitin is the second most abundant renewable resource available in nature. Marine invertebrates and fungal biomass are the two main sources of chitinous waste, which is commercially exploited. The enzymes involved in chitin degradation have been particularly well studied. Such enzymes have applications in ultrastructural studies, in the preparation of chitooligosaccharides which show anti-tumour activity, as biocontrol agents and in single-cell protein production. Here, the contribution chitin enzymology can make to basic and applied research is discussed.     

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT₅₀ of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60–75 g/kg rice), faster sedimentation time (ST₅₀) (2.3–2.65 h in 0.1% (w/v) Tween 80) and lower LC₅₀ (1.4–5.7×10³ conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).     

Production of chitinolytic enzymes from a novel species ofAeromonas

A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source. The isolated bacterium, designated asAeromonas sp No. 16 exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml^–1. A slightly higher enzymatic activity of 1.5 U ml^–1 was obtained when cultivation was carried out in a 5-liter jar fermentor using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of -N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

The peritrophic membrane: Ultrastructural analysis and function as a mechanical barrier to microbial infection in Orgyia pseudotsugata

The feasibility of freeze-etching as a method for structural analysis of the peritrophic membrane (PM) has been demonstrated, and this method and other electron microscopical techniques have been employed to investigate the possible function of the PM as a mechanical barrier to infection of the larval midgut. Thin sections show that the PM consists of granular and microfibrous layers typical of PMs from other insects. Freeze-etch studies show that the membrane consists of at least three structurally distinct layers. The major component is a layer of chitin microfibrils that appear to be arranged in oriented layers. The fibrils exhibit a periodicity of 4 nm and are embedded in a granular matrix. The fibrils coalesce into 100-nm fibers which run throughout the microfibrillar layer. Adjacent to this layer is a very rough layer, with randomly dispersed granules 100 nm in diameter and covered with particles 32 nm in diameter. Adjacent to the midgut lumen is a finely granular layer which gives the PM a smooth appearance. No pores or other discontinuities through which a bacterial or viral pathogen could penetrate were detected in the PM, indicating the PM of the Douglas fir tussock moth could function as a mechanical barrier to microbial infection. The importance of chitin and protein to the apparent strength of the Douglas fir tussock moth PM was partially elucidated by studies with chitinases and proteases. Both types of enzymes degraded the PM, causing release of the products of hyrolysis or structural changes in the membrane.     

An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.     

Effect of repeated in vitro sub-culturing on the virulence of Metarhizium anisopliae against Helicoverpa armigera (Lepidoptera: Noctuidae)

The effect of repeated conidial sub-culturing of Metarhizium anisopliae on its virulence against Helicoverpa armigera (Hübner) was studied. The LT₅₀ observed against third instar larvae of H. armigera for the first sub-culture was 3.4 days; it increased to 4.5 and 5.6 days for the 20th and the 40th sub-cultures, respectively. The LT₅₀ values after passage of the 40th sub-culture on H. armigera decreased to 4.4 and 3.7 days for the 40th (first in vivo) and the 40th (fifth in vivo) passages, respectively. Similarly, the LC₅₀ of M. anisopliae towards third instar larvae of H. armigera increased from the first sub-culture (0.17×10⁴) to (3.0×10⁴) for the 40th conidial transfers on potato dextrose agar and again decreased to 0.74×10⁴ and 0.23×10⁴ in the 40th (first in vivo) and the 40th (fifth in vivo) passage, respectively. Similar trends for LC₅₀ and LT₅₀ values were seen when sugarcane woolly aphid, Ceratovacuna lanigera Zehntner was used as a host. Significant variation in appressorium formation and cuticle-degrading enzyme production such as chitinase, chitin deacetylase, chitosanase and protease during subsequent sub-culturing and passage through H. armigera was observed. Though there was no effect on internal transcribed spacer (ITS) sequence pattern, interestingly, in randomly amplified polymorphic DNA (RAPD), significant differences in the band intensities and in the banding pattern for different sub-cultures of M. anisopliae were observed. As stable virulence towards the insect pest is desirable for commercialisation of a mycoinsecticide, such changes in virulence due to repeated in vitro transfer need to be monitored and minimised.     

Identification of isobavachalcone as a potential drug for rice blast disease caused by the fungus Magnaporthe grisea

The rice blast disease caused by the fungus Magnaporthe grisea is one of the most devastating rice diseases, but there is no effective fungicide toward chitinase which is a key enzyme of M. grisea. In this study, we observed that distortion and cell-wall damage of M. grisea hyphae were significantly under the scanning electron micrograph after a 24-h treatment with 10?mg/L isobavachalcone (IBC) extracted from Psoralea corylifolia L. To further explore the effect of IBC on the cell wall of M. grisea, we examined changes in enzymes associated with cell wall degradation by enzyme activity experiments, treated liquid culture mycelia with 10?mg/L IBC for 1?h. Results displayed that chitinase was obviously more active than control group. To illustrate the interactions between IBC and chitinase, the studies of homology modeling and molecular docking were carried out successively. The results revealed that IBC had hydrogen bonds with residues ASP267 and ARG276 of chitinase. Besides, these nonpolar residues TYR270, PRO271, VAL272, LEU310, PRO311, TYR316, and LEU317 were able to form strong hydrophobic interactions. Binding energies of the chitinase-IBC complexes were calculated by MM-GBSA showed that the ΔG_bind score of molecular dynamics had lower binding energy and more stable than docking complexes. All above, IBC owns significant agonistic activity in chitinase and would be a potent fungicide to inhibit the growth of M. grisea. We hope the above information provides an important insight for understanding the interactions between IBC and chitinase, which may be useful in the discovery of a novel potent agonist.

Communicated by Ramaswamy H. Sarma     

Isolation and identification of cytokinins in a new commercial seaweed product made from Fucus serratus L.

Marine Adenine Cytokinins (MAC) is a new commercial seaweed product made from Fucus serratus L. Cytokinins were isolated from this extract by cation exchange, paper and high performance liquid chromatography. Cytokinins were detected and isolated by bioassay guided fractionation using the soybean callus assay. Trans-zeatin, dihydrozeatin and iso-pentenyladenosine were identified by GC-MS as being the dominant cytokinins present in MAC. This revised version was published online in August 2006 with corrections to the Cover Date.     

An efficient synthesis of argifin: a natural product chitinase inhibitor with chemotherapeutic potential

The first synthesis of the cyclopentapeptide family 18 chitinase inhibitor argifin has been achieved by a combination of solid phase and solution chemistry. Synthetic argifin is a nanomolar inhibitor of chitinase B1 from Aspergillus fumigatus and the high-resolution X-ray structure of the synthesized material in complex with the same enzyme is identical to that previously obtained for the natural product.     

Regulation of human immune gene expression as influenced by a commercial blended Echinacea product: preliminary studies

Consumption of Echinacea at the first sign of symptoms has been clinically shown to reduce both the severity and duration of cold and flu. Quantitative polymerase chain reaction optimized for precision and reproducibility was utilized to explore in vitro and in vivo changes in the expression of immunomodulatory genes in response to Echinacea. In vitro exposure of THP-1 cells to 250 microg/ml of Echinacea species extracts induced expression (up to 10-fold) of the interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, interleukin-8, and interleukin-10 genes. This preliminary result is consistent with a general immune response and activation of the nonspecific immune response cytokines. In vivo gene expression within peripheral leukocytes was evaluated in six healthy nonsmoking subjects (18-65 years of age). Blood samples were obtained at baseline and on Days 2, 3, 5, and 12 after consuming a commercial blended Echinacea product, three tablets three times daily (1518 mg/day) for two days plus one additional dose (506 mg) on day three. Serum chemistry and hematological values were not different from baseline, suggesting that liver or bone marrow responses were not involved in acute responses to Echinacea. The overall gene expression pattern at 48 hr to 12 days after taking Echinacea was consistent with an antiinflammatory response. The expression of interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, and interleukin-8 was modestly decreased up through Day 5, returning to baseline by day 12. The expression of interferon-alpha steadily rose through Day 12, consistent with an antiviral response. These preliminary data present a gene expression response pattern that is consistent with Echinacea's reported ability to reduce both the duration and intensity of cold and flu symptoms.     

N‐terminal fusion of a hyperthermophilic chitin‐binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes

Immobilization of a thermostable D ‐xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N‐terminal fusion with a chitin‐binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD‐TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD‐TNXI were greater than the wild‐type enzyme: k_cat (min^?1) was ～1,000, 3,800, and 5,800 at 80°C compared to 1,140, 10,350, and 7,000 at 90°C, for the wild‐type, unbound, and immobilized enzymes, respectively. These k_cat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi‐phasic inactivation model, showed that the CBD‐TNXI bound to chitin had a half‐life approximately three times longer than the soluble wild‐type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD‐TNXI had a significantly longer half‐life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N‐terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD‐TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate‐binding affinities and generating higher turnover rates in the unbound fusion protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Plectin: general overview and appraisal of its potential role as a subunit protein of the cytomatrix

Plectin has recently been identified as a widespread and abundant cytoplasmic protein of mammalian cells. In this article the available data on plectin are reviewed, focusing on plectin's occurrence and localization in various cell types and tissues, its biochemical characterization, and its molecular interaction partners. Furthermore, the putative role of this protein has a multifunctional connecting link of the cytomatrix and its structural as well as functional relationship to other cytoskeletal proteins is discussed. It is concluded that plectin is potentially the most versatile crosslinking element of the cytomatrix reported to date.     

Molecular Engineering Using an Anthanthrone Dye for Low‐Cost Hole Transport Materials: A Strategy for Dopant‐Free,High‐Efficiency,and Stable Perovskite Solar Cells

In this report, highly efficient and humidity‐resistant perovskite solar cells (PSCs) using two new small molecule hole transporting materials (HTM) made from a cost‐effective precursor anthanthrone (ANT) dye, namely, 4,10‐bis(1,2‐dihydroacenaphthylen‐5‐yl)‐6,12‐bis(octyloxy)‐6,12‐dihydronaphtho[7,8,1,2,3‐nopqr]tetraphene (ACE‐ANT‐ACE) and 4,4′‐(6,12‐bis(octyloxy)‐6,12‐dihydronaphtho[7,8,1,2,3‐nopqr]tetraphene‐4,10‐diyl)bis(N,N‐bis(4‐methoxyphenyl)aniline) (TPA‐ANT‐TPA) are presented. The newly developed HTMs are systematically compared with the conventional 2,2′,7,7′‐tetrakis(N,N′‐di‐p‐methoxyphenylamino)‐9,9′‐spirbiuorene (Spiro‐OMeTAD). ACE‐ANT‐ACE and TPA‐ANT‐TPA are used as a dopant‐free HTM in mesoscopic TiO₂/CH₃NH₃PbI₃/HTM solid‐state PSCs, and the performance as well as stability are compared with Spiro‐OMeTAD‐based PSCs. After extensive optimization of the metal oxide scaffold and device processing conditions, dopant‐free novel TPA‐ANT‐TPA HTM‐based PSC devices achieve a maximum power conversion efficiency (PCE) of 17.5% with negligible hysteresis. An impressive current of 21 mA cm^?2 is also confirmed from photocurrent density with a higher fill factor of 0.79. The obtained PCE of 17.5% utilizing TPA‐ANT‐TPA is higher performance than the devices prepared using doped Spiro‐OMeTAD (16.8%) as hole transport layer at 1 sun condition. It is found that doping of LiTFSI salt increases hygroscopic characteristics in Spiro‐OMeTAD; this leads to the fast degradation of solar cells. While, solar cells prepared using undoped TPA‐ANT‐TPA show dewetting and improved stability. Additionally, the new HTMs form a fully homogeneous and completely covering thin film on the surface of the active light absorbing perovskite layers that acts as a protective coating for underlying perovskite films. This breakthrough paves the way for development of new inexpensive, more stable, and highly efficient ANT core based lower cost HTMs for cost‐effective, conventional, and printable PSCs.     

国家公园生态系统生产总值核算——以海南热带雨林国家公园为例

海南热带雨林国家公园拥有我国最具代表性、连片面积最大的热带雨林,生态产品丰富,在开展国家公园生态产品价值实现机制探索上具有得天独厚的优势。本研究基于《陆地生态系统生产总值(GEP)核算技术指南》框架,构建符合热带雨林国家公园特色的GEP核算体系,对2019年海南热带雨林国家公园GEP进行核算。结果表明: 2019年,海南热带雨林国家公园GEP为2045.13亿元,单位面积GEP为0.46亿元·km^-2。其中,物质产品价值为48.50亿元,占国家公园GEP总量的2.4%;生态系统调节服务价值为1688.91亿元,占82.6%;文化服务价值为307.72亿元,占15.0%。从不同生态系统类型来看,以山地雨林、低地雨林、落叶季雨林、热带云雾林等为代表的热带雨林生态系统的单位面积价值量远高于人工林及其他生态系统,表明热带雨林生态系统在提供生态系统服务功能方面具有显著作用。此外,本研究还基于国家公园GEP核算结果,提出进一步探索生态产品价值实现路径和实现机制的相关建议。     

Altered product specificity of a cyclodextrin glycosyltransferase by molecular imprinting with cyclomaltododecaose

Cyclodextrin glycosyltransferases (CGTases), members of glycoside hydrolase family 13, catalyze the conversion of amylose to cyclodextrins (CDs), circular α‐(1,4)‐linked glucopyranose oligosaccharides of different ring sizes. The CD containing 12 α‐D‐glucopyranose residues was preferentially synthesized by molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose (CD₁₂) as the template molecule. The imprinted CGTase was stabilized by cross‐linking of the derivatized protein. A high proportion of CD₁₂ and larger CDs was obtained with the imprinted enzyme in an aqueous medium. The molecular imprinted CGTase showed an increased catalytic efficiency of the CD₁₂‐forming cyclization reaction, while decreased k_cat/K_m values of the reverse ring‐opening reaction were observed. The maximum yield of CD₁₂ was obtained when the imprinted CGTase was reacted with amylose at 40°C for 30 min. Molecular imprinting proved to be an effective means toward increase in the yield of large‐ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Modelling carbon stocks and fluxes in the wood product sector: a comparative review

In addition to forest ecosystems, wood products are carbon pools that can be strategically managed to mitigate climate change. Wood product models (WPMs) simulating the carbon balance of wood production, use and end of life can complement forest growth models to evaluate the mitigation potential of the forest sector as a whole. WPMs can be used to compare scenarios of product use and explore mitigation strategies. A considerable number of WPMs have been developed in the last three decades, but there is no review available analysing their functionality and performance. This study analyses and compares 41 WPMs. One surprising initial result was that we discovered the erroneous implementation of a few concepts and assumptions in some of the models. We further described and compared the models using six model characteristics (bucking allocation, industrial processes, carbon pools, product removal, recycling and substitution effects) and three model‐use characteristics (system boundaries, model initialization and evaluation of results). Using a set of indicators based on the model characteristics, we classified models using a hierarchical clustering technique and differentiated them according to their increasing degrees of complexity and varying levels of user support. For purposes of simulating carbon stock in wood products, models with a simple structure may be sufficient, but to compare climate change mitigation options, complex models are needed. The number of models has increased substantially over the last ten years, introducing more diversity and accuracy. Calculation of substitution effects and recycling has also become more prominent. However, the lack of data is still an important constraint for a more realistic estimation of carbon stocks and fluxes. Therefore, if the sector wants to demonstrate the environmental quality of its products, it should make it a priority to provide reliable life cycle inventory data, particularly regarding aspects of time and location.