昆虫来源的几丁质酶的分离纯化及酶学性质

几丁质酶在真菌和昆虫的生理和发育过程中起着关键作用,该酶本身及其酶抑制剂是获取生物农药的重要途径。本研究从蚕蛹体内提取几丁质粗酶,经硫酸铵分级沉淀和Sephadex G-150分离得到几丁质酶。用SDS-PAGE测得该酶的分子量为88kDa。水解胶体几丁质的Km值为22.3μmol/L。酶反应的最适温度为45℃,最适pH值为6.0,金属离子和有机试剂对几丁质酶活性都有影响,其中高浓度的Mn2+对酶有较强的激活作用,而Cu2+、SDS则有较强的抑制作用。研究结果为基于几丁质酶的生物农药筛选研究奠定了基础。     

家蚕的各种应用

家蚕和人类相伴生存了5000年之久,除了取茧、缫丝获取精美的丝绸之外,家蚕自身许多独特的作用也被人们——挖掘,从卵、幼虫、蛹、蚕蜕、茧及粪便等均被研究出具有药用价值,可以毫不夸张地说家蚕浑身都是宝。蚕蛹蚕蛹中含有丰富的蛋白质和脂肪,包括人体所必需的8种氨基酸;蛹油的脂肪酸中不饱和脂肪酸占75%,其营养价值可与猪肉、禽蛋相媲美。具有很高的食用价值。过去,蚕蛹是蚕茧缫丝后缫丝厂的下脚物,蚕区多作为禽畜及塘鱼饲料。     

昆虫蛹油的索氏提取法

昆虫蛹广泛存在于自然界中,有野生的亦有人工饲养的,尤以人工饲养的来源较为广泛,如蚕蛹等。通常蚕蛹作为缫丝加工后的下脚料用作牲口等的饲料。昆虫蛹体内富含粗脂肪(桑蚕蛹含30%左右),从蛹中提取出的蛹油可作为工业、医药用原材料,经加工制成肥皂、增塑剂、土耳其红油、润滑剂、甘油、亚油酸及一系列高级脂肪酸等,最引人注目的是能以蛹油为原料分离制成脑白金的有效成分DHA(二十二碳六烯酸)。近年来国内外研究者就昆虫蛹油的提取开发做了许多有益的探索。笔者以蚕蛹为例介绍一种提取蛹油的实验方法——索氏提取法。1　蛹油的组成特点蛹…     

几丁质酶抑制剂的研究进展

几丁质和几丁质酶在工农业、环保、医药、食品等行业具有广泛的用途。几丁质酶抑制剂是指一类能特异性地与几丁质酶结合,并对几丁质酶产生抑制作用的化合物分子。研究几丁质酶抑制剂,不仅对于深入研究几丁质酶的结构与功能,而且还可能为发现新型的几丁质酶抑制剂提供依据,并为杀菌剂、杀虫剂、化疗药物等的发展提供新的导向。     

油葫芦的营养价值(英文)

本研究旨在确定油葫芦Gryllus testaceus Walker的主要营养成分和营养价值。研究发现油葫芦体内主要含有蛋白质、脂类和几丁质三类物质,它们的含量(干重比)分别是58.3％,10.3％和8.7％。其中必需氨基酸的含量除了半胱氨酸和蛋氨酸外,非常符合FAO／WHO所确定的人体对氨基酸需求标准。脂肪酸分析显示,不饱和脂肪酸的含量很高,仅油酸、亚油酸和亚麻酸的含量就占总脂肪含量的77.51％。油葫芦的甲壳素含量为8.7％,而且从该虫提取甲壳素／壳聚糖的品质在甲壳素色泽及残留灰分含量、壳聚糖粘度等方面比常规甲壳素原料虾蟹壳的好。因此,油葫芦可以作为很好的食品或饲料添加剂,或医药原料。     

蚕蛹蛋白的营养价值及其提取方法

本文测定了家蚕蛹的化学成分及其氨基酸的组成,得到的结果是蚕蛹中有17种氨基酸,而其中8种人体必须氨基酸占全部氨基酸的44.11%,分析了营养价值,初步获得用酸矸法提取蚕蛹蛋白的方法。     

耐除草剂转基因大豆ZH10-6和亲本大豆中黄10的营养成分比较

目的：比较北京、石家庄、三亚三个产地的耐除草剂转基因大豆ZH10 6和亲本大豆中黄10的营养成分。方法：对三产地耐除草剂转基因大豆ZH10 6和亲本大豆中黄10的营养成分：水分、灰分、蛋白质、脂肪、膳食纤维、氨基酸、维生素、矿物质、脂肪酸等进行检测和分析。结果：三产地转基因大豆的钙、钾、叶酸含量高于亲本大豆,但均在ILSI推荐的参考范围内;个别产地、个别营养成分转基因大豆与亲本大豆营养成分存在差异,但属于自然变异;其余各营养成分转基因大豆和亲本大豆之间均无显著性差异。结论：耐除草剂转基因大豆ZH10 6和亲本大豆中黄10在营养成分上具有实质等同性。     

21科41种(变种)植物叶片几丁质酶系的研究

对广州地区常见的21科41种（变种）植物叶片几丁质酶系研究的结果表明,所有受试植物都具有几丁质酶活性。几丁质酶不仅存在于被子植物的双子叶植物和单子叶植物中,而且也存在于裸子植物及蕨类植物中。几丁质酶活性及比活性均较高的有蕹菜、葱、蕨类植物、蒜、茄科植物、玉米、菜豆、番木瓜等。植物一般都具有两种几丁质酶:外切酶和内切酶。几丁质外切酶活性及比活性均较高的有蕨类植物、葱、茄科植物等。几丁质内切酶活性及比活性均较高的有蕹菜、裸子植物等。不同植物几丁质外切酶与内切酶的比例相差较大。有些植物的几丁质酶系以外切酶为主,如茄科植物、大部分豆科植物、番木瓜等;有些植物以内切酶为主,如裸子植物、伞形科植物、蕹菜等;有些植物的外切酶与内切酶含量相差不大。     

转几丁质酶基因防植物病害研究:进展、问题与展望

综合评述了近几年转几丁质酶基因防植物病害研究中的发现与进展：（１）至少３１种病原真菌（含变种和专化型）可诱导植物几丁质酶,（２）据称已提纯的植物几丁质酶对立枯丝核菌等真菌呈现了体外抑菌活性;（３）一些生防细菌的防病作用依赖于其几丁质酶基因的存在;（４）数种植物几丁质酶基因和一种细菌几丁质酶基因已被转入水稻、烟草、番茄等植物,一些转基因株系抗病性显著增强;（５）粘质沙雷氏菌的一种几丁质酶基因已被转入荧光假单胞菌和根瘤菌中,转基因细菌对立枯丝核菌有显著抑制作用。就几丁质酶抑菌谱、转基因的策略和研究中存在的疑点进行了分析讨论。建议今后的研究方向应集中在以下几方面：（１）加速转几丁质酶基因或几丁质酶基因与其它基因组合的植物实用化;（２）进一步研究不同基因组合的增效作用及增效机理;（３）查明个别几丁质酶基因的抑菌谱及选择性抑菌机制;（４）转几丁质酶基因或与其它基因的组合于植物内生细菌。     

黄精种子营养成分的测定与分析

采用化学分析的方法,对黄精(Polygonatum sibiricum Red.)种子主要营养成分--粗淀粉、粗脂肪、粗蛋白的含量进行了测定,并采用气相色谱法、氨基酸自动分析仪分别对其脂肪酸和氨基酸的组分作了分析,以明确黄精种子的营养成分及其利用价值.结果显示:(1)黄精种子的营养成分以淀粉(73.70%)为主,脂肪含量为(10.74%),蛋白质含量为(8.21%);(2)脂肪酸中以不饱和脂肪酸(89.52%)为主,其中人体必需的多不饱和脂肪酸亚油酸占49.77%;(3)氨基酸种类齐全,其中人体必需氨基酸蛋氨酸、赖氨酸、异亮氨酸的含量比小麦、玉米等作物的含量高.结果说明,黄精种子具有较高的营养价值,在食品、医疗保健方面具有一定开发前景.     

家蚕前部丝腺特异表皮蛋白Bm11721的鉴定及表达

家蚕的丝腺是其丝蛋白合成和分泌的器官,根据其形态和功能的不同分为前部、中部和后部丝腺,前部丝腺不具有合成丝蛋白的能力,是丝蛋白构象发生转变的场所。剪切力在丝蛋白构象转变中起到重要的作用,其在家蚕前部丝腺主要由前部丝腺逐渐变细的管腔结构和富含几丁质及表皮蛋白的坚硬的内壁提供。鉴定家蚕前部丝腺新的几丁质结合蛋白,并调查其在家蚕幼虫不同组织的表达特征。通过几丁质亲和层析的方法在前部丝腺筛选并鉴定到一个新的具有几丁质结合功能的表皮蛋白Bm11721,其编码基因编号为BGIBMGA011721(Gen Bank Accession No.NM-001173285.1)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm11721的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm11721均只在前部丝腺特异表达,且Bm11721蛋白在5龄期的前部丝腺中恒定表达。免疫荧光定位结果显示Bm11721蛋白定位在前部丝腺的内膜中,推测其可能与前部丝腺的机械硬度有关,为丝蛋白的构象转变提供剪切力。     

Comparative Proteome Analysis Reveals that Cuticular Proteins Analogous to Peritrophin‐Motif Proteins are Involved in the Regeneration of Chitin Layer in the Silk Gland of Bombyx mori at the Molting Stage

The silk gland of silkworm produces silk proteins during larval development. Many studies have long focused on the silk gland of the fifth instar larvae, but few have investigated this gland at other larval stages. In the present study, the silk gland proteomes of the fourth instar and fourth molt are analyzed using liquid chromatography–tandem mass spectrometry. In total, 2654 proteins are identified from the silk gland. A high abundance of ribosomal proteins and RR‐motif chitin‐binding proteins is identified during day 2 of the fourth instar (IV‐2) larval developmental stage, and the expression of cuticular proteins analogous to peritrophin (CPAP)‐motif chitin‐binding proteins is higher during the fourth molt (IV‐M). In all, nine enzymes are found to be involved in the chitin regeneration pathway in the silk gland. Among them, two chitinase and two chitin deacetylases are identified as CPAP‐motif proteins. Furthermore, the expression of CPAP3‐G, the most abundant CPAP‐motif cuticular protein in the silk gland during the IV‐M stage, is investigated using western blot and immunofluorescence analyses; CPAP3‐G shows a reverse changing trend with chitin in the silk gland. The findings of this study suggest that CPAP‐motif chitin‐binding proteins are involved in the degradation of the chitin layer in the silk gland. The data have been deposited to the ProteomeXchange with identifier PXD008677.     

Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm,Bombyx mori

In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5–2.0 and 5.5–6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84 % of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.     

Oral Administration of a Fusion Protein between the Cholera Toxin B Subunit and the 42-Amino Acid Isoform of Amyloid-β Peptide Produced in Silkworm Pupae Protects against Alzheimer's Disease in Mice

A key molecule in the pathogenesis of Alzheimer''s disease (AD) is a 42-amino acid isoform of the amyloid-β peptide (Aβ42), which is the most toxic element of senile plaques. In this study, to develop an edible, safe, low-cost vaccine for AD, a cholera toxin B subunit (CTB)-Aβ42 fusion protein was successfully expressed in silkworm pupae. We tested the silkworm pupae-derived oral vaccination containing CTB-Aβ42 in a transgenic mouse model of AD. Anti-Aβ42 antibodies were induced in these mice, leading to a decreased Aβ deposition in the brain. We also found that the oral administration of the silk worm pupae vaccine improved the memory and cognition of mice, as assessed using a water maze test. These results suggest that the new edible CTB-Aβ42 silkworm pupae-derived vaccine has potential clinical application in the prevention of AD.     

The preparation and applications of functional fibres from crab shell chitin

Novel chitin–silk fibroin fibres and chitin fibres were prepared by an environmental friendly wet-spinning method. Each aqueous solution of sodium chitin (N-acetylchitosan) salt and its blends of silk fibroin in aqueous 14% sodium hydroxide was spun through a viscose-type spinneret into an aqueous 10% sulfuric acid solution saturated with ammonium sulfate (about 43%), and the corresponding white filament was obtained. The tenacity and elongation values of the chitin–silk fibroin filament decreased with an increase of fibroin content up to 33% by weight. A scanning electron microscopy analysis revealed that both the chitin filament and the chitin–silk fibroin (67:33, w/w) filament had vertical strips with faint scale structures on their surfaces. Some applications of these staple fibres were also reported.     

Chitin synthesis in larval and pupal epidermis of the Indian meal moth,Plodia interpunctella (Hübner), and the greater wax moth,Galleria mellonella (L.)

The level of chitin synthesis was determined in whole last-instar larvae and in pupae of Plodia interpunctella, and in epidermal tissue from similar stages of Galleria mellonella. The incorporation of radiolabelled N-acetyl-d-glucosamine into chitin was used to measure synthesis. Chitin production was similar in both species with peaks of synthesis occurring at the beginning of the last larval instar, in prepupae, in white pupae and prior to adult emergence. P. interpunctella also exhibited an additional small increase at mid-instar. Exposure of larval epidermis of P. interpunctella to 20-hydroxyecdysone in vitro stimulated chitin synthesis, but only after a 24 hr lag period subsequent to exposure to the hormone. This hormonal stimulation of chitin synthesis was inhibited by actinomycin-D and cycloheximide which suggested that 20-hydroxyecdysone-stimulated production of chitin depended on synthesis of RNA and protein. Comparison of the synthesis of chitin in epidermis of G. mellonella with previously published ecdysone titres, indicated that chitin production in vivo is preceded by an elevated ecdysone titre.     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应，利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异； 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa，该亚基在脂肪体中未出现降解现象，而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带，说明其降解可能先于未寄生对照。就含量而言，寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照，芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照，其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹，且寄生后2，12，24 h的差异达显著水平；芳基蛋白的含量均有低于未寄生的趋势，其中寄生后12 h的含量为未寄生的17.0%。综合认为，丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。