蜜环菌子实体发生条件的初探

目的蜜环菌在人工培养过程中子实体发生条件的摸索;方法固体培养并给予不同的温度、光照、通气条件;结果在给予黑暗交替光照及充足空气的条件下蜜环菌可于18±1℃在三角瓶中萌发子实体;结论在湿度适宜的情况下,蜜环菌子实体的发生温度、光照、通气起关键作用。     

大曲淀粉酶同工酶分析

从不同发酵期的大曲曲样中分离出产淀粉酶的菌株共７２株，其中细菌３７株，霉菌２７株，酵母８株，分别培养分析其淀粉酶同工酶，对酶谱作了比较。该研究对阐明发酵过程中各微生物菌群间的相互关系有重要意义。     

汾酒大曲酯酶和淀粉酶同工酶的分析

利用同工酶技术进行了清茬、后火和红心3种汾酒大曲酯酶和淀粉酶同工酶的比较研究。大曲电泳参数研究表明:酯酶和淀粉酶同工酶电泳分离胶浓度分别为12%和10%,上样蛋白浓度分别为5.5～11 mg/mL和0.34～0.69 mg/mL,连续抽提10批储存9 d,同工酶电泳图谱一致稳定。3种大曲同工酶酶谱分析表明:每种大曲都含有丰富的酯酶和淀粉酶同工酶酶带。3种大曲酯酶同工酶谱主带的数量和位置基本相同,曲皮、曲心和整曲也基本一致,仅在弱带区有所不同。3种大曲淀粉酶同工酶谱明显不同,后火曲有一条特征主带。曲心明显不同于曲皮和整曲,曲心的淀粉酶同工酶酶谱明显将3种大曲鉴别,其中有一条酶带可作为后火曲的同工酶标记。10批混合生产用曲的同工酶分析,酯酶和淀粉酶同工酶酶谱基本一致,淀粉酶同工酶谱分析混合曲中有后火曲存在。     

硒对蜜环菌的作用规律及其在多糖中的积累

采用不同硒浓度对蜜环菌进行了补硒培养,研究了硒对蜜环菌生长、蜜环菌多糖产率的影响以及蜜环菌对硒的积累。结果表明:浓度为10mg/L以下的硒能够促进蜜环菌的生长,超过该浓度之后,硒对蜜环菌的生长产生阻碍;在所设计的浓度范围内,硒能够促进蜜环菌胞内外多糖的合成和分泌。蜜环菌胞内外多糖中硒质量分数都随着硒处理浓度的增加而增加,但硒积累量变化趋势不尽相同。胞内多糖的硒积累量在硒处理浓度为10mg/L时后趋于稳定;胞外多糖硒积累量在硒处理浓度超过5mg/L后仍有增长趋势。     

蜜环菌水溶性多糖的分离鉴定与发酵条件优化

目的:筛选出产蜜环菌多糖和菌丝体生长的最适碳氮源,对蜜环菌水溶性多糖进行结构和组成分析。方法:改变蜜环菌发酵的不同种类碳氮源,测定多糖含量和菌体干重,经热水浸提、除蛋白等步骤分离纯化到水溶性多糖,利用紫外、红外光谱、气相衍生化法分析其基本结构信息。结果:红薯粉和豆粕的组合最适合产多糖,玉米浆和玉米粉的组合菌丝体得率最高;多糖结构由吡喃葡萄糖组成。结论:在富含天然类成分的培养基中,蜜环菌菌丝体生长和产多糖能力较高;提取到的水溶性多糖,成分单一;糖醇乙酸酯气相衍生化法可简便、准确地测出蜜环菌多糖中所含的各种单糖。     

利用过氧化物同工酶进行葡萄品种及霜霉病抗性鉴定研究

采用聚丙烯酰胺垂直板凝胶电泳,分析了22个葡萄品种的叶片中过氧化物(POD)同工酶酶谱特征.结果表明,22个品种共电泳出6种过氧化物同工酶酶带,酶谱类型17种,表明POD同工酶酶谱不能将葡萄品种鉴别开来,每种过氧化酶酶谱类型具3～5条酶带;Rf为0.6089的酶带为22个葡萄品种共有的特征谱带;Rf为0.3333酶带,为感或中感霜霉病的品种的特征谱带;Rf为0.2978、0.2661和0.4455的酶带,为抗或中抗霜霉病品种的特征谱带;SPSS分析和UPGMA聚类分析结果表明,供试品种可分为五大类群:第一、二类群为森田尼无核、无核8611等12个欧亚品种,表现感至中感霜霉病;第三、四类群为超藤、饭刚黑、巨峰等10个欧美杂交种,表现抗至中抗霜霉病.用POD同工酶特征谱带鉴定葡萄品种抗感霜霉病类型是比较有效的方法,但不能作为葡萄品种鉴定的有效方法.     

长白山地区蜜环菌菌种的分离与筛选

本文主要对从长白山地区采集到的天麻和蜜环菌的了实体分离鉴定，得到了16个蜜环菌菌株。通过固体培养和液体培养，开展了各菌株的菌丝和菌索萌发时间及其形态特征、荧光特性、液体培养时菌丝球的形态、大小，发酵液色泽，荧光强度，产物的生物量等方面的实验研究，进一步筛选出生长性状良好的8个菌株Ar-02，Ar-03，Ar-06，Ar-08，Ar-09，Ar-11，Ar-13，Ar-31。     

叠氮化钠对花生下胚轴酯酶同工酶的影响

用不同浓度的叠氮化钠处理花生４个品种，通过聚丙烯酰胺凝胶电泳对花生下胚轴的酯酶同工酶谱进行测定。实验结果表明，ＮａＮ３对花生酯酶同工酶谱主要影响的酶带有Ｍ１，Ｍ放Ｓ１带。汕油２７品种经０．１，１．５和１０ＭＭＯＬ．ｌ＾－１ＮａＮ３处理，其酶变出现新的酶带。     

不同陈化时期的烟叶酶活性变化及其同工酶酶谱分析

以3个烤烟品种为试验材料,对不同陈化时期烟叶中多酚氧化酶、过氧化物酶活性进行了分析,同时利用电泳技术研究了不同陈化时期烟叶中多酚氧化酶和过氧化物酶同工酶酶谱的变化。结果表明随着陈化时间的延长,多酚氧化酶、过氧化物酶活性呈现先升高后降低的趋势;多酚氧化酶和过氧化物酶同工酶酶谱在陈化过程中发生了较为显著的变化,表现为陈化3个月以前酶带较少,酶带的亮度较弱,陈化6个月时酶带最多,亮度最强,6个月以后酶带数量开始减少,亮度也有所下降。通过酶谱分析,也进一步验证了不同陈化时期烟叶中多酚氧化酶和过氧化物酶活性变化规律。     

蜜环菌抑菌作用的初步研究

采用滤纸片法、液体培养法和活菌计数法,研究了蜜环菌菌索提取液对常见微生物的抑制作用。通过测定MIC值,蜜环菌的抑菌能力依次为:大肠杆菌>金黄色葡萄球菌>枯草芽孢杆菌>啤酒酵母菌。15%的蜜环菌浸提液可抑制细菌生长,高于20%的蜜环菌浸提液可对霉菌和酵母菌产生抑制作用。     

Comparative analysis of peroxidase profiles in Chinese kale (Brassica alboglabra L.): Evaluation of leaf growth related isozymes

Plant peroxidases (EC 1.11.1.7) with different isoforms catalyze various reactions in plant growth and development. However, it is difficult to elucidate the function of each isozyme in one plant. Here, we compared profiles of entire isozyme in young seedling and mature leaves of Chinese kale (Brassica alboglabra L.) on zymogram and ion exchange chromatography in order to investigate leaf growth related peroxidase isozymes. The results showed that four isozymes were constitutively expressed in kale leaves, whereas other two isozymes were induced in the mature leaves. The Mono Q ion exchange chromatography separated the six isozymes into two major groups due to the difference in their isoelectric points. The results suggested that although there were several isozymes in the leaves of Chinese kale, one isozyme functioned mainly through the leaf development. Two anionic isozymes with molecular weights lower than 32 kDa were considered mature related.     

PURIFICATION AND CHARACTERIZATION OF LIPOXYGENASE ISOZYMES FROM CANOLA (Brassica napus cv, WESTAR) SEED

Four isozymes, I', II'a, II'b and III’ of lipoxygenase (EC 1.13.11.12) from Canola (Brassica napus, cv Westar) seed were purified by successive chromatography on ion-exchange and size-exclusion columns using a Fast Protein Liquid Chromatograph (FPLC). The homogeneity of each isozyme was demonstrated by a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weights of isozymes I', II'a, II'b and III’ were 72, 000, 106, 000, 78, 000 and 62, 000, respectively. The optimum pH for lipoxygenase activity was 6.5 for isozyme I’ and 6.0 for isozymes II'b, II'b and III'. Apparent K_m value for isozymes I', II'a, II'b and III’ were 5.5 × 10^?4 M, 3.4 × 10^?4 M, 4.0 × 10^?4 M and 3.8 × 10^?4 M, respectively. Isozyme I’ displayed preferential activity towards monolinoleate and dilinoleate, while isozyme II'a demonstrated preferential activity towards dilinoleate followed by mono- and trilinoleate. No enzymatic activity was observed with both isozymes I’ and II'a toward free linoleic acid. Isozyme II'b showed activity towards free linoleate as well as mono-, di- and trilinoleate. Isozyme III’ showed preferential activity towards free linoleate. The activity of isozymes I’ and II'a was inhibited completely by the addition of 10 mM and 4 mM KCN, respectively, while the addition of 3 mM and 10 mM KCN to isozymes II'b and III', respectively, increased activity by approximately 20%.     

PURIFICATION AND CHARACTERIZATION OF THREEISOZYMES OF PECTIN METHYLESTERASE FROM TOMATO FRUIT

Three isozymes of pectin methylesterase (EC 3.1.1.11) have been purified to homogeneity from tomato (var. S. marzano). The isozymes were separated by affinity chromatography on Heparin-Sepharose column. They exhibited a molecular mass of 31 kDa when analyzed in sodium dodecyl sulfate gel electrophoresis and of 35 kDa in gel-filtration chromatography in native conditions. The isoelectric points of all three isozymes were found to be higher than 9.3. The K_ms calculated for the three isozymes were different toward citrus pectin used as substrate; one had a K_m of 9.7 mM (by expressing the pectin concentration as mmoles/L of methoxy groups) and the other two had similar K_ms of 3.0 and 2.6 mM, respectively. The isozyme having the higher K_m for substrate was inhibited by citrus pectin (which had a degree of methylation of 70%) at concentrations higher than 5 mM, but no inhibition was found using a pectin with a degree of methylation of 30% at concentrations up to 13 mM (i.e. 9 mg/ml) with a K_m of 14.7 mM. Furthermore, this isozyme showed a more broad range of activity in a pH range 5–10 with respect to that exhibited by the other two isozymes. All three isozymes were found to be glycosylated, although to different extents.     

接种PVY^N后烟草叶片SOD活性和MDA含量变化研究

对接种马铃薯Ｙ病毒脉坏死株系后烟草叶片内超氧化物岐化酶活性及其同工酶谱带和丙二醛含量进行了分析。结果表明,接种ＰＶＹＮ后烟草叶片内超氧化物岐化酶活性发生明显变化,接种后１４ｄ内其活性值均较对照增高,其中接种后第８ｄ达高峰值,约比对照增加７２．３％,１４ｄ后超氧化物岐化酶活性开始下降,至接种后１６ｄ约低于对照２８．０％。接种后ＳＯＤ同工酶谱带与对照相比差别不大,仅在接种后第８ｄ出现了一条新带。接种后烟草叶片内丙二醛含量逐渐增加。     

Isolation of a novel alkaline-induced laccase from Flammulina velutipes and its application for hair coloring

Laccase is a member of the multi-copper oxidase family and a promising for hair coloring. In this study, we isolated a novel alkaline-induced laccase from the white-rot fungus Flammulina velutipes and studied the possibility to apply the enzyme for hair coloring. Laccase activity detected in the culture supernatant of F. velutipes was found to significantly increase when exchanging the medium to laccase inducing one whose pH was adjusted to 9.0. Three isozymes were detected by activity staining on non-denaturing SDS-PAGE. The major isozyme, Flac1, was purified from the culture supernatant after being induced at pH 9.0 by ion-exchange column chromatography. The N-terminal peptide sequence of Flac1 was determined, revealing clear homology with laccases from other white-rot fungi. Optimum pH of oxidation was found to be around pH 5.0-6.5 regardless of several different substrates used. Oxidation activities of Flac1 to several hair dye agents as substrate showed the higher activity at pH 6.5 than that at pH 9.0. Oxidation activity was also detected at pH 9.0 which was suitable for hair coloring. When the purified Flac1 was applied for hair coloring system without using hydrogen peroxide, effective coloring was observed at the protein amount of 0.25mg/1g of hair used. These results indicated that this alkaline-induced novel laccase isolated from the culture supernatant of F. velutipes might be a useful enzyme for hair color.     

TOTAL AND INDIVIDUAL BARLEY (1–3), (1–4)-β-D-GLUCANASE ACTIVITIES IN SOME GREEN AND KILNED MALTS

Total β-glucanase activity was measured in extracts of malts prepared from three winter and three spring cultivars of barley. Samples were taken at intervals during modification and, after 116 hr, from malt kilned to 65°C. Good malting varieties showed highest levels of total β-glucanase activity, with a high correlation (r = 0.926) with HWE. Angora had the highest activity in the intermediate stages of malting, least loss of activity after heating extracts to 48°C for 10 mins and least loss of activity on kilning. Separation of isozymes by HPLC⁹ confirmed the greater heat stability of isozyme II, but, unlike previous studies on Australian cultivars, we found considerable activity of isozyme I after kilning, even up to 85°C. However, total β-glucanase activity was destroyed by heating extracts of all varieties to 60°C. Angora showed the highest proportion of total activity as isozyme II after kilining. Cultivar differences suggested some scope for breeding varieties with increased total activity by combining high activities of each isozyme. The high correlation of total activity with HWE suggests β-glucanase activity as a rapid test of malting quality.     

过氧化物酶活性与烟草对马铃薯Y病毒抗性关系的研究

本文测定了接种马铃薯Y病毒脉坏死株系后烟草抗病转基因品系9608(转PVY-NIb的NC89)和非转基因NC89叶片组织透性和过氧化物酶活性,并对过氧化物酶同工酶带谱进行了分析。结果表明,抗病转基因9608和感病NC89叶片的组织透性与叶片内过氧化物酶活性的变化趋势一致,两者在接种后均呈增高趋势,但感病NC89的增高幅度明显高于抗病转基因9608;接种后,转基因9608和非转基因NC89的过氧化物同工酶均出现了一条新酶带,9608的新酶带在接种后第2d开始出现,而NC89的新酶带则在接种后第6d开始出现,且明显强于9608。      

大球盖菇提取物对CCl4所致急性肝损伤小鼠的抗氧化酶活性及同工酶的影响

目的：研究大球盖菇提取物(SRE)对小鼠肝和主要脏器的抗氧化酶活性及同工酶的影响。方法：小鼠连续30d 给予低(100mg/(kg bw·d))、中(200mg/(kg bw·d))、高(400mg/(kg bw·d))剂量SRE 后,灌胃给予CCl4(80mg/(kg bw·d))建立动物急性肝损伤模型,动物处死后取肝及主要脏器测定抗氧化酶活性,采用聚丙烯酰胺梯度凝胶电泳分析CAT 和SOD 同工酶。结果：与模型组比,高剂量SRE 能显著提高肝总SOD、Mn-SOD、CuZn-SOD 及CAT、GPx 活性。主要脏器抗氧化酶活性测定表明,给予SRE 后,心和肾SOD、CAT、GPx 活性比模型组显著提高;造模后肾Mn-SOD 表现出代偿性增高,给予SRE 后能使其恢复到对照组水平。各脏器CAT 同工酶都为1 条,在造模后酶带染色密度明显降低,在给予SRE 后能恢复到正常水平。肝有1 条Mn-SOD带和3 条CuZn-SOD 带,在给予SRE 后能使这些酶蛋白含量达到或高于正常水平;心、肾有1 条Mn-SOD 带和4 条CuZn-SOD 带,SRE 能增加心、肾CuZn-SOD 酶蛋白染色密度。结论：SRE 能通过提高抗氧化酶的活性和同工酶酶蛋白含量,有效预防急性肝损伤小鼠肝脏和主要脏器的氧化损伤作用。     

Isolation and Characterization of Chitinase‐Producing Bacillus and Paenibacillus Strains from Salted and Fermented Shrimp,Acetes japonicus

Chitinases catalyze the conversion of chitin and are produced by a wide range of bacteria. The biological applications of these enzymes have been exploited in food and pharmaceutical industries. We isolated 2 halophilic chitinase‐producing novel strains of bacteria—SCH‐1 and SCH‐2 from Saeu‐jeot, a traditional Korean salted and fermented food made with shrimp (Acetes japonicus). The isolated strains‐ SCH‐1 and SCH‐2 were Gram‐positive, rod‐shaped, endospore‐forming facultative anaerobes, with strain SCH‐2 showing peritrichous flagella. Molecular characterization of the 16S rRNA gene identified the strains SCH‐1 and SCH‐2 as Bacillus sp. and Paenibacillus sp. respectively. Basic Local Alignment Search Tool and subsequent phylogenetic analysis of strain SCH‐1 showed an identity of 97.83% with Bacillus cereus ATCC 14579 (NR_074540), whereas strain SCH‐2 showed an identity of 99.16% with Paenibacillus lautus JCM 9073 (NR_040882). Furthermore, the SCH‐1 strain could use glucose, N‐acetyl glucosamine, esculin, and maltose as carbon source substrates. Cellular fatty acid analysis showed that iso‐C_15:0 and anteiso‐C_15:0 are the major acids in strain SCH‐1 and SCH‐2, respectively. The SCH‐1 strain showed a higher chitinase activity at 15.71 unit/mg protein compared with SCH‐2 strain. Chitinase isozymes of Bacillus sp. SCH‐1was expressed as 2 bands having sizes of 41 and 50 kDa, and as 4 bands with sizes of 30, 37, 45.7, and 50 kDa in Paenibacillus sp. SCH‐2. The rich chitinase activity with the isozyme profiles of the isolated Bacillus and Paenibacillus strains provide advancement in the study of fermentation and may play putative functions in the chitin bioconversion of sea crustacean foods.