Cloning, expression, and characterization of two beta-glucosidases from isoflavone glycoside-hydrolyzing Bacillus subtilis natto

On the basis of the genomic sequence of Bacillus subtilis 168, two beta-glucosidase genes (bglH and yckE) from B. subtilis natto, which has been reported to have high isoflavone glucoside-hydrolyzing activity, were cloned and overexpressed in E. coli M15. The temperature for the optimal p-nitrophenyl-beta-D-glucoside hydrolyzing activity of both enzymes was between 37 and 45 degrees C, but BglH had a higher thermal stability than YckE. Both showed high activity at pH 6.0, but YckE was stable over a wider pH range than BglH. Recombinant BglH was inhibited 73%, 63%, and 43% by 1.0 mM Cd(2+), Fe(2+), or Cu(2+), respectively, while other divalent metal ions resulted in 0-23% inhibition, whereas YckE was inhibited by less than 20% by any of the divalent metal ions we tested. Among the substrate we used, BglH showed the highest affinity for genistin and YckE showed the highest affinity for p-nitrophenyl-beta-D-fructopyranoside. Both BglH and YckE hydrolyzed genistin and daidzin into their isoflavone aglycones, genistein and daidzein, but BglH was more efficient than YckE in isoflavone glucoside hydrolysis (20-fold higher kcat). Our results suggest that recombinant BglH may be applicable in the process of isoflavones deglycosylation.     

Effect of phytocompounds from the heartwood of Acacia confusa on inflammatory mediator production

Acacia confusa Merr. (Leguminosae) is traditionally used as a medicinal plant in Taiwan. In the present study, anti-inflammatory activity of extracts from the heartwood of A. confusa were investigated for the first time. Results demonstrated that ethanolic extracts of A. confusa heartwood strongly suppressed NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Among all fractions derived from ethanolic extracts, the EtOAc fraction exhibited the best inhibitory activity. Following column chromatography and reverse-phase high-performance liquid chromatography, 13 specific phytocompounds including 5 new flavonoids (i.e., 7,8,3',4'-tetrahydroxy-4-methoxyflavan-3-ol, 7,8,3',4'-tetrahydroxyflavone, 7,8,3'-trihydroxy-3,4'-dimethoxyflavone, 7,3',4'-trihydroxyflavone, and 7,3',4'-trihydroxy-3-methoxyflavone) were isolated and identified from the EtOAc fraction. In addition, melanoxetin (3,7,8,3',4'-pentahydroxyflavone), a major compound in the EtOAc fraction, markedly suppressed LPS-induced NO and prostaglandin E 2 (PGE 2) production. Moreover, melanoxetin completely suppressed gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) at 50 and 100 microM, respectively. This is the first report to identify the inhibitory bioactivities of melanoxetin on iNOS and COX-2.     

Black soybean promotes the formation of active components with antihepatoma activity in the fermentation product of Agaricus blazei

The antihepatoma activity and related active components in the fermentation products of Agaricus blazei (AB) cultured in the medium containing soybean (S) or black soybean (BS) were investigated. AB(BS)-pE and AB(S)-pE were the ethanolic extracts from the fermentation products of AB(BS) and AB(S), respectively. According to the IC 50 values, AB(BS)-pE (161.1 and 24.0 microg/mL for Hep 3B and Hep G2 cells, respectively) exhibited stronger cytotoxicities against hepatoma cells than AB(S)-pE (>200 and 99.9 microg/mL for Hep 3B and Hep G2 cells, respectively). AB(BS)-pE was separated by silica gel column chromatography and eluted with n-hexane/ethyl acetate/methanol gradient solvent system into 21 fractions. Fraction 3 [AB(BS)-pE-F3], eluted with n-hexane/ethyl acetate (97:3 and 19:1, v/v), was the most active fraction having inhibitory activity on the proliferation of Hep 3B and Hep G2 cells (IC 50 of 3.6 and 1.9 microg/mL, respectively). Three major compounds, compounds 1- 3, were further isolated from the AB(BS)-pE-F3 fraction by reversed-phase semipreparative high-performance liquid chromatography. Compounds 2 and 3 gave better antihepatoma activity than that of compound 1. The IC 50 values of compounds 2 and 3 were 2.8 and 4.5 microg/mL for Hep 3B cells and 1.4 and 2.0 microg/mL for Hep G2 cells, respectively. The structures of compounds 2 and 3 were identified by UV, IR, electron impact mass spectrometry, and (1)H and (13)C NMR to be blazeispirols A and C, respectively. Blazeispirols A and C existed in the mycelia but not in the broth and were more in AB(BS)-pE (49.9 +/- 8.9 and 14.2 +/- 2.4 mg/g, respectively) than AB(S)-pE (15.9 +/- 1.7 and 3.9 +/- 0.6 mg/g, respectively). Additionally, the result shows that the production of blazeispirols A and C was increased after cultivation in the medium containing black soybean on day 6 and reached the maximum on day 12, and the contents of blazeispirols A and C were negatively correlated with Hep 3B and Hep G2 cell viabilities ( r = -0.84 to -0.93, P < 0.01). It suggests that blazeispirols A and C could be used as biomarkers to produce the fermentation product of A. blazei with antihepatoma activity.     

苹果管道气力输送装置参数优化与试验

针对苹果在管道输送过程中的撞击损伤问题,对管道气力输送装置的参数组合进行研究。以输送管道内径、管型和进风口风速为影响因素,以苹果迎风面风速为响应值,通过单因素仿真试验确定各因素对响应值的影响规律;基于单因素仿真结果利用多因素仿真试验获得最优输送参数组合,通过验证试验检验最优组合的可靠性。单因素仿真试验结果表明:当管道内径分别为88、96、104、112和120 mm时,随着输送管道内径的增大,苹果迎风面风速呈先增大后减小的趋势;管型对苹果迎风面风速大小影响的排序为,下圆弧管>螺旋管>S型管>上圆弧管>反S型管;当进风风速分别为14.07、15.83、17.59、19.35和21.11 m/s时,随着进风风速的增大,苹果迎风面风速呈先增大后减小的趋势。多因素仿真试验结果表明:最优输送参数组合为,管道内径112 mm、下圆弧型管型、进风风速19.35 m/s,此时苹果迎风面风速为18.46 m/s。验证试验结果表明,基于最优输送参数组合的管道气力输送装置输送苹果时苹果损伤率为14%,苹果损伤面积0~14.82 mm²,损伤体积0~23.84 mm³,86%的苹果为一等及以上果,14%的苹果为二等果。管道气力输送装置可有效降低苹果采摘后落入果筐时的损伤。     

棉花烂铃及炭疽病的研究

长江流域棉区常见的烂铃病菌有:炭疽病菌(Glomerella gossppii)、角斑病菌(Xanthomonas malvacearum)、花腐病菌(Choanephora cucurbitarum)、黑果病菌(Diplodia gossypina)、印度炭疽病菌(Colletotrichum indicum)、面包霉菌(Rhizopus nigricans)、红粉病菌(Cephalothecium roseum)、红腐病菌(Fusarium spp.)、疫菌(Phytophthora sp.)、轮纹斑病菌(Alternaria spp.)、褐斑病菌(Phyllosticta spp.)、青霉菌(Penicillium spp.)、黑子菌(Nigrospora sp.)、斑纹病菌(Pestalozzia sp.)、(?)腐病菌(Aspergillus spp.)、蠕子菌(Helminthos-porium cuverlaria)、黑斑病菌(Ceratostoma sp.)、汚叶病菌(Cladosporium sp.)等18种,某些年分以炭疽、角斑、红腐等为最主要。1950—51年田间观察结果表明,棉花生长期8、9二个月久雨多湿是引起炭疽病及其他烂铃发生的重要因子。田间接种试验证明:气温平均在25—30℃以及长期保湿,炭疽病菌能直接侵害棉铃。机械损伤、虫孔、角斑病等都是病菌侵害的有利条件。以生长10天的幼铃抗力最强。接近吐絮时的自然裂口,在高湿多雨时,也可成为各种菌类侵害的重要途径。适当密植,合理施用氮肥,进行整枝摘叶,使株行间通风透光,减低湿度,对减轻烂铃损失有一定的作用。     

Nitrogen mineralization and availability of mixed leguminous and non-leguminous cover crop residues in soil

Whereas non-leguminous cover crops such as cereal rye (Secale cereale) or annual ryegrass (Lolium multiflorium) are capable of reducing nitrogen (N) leaching during wet seasons, leguminous cover crops such as hairy vetch (Vicia villosa) improve soil N fertility for succeeding crops. With mixtures of grasses and legumes as cover crop, the goal of reducing N leaching while increasing soil N availability for crop production could be attainable. This study examined net N mineralization of soil treated with hairy vetch residues mixed with either cereal rye or annual ryegrass and the effect of these mixtures on growth and N uptake by cereal rye. Both cereal rye and annual ryegrass contained low total N, but high water-soluble carbon and carbohydrate, compared with hairy vetch. Decreasing the proportion of hairy vetch in the mixed residues decreased net N mineralization, rye plant growth and N uptake, but increased the crossover time (the time when the amount of net N mineralized in the residue-amended soil equalled that of the non-amended control) required for net N mineralization to occur. When the hairy vetch content was decreased to 40% or lower, net N immobilization in the first week of incubation increased markedly. Residue N was significantly correlated with rye biomass (r=0.81, P<0.01) and N uptake (r=0.83, P<0.001), although the correlation was much higher between residue N and the potential initial N mineralization rate for rye biomass (r=0.93, P<0.001) and N uptake (r=0.99, P<0.001). Judging from the effects of the mixed residues on rye N Concentration and N uptake, the proportion of rye or annual ryegrass when mixed with residues of hairy vetch should not exceed 60% if the residues are to increase N availability. Further study is needed to examine the influence of various mixtures of hairy vetch and rye or annual ryegrass on N leaching in soil. Received: 10 March 1997     

双酶法水解罗非鱼下脚料制备降血糖肽的工艺研究

以水解度和α-葡萄糖苷酶抑制率为评价指标,确定双酶复合水解罗非鱼下脚料的方案,并通过单因素试验和正交试验进行优化,最后得到最适酶解工艺参数为先在碱性蛋白酶在温度50℃,加酶量10000 U/g,pH值9.5,底物质量分数6%条件下水解;再在胰蛋白酶在温度37℃,加酶量10000 U/g,pH值8条件下水解100 min。此工艺条件下罗非鱼下脚料水解度、水解产物的α-葡萄糖苷酶抑制率分别为48.26%和41.46%。     

Characteristics of Medical Waste in Taiwan

The purpose of this study was threefold: to investigate the types and amounts of medical waste from various hospitals in Taiwan; to analyze residues from incinerated medical waste for heavy metal content; and to measure the amount of infectious and general medical waste per bed per day and determine the ratio of infectious to general medical waste. Information was first obtained on the amounts of medical waste produced per bed per day and on waste handling and treatment. Next, residue samples were taken from incinerators and analyzed for heavy metal content. Third, daily records concerning waste content and weight from one hospital were kept over the period of one year. Chromium (Cr) and zinc (Zn) concentrations were significantly higher in medical waste than in general waste. Medical waste fluctuated significantly over the one-year period. Records showed that wastes containing dialysis material and wood/cotton materials were found to have the highest weight and wastes containing human organs had the lowest weight. Results of the present study underscore the need for all hospitals to effectively separate waste for proper treatment and disposal, particularly waste that produces residues which may be hazardous to the environment. In addition, hospitals also need to recycle or reuse certain materials in order to reduce waste production.     

沿浦湾秋茄种植前后大型底栖动物生态位和功能群变化

基于沿浦湾2014年10月(秋季)和2016年10月(秋季)的潮间带大型底栖动物调查资料,运用生态位宽度、生态位重叠、功能群分类和冗余分析(RDA)等指标与方法,分析秋茄(Kandelia candel)种植前、后大型底栖动物的生态位、功能群变化。研究结果表明,秋茄种植后广生态位种和中生态位种物种数占比与种植前相比较均有所增加,窄生态位种物种数占比减少。种植前生态位重叠值最大的种对为大青弹涂鱼(Scartelaosgigas)和细螯虾(Leptochela gracilis)(0.94),种植后则分别为豆形拳蟹(Pyrhila pisum)和微黄镰玉螺(Lunatica gilva)(0.99)、堇拟沼螺(Assiminea violacea)和光滑河篮蛤(Potamocorbula laevis)(0.99)。种植后高、中潮带功能群多样性较种植前上升,而低潮带功能群多样性下降;种植后浮游生物食者(Pl)、植食者(Ph)和杂食者(Om)的密度较种植前均增加,而肉食者(Ca)的密度略有减少。秋茄种植前3个潮带之间无显著性差异(P0.05);秋茄种植后高潮带与低潮带,以及中潮带与低潮带均有显著性差异(P0.05)。冗余分析(RDA)得出,对大型底栖动物群落具有较大影响的因子由种植前的铅、镉和石油类变为种植后的锌、镉和砷。总之,苍南沿浦湾秋茄林种植对大型底栖动物生态位和功能群总体上具有积极的改善作用。