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Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of chitinase Isozymes from the Liver of Golden Cuttlefish <i>Sepia esculenta</i>

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Advances in Bioscience and Biotechnology 2017年第10期8卷 361-377页

作者： Ryo Nishino Hiromi Kakizaki Hideto Fukushima Masahiro Matsumiya Department of Marine Science and Resources College of Bioresource Sciences Nihon University Fujisawa Kanagawa Japan

The distribution of chitinolytic enzymes in eight organs of the golden cuttlefish Sepia esculenta was determined. chitinase activity (activity of endo-type chitinolytic enzyme) was measured using pNP-(GlcNAc)n (n = 2, 3) as substrates, with high activity detected in the liver, posterior salivary gland, and stomach. β-N-acetylhexosaminidase (Hex) activity (activity of exo-type chitinolytic enzyme) was determined using pNP-(GlcNAc) as a substrate, and high activity was observed in six organs, including the liver, branchial heart, posterior salivary gland, and stomach. In addition, two chitin-binding proteins (CBP-A, CBP-B) were isolated from the liver using a chitin affinity column. Two full-length cDNAs (SeChi-1: 1484 bp;SeChi-2: 1748 bp) encoding chitinases were obtained from the liver of S. esculenta. SeChi-1 contained a 1377-bp open reading frame (ORF) encoding 459 amino acids, and SeChi-2 contained a 1656-bp ORF encoding 552 amino acids. Domain structures predicted from the deduced amino acid sequences of SeChi-1 and SeChi-2 (SeChi-1, SeChi-2) contained signal peptides, a GH Family 18 catalytic domain, one chitin binding domain (CBD) in SeChi-1, and two CBDs in SeChi-2. Proteome analysis revealed that 125 peptide residues of CBP-A were present in SeChi-1, and 116 peptide residues of CBP-B were present in SeChi-2. Organ expression analysis revealed that SeChi-1 and SeChi-2 were expressed only in the liver of S. esculenta. Phylogenetic analysis of SeChi-1, SeChi-2, and GH family 18 chitinases revealed that SeChi-2 belongs to a group of previously reported squid chitinases, while SeChi-1 does not belong to any previously reported group of mollusk chitinases.

关键词： Chitinolytic Enzyme chitinase Distribution cDNA Cloning Golden Cuttlefish Sepia esculenta

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Cloning and Analysis of chitinase Gene in Bacillus subtilis HAS

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Plant Diseases and Pests 2019年第2期10卷 4-6页

作者： Xiong Guoru Zhao Gengfeng Cai Wenwei Wang Jungang Yang Benpeng Zhang Shuzhen Institute of Tropical Biotechnology/Sugarcane Research Center CATAS Department of Biochemistry Hainan Medical College

[Objective] The paper was to study the possible mechanism of chitinase gene. [Method] The chitinase gene with inhibitory effect against a variety of pathogenic fungi was cloned and analyzed in this study. [Result] The genome of HAS strain contained an ORF of 834 bp. The homology of coding protein corresponding to ORF with chitosanase [Bacillus subtilis subsp. subtilis str. 168](ref|NP_390566.1) was the highest of 98%(272/277).[Conclusion]Through the analysis of amino acid sequence and secondary structure of the protease, tertiary structure prediction and analysis, HAS strain may play a pivotal role in controlling Sporisorium scitaminea Syd.

关键词： Bacillus subtilis HAS Sporisorium scitaminea Syd. Antifungal protein chitinase gene

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Optimization of the chitinase Production by Different Metarhizium anisopliae Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD

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Advances in Microbiology 2012年第3期2卷 268-276页

作者： Cynthia Barbosa Rustiguel Joao Atílio Jorge Luis Henrique Souza Guimaraes Department of Biology Faculdade de FilosofiaCiencias e Letras de Ribeirao PretoUniversidade de Sao PauloSao PauloBrazil

Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control various insect pests. These fungi attack the integument of the host using an enzymatic complex. Among the enzymes found in this complex, chitinase is an important component. However, the relation between the chitinase production and the virulence from different M. anisopliae strains has not been analyzed. In this manuscript it is presented the chitinase production by four M. anisopliae strains with different potential of virulence in Solid-State Fermentation using silkworm chrysalis as substrate. The higher chitinase level was obtained with the strain IBCB 360 (7.14 U/g of substrate) with potential virulence of 68% on Diatrea saccharalis. The enzyme production was optimized for all strains using a factorial planning (CCRD) considering the cultivation time and medium humidity as independent variables. The maximal production of chitinase was obtained at a range from 8 to 12-days old cultures and from 45% to 62% of moisture according to the surface response plot, with high R2 value. The enzyme production by the strain IBCB 167 was increased two-folds under optimized conditions, while for the strains IBCB 360 and 425 the chitinase production was increased four-folds and nine-folds for the strain IBCB 384.

关键词： Entomopathogenic Fungus Metarhizium anisopliae chitinase Solid-State Fermentation

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Changes in Protein Content and chitinase and β-1,3- glucanase Activities of Rice with Blast Resistance Induced by Ag-antibiotic 702

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Plant Diseases and Pests 2017年第4期8卷 33-36页

作者： Hu Neng Tu Xiaorong Li Kuntai Ding Hao Li Huan Zhang Huawei Tu Guoquan Huang Lin College of Bioscience and Bioengineering Jiangxi Agriculture UniversityNanchang 330045China

[Objective] The paper was to explore the effect of rice blast resistance induced by Ag-antibiotics 702 on protein content and chitlnase and β-1,3-glucanase activities in rice. [ Method] At the fourth leaf stage of Luliangyou 996, 15 μg/mL Ag-antibiotic 702 was sprayed, while Validamycin and distilled wa- ter were sprayed as positive control and negative control, respectively. All treatments were inoculated with spore fluid of Magnaporthe grisea at 48 h post spraying, and the rice inoculated with only distilled water was used as blank control. The enzymes activities （endochitinase, exochitinase and β-1,3-glucanase） and total pro- tein content in rice leaves were determined every 24 h within 168 h post spraying. [ Result] Compared with the blank control, the rice inoculated with spore fluid of M. grisea could significantly increase the total protein content and the activities of β-1,3-glucanase and chitinase. The induction effect of Ag-antibiotics 702 exceeded that of Validamycin treatment. And the changes in activities of β-1,3-glucanase and chitinase had obvious synchronicity. [ Conclusion] Ag-antibiotic 702 can significantly improve the total protein content and the activities of β-1,3-glucanase and chitinase, thus enhancing the resistance to rice blast.

关键词： Ag-antibiotics 702 Luliangyou 996 Rice blast chitinase β-1,3-glucanase

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chitinase转基因拟南芥对棉铃虫核型多角体病毒（HearNPV）增效作用研究

Chitinase转基因拟南芥对棉铃虫核型多角体病毒（HearNPV）增效作...

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作者：王毛西北农林科技大学

学位级别：硕士

棉铃虫核型多角体病毒（Hear NPV）含有的几丁质酶在昆虫液化和病毒与寄主传播水平上有着重要作用。此外,原核表达的几丁质酶还可以增强Hear NPV的感染效率和杀虫毒力。而几丁质是昆虫消化道一个重要的结构,是昆虫体内中肠中天然屏障围... 详细信息

棉铃虫核型多角体病毒（Hear NPV）含有的几丁质酶在昆虫液化和病毒与寄主传播水平上有着重要作用。此外,原核表达的几丁质酶还可以增强Hear NPV的感染效率和杀虫毒力。而几丁质是昆虫消化道一个重要的结构,是昆虫体内中肠中天然屏障围食膜的重要组成成分,即在昆虫自身保护其免受肠内容物的伤害中扮演重要角色。在这项研究中,以拟南芥（Arabidopsis thaliana）为植物载体,利用根癌农杆菌（Agrobacterium tumefaciens）介导的花序侵染法对拟南芥转化了chitinase外源基因,并进行了其对棉铃虫核型多角体病毒（Helicoverpa armigera nucleopolyhedrovirus,Hear NPV）增效作用研究,取得如下结果:1、利用花序侵染法转化拟南芥,获得了6株表现型为阳性的chitinase转基因拟南芥株系,通过PCR检测初步确定为阳性的转化拟南芥株系。2、通过RT-PCR检测,表明目的基因chitinase在6个转基因株系A-F中都有表达,并且株系A的相对表达量最高,株系D的相对表达量最低。3、chitinase转基因拟南芥对Hear NPV的增效作用试验表明,饲喂株系A的棉铃虫感染Hear NPV后的LT50和LT90比取食野生型拟南芥对照组分别缩短了36.17%和40.33%。饲喂株系D的棉铃虫感染Hear NPV后的LT50和LT90比对照组分别缩短了18.08%和25.14%。4、感染相同剂量的棉铃虫在取食转基因植物后病毒在细胞中DNA拷贝数显著高于取食野生型植物的棉铃虫,其中取食转基因株系A的棉铃虫感染Hear NPV后其细胞体内病毒复制能力最强也最快,其次是取食株系D的棉铃虫。5、取食转基因拟南芥的棉铃虫病毒产量明显高于取食野生型拟南芥,且取食转基因拟南芥株系A的棉铃虫病毒产量明显高于取食转基因株系D的棉铃虫。6、感染HearNPV的棉铃虫无论取食野生型拟南芥、株系A或株系D,其日均虫重均无显著性差异;单取食转基因和野生型拟南芥未感染病毒的棉铃虫日累积增重没有显著差异,说明转基因植物单独饲喂对棉铃虫不会有很大影响。结果表明,chitinase转基因拟南芥在饲喂同时感染Hear NPV的棉铃虫时有利用病毒粒子更快更多的进入昆虫中肠细胞,且有助于病毒Hear NPV在棉铃虫体内的增值,以及提高病毒对棉铃虫的杀虫速度和杀虫效率。

关键词：拟南芥 chitinase 转基因植物 HearNPV 棉铃虫增效

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Bioluminescence and chitinase Production during Chitin Fermentation by Vibrio harveyi

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Journal of Life Sciences 2013年第5期7卷 491-494页

作者： Badireddy Madhusudana Rao Floyd L. Inman Ⅲ Leonard D. Holmes Sartorius Stedim Biotechnology Laboratory Biotechnology Research and Training Center University of North Carolina NorthCarolina 28472 USA Central Institute of Fisheries Technology Visakhapatnam Research Centre Andhra University Visakhapatnam 530003 India

Vibrio harveyi, like other luminescent bacteria, is capable of producing extracellular chitinases. Microbial chitinases are utilized to depolymerize chitin into chitooligosaccharides and N-acetylglucosamine for the acquisition of carbon and possibly nitrogen, needed for survival. For many luminous marine bacteria (Vibrio spp.), quorum-sensing is highly speculated to be responsible for bioluminescence; however, in terrestrial species (Photorhabdus spp.) luminosity seems to be controlled through unknown mechanism of phase variation. In the present work, the correlation between bacterial luminosity and chitinase production of V. harveyi was studied. The utilization of bioluminescence could prove to be an easier and more convenient method to monitor chitin fermentations that employ luminous bacteria. Results from the fermentation study indicate that luminosity of V. harveyi inversely correlates with chitinase production. In other words, during chitin fermentation, chitinase production was seen to increase while luminosity decreased with respect to growth and growth conditions. Furthermore, the results also suggest that V. harveyi may utilize an alternate mechanism that can counter quorum-sensing mechanisms to ensure bacterial survival under deteriorating growth conditions. The inverse relationship observed in this study may lead to a basic understanding of monitoring and studying chitin fermentations and anti-quorum-sensing/phase variation mechanisms exhibited by luminous bacteria.

关键词： Vibrio harveyi bioluminescence chitinase chitin fermentation

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小拟南芥chitinase基因原核表达载体的构建及其在大肠杆菌中的表达

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植物研究 2005年第4期25卷 419-422页

作者：朱新霞田丽萍高剑峰祝建波新疆兵团绿洲生态农业重点实验室石河子832003 石河子大学生物工程学院石河子832003

以野生资源小拟南芥(Arabidopsis pum ila)chitinase基因的cDNA为基础,采用基因重组技术,将该基因按正确的阅读框架定向克隆于原核表达载体pET-30a(+)中,转化大肠杆菌BL21(DE3),用IPTG诱导表达,并对表达产物进行SDS—PAGE分析。结果表明... 详细信息

以野生资源小拟南芥(Arabidopsis pum ila)chitinase基因的cDNA为基础,采用基因重组技术,将该基因按正确的阅读框架定向克隆于原核表达载体pET-30a(+)中,转化大肠杆菌BL21(DE3),用IPTG诱导表达,并对表达产物进行SDS—PAGE分析。结果表明:重组小拟南芥chiti-nase基因在大肠杆菌中获得高效表达,其分子量约为40 KD。小拟南芥chitinase基因原核表达载体的成功构建和重组小拟南芥chitinase蛋白在大肠杆菌中的高效表达,为进一步研究其生物学功能奠定了基础。

关键词：小拟南芥 chitinase 基因原核表达载体大肠杆菌基因表达生物学功能

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Purification and characterization of an antifungal chitinase from Bacillus sp. SL-13

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生物技术世界 2014年第12期11卷 244-245页

作者： Chen Shan Tianjin Modern Vocational Technology College

Bacillus sp.SL-13 produced antifungal proteins.The growth of the plant-pathogenic fungi Rhizoctonia solani was considerably inhibited by the presence of the SL-13 culture supernatant.The proteins were purified by DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration,and the main antifungal protein was purified to be chitinase.The molecular weight of chitinase was estimated to be 36 kD by 12%SDS PAGE.The optimal pH and temperature for the chitinase was 7.0 and 50℃.It demonstrated that the enzyme was stable from pH 5 to 9 and form 40?C to 60℃.The enzyme still kept 70%activity when incubated at 121℃,0.11MPa up to 20 minutes and the enzyme is also not lost the activity when treated with protease K and ultraviolet radiation for 1.5hours.It is very suitable for the use in a relatively unstable environment,exhibiting effective biological control.

关键词： Bacillus sp. Antifungal chitinase Purification

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Characterization of an antifungal chitinase from Bacillus sp.SL-13

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生物技术世界 2014年第11期11卷 206-206,208页

作者： Chen Shan Tianjin Modern Vocational Technology College

关键词： Bacillus sp. Antifungal chitinase

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Phenological Stage, Plant Biomass, and Drought Stress Affect Microbial Biomass and Enzyme Activities in the Rhizosphere of Enteropogon macrostachyus

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Pedosphere 2019年第2期29卷 259-265页

作者： Kevin Z.MGANGA Bahar S.RAZAVI Muhammad SANAULLAH Yakov KUZYAKOV Department of Soil Science of Temperate Ecosystems Institute of Soil Science University of G?ttingen Department of Range and Wildlife Sciences South Eastern Kenya University Department of Agricultural Soil Science Institute of Soil Science University of G?ttingen Institute of Soil and Environmental Sciences University of AgricultureFaisalabad Institute of Environmental Sciences Kazan Federal University

Indigenous grasses have been effectively used to rehabilitate degraded African drylands. Despite their success, studies examining their effects on soil bioindicators such as microbial biomass carbon(C) and enzyme activities are scarce. This study elucidates the effects of drought stress and phenological stages of a typical indigenous African grass, Enteropogon macrostachyus, on microbial biomass and enzyme activities(β-glucosidase, cellobiohydrolase, and chitinase) in the rhizosphere soil. Enteropogon macrostachyus was grown under controlled conditions. Drought stress(partial watering) was simulated during the last 10 d of plant growth, and data were compared with those from optimum moisture conditions. The rhizosphere soil was sampled after 40 d(seedling stage), 70 d(elongation stage), and 80 d(simulated drought stress). A high root:shoot ratio at seedling stage compared with elongation and reproduction stages demonstrated that E. macrostachyus invested more on root biomass in early development, to maximise the uptake of nutrients and water. Microbial biomass and enzyme activities increased with root biomass during plant growth. Ten-day drought at reproduction stage increased the microbial biomass and enzyme activities, accompanying a decrease in binding affinity and catalytic efficiency. In conclusion, drought stress controls soil organic matter decomposition and nutrient mobilization, as well as the competition between plant and microorganisms for nutrient uptake.

关键词： African grass catalytic efficiency cellobiohydrolase chitinase β-glucosidase nutrient uptake optimum moisture soil bioindicators

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