3株不同表型马尔尼菲青霉外分泌蛋白酶活性比较

目的探讨如何有效诱导马尔尼菲青霉蛋白酶分泌及比较3株不同表型的马尔尼菲青霉菌株之间外分泌蛋白酶活性差异。方法选用临床分离的马尔尼菲青霉B-6323株(P株)及其两个不同表型的突变株M/M株和M/Y株,用以牛血红蛋白为底物的酵母碳基琼脂培养基(蛋白酶诱导培养琼脂基)诱导其外分泌蛋白酶分泌。将pH值为4.0、5.6及7.2的蛋白酶诱导培养琼脂基平板打孔后分别等量接种上述3株菌,分别置于25℃及37℃培养6 d后,行考马斯亮兰G-250染色,测定各菌株周围的透亮圈(蛋白分解环)直径。结果以牛血红蛋白为底物的酵母碳基琼脂培养基可有效诱导马尔尼菲青霉蛋白酶分泌;3株马尔尼菲青霉在任意3种pH值培养基中培养,37℃培养下菌周透亮圈直径大于25℃培养下,差异经t检验有统计学意义。经方差分析,37℃,pH4.0、5.6、7.2培养条件下马尔尼菲青霉菌周透亮圈直径之间差异有统计学意义(F值为97.198,P=0.000 1),用SNK法进行两两比较发现pH4.0与pH7.2菌周透亮圈直径及pH5.6与pH7.2菌周透亮圈直径差异均有统计学意义。当温度为37℃或25℃时,在任意一个pH值下,均为M/M株的菌周透亮圈直径均数最大,其次为P株,M/Y株最小。结论马尔尼菲青霉在37℃,酸性培养条件(pH4.0、5.6)下外分泌蛋白酶活性大,且不同表型株之间外分泌蛋白酶活性有差异,M/M株及P株为高外分泌蛋白酶活性株,M/Y株为低外分泌蛋白酶活性株。     

高产蛋白酶菌株Bacillus cereus KM-4的产酶实时动态研究

从昆明豆豉样品分离得到一株蛋白酶活性较高的菌株,其酶活达14.58 U/mL.采用SDS-gelatin-PAGE蛋白电泳及Zymography染色,结合茚三酮法测定其蛋白酶活性,对KM-4菌株分泌的蛋白酶进行酶谱分析.结果表明,培养pH6.0有利于该菌株分泌分子量约为116 kD的蛋白酶,经明胶诱导培养后,其酶活明显提高,推测其所分泌的蛋白酶可能需要蛋白质诱导.     

马拉色菌蛋白酶活性测定方法的建立及其应用

为建立检测马拉色菌蛋白酶的方法并检测不同来源菌株的蛋白酶活性 ,使用全脂牛奶平板法、BSA平板法检测 7株标准株 ,3 3株M .furfur、12株M .sympodialis、4株M .obtusa临床分离株和 2 8株M .furfur正常皮肤分离株蛋白酶活性 ,并检测温度、pH值和蛋白酶抑制剂对蛋白酶活性的影响。 7株标准株均检出蛋白酶活性 ,M .furfur临床分离株蛋白酶活性高于正常皮肤分离株 (p <0 .0 1) ,最高蛋白酶活性在 3 2℃、pH5.5时表达 ,EDTA能抑制其蛋白酶活性。全脂牛奶平板法为简便、可靠的测定马拉色菌蛋白酶活性的方法 ,蛋白酶活性与菌株的致病性相关     

成纤维生长因子-2与肝细胞生长因子诱导骨髓间充质干细胞分化为肝细胞样细胞的作用比较

比较成纤维生长因子-2(FGF-2)与肝细胞生长因子(HGF)诱导大鼠骨髓间充质干细胞(BM-MSCs)向肝细胞分化的能力,并进一步的研究BM-MSCs诱导成肝细胞所需的最佳诱导因子以及其用量。体外获取、培养大鼠BM-MSCs,将第3代BM-MSCs采用不同剂量的FGF-2和HGF诱导。诱导后,在显微镜下观察细胞形态学的改变;Shiff染色法检测糖原的分泌;ELISA法检测AFP的分泌;免疫细胞化学法检测白蛋白和CK19的分泌。诱导后BM-MSCs由梭形向多角形、卵圆形方向变化,AFP第3天就有分泌,第12天达到高峰,以后逐渐减少;白蛋白、CK19和糖原第12天即有阳性表达,以后随着诱导时间的延长阳性率逐渐升高。第2组和第4组比其他组分泌的白蛋白、CK19和糖原均多。FGF-2比HGF具有更强的诱导BM-MSCs向肝细胞分化的能力。     

放线菌株F培养条件优化和抗菌物质的稳定性研究

目的:研究放线菌株F菌的培养条件,优化其发酵条件,分析其抗菌物质的稳定性.方法:以平板对峙法检测抑菌活性,通过比较抑菌圈直径和抑菌率,优化培养条件.结果:F菌株对黄瓜枯萎病和番茄灰霉病均有一定的抑制作用.菌株F的最佳接种量为10mL/100mL,最佳碳源为葡萄糖,最佳氮源为硝酸钾.最适发酵条件为:葡萄糖20g,硝酸钾15g,酵母膏5g,蛋白胨2g,CaCO3 4g,NaCl 4g,蒸馏水1 000mL,pH 7.2～7.4.F菌株抗菌物质在80℃时仍保持很好的抑菌活性,但温度继续升高时活性下降.不同pH间抑菌率存在差异,pH值为6时抑菌率出现最大值60.46%,pH值为9时抑菌率最小,为57.93%.在发酵培养液培养以pH 6为宜.结论:F菌株无菌发酵液具有较好的温度、光照和酸碱性的稳定性.     

pH对毕赤酵母表达重组人复合α干扰素的降解影响

使用Pichiapastoris表达重组人复合α干扰素(cIFN)会发生降解、聚合等不均一表达的现象.在5 L发酵罐中考察了不同诱导pH对cIFN表达产生降解的影响,结果发现在适合酵母生长的pH 3.0～7.0范围内,当诱导pH为4.0～5.0时,cIFN不均一表达现象最少,生物活性达到2.5×108 IU/mL.通过测定发酵液中总蛋白酶活和细胞活性寻找了cIFN降解出现的原因:发现低诱导pH下细胞死亡率升高释放更多酶系,高诱导pH下蛋白酶活性明显增大,两者都使蛋白酶作用加强,加剧cIFN的降解;特别是诱导pH为7.0时,适宜的pH使蛋白酶酶活陡升,将cIFN完全降解.     

黑木耳原种胞外酶活性的研究

目的：研究黑木耳菌株原种胞外酶活分泌特性,为进行大规模生产用种早期判定提供检测手段。方法：以9个黑木耳栽培菌株的原种为实验材料,分别测定了其胞外漆酶、多酚氧化酶、羧甲基纤维素酶、滤纸维素酶的酶活性变化。结果：胞外漆酶、多酚氧化酶、纤维素酶活性的变化趋势大致相似,酶活随培养时间呈规律性的升高、降低,除个别菌株（3、6）外,不同菌株同种酶活性差别不大,酶活相对较高的菌株有2、3、6、9号。不同菌株酶活高峰出现的时间有差异,较早的出现在培养的第10d,较晚的出现在培养的第70d。结论：被测菌株胞外酶活存在一定的规律性,利用该规律性可检测菌种退化、老化和不利变异。     

鹿茸血制备ACE抑制肽的酶解条件优化

以天山马鹿的鹿茸血为原料,通过体外检测方法检测不同酶及酶解条件下的产物的ACE抑制肽活性及抗氧化活性。结果表明,以A lcalase碱性蛋白酶水解,底物浓度6%、加酶量40μl/g蛋白、pH 8.0、温度55℃的条件下,酶解3 h,水解度为28.0%,酶解产物的血管紧张素转化酶(angiotensin converting enzym e,ACE)抑制活性可达93.55%,与抗氧化活性呈正相关关系。     

栗疫病菌不同毒力菌株胞外酶活性和草酸产量的比较

对栗疫病菌不同毒力菌株产生胞外酶的种类、活性和草酸产量以及草酸对多聚半乳糖醛酸酶水解聚果胶酸钙的影响进行了研究。所有供试菌株均未能检测到淀粉酶活性。栗疫病菌在培养中可分泌漆酶,多聚半乳糖醛酸酶、蛋白酶、纤维素酶和脂酶,但不同毒力菌株产生这些酶的能力不同。总的来说,强毒力菌株均可分泌这些酶,且活性强,但弱毒力菌株的酶活性较弱或不分泌这些酶。菌丝产量和草酸产量分析表明,强毒力菌株的草酸产量明显高于弱毒力菌株。菌丝产量与草酸产量没有相关性。在没有草酸盐存在的条件下,多聚半乳糖醛酸酶不能降解聚果胶酸钙。     

光照和pH对药用真菌二型附毛孔菌液体培养过程中抗氧化活性的影响

本文以多酚含量、总抗氧化能力(total antioxidant capacity,T-AOC)、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除能力和超氧化物歧化酶(superoxide dismutase,SOD)活性为测定指标,研究了不同光照和pH条件对药用真菌二型附毛孔菌Trichaptum biforme液体培养过程中抗氧化活性的影响。结果显示,二型附毛孔菌在液体培养过程中具有抗氧化活性。一定的光照可提高其分泌次级代谢产物的能力及抗氧化活性,14d光照条件下,多酚含量和T-AOC分别在第12天和第10天达到峰值,为1.85mg/m L和3.99U/m L;1d黑暗、1d光照交替的光照条件下,DPPH自由基清除率和SOD活性均于第2天达到最大值,分别为87.44%和0.63U/m L。不同pH条件对二型附毛孔菌液体培养过程中的抗氧化活性影响更大。当pH为9.0时,二型附毛孔菌在液体培养过程中分泌的多酚含量、T-AOC、SOD活性和DPPH自由基清除能力均最高,分别于第2天、第10天、第2天和第2天达到2.92mg/m L、5.22U/m L、0.70U/m L和95.07%。其次是pH 3.0,最后是pH 5.0和7.0,说明偏碱或偏酸条件更能刺激药用真菌二型附毛孔菌通过分泌大量次级代谢产物以抵抗来自微环境的氧化压力。     

Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer.     

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.     

Unraveling biochemical properties of cockroach (Periplaneta americana) proteinases with a gel X-ray film contact print method

Eleven proteinase activity bands were detected in American cockroach (Periplaneta americana) gut. These were partially purified and characterized using a gel X-ray film contact print method. Cockroach gut proteinases (CGPs) show activity over a broad range of pH with maximum activity between pH 6 and 10, and optimal activity at 50-70 degrees C. CGPs were partially purified by preparative gel electrophoresis and analyzed using synthetic substrates and inhibitors. Four of the proteases exhibited chymotrypsin-like (C1 to C4) activity and seven trypsin-like (T1 to T7) activity. Accuracy of the gel X-ray film contact print method is confirmed by including bovine chymotrypsin in CGP analysis. Inhibition of CGPs with different plant proteinaceous proteinase inhibitors allowed identification of potential CGP inhibitors. Our results imply that presence of several CGP activity bands, and their stability and activity over a broad pH and temperature range might contribute to adaptation of P. americana to extreme environmental conditions and the polyphagous nature of the species.     

Production of polygalacturonase byByssochlamys fulva

Summary Byssochlamys fulva was grown in two fermentation media using shake flasks, stirred fermentor and disc fermentor under conditions to give maximum production of pectolytic enzymes. Only polygalacturonase activity was detected in the culture filtrates during all fermentations. In all production conditions studied, no evidence of pectin methylesterase, pectin lyase, cellulase or proteinase activities were found. The maximum polygalacturonase activity (4.5 units/ml) was achieved when the microorganism was grown on medium II in shake flasks at pH 4.0–4.5 and 30°C after 12 days of fermentation.     

The effect of dilution rate and pH on biomass and proteinase production by Micrococcus sedentarius grown in continuous culture.

Micrococcus sedentarius, an organism associated with pitted keratolysis, produced two proteinases in culture supernatant fluids, as shown by non-denaturing PAGE with overlaying with a casein substrate. A mixture had optimal activity at pH 10 with azocasein substrate. At pH 7.1 and 8.1 in continuous culture with varying dilution rates high proteinase production occurred at relative specific growth rates (mu rels) 0.39 and 0.77 and biomass concentrations decreased with increasing dilution rate. One proteinase was constitutive and varied little in production with different growth rates. The other proteinase was under control with high production at low growth rates and no production at high growth rates. With varying pH at mu rels 0.39 and 0.77 maximum biomass concentration and proteinase production occurred between pH 8.0 and 9.0 as did the highest specific growth rate. These results support the hypothesis that Mic. sedentarius produces pitting in the stratum corneum when the skin is hydrated and the pH rises above neutrality.     

Centroid search optimization of cultural conditions affecting the production of extracellular proteinase by Pseudomonas fragi ATCC 4973

The production of extracellular proteinase by Pseudomonas fragi ATCC 4973 grown in a defined citrate medium, containing glutamine as the sole nitrogen source, was determined under varying cultural conditions. Simultaneous evaluation of cultural conditions using a 'centroid search' optimization technique showed that the optimum cultural conditions for proteinase production by Ps. fragi were: incubation temperature, 12.5 degrees C; incubation time, 38 h; initial pH, 6.8; organic nitrogen concentration, 314 mmol nitrogen/l (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by Ps. fragi. The results may have applications in the storage of fluid milk. Centroid search optimization was shown to be suitable for microbiological experiments.     

Investigation of acid proteinase biosynthesis by the fungus Humicola lutea 120-5 in an airlift bioreactor

Acid proteinase production using filamentous fungus Humicola lutea 120-5 was studied under batch and continuous fermentation conditions in an airlift bioreactor. A comparison with proteinase production by fungal cells, cultivated in stirred tank bioreactor was made. The process performance in both fermentation devices was similar with respect to substrate utilization, biomass, and enzyme concentration. Continuous acid proteinase production was achieved for 14 days at an optimal dilution rate of 0.05/h with maximum specific activity of 90 U/mg DW of mycelia and yield of 38 U/mg glucose. The volumetric productivity (50 U/ml. h) was approximately 3 times higher than this of the batch system. All continuous experiments were carried out without any bacterial contamination, due to the low pH (3.0-3.5) during the process. The "pellet" type growth of the fungus in the airlift reactor prevented the system from plugging with filaments.     

Identification of yolk platelet-associated hydrolases in the oocytes of Rhodnius prolixus.

The yolk platelets from Rhodnius prolixus, a blood-sucking bug, are composed mostly of vitellin and here are shown to contain at least two hydrolytic enzymes, a phosphatase and a cathepsin D-like proteinase. Both the proteinase and the phosphatase have an acid pH optimum. No hydrolytic activity was observed under alkaline or neutral conditions. Among several proteinase inhibitors tested, only pepstatin could abolish vitellin breakdown in vitro. The proteinase appears to be bound to the yolk platelet membranes. The phosphatase activity, using p-nitrophenyl phosphate as substrate, was enhanced after disruption of the platelet membrane by Triton X-100. This activity could be inhibited by tartrate but not by p-cloromercuribenzoate.     

Extracellular alkaline proteinase of Colletotrichum gloeosporioides

The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.     

Isolation and properties of extracellular proteinases of Penicillium marneffei

Penicillium marneffei is a dimorphic fungus native to Southeast Asia. Disease caused by this organism, until recently a very rare condition, has increased dramatically in parallel with the increase in the number of individuals in the region immunocompromised by AIDS and other conditions. While much research has been performed on the control of dimorphic switching in P. marneffei, there is a relative dearth of information regarding the proteinases secreted by this pathogen. Our laboratory has purified and characterized two proteinases produced by this organism in liquid culture and cloned the gene of a third. Both the recombinant enzyme expressed from the cloned gene and one of those purified from culture supernatants have been identified as members of the eqolisin family, a group of pepstatin-insensitive acid proteinases. The other enzyme purified from a culture supernatant is a serine proteinase with activity in the neutral pH range. These enzymes appear to be differentially expressed, depending on culture conditions.