Temporal changes in digestive enzyme activities in the gastrointestinal tract of European eel (Anguilla anguilla) (Linneo 1758) following feeding

Changes occurring after feeding in the digestive enzyme activities of European eel were investigated to provide some insights into the digestive physiology of this fish. Total and specific proteases, amylase and lipase activities were measured using standard biochemical assays over a 24 h cycle in fed eels, compared to starved ones, under the same rearing conditions. In the gastrointestinal tract of fed eels quantitative changes started 4 h after feeding and continued later on; conversely, in starved eels enzyme activities remained unchanged over time. In fed eels, total and specific protease activities showed an overall increasing trend in the intestine, while in the stomach they progressively decreased to values 22–50% lower than those measured at the pre-feeding time; this behaviour probably reflected the progression of digesta along the intestinal tract. The prolonged secretory response of European eel to food ingestion proved its extended activity in the digestive process.     

桡足类消化酶活力的影响因子

桡足类消化酶活力的影响因子汤鸿,李少菁（厦门大学海洋系,３６１００５）ＦａｃｔｏｒｓＩｎｆｌｕｅｕｃｉｎｇｔｈｅＤｉｇｅｓｔｉｖｅＥｎｚｙｍｅＡｃｔｉｖｉｔｙｏｆＣｏｐｅｐｏｄｓ．￥ＴａｎｇＨｏｎｇ;ＬｉＳｈａｏｃｈｉｎｇ（Ｏ－ｃｅａｎｏｌｏｇｙＤｅ...     

Comparative study of digestive enzymes of squid (Todarodes pacificus) viscera after supercritical carbon dioxide and organic solvent extraction

Three major classes of digestive enzymes of squid viscera were characterized following extraction of oil by supercritical carbon dioxide (SCO₂) and organic solvent, n-hexane. Squid viscera were extracted at temperature, 35∼45°C and pressure, 15∼25 MPa for 2.5 h by SCO₂ with a constant flow rate of 22 g/min. Oil extraction yield increased with the increasing of extraction pressure and temperature. The highest oil extracted residues of squid viscera were used for characterization of digestive enzymes. The activities of protease, lipase, and amylase were highest in n-hexane treated squid viscera samples and lowest in SCO₂ treated samples. The crude extracts of SCO₂ and n-hexane treated squid viscera samples showed almost same optimum pH and pH stability for each of the digestive enzymes. The optimum temperature of protease, lipase, and amylase were found to almost similar in SCO₂ and n-hexane treated samples. But the thermal stability for each digestive enzyme in SCO₂ treated squid viscera were slightly higher than that of n-hexane treated squid viscera. Studies using SDS-PAGE showed no significant differences in protein patterns of the crude extracts of untreated and SCO₂ and n-hexane treated squid viscera indicating no denaturation of proteins.     

A comparison of screening methods for antioxidant activity in seaweeds

The inhibition of lipid peroxidation and radical scavenging effects were studied to evaluate the antioxidant activity for extracts of 17 species of seaweed. The antioxidant effect was evaluated by determination of lipoxygenase activity and by α, α-diphenyl-β-picrylhydrazyl (DPPH) decolorization. Lipoxygenase activity was depressed in the presence of aqueous and ethanol extracts of 4 algal species; Sargassum species had the highest antioxidant activity of all the species examined. The ethanol extracts of one Sargassum species showed competitive inhibition with the substrate. The same species also showed radical scavenging activity in the DPPH decolorization test. Comparison of these results shows no relationship between enzyme inhibition and radical scavenging activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Momordica charantia trypsin inhibitor II inhibits growth and development of Helicoverpa armigera

Abstract  Bitter gourd ( Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs), which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera . In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-II, its cloning and expression as a recombinant protein using Pichia pastoris have been reported. Recombinant McTI-II inhibited bovine trypsin at 1: 1 molar ratio, as expected, but did not inhibit chymotrypsin or elastase. McTI-II also strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H. armigera larvae. The insect larvae fed with McTI-II-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding. Moreover, ingestion of McTI-II resulted in 23% mortality in the larval population. The strong antimetabolic activity of McTI-II toward H. armigera indicates its probable use in developing insect tolerance in susceptible plants.     

Production,purification and application-relevant characterisation of an <Emphasis Type="Italic">endo</Emphasis>-1,3(4)-β-glucanase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

Growth on a wheat bran media induced production of an extracellular β-glucanase by Rhizomucor miehei (DSM 1330). The enzyme was purified to homogeneity. Substrate specificity studies coupled with protein database similarity searching using mass spectrometry-derived sequence data indicate it to be an endo-1,3(4)-β-glucanase (EC 3.2.1.6). The enzyme was characterised in terms of potential suitability for use in animal (poultry) feed. Significant activity was observed over the entire pH range typical of the avian upper digestive tract (pH 2.6–6.5). The enzyme was also found to be more thermostable than current commercialized β-glucanases, particularly when heated at a high enzyme concentration, and retained twice as much residual activity as the latter upon exposure to simulated avian digestive tract conditions. There are no previous reports of the production, purification or characterization of a β-glucanase from a Rhizomucor, and the enzyme’s application-relevant physicochemical characteristics render it potentially suited for use in animal feed.     

丽斗蟋翅二型雄虫食物消化能力及消化酶活性比较

【目的】丽斗蟋Velarifictorus ornatus具明显的翅二型现象,为探讨翅型分化对丽斗蟋翅二型雄虫消化能力及中肠内消化酶活性产生的影响,对长翅型与短翅型雄虫食物消化能力及中肠内消化酶活性进行了检测比较。【方法】我们采取重量营养指数测定了羽化后12 d内丽斗蟋两型雄成虫增长量、相对增长率、取食量、食物利用率、近似消化率和食物转化率。为进一步明确丽斗蟋翅二型成虫食物消化能力与中肠内消化酶活性的关系,我们采用4种专用底物测定了中肠内用于分解蛋白质、脂肪和碳水化合物的总蛋白酶、胰蛋白酶、脂肪酶和淀粉酶的活性。【结果】结果表明,丽斗蟋两型雄虫取食量、食物转化率、食物利用率与增长量均无统计差异,但中肠内消化酶活性变化规律不同。成虫羽化后4 d时,长翅型雄虫中肠内总蛋白酶与胰蛋白酶活性显著高于短翅型雄虫,相反,羽化后0 d时,短翅型雄虫中肠内总蛋白酶与胰蛋白酶活性则显著高于长翅型雄虫,而羽化后12 d时,虽然短翅型雄虫总蛋白酶活性高于长翅型雄虫,但胰蛋白酶活性在两型雄虫间并无差异。成虫羽化后0 d时,两型雄虫脂肪酶活性无差异,但无论是羽化后4 或 12 d,长翅型雄虫中肠内脂肪酶活性皆显著大于短翅型雄虫。成虫羽化后4 d时,短翅型雄虫中肠内淀粉酶活性显著高于长翅型雄虫,而羽化后0与12 d时,两型雄虫间无显著差异。【结论】丽斗蟋翅二型雄虫食物消化能力无显著差异,但羽化后不同时间,中肠内消化酶活性存在差异,该差异可能与成虫羽化后不同时期,翅二型雄虫在飞行与繁殖投资中对不同能源物质的需求有关。     

Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

幼虫密度对草地螟食物利用率及消化酶活性的影响

为了阐明草地螟Loxostege sticticalis大发生种群幼虫取食行为特征, 在室内条件下（温度22±1℃, 相对湿度70%）对不同幼虫密度［1, 10, 30头/瓶（650 mL）］饲养草地螟幼虫的食物利用率及消化酶活性进行了研究。结果表明： 幼虫中等（或高）密度对草地螟幼虫相对中肠重量、 相对取食量、 粪便干重、 食物利用率和近似消化率及总蛋白酶和亮氨酸氨肽酶活性影响显著。幼虫相对中肠重量以10头/瓶的幼虫密度最大, 1头/瓶的幼虫密度最小。随着幼虫密度的增加, 幼虫相对取食量和粪便干重增加, 而虫体干重减轻, 幼虫食物利用率降低。幼虫密度30头/瓶的幼虫相对取食量和粪便干重显著高于1和10头/瓶的, 而30头/瓶的幼虫食物利用率显著低于1头/瓶的。幼虫近似消化率随幼虫密度的逐渐增加而显著降低。幼虫密度10头/瓶的幼虫总蛋白酶和亮氨酸氨肽酶的活性显著高于1和30头/瓶的, 而淀粉酶的活性受幼虫密度影响不显著。随幼虫密度的增加, 幼虫相对中肠重量与总蛋白酶和亮氨酸氨肽酶活性变化趋势较为一致, 消化酶活性的变化可能与相对中肠重量大小有关。因此, 幼虫密度是影响草地螟幼虫取食行为的重要因子, 这些结果为阐明草地螟大发生种群与一般种群的为害特征提供了重要理论依据。     

北草蜥和中国石龙子几种消化酶活力比较研究

应用酶学分析法测定了越冬后北草蜥和中国石龙子的胃、肠、胰组织中蛋白酶、淀粉酶、纤维素酶的活力。结果表明：不同组织中的消化酶活力差异显著;同一组织中不同消化酶的活力差异显著;不同动物的同一组织中消化酶活力也有差异。这些差异说明消化酶的活力与动物种类和器官的分化有关,并受食物组成和遗传因素的影响,产生了不同的酶活力分布。这也是生物长期适应环境,形成不同的代谢水平的结果。     

Reaction of microorganisms to the digestive fluid of earthworms

The reaction of soil bacteria and fungi to the digestive fluid of the earthworm Aporrectodea caliginosa was studied. The fluid was obtained by centrifugation of the native enzymes of the digestive tract. The inhibition of growth of certain bacteria, spores, and fungal hyphae under the effect of extracts from the anterior and middle sections of the digestive tract of A. caliginosa was discovered for the first time. In bacteria, microcolony formation was inhibited as early as 20–30 s after the application of the gut extracts, which may indicate the nonenzymatic nature of the effect. The digestive fluid exhibited the same microbicidal activity whether the earthworms were feeding on soil or sterile sand. This indicates that the microbicidal agents are formed within the earthworm’s body, rather than by soil microorganisms. The effect of the digestive fluid from the anterior and middle divisions is selective in relation to different microorganisms. Of 42 strains of soil bacteria, seven were susceptible to the microbicidal action of the fluid (Alcaligenes faecalis 345-1, Microbacterium sp. 423-1, Arthrobacter sp. 430-1, Bacillus megaterium 401-1, B. megaterium 413-1, Kluyvera ascorbata 301-1, Pseudomonas reactans 387-2). The remaining bacteria did not die in the digestive fluid. Of 13 micromycetes, the digestive fluid inhibited spore germination in Aspergillus terreus and Paecilomyces lilacinus and the growth of hyphae in Trichoderma harzianum and Penicillium decumbens. The digestive fluid stimulated spore germination in Alternaria alternata and the growth of hyphae in Penicillium chrysogenum. The reaction of the remaining micromycetes was neutral. The gut fluid from the posterior division of the abdominal tract did not possess microbicidal activity. No relation was found between the reaction of microorganisms to the effects of the digestive fluid and the taxonomic position of the microorganisms. The effects revealed are similar to those shown earlier for millipedes and wood lice in the following parameters: quick action of the digestive fluid on microorganisms, and the selectivity of the action on microorganisms revealed at the strain level. The selective effect of the digestive gut fluid of the earthworms on soil microorganisms is important for animal feeding, maintaining the homeostasis of the gut microbial community, and the formation of microbial communities in soils.     

Engineering of a fungal β-galactosidase to remove product inhibition by galactose

β-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable β-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. β-galactosidase crystal structure with bound β-galactose. This led to targeted mutagenesis of an Asp²⁵⁸-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant β-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K _i of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K _m (3.76 mM compared to 2.21 mM) and reduced V _max (110.8 μmol min⁻¹ mg⁻¹ compared to 172.6 μmol min⁻¹ mg⁻¹) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.     

转Bt基因抗虫棉对棉铃虫拒食作用及其机理研究

转Bt基因抗虫棉对 3 ,4,5龄棉铃虫幼虫的抗性表现形式为拒食作用 ,且随幼虫龄期的增加拒食作用明显降低 ,其中对 5龄幼虫的拒食作用很低。取食Bt棉后 ,3 ,4,5龄棉铃虫幼虫中肠消化酶比活力均较对照有所减退 ,且随幼虫龄期的增加减退率明显降低 ,其中对 5龄幼虫减退率最低。由此 ,解释了Bt棉对 3龄及 3龄以上棉铃虫幼虫抗性表现形式、抗性随不同幼虫龄期的差异性 ,及其抗性差异性的消化机理。     

慈菇蛋白酶抑制剂对亚洲玉米螟消化酶的影响

亚洲玉米螟幼虫中肠消化酶主要为胰蛋白酶：利用慈菇蛋白酶抑制剂研究它对玉米螟消化酶的影响,发现这种抑制剂体外强烈抑制玉米螟幼虫中肠消化酶：同时我们又比较了长时间取食（三龄至五龄第二天）和短时间取食(四龄未取食48小时)抑制剂的幼虫体内胰蛋白酶活性,当它们短时间取食抑制剂后,体内消化酶水平迅速下降,取食更长时间时,胰蛋白酶活力下降更明显,仅为对照的27．2%。由此可见,慈菇蛋白酶抑制剂严重影响了玉米螟的正常消化,阻断了玉米螟营养与食物间的联系．使玉米螟生长发育不良。     

北草蜥几种消化酶活力比较

应用酶学分析法测定了越冬后北草蜥胃、肠组织中蛋白酶、淀粉酶、纤维素酶的活力。结果表明 ,不同年龄、性别的北草蜥同一组织中消化酶活力有显著差异 ;不同地理种群的北草蜥同一组织中消化酶活力有显著差异 ;不同消化酶在北草蜥同一组织中的活力有显著差异 ;在北草蜥不同的组织中同一消化酶的活力有显著差异。说明北草蜥消化酶的活力与年龄、性别、部位和地理环境等因素有关 ,受食物组成、能量需求和遗传等因素的影响 ,产生了不同的酶活力和分布。这也说明生物长期适应环境 ,形成了不同的代谢水平     

Digestive enzyme activity of the Japanese grenadier anchovy Coilia nasus during spawning migration: influence of the migration distance and the water temperature

In this study, we investigated the activity levels of two major digestive enzymes (pepsin and lipase) in the commercially important Japanese grenadier anchovy Coilia nasus during its upstream migration to analyse the digestive physiological responses to starvation and to analyse the influence of the water temperature on enzyme activity. Water temperature had a significant effect on pepsin activity, while long-term starvation resulted in a significant decrease in pepsin activity. As starvation continued, however, a slight increase in pepsin activity between the Wuhu (440 river km) and Anqing (620 river km) regions may indicate that C. nasus had refeeding behaviour due to its large expenditure of energy reserves. In contrast, lipase activity was not significantly affected by the water temperature but the effect of fasting increased as much as 13% of lipase activity from the Chongming region (20 river km) to Anqing region, suggesting that the stored lipids of grenadier anchovy were mobilised to meet energy requirements of upstream migration activity and gonad development. Lipid mobilisation activated lipoprotein lipase (LPL; proteins with lipase activity) to hydrolyse triacylglycerides (TAG), which is the first step of lipid assimilation and obtained energy from fatty acids under fasting conditions. Therefore, the increased lipase activity is attributed mainly to the lipase that is involved in endogenous lipid hydrolysis. Grenadier anchovy appears to adapt to long-term starvation during migration and the increased lipase activity may indicate a crucial effect on lipid metabolism. This study demonstrated that distinct alterations occur in pepsin and lipase activities during the spawning migration of grenadier anchovy due to exogenous nutrition and endogenous metabolism. Furthermore, it provides a basis for further research on the digestive physiology and energy metabolism in this species.     

12a-羟基鱼藤酮对斜纹夜蛾生殖力的影响及其作用机理

为探讨12a-羟基鱼藤酮对斜纹夜蛾Prodenia litura (Fabricius)生殖力的影响及其作用机理, 本文测定了该虫取食12a-羟基鱼藤酮后的产卵量。在用高效液相色谱仪确定其卵巢组织中存在12a-羟基鱼藤酮后, 通过MTT法测定该化合物对斜纹夜蛾卵巢细胞（PL细胞）的细胞毒性,并利用流式细胞仪检测该化合物对PL细胞细胞周期、膜电位、胞内钙离子浓度、线粒体膜电位的影响。结果表明:斜纹夜蛾幼虫取食12a-羟基鱼藤酮后产卵量下降80％以上, 并从卵巢组织中检测出该化合物。12a-羟基鱼藤酮对PL细胞增殖具有抑制活性, 其IC₅₀为6.6 mg/L。该化合物将PL细胞周期阻滞于S期, 导致PL细胞膜电位、线粒体膜电位和细胞内游离钙离子浓度均显著升高。该化合物导致斜纹夜蛾繁殖力降低。12a-羟基鱼藤酮将PL细胞增殖阻滞于细胞周期的S期。同时, 该化合物对卵巢细胞具有较弱的毒杀活性, 导致一些卵巢细胞死亡。由于上述原因, 卵巢细胞数量逐渐减少而导致卵巢管萎缩。卵巢管的萎缩使卵巢管内的卵母细胞不能发育成卵细胞, 故其生殖力下降。     

Effects of Dietary Pb on Accumulation,Histopathology, and Digestive Enzyme Activities in the Digestive System of Tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-μg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.     

云斑尖塘鳢消化酶活力的研究

本文采用酶学分析方法研究了云斑尖塘鳢在正常摄食状态与饥饿的状态下胃、肠及肝胰脏组织中蛋白酶、淀粉酶和脂肪酶的活性。结果显示,在30℃的条件下,正常摄食组样本在酸性条件下的蛋白酶活力表现为:胃后肠肝胰脏前肠,中性和碱性条件下:后肠肝胰脏前肠及胃;饥饿组样本仅有胃表现出较高的酸性蛋白酶活性,其他器官的蛋白酶活性均很低。在正常和饥饿实验组中肝胰脏的淀粉酶活性均高于其他器官,胃肠的淀粉酶活性均较低。正常摄食组中脂肪酶活力后肠肝胰脏;而在饥饿组中仅有肝胰脏检测到脂肪酶活性。结果表明,云斑尖塘鳢适度饥饿组较正常摄食组消化酶活性大幅降低;其高蛋白酶活力及中等脂肪酶活力与其肉食性相一致;此外云斑尖塘鳢也具备少量的淀粉消化能力。