Digestion of peptidic residues in humic substances by an alkali-stable and humic-acid-tolerant proteolytic activity in the gut of soil-feeding termites

Previous feeding experiments have shown that soil-feeding termites (Termitidae: Termitinae) preferentially mineralize the peptidic component of synthetic humic acids, but nothing was known about the mechanism involved in digestion. Here, we studied the hydrolysis of humus-stabilized peptides in gut extracts of Cubitermes orthognathus by measuring the release of radiolabel from ¹⁴C-peptide-labeled synthetic humic acids. Gut extracts exhibited proteolytic activity over a wide pH range (from 4 to 12) with a maximum at about pH 8. The highest activity was located in the gut section containing the midgut and the extremely alkaline (up to pH 12) mixed segment. Chemical hydrolysis at in situ pH (up to pH 12) was negligible. Proteolytic activity in the hindgut fluid was generally relatively low, but alkaline proteases dominated in the anterior hindgut. When compared to other alkaline proteases, the proteolytic activity of gut extracts had a higher alkali-stability and tolerance to humic acids than subtilisin and an alkaline protease of Streptomyces griseus. Gut extracts also hydrolyzed the peptidic component of synthetic humic acids more efficiently than the commercial enzymes. Together with previous results, this study strongly supports the hypothesis that soil-feeding termites mobilize and digest the peptidic component of organic nitrogen in soil humic substances by a combination of proteolytic activities and extreme alkalinity in their intestinal tract.     

Gross nitrogen mineralisation rates in pastural soils and their relationships with organic nitrogen fractions, microbial biomass and protease activity under glasshouse conditions

In this study, gross nitrogen (N) mineralisation rates were determined in six pasture soils (Fleming, Kairanga, Karapoti, Lismore, Templeton and Waikoikoi) from three different regions of New Zealand. The soils were kept under controlled soil water potential (–10 to –30 kPa) and temperature (12–20°C) conditions in a glasshouse. The gross N mineralisation rates ranged from 0.76 to 5.87 g N g^–1 soil day^–1 in the six soils and were positively correlated with the amount of amino acid-N (AA-N), ammonia-N (NH₃-N), total hydrolysable-N (TH-N), microbial biomass-carbon (MB-C), microbial biomass-N (MB-N), protease activity and organic C and N. A stepwise regression was used to generate equations that could best describe gross N mineralisation rates. Microbial biomass-carbon and AA-N were included in the equation that best described the gross N mineralisation rate:

Studies on digestibility and digestive enzyme activities in Labeo rohita (Hamilton) juveniles: effect of microbial α-amylase supplementation in non-gelatinized or gelatinized corn-based diet at two protein levels

A 60-days feeding trial was conducted to delineate the effect of both gelatinized and non-gelatinized corn with or without supplementation with exogenous α-amylase at two dietary protein levels (35% and 28%) on dry matter digestibility, digestive enzymes and tissue glycogen content of Labeo rohita juveniles. Three hundred and sixty juveniles (average weight 10±0.15 g] were randomly distributed into 12 treatment groups with each of two replicates. Twelve semi-purified diets containing either 35% or 28% crude protein were prepared by including gelatinized (G) or non-gelatinized (NG) corn as carbohydrate source with different level of microbial α-amylase (0, 50, 100 and 150 mg kg⁻¹). The dry matter digestibility of G corn fed groups was significantly higher (P < 0.05) than that of the NG corn fed groups. Hepatosomatic index (HSI), liver glycogen and intestinal amylase activity of G starch fed groups were significantly higher (P < 0.05) than those of the NG corn fed groups. However, the reverse trend was found for gastrosomatic index (GSI), muscle glycogen and intestinal protease activity. Addition of 50 mg α-amylase kg⁻¹ feed improved the dry matter digestibility of NG starch fed groups, which was similar to that of the G corn fed groups or NG corn supplemented with 100/150 mg α-amylase kg⁻¹ feed. HSI, liver glycogen and intestinal amylase activity were significantly increased (P < 0.05) at minimum level of α-amylase in the feed (50 mg kg⁻¹) and did not increase due to further inclusion of amylase in the diet. Supplementation with α-amylase at 50 mg kg⁻¹ increased the intestinal amylase activity beyond which no significant changes were observed. Protease activity of liver and intestine was highest (P < 0.05) in higher crude protein (CP) fed groups, but protease activity of the intestine was significantly higher in the α-amylase supplemented groups. Hence, it was concluded that feed with 28% CP containing either G corn without α-amylase or NG corn with 50 mg α-amylase kg⁻¹ may be used as the alternative carbohydrate source for L. rohita juveniles.     

“东—号”剑麻蛋白酶应用于猪皮脱毛的初步研究

本文在实验室和工厂进行猪皮酶法脱毛的试验，结果表明，“东一号”剑麻蛋白酶对猪皮脱毛效果好，皮革成品质量高，为龙舌兰麻的综合利用提供了依据。     

百草枯对土壤中蛋白酶和过氧化氢酶活性影响研究

［目的］研究百草枯对土壤中蛋白酶和过氧化氢酶活性的影响。［方法］设4个百草枯处理水平,分别于培养2、6、12、20、30、45和60 d时取样测定蛋白酶和过氧化氢酶的活性。［结果］1 000 μg/g百枯草处理的酶活性低于其他4个处理。100 μg/g百枯草处理的蛋白酶活性的最低值出现在第20天,其余3个百草枯处理的蛋白酶活性的最低值均出现在第12天。1 000 μg/g百枯草处理的蛋白酶抑制率在第20天达51.08%,在第60天为41.48%。200、500和1 000 μg/g百枯草处理均不同程度地抑制过氧化氢酶活性,且其抑制作用随着百草枯浓度的增加而增强。1 000 μg/g百枯草处理对过氧化氢酶的抑制率在第12天达66.18%,在第60天为39.18%。［结论］百草枯对土壤中蛋白酶和过氧化氢酶的活性有较大的影响。     

投饲蚕豆对异育银鲫生长、肉质及肠道蛋白酶活力的影响

分别以配合饲料、浸泡蚕豆和发芽蚕豆饲喂平均体重23.7 g的异育鲫鱼(Allogynogenetic crucian carp),观察对其生长性能、肌肉品质和前肠蛋白酶活力的影响。经44 d养殖实验后,配合饲料组,浸泡蚕豆组和发芽蚕豆组的鱼体增重率分别为95.64%、33.54%和42.62%,饵料系数为1.97、5.09和4.15,投饲蚕豆显著降低了异育银鲫的增重率,提高了饵料系数(P<0.01);对肉质的分析表明,浸泡蚕豆组和发芽蚕豆组的肌肉失水率,肌原纤维耐折力和肌纤维直径显著提高(P<0.01),肌肉粗脂肪含量由1.92%(配合饲料组)降至1.05%和1.14%(P<0.01)。对肠道蛋白酶活力的分析表明,投饲蚕豆显著降低了前肠蛋白酶的活性(P<0.01)。上述研究表明,饲喂蚕豆可改善异育银鲫的肉质,但对其生长有抑制作用。     

Modeling growth factor activity during proinflammatory stress: methodological considerations in assessing cytokine modulation of IGF binding proteins released by cultured bovine kidney epithelial cells

The present research was conducted to model potential mechanisms through which IGFBPs might be affected by a key proinflammatory response initiating cytokine tumor necrosis factor (TNF-)-. Madin–Darby bovine kidney epithelial (MDBK) cells, known to release IGFBPs in response to several stimuli, were grown under several conditions and challenged with forskolin (F) or recombinant TNF- for 24 h. Forskolin increased IGFBP-3 gene expression and media content of BP-3 protein. TNF- increased basal and augmented F-mediated IGFBP-3 gene expression. However, TNF- effects on the measurable media content of IGFBPs were influenced by culture conditions; in the absence of added protease inhibitors (PIs) or sufficient media albumin concentration (high BSA, 1 mg/ml), the effect of TNF- was to decrease (P < 0.02) measurable IGFBPs. In the presence of PI and high BSA, media IGFBP-3 levels were shown to be increased by TNF- consistent with the gene expression data. Changes in media IGFBP-3 protease activity were examined further to explain the observed effects of TNF- on production and destruction of IGFBPs in media. When recombinant human IGFBP-3 (500 ng/ml) was added to PI-free, low BSA 100 μg/ml) media from TNF-treated MDBK cells, less than 10% of the BP-3 was recognizable by Western blot in 30 min; conversely, inclusion of High BSA and PI in media resulted in attenuation of the protease effect on the IGFBPs. The data suggest that the MDBK model of cellular response to proinflammatory stimulus is affected by culture conditions and that TNF- affects media content of IGFBPs through effects on IGFBP gene expression coupled with degradation of IGFBPs via enhanced proteolytic enzyme release.     

瘤胃微生物Fosmid文库蛋白酶活性克隆的筛选与序列分析

在日粮蛋白质的降解过程中,蛋白酶是把蛋白质水解为肽或氨基酸的关键酶,由于受到纯培养技术的影响,瘤胃内产生蛋白酶活性的细菌和各种蛋白酶的遗传信息知之甚少。本实验旨在利用蛋白酶选择性培养基从瘤胃微生物 Fosmid 文库中筛选出含蛋白酶活性的克隆子,通过生物信息学分析获得这些克隆子的遗传信息。应用脱脂乳粉和大豆蛋白粉两种蛋白酶选择性培养基,从 30 000 个克隆中筛选得到 14 个具有蛋白酶活性的活性克隆。利用福林酚试剂法检测 14 个蛋白酶克隆子的酶活力,结果表明,每个克隆子分别具有不同的蛋白质分解能力。以酪蛋白为底物的克隆子酶活力介于 0.59~2.74 U/mg 之间,以大豆蛋白粉为底物的克隆子酶活力在 0.70~7.19 U/mg 之间,而且同一克隆对于不同的底物所表现的酶活力也不同。随机挑选 10 个活性克隆进行末端测序(GenBank 登录号:JY084410~JY084429),经 Blast 比对后发现,45%的基因序列与已知编码基因无法匹配,pro10F 末端序列与金属肽酶匹配度为 54%,属于肽酶 M13 家族,且该克隆蛋白酶最适 pH 值为 7.0,为下一步研究该克隆的酶学性质和序列特征分析提供了基础资料。     

罗非鱼鱼排蛋白酶解液美拉德反应生香工艺优化研究

为了对罗非鱼鱼排蛋白酶解液美拉德反应生香工艺进行研究,以风味总体接受性为指标,对反应温度、时间、pH值、还原糖添加量4个因素采用响应面法进行多元回归拟合,优化美拉德反应条件,并用HPLC、GC-MS对反应产物进行分析。结果表明：美拉德反应最佳工艺条件为反应时间57 min,反应温度111℃,pH 6.0,还原糖（葡萄糖:木糖=2:1）添加量2.0%;反应产物中有机酸含量较反应前酶解液中有机酸含量丰富,核酸关联物较反应前多了呈味的鸟苷酸;反应产物中挥发性成分包括68种风味化合物,分别是醇类6种、醛类7种、酮类5种、芳香族7种、酯类11种、呋喃8种、吡嗪9种、吡咯1种、烃类7种、酸类5种和含硫化合物2种。罗非鱼鱼排蛋白酶解液经美拉德反应,产物呈透明红褐色,具有独特的鱼香味（香味浓郁）。     

Digestive Proteases of Pacu,Piaractus mesopotamicus,Fed on Distinct Protein-Starch Diets

Abstract

Fish adjust the enzyme profile to dietary protein and carbohydrate. A balance of nutrients optimizes the enzyme profile and metabolic performance offish. Dietary levels (10, 20, 30 and 40%) were combined to four levels of starch (57, 32, 21 and 4%) to feed juvenile pacu, Piaractus mesopotamicus. Acid and alkaline proteases were color-imetrically determined by hemoglobin and azocasein hydrolysis, respectively. Liver, white and red muscle glycogen, glucose and free amino acids were colorimetrically quantified. Those enzyme activities were induced by dietary protein. Enzyme responses to the feeding and the metabolic profile of the tissues suggest the best protein/starch ratio as 30%/21%. The highest levels of protein (30%-40%) impaired nutrient uptake.