黑眶蟾蜍皮肤白蛋白的纯化及胰蛋白酶抑制活性

[目的]探讨黑眶蟾蜍皮肤白蛋白的纯化及胰蛋白酶抑制活性。[方法]通过离子交换层析和凝胶过滤层析,从黑眶蟾蜍皮肤中得到其白蛋白,命名为BufomA-skin。65nmol／L的BufomA-skin分别与30nmol／Ltrypsin在25℃不同保温时间（0．25—24h）下测定胰蛋白酶抑制活性,计算剩余酶活力。[结果]BufomA-skin为单链蛋白,分子量约为66．7kDa。但在非还原状态下,50～66kDa条带呈弥散样。该蛋白有抑制胰蛋白酶水解小肽底物的活性,但对其他丝氨酸蛋白酶如凝血酶、糜蛋白酶、弹性蛋白酶及枯草杆菌蛋白酶无抑制。BufomA-skin与胰蛋白酶保温24h后,胰蛋白酶活性无恢复。[结论]黑眶蟾蜍皮肤白蛋白具有稳定的胰蛋白酶抑制活性,在黑眶蟾蜍防御天敌捕食的过程中发挥重要作用.     

紫花芸豆胰蛋白酶抑制剂部分性质研究

[目的]研究紫花芸豆（Phaseolus vulgaris）胰蛋白酶抑制剂（PVTI）的部分性质。[方法]探索了不同温度、不同还原剂和变性剂处理对PVTI抑制活性和蛋白构象的影响。[结果]PVTI是一种对热稳定蛋白;部分二硫键对活性中心有重要贡献;经尿素处理,PVTI的荧光光谱没有明显变化,但残留了较少的抑制活性,推测亚基的解离影响了抑制活性而每个亚基的内部结构未受到影响;经盐酸胍处理,Trp残基完全暴露于极性环境中,整个蛋白质分子处于一种松散伸展的状态,PVTI活性全部丧失。[结论]该研究可为PVTI的功能研究及其进一步应用奠定基础。     

Effect of Acibenzolar-S-methyl (ASM) pre-treatment in inducing resistance against Pythium aphanidermatum infection in Curcuma longa

In vitro experiments were carried out to test the efficacy of plant activator Acibenzolar-S-methyl (ASM, a benzothiadiazole derivative; trade name Bion 50WG) against rhizome rot disease of turmeric (Curcuma longa L.) caused by Pythium aphanidermatum. The plant activator was applied as a liquid rhizome pre-treatment followed by inoculation with P. aphanidermatum. Cell death, activities of pathogenesis related (PR) proteins such as cysteine protease (EC 3.4.22), peroxidases (EC 1.11.1.7) both soluble and ionically bound (IB), trypsin inhibitor (EC 3.4.21.1) and chymotrypsin inhibitor (EC 3.4.21.4) were monitored. Rhizome pre-treatment was effective in controlling P. aphanidermatum infection. Anatomical observation of turmeric rhizomes indicated the presence of calcium oxalate deposits in infected tissue and an accumulation of starch grains in response to infection by P. aphanidermatum. Pathogen infection also induced new basic polypeptides corresponding to 18.0 and 41.0 kDa. Induction of protease, protease inhibitors, soluble and ionically bound peroxidase activity was observed after ASM pre-treatment and P. aphanidermatum infection. ASM treatment also enhanced activities of proteases and peroxidase in rhizomes already infected with P. aphanidermatum. Increases in enzyme activities and protease inhibitors occurred much more rapidly and were enhanced in P. aphanidermatum infected rhizomes that were previously treated with ASM suggesting that increased activities of peroxidases and protease inhibitors may play a key role in restricting the development of disease symptoms on the rhizomes infected with P. aphanidermatum as evidenced by a reduction in cell death. Hence, pretreatment with ASM suppress the P. aphanidermatum induced oxidative damage through higher accumulation of peroxidases and induced defense through activities of protease inhibitors thereby, protected turmeric rhizomes from rhizome rot disease.     

The tryptophan-rich domain of puroindoline is directly associated with the starch granule surface as judged by tryptic shaving and mass spectrometry

The starch granule surface is a frontline of microbial attack and defence, operating in the background of normal starch granule metabolism. Puroindoline, a wheat protein which binds starch granule surfaces, contains a unique tryptophan-rich domain likely responsible for this property, though direct evidence is lacking. To test puroindoline’s tight association, prime starch granule extracts were water-washed 8 or 20 times and residual puroindoline removed using a solution of 50% isopropanol/50 mM NaCl. We found that this solvent was consistent in the amount of protein extracted from wheat flour and washed starch, regardless of initial protein content. Relative quantification of puroindoline following water-washing was performed using dot blot. Washing more than 8 times did not further reduce puroindoline content of starch granules suggesting a strong association with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, a combination of in situ tryptic digestion and mass spectrometry was used. Following digestion and water-washing, 50% isopropanol/50 mM NaCl was used to remove tightly-associated peptides for identification by mass spectrometry. Using this method, we identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat.     

Physicochemical characteristics of flours of faba bean as influenced by processing methods

Wet- and dry-processing with and without heating treatments were used to dehull faba beans for preparation of flours from the cotyledons. Flour qualities were assessed by levels of tannin and trypsin inhibitor and other measures of proximate composition. High roasting temperature and shorter process time that improved the recovered cotyledon yields were verified significant by path analysis methodologies. In turn, the higher cotyledon recoveries correlated with higher protein levels and inversely with the measured tannin levels. Ash reductions were correlated to the wet processing options in hull removal while reductions in the insoluble dietary fiber were notably influenced by level of heating temperatures applied. Trypsin inhibitor levels ranged from 42 to 56% of the original with reductions tied to applications of wet and/or heat processing in each case improved by increased durations and temperatures of treatments.     

Inactivation of trypsin inhibitors in sweet potato and taro tubers during processing

In order to understand the extent of elimination of trypsininhibitors during processing of sweet potato (Ipomoea batatas) andtaro (Colocasia esculenta) tubers, a detailed study was conductedusing tubers processed by oven drying, cooking, and microwavebaking. Between 80 and 90% trypsin inhibitor (TI) activity wasretained in sweet potato chips up to 2h at 70 ^°C. Among thefour cultivars of sweet potatoes, RS-III-2 trypsin inhibitors were more heatlabile. Heating at 100 ^°C led to rapid inactivation of TI of sweetpotatoes. Varietal differences in thermal stability were more pronouncedfor the trypsin inhibitors of taro than sweet potatoes. Taro inhibitors werealso more rapidly inactivated than sweet potato TI. Between 17 and31% TI activity was retained in cooked tuber pieces of sweet potatoes,while only 3–10% were retained in taro cultivars. Very effectiveinactivation of trypsin inhibitors of sweet potatoes and taro could beobtained through microwave baking. Flour prepared from taro was devoidof TI activity, while 5–12% TI activity was retained in the flour preparedfrom sweet potatoes. The study clearly established that among the fourtechniques used, microwave baking and flour preparation were the best methods to eliminate TI from sweet potatoes and taro.     

Studies on protein quality of green gram (Phaseolus aureus)

Raw green gram contains trypsin inhibitor (5.6 × 10^–3 units/0.1 g) while cooking destroyed the trypsin inhibitor in green gram. No destruction was observed in the case of lysine, methionine, cystine, threonine and tryptophan. Sulphur containing amino acids have been found to be the only limiting amino acids in green gram from studies on blood levels of amino acids and PER experiments. Raw green gram, when fed as a sole source of protein promoted an extremely low growth rate, (13.2 g/4 weeks) and PER (0.77) in rats. Cooking slightly improved the nutritive value of the gram, as indicated by increase in gain in body weight (24 g/4 weeks) as well as PER (1.22). The presence of inhibitors in green gram did not appear to affect some of the essential amino acids as observed from the blood amino acid levels.     

Effect of cooking on proteinase inhibitors ofDolichos lablab bean (Dolichos lablab perpureus L.)

Proteinase inhibitory activity in ten different varieties ofDolichos lablab perpureus. L. was determined. All the varieties tested exhibited appreciable level of proteinase inhibitory activity (PIA). The trypsin inhibitory activity (TIA) (Mean:20170 TIU/g) was relatively higher than the chymotrypsin inhibitory activity (CIA) (Mean: 15380 CIU/g). Effect of temperature and cooking on PIA was studied. The nature of cooking medium and duration of cooking had profound effect on the PIA. The dry fried seeds lost their PIA very rapidly (91% in 20 min). Seeds cooked in slightly alkaline medium lost their PIA quickly (89% in 30 min) compared to those cooked in acidic (80% in 30 min) and neutral pH (83% in 30 min). The PIA in green pods was also determined and they had only one third of the PIA (8200 TIU/g and 8125 CIU/g) found in the dry seeds.     

Changes in trypsin and chymotrypsin inhibitory activity on soaking of sorghum (Sorghum bicolor L. Moench)

Changes in trypsin and chymotrypsin inhibitory activity of sorghum (Sorghum bicolor L. Moench) seeds on soaking in distilled water, different salt solutions and mixed salt solution were investigated. A greater reduction in proteinase inhibitory activity was observed on soaking in salt solution than on soaking in distilled water. Maximum loss of trypsin and chymotrypsin inhibitory activity was observed on soaking seeds in mixed salt solution.     

Novel insecticide polymer chemistry to reduce the enzymatic digestion of a protein pesticide, trypsin modulating oostatic factor (TMOF)

A limiting factor in the use of proteins as insecticides, especially when the site of action is in the insect hemocoel, is protease degradation in the digestive system and hemolymph and movement across the midgut ventriculus. Trypsin modulating oostatic factor (TMOF) is a per os mosquito peptidic larvacide which moves across the digestive system and binds to receptors on the hemolymph side of the gut where the hormone inhibits protease synthesis and food utilization ultimately causing death. In the current study, the in vitro degradation of TMOF by the digestive enzyme, leucine aminopeptidase, was inhibited by conjugation of TMOF-K with aliphatic polyethylene glycol (PEG) polymers. Structure activity studies demonstrated a correlation between the molecular weight of the PEG polymer and resistance to digestion and show proof of concept that aliphatic-PEG protein polymerization can be used to prevent protease degradation of a protein insecticide.