一株产海藻糖合成酶嗜中温芽孢杆菌的鉴定及其酶学性质

由长白山温泉附近土壤中筛选得到产海藻糖舍酶菌株，为革兰氏阳性菌，具有芽孢和鞭毛，其最适生长温度为55℃，对这一菌株进行生理生化指标测定。进一步对该菌株所产海藻糖合酶的酶学性质进行研究，酶反应最适作用pH值为7．0，在pH6．6～7．4时该酶稳定，最适作用温度为35℃，在25-45℃时该酶可稳定保存。     

β-葡萄糖苷酶产生菌的选育

对β－葡萄糖苷酶活力为０．４９７μｍｏｌ／（ｓ·ｇ）的无花果曲霉１０１菌株进行Ｃｏ６０及ＵＶ和ＮＴＧ诱变，得到一株产酶提高１．７２倍的Ｌ２菌株；再经发酵培养基及培养条件，包括碳源、氮源、有机酸、表面活性剂、金属离子、产酶诱导剂、培养温度、水分、初始ｐＨ值、通风量、接种形式及接种量等的优化，以优化得到的最佳条件固态发酵６ｄ，产酶稳定在４．１７～４．６７μｍｏｌ／（ｓ·ｇ）．     

拮抗性酵母几丁质酶的纯化、性质及抗菌活性

从番茄果实表面分离到一株陆生伊萨酵母（Issatchenkia terricola），它在几丁质诱导下能产生较高活性的几丁质酶。采用硫酸铵盐析、DEAE-纤维素（DE32）离子交换柱层析、SephadexG-100分子筛柱层析，获得了凝胶电泳均一的几丁质酶。用SDS-PAGE测得该酶的相对分子质量为42400；以胶体几丁质为底物的Km和Vmax分别为6．91mg／mL和12．95μmol／min；该酶反应的最适温度和最适pH值分别为50℃和7．0。在50℃条件下，酶蛋白在pH5．0～9．0较为稳定；抗菌活性实验表明，该酶对供试病原菌有不同程度的抑制作用。     

嗜热芽孢杆菌2004产β-甘露聚糖酶产酶条件优化和酶学性质

对芽孢杆菌2004 β-甘露聚糖酶进行了产酶条件优化、初步纯化及其酶学性质的研究.条件优化结果:1.5 g/dL槐豆胶,2 g/dL蛋白胨,培养温度为40 ℃,250 mL三角瓶装液量为70 mL,其他因素影响不大.此最佳产酶条件下,嗜热芽孢杆菌2004 12 h的产酶水平平均为41.92 U/mL,比原来提高了30倍.培养液离心得到上清液,经硫酸铵沉淀,Sephadex G-200分子凝胶过滤和DEAE纤维素离子交换,酶比活达到722 U/mg,纯化了23.94倍,收率为35.1 %.该酶的最适反应温度为70～80 ℃;反应的最适pH值为5.8～6.0;pH值稳定范围为4.0～9.0.金属离子Mg2 和K 对酶略有激活作用.适当浓度的Mg2 不仅对酶有激活作用,还对酶的热失活具有保护作用.     

酶法浸提枸杞的研究

通过对外源酶酶解枸杞效果的研究，结果表明：外源酶能显著提高枸杞浸提液中可溶性固形物和营养成分的含量，其单一纤维素酶和木瓜蛋白酶浸提枸杞的适宜pH值分别是5．0～5．5和pH值6．0，适宜的酶质量浓度分别为0．20g／dL和0．40～0．60g／dL，温度分别为55．C和65℃，适宜时间都为50-60min；两种酶组合浸提枸杞的较适pH值、温度、时间和酶组合分别为pH值5．5～6．0，55-60℃．50min和0．20g／dL纤维素酶＋0．40g／dL木瓜蛋白酶。     

节杆菌10137产β-呋喃果糖苷酶条件及酶学性质

研究表明,节杆菌10137产β 呋喃果糖苷酶的最佳条件为:温度为30℃,pH值7 5,接种体积分数为2%,装液体积为40mL.该条件下β 呋喃果糖苷酶的转移酶活为177.78U/mL,酶作用最适pH值为6.5,最适温度为30℃,在pH值为6.0～8.0和45℃以下稳定.Ag+和Cu2+对该酶有较强烈的抑制作用,EDTA和Mg2+对酶活也有一定影响.     

重组大肠杆菌产β-胡萝卜素的发酵条件

对重组大肠杆菌产β-胡萝卜素发酵条件进行了研究．在M9培养基中添加Span-200．7g／L有利于细胞生长和β-胡萝卜素表达，初始pH值6．0、接种体积分数5％、发酵温度28℃、抗生素氨苄青霉素质量浓度80μg／mL和氯霉素质量浓度40μg／mL为最佳发酵条件，在7L发酵罐进行发酵动力学试验表明，重组菌最适发酵周期为22h，细胞干重最高可达1．55g／L，β-胡萝卜素表达量可达0．75μg／mL。     

重组过氧化氢酶与一种商品过氧化氢酶的性质比较

将重组过氧化氢酶和市售一种商品过氧化氢酶的性质进行了比较。重组酶的最适温度为70℃，某商品过氧化氢酶的最适温度为40℃；与这种商品过氧化氢酶相比，重组酶的热稳定性更好。重组过氧化氢酶和这种商品过氧化氢酶的最适pH都是7．0；重组酶和这种商品过氧化氢酶在pH3-8的范围内均较为稳定；重组酶在室温放置1个月后酶活力基本保持不变，而此种商品过氧化氢酶活力损失约70％；金属离子对重组酶和这种商品过氧化氢酶都有抑制作用，抑制程度有所不同；EDTA抑制这种商品过氧化氢酶的活性，但不抑制重组酶的活性。     

嗜热古菌Thermococcus sp.HJ21产高温普鲁兰酶条件和酶学性质

对一株分离自热液口的超嗜热古菌(Thermococcus sp. HJ21)菌株产普鲁兰酶的条件及酶学性质进行了研究.结果发现:该菌株在18 h产酶量达到最高.在发酵温度为88 ℃、培养基初始pH 6.5,以及NaCl质量浓度为2.5 g/dL时,产酶较高.麦芽糖、酵母粉和蛋白胨有利于普鲁兰酶的产生.该酶的最适作用温度为95 ℃,在80～100 ℃之间仍可保持较高的酶活性;该酶具有较好的热稳定性,100 ℃保温2 h,仍有50%以上的残余酶活.该酶的最适作用pH为6.0,并且在pH 5.0～7.0之间可以保持较高酶活性;在pH 5.0～7.0之间较稳定,在pH 6.5时稳定性最好.Ca2+和Na+对普鲁兰酶具有较强的激活作用,而Al3+、Ni2+、Hg2+、Cu2+等则强烈抑制酶的活性.     

耐热纤维素酶产生菌的筛选、鉴定及产酶条件优化

从温泉热源地区采集的大量泥土和水样中，筛选出一株在60℃生长的纤维素酶产生菌SH2。结合菌株的生理生化特性分析与Biolog微生物自动鉴定仪的鉴定结果，命名为热葡糖苷酶地芽孢杆菌（Geobacillus thermoglucosidasius）SH2，该菌株兼性好氧，在45～60℃能较好地生长。对菌体生长与产酶培养条件优化表明：初始pH值为5．5，碳、氮源分别为蔗糖和玉米浆时有利于产酶，经48h发酵后纤维素酶酶活达0．36IU／mL。纤维素酶反应条件研究表明，该纤维素酶的最适pH值为6．0，在pH4．0～10．0范围具有较强的耐受性；在45～65℃间酶活差异仅在5％之内，显示了很好的温度耐受性。     

雅致放射毛霉蛋白酶

借助超声波、电泳和高压液相色谱技术对雅致放射毛霉胞外和胞内蛋白酶进行了研究．结果表明：腐乳前期培菌时，雅致放射毛霉同时分泌胞外及胞内蛋白酶，两者酶活力变化趋势基本相同，在培菌45h时，达到最大酶活力；胞外和胞内蛋白酶均为蛋白酶系，相对分子质量组成相同；以大豆蛋白为底物时，主要水解产物相同．     

EXCRETION OF BIKUNIN AND ITS FRAGMENTS IN THE URINE OF PATIENTS WITH RENAL STONES

PURPOSE: Bikunin is a Kunitz-type protease inhibitor found in serum and urine. It has been implicated in urinary stone formation. This study was designed to investigate the role of urinary bikunin in stone formation. MATERIALS AND METHODS: Urinary concentrations of bikunin were measured in 18 male formers of urinary stones 28 to 74 years old and in 77 healthy controls, including 39 males and 38 females, without urological abnormality. A sensitive competitive solid phase enzyme immunoassay was established for urinary bikunin. Bikunin was also qualitatively assessed by Western blot analysis. RESULTS: The mean urinary bikunin-to-creatinine ratio plus or minus standard deviation in stone formers was significantly elevated compared with that in healthy male and female controls (52.9 +/- 46.0 microg./mg. creatinine versus 28.0 +/- 30.4 and 26.5 +/- 21.7, p = 0.005 and 0.006, respectively). By Western blot analysis all urine samples contained authentic 40 kDa. bikunin species. However, a significantly higher proportion of patients was found to have aberrant 25 kDa. bikunin species compared with controls (10 of 18 or 55.6% versus 15 of 77 or 19.5%, p = 0.002). Experiments on de-glycosylation with chondroitinase ABC, amino acid sequencing of the aberrant bikunin species and calcium oxalate crystal growth inhibition assay demonstrated that the 25 kDa. bikunin fragment was identical to de-glycosylated bikunin and less inhibitory on calcium oxalate crystal growth. CONCLUSIONS: If urinary bikunin is important in the pathogenesis of urolithiasis, its effect is probably attributable to the concentration and degree of glycosylation.     

假单胞菌JW12发酵液的脂肪酶稳定性

通过对假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｓｐ．）ＪＷ１２发酵液脂肪酶稳定性的研究，表明了发酵液脂肪的稳定性主要受蛋白酶的影响。当用吐温８０作为碳源时，通过对发酵培养基组成的调整，达到降低蛋白酶的目的，提高发酵液脂肪酶酶活。     

人类精子表面附睾蛋白激酶抑制剂Eppin和精囊凝固蛋白Semenogelin的相互作用研究

目的:探讨人类精子表面一种附睾蛋白激酶抑制剂Epp in和精囊凝固蛋白Sem enogelin(Sg)的相互关系。方法:①用抗Epp in的抗体与精子和精浆中Epp in蛋白进行免疫沉淀反应;②免疫荧光法在精浆和精子表面共同显色定位;③重组Epp in(rEpp in)和重组精囊凝固蛋白Sg(rSg)共同孵育,用抗Epp in抗体或抗Sg抗体进行免疫共沉淀;④W estern印迹检测rEpp in和rSg相互作用;⑤放射性125I-rSg与rEpp in结合达饱和后,用未标记的rSg竞争性结合分析;⑥放射性125I-rSg直接与印迹膜上的rEpp in行放射性自显影。结果:人精子表面蛋白Epp in与精囊蛋白Sg发生特异性结合,Sg分子结构中的半胱氨酸残基Cys-239是唯一的结合位点,Cys-239的还原及羧甲基化将阻碍125I-rEpp in与rSg的结合。结论:Epp in与Sg的结合是靠分子结构中二硫化键的参与。在人类射精后的精子表面,精囊蛋白Sg结合在精子表面的Epp in上。     

Adenocarcinoma of the canine prostate: immunohistochemical examination for secretory antigens

Thirty-one canine adenocarcinomas of prostatic origin were examined immunohistochemically with antibodies generated against three prostate-specific antigens by using a peroxidase-antiperoxidase technique. Primary antibodies included two generated with human prostatic antigens (PSA, PSAP) and one generated with a canine prostatic antigen (CPSP). Eight of 31 adenocarcinomas were positive with CPSP, two were positive with PSA, and three were PSAP positive. Normal and hyperplastic canine prostatic epithelium was strongly positive with all three markers. These results contrast with those in human studies in which PSA and PSAP stain normal and neoplastic epithelium with comparable intensity in all but the most anaplastic tumors.     

附睾蛋白酶抑制剂Eppin的研究进展

附睾蛋白酶抑制剂Eppin是一种睾丸和附睾特异性分泌的蛋白质,精液中含量极为丰富,精子表面大量存在,用重组Eppin免疫的猴子出现不育,Eppin有望成为人类有效的可逆性的免疫避孕疫苗。现就Eppin基因及其蛋白质结构功能特点,Eppin引起免疫性不育的分子机制以及Eppin调控PSA水解精囊蛋白Semenogelin的研究进展进行了综述。     

Protease inhibitors: possible anticarcinogens in edible seeds

Protease inhibitors, which are common constituents of seeds (rice, beans, and maize) have been shown to inhibit breast, colon, and skin cancers in animal experiments. Epidemiological studies have shown that diets rich in these components (ie seed proteins) decrease the occurrence of prostatic, breast, and colon cancers in man. We have demonstrated that a typical purified inhibitor from soybeans, the Bowman-Birk inhibitor, is excreted in the feces complexed with digestive proteases from the duodenum. This results in greater excretion of proteins, thus effectively decreasing the amount of protein available for digestion. Excess protein in the diet in addition to fats may contribute to increased cancer. Thus, the addition of seeds (chick-peas, beans, etc) to the diet rather than removal of meat or fat offers the opportunity of preventing these cancers, because the protease inhibitors present would effectively remove a portion of the proteins being consumed. Protease inhibitors also act directly as antioxidants in relation to tumor promoters and ionizing radiation. To what extent this contributes to the nutritional effects of eating seeds containing protease inhibitors remains to be determined.     

Comparative study of neutral proteoglycanase activity by growth plate zone

The degradation of proteoglycans has been considered an essential step in the process of endochondral ossification. Neutral proteases, described in the growth plate, have been implicated in this process. If these neutral proteases are important in the endochondral process, their level of activity should be highest at the point where calcification is occurring. This study measures neutral protease activity with a quantitative viscometric assay throughout all zones of the bovine growth plate. The results demonstrate that the enzyme is present throughout all zones of the growth plate. However, enzyme levels in the lower hypertrophic and calcifying zones are significantly higher than anywhere else. The enzymes came from the cartilage cells and not from the invading vasculature. This implied proof of the role of neutral proteases in the endochondral process implies that by inhibiting this class of enzymes, one should be able to block endochondral ossification.     

Molecular cloning and expression of a variant form of hippostasin/KLK11 in prostate

BACKGROUND: Hippostasin is a kallikrein-like serine protease, which has two alternatively spliced isoforms, brain-type and prostate-type. We previously reported alternative expression of hippostasin in prostate cancer cell lines. METHODS: We studied the expression of a variant-form hippostasin (isoform 3) mRNA by RT-PCR. Localization of the isoform 3 protein was examined by immunohistochemistry. The enzymatic activity of the recombinant protein was measured with synthetic substrates. RESULTS: A novel isoform of hippostasin contains 25 additional amino acids in the catalytic triad of brain-type hippostasin. Its mRNA was expressed in normal prostate tissue, BPH, and prostate cancer cell lines. The protein was localized in the prostate secretory epithelium. The enzyme activity was similar to that of brain-type hippostasin, which has kallikrein-like activity. CONCLUSIONS: In this study, we have identified a third isoform of hippostasin, which was designated variant-form (isoform 3). Hippostasin isoform 3 may play a role in the prostate, including reproductive and/or tumorigenic functions.