Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2

The truncated forms of tissue inhibitor of metalloproteinase-1and -2 (TIMP-1 and -2), comprising the N-terminal active domain,are ideal molecules for structural analysis by intrinsic fluorescenceas each contains a single conserved tryptophan residue. In thispaper we describe studies on their conformational stability,unfolding/refolding kinetics and the environment of the uniquetryptophan as judged by its fluorescence properties in the nativestate and exposure to an external quencher, acrylamide. Twoforms of TIMP-2 were studied: TIMP-2 T21 derived from the full-lengthcDNA clone isolated from a mixed-tumour library, and TIMP-2A21 containing the highly conserved V18IRAK22 sequence. In allthree TIMP proteins the tryptophan environments in the nativestate appeared to be similar, but substantial differences wereseen in their conformational stabilities and refolding kinetics.TIMP-1 was approximately twice as stable as TIMP-2 T21 and 1.4-foldmore stable than TIMP-2 A21. This stability difference betweenTIMP-1 and TIMP-2 was shown to be independent of N-linked glycosylation.TTMP-1 and TIMP-2 A21 both showed simple two-state refoldingkinetics, whereas TIMP-2 T21 refolding was more complex andbiphasic in character. These differences between TIMP-2 T21and A21 suggest that residue 21 is a structurally importantsite in the TIMP protein.All three truncated molecules can beconsidered as stable independent folding domains ideally suitedfor further structural analysis     

柞蚕丝织物的柔软整理方法分析

采用蛋白酶和有机硅柔软剂对柞蚕丝织物进行柔软整理,通过正交试验,确定了两种整理的最佳工艺条件,并对比分析了两种整理方法在柔软效果、工艺条件和整理成本上的差异,得出柞蚕丝织物采用有机硅柔软整理优于蛋白酶整理的结论。     

天冬氨酸蛋白酶的研究进展

天冬氨酸蛋白酶（aspartic proteinase）是一类重要的蛋白水解酶,参与机体的新陈代谢及生物调控作用.它的活性中心由两个催化性天冬氨酸残基组成.天冬氨酸蛋白酶包括：胃蛋白酶亚型、逆转录蛋白酶亚型、天冬氨酸膜内裂解蛋白酶亚型.天冬氨酸蛋白酶家族中的胃蛋白酶亚型需要去除前肽区域并在酸性环境的诱导下自身催化;而肾素类亚型则是由其他的酶催化.研究这些亚型的结构和活化的机制,有助于研究天冬氨酸蛋白酶家族在病理和生理情况下的变化,在某些疾病的治疗上提供了新的研究方向.     

虾夷扇贝生殖腺多肽的制备及分离

为了对扇贝加工副产物进行高值化利用,采用中性蛋白酶,分别以变性(100℃,10 min)和未变性虾夷扇贝生殖腺匀浆为底物,经50℃酶解3h后制备不同的生殖腺水解液.结果表明,中性蛋白酶水解雌性虾夷扇贝生殖腺的效果优于雄性生殖腺,并且虾夷扇贝生殖腺经变性处理后,水解度和肽得率得到显著提高.与未变性处理组雌性虾夷扇贝生殖腺...     

Influence of cabbage proteinase inhibitorsin situ on the growth of larvalTrichoplusia ni andPieris rapae

Trypsin and chymotrypsin inhibitors are proteins that are developmentally regulated in foliage of cabbage plants, appearing at high concentrations in young foliage on mature plants. This temporal and spacial regulation of foliar proteinase inhibitors is synchronized with the appearance and distribution of foliar feeding Lepidoptera. When insects were allowed to select their feeding sites, larvalPieris rapae fed on the young foliage of cabbage plants, while larvalTrichoplusia ni fed on the mature foliage on cabbage plants. LarvalP. rapae that fed on mature plants were significantly smaller than larvae feeding on young plants, while there was no significant difference between larvalT. ni feeding on mature plants and those feeding on young plants. Thus, there was a significant inverse correlation between the level of proteinase inhibitory activity in cabbage foliage and larval growth. WhenP. rapae andT. ni were provided with an artificial diet containing total protein (including significant levels of proteinase inhibitors) that was extracted from cabbage foliage, there was a significant reduction in growth and development of both species of Lepidoptera.     

玉米生粉发酵生产L-乳酸的研究

研究了以α 淀粉酶为主的多种酶配合作用对玉米生粉乳酸发酵的影响。添加纤维素酶、酸性蛋白酶和脂肪酶能够加强液化酶和糖化酶对玉米生粉的水解作用 ,提高产酸水平和底物利用率。各种酶的最适添加量为α 淀粉酶 :8u/g干淀粉 ;纤维素酶 :5u/g原料 ;酸性蛋白酶 :2u/g原料 ;脂肪酶 :1u/g原料。在此条件下 ,当玉米粉初始浓度为 14 0g/L时 ,摇瓶产酸 6 5 .72 g/L ,发酵罐中产酸可达 71.6 5 g/L ,达到并超过高温蒸煮乳酸发酵的水平。同时研究了底物浓度对产酸的影响。     

AS1.398中性蛋白酶的固定化研究

利用壳聚糖为载体，戊二醛为交联剂，对AS1．398中性蛋白酶进行了固定化研究，固定化反应的最佳条件是采用1％的戊二醛浓度，壳聚糖和戊二醛的交联反应时间为4h，加入酶固定化反应12h，固定化酶的最适温度35℃，最适pH值8．0。得到的固定化酶的活力回收平均为82．5％，固定化酶的稳定性能好于游离酶。     

Purification,structural characterization,and technological properties of an aspartyl proteinase from submerged cultures of Mucor mucedo DSM 809

An extracellular aspartyl proteinase from Mucor mucedo DSM 809 submerged cultures was purified by a two-steps chromatographic procedure. The enzyme had a molecular weight (MW) of 32.7 kDa, and an isoelectric point (pI) value of 4.29; no evidence of N-linked glycosylation was found. As judged by mass spectrometry, the primary structure of the M. mucedo enzyme presented homology with Rhizopus spp. proteinases. The secondary structure showed 4% α-helix, 48% β-sheet and 48% random coil structure in 20 mM phosphate buffer (pH 5.8), as evidenced by circular dichroism spectroscopy. When acting on milk to provoke curd formation, the proteinase showed maximum potency at pH 5.0 and at 40 °C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55 °C for 10 min. These results indicate that the milk-clotting enzyme from M. mucedo can be considered as a potential substitute for bovine chymosin in cheese manufacturing.