醇化烟叶产蛋白酶菌株的分离鉴定及其对烟叶内在品质影响

目的从采自云南玉溪的醇化烤烟中分离得到15株产蛋白酶菌株,通过16SrRNA基因分析鉴定为芽胞杆菌属的菌株。方法为改善玉溪低次等烟叶的内在品质,利用分离到的15株产蛋白酶细菌进行发酵,发酵液经超滤制备得到的粗蛋白酶液,在室温条件下处理云南初烤烟叶,然后通过感官评吸进行评价。结果不同菌株来源的蛋白酶对烟叶感官质量改善效果不同,酶液P7、P9和P10可明显改善烟叶感官质量,烟叶香气质改善,杂气减轻,均衡性好;而酶液P5和P11则使烟叶的感官质量下降。结论研究为蛋白酶应用于烟草生产进行了有益的探索。     

微生物对烟叶蛋白质含量的影响

经菌株不同浓度、不同菌株、及同浓度不同菌株的等量组合处理过的烟叶放置一段时间未经发酵与发酵后对蛋白质检测,结果表明降解K326中部碎烟叶蛋白质的处理中Y菌株106浓度降解率最高,为18.81%;处理烟叶发酵后,降解率最高的处理是S5菌株107浓度,高达24.46%。K326上部碎烟叶处理后检测结果表明,对蛋白质降解率最高的处理是Y与S8混合,降解率为8.98%;K326上部碎烟叶处理发酵后检测结果表明,S8菌株108浓度处理降解率最高,达24.09%,其次为S5菌株108浓度处理降解率达23.48%。整叶处理结果表明S8降解蛋白质效果最好,降解率达5.17%;整叶处理烟叶发酵后检测S5对烟叶降解率最高,为5.17%。     

真菌菌剂改善烟叶品质的初步研究*

利用分离自不同生境烟叶上的7个真菌菌株菌剂处理上部叶烤烟烟丝,分析表明,处理后烟叶内与品质相关的主要化学成分含量发生了改变,烟叶品质得到改善。其中菌株BF03、BF06及BF63使烟叶内糖、氮、烟碱、蛋白质等主要化学成分含量及各种成分之间比例趋于平衡。评吸结果表明,经供试菌剂处理的烟叶香气质提高,香气量增加,刺激性减小,余味舒适,烟叶品质明显高于对照。     

1株褐藻胶降解菌的筛选、鉴定及产酶条件优化

为获得可产生褐藻胶裂解酶并高效降解褐藻胶的菌株,以海藻酸钠为唯一碳源配制培养基,以透明圈法进行初筛,DNS法复筛,从海洋生物中筛选得到1株高酶活力褐藻胶降解菌株B12,经16S rDNA序列分析、生理生化试验、电镜观察,确定该菌为弧菌属(Vibrio sp.)。通过单因素试验及响应面优化试验对影响菌株生长和产酶条件的5个因素(发酵初始pH值、发酵温度、NaCl质量浓度、接种量和装液量)进行优化。得到该菌株最佳产酶条件:pH 6.52,发酵温度28.2℃,NaCl质量浓度20.1 g/L,接种量2.1%,装液量59.5 mL。在最佳发酵条件下,B12菌株酶活力可达91.68 U/mL,相比于优化前提高了38.5%。菌株开始产酶时间提前6 h, 4℃冷藏酶活力稳定性较好。     

褐藻表面可培养褐藻酸降解菌的原位筛选

【背景】褐藻酸经酶解后生成的褐藻寡糖具有抗氧化、抗肿瘤、诱导免疫调节、调节植物生长等多元化的生物学功能,在食品、医药领域应用前景广阔。大量筛选褐藻酸降解菌有利于获得新结构、新功能的褐藻寡糖,有利于积极推动寡糖产业进程。【目的】高效筛选褐藻酸降解菌株,发掘具有开发前景的海藻原位微生物资源。【方法】利用褐藻酸唯一碳源培养基对天然铜藻表面微生物进行筛选;革兰氏碘液显色反应指示微生物降解褐藻酸的特性;牛津杯法初步测定产褐藻酸裂解酶菌株的酶活大小。【结果】从铜藻表面共获得81个菌落,经透明圈显色筛选出28株菌,通过16S rRNA基因测序分析得到7株褐藻酸降解菌,分别属于芽孢杆菌属、节杆菌属、德库菌属、短杆菌属和链孢子囊菌属。除芽孢杆菌属外,其余菌属的菌种此前均未被报道过有产褐藻酸裂解酶的能力。进一步分析表明,T-1菌株的产酶能力最强、酶活力最高。【结论】利用革兰氏碘液显色结合牛津杯法筛选到7株产酶菌株,并比较了各菌株的酶活大小,表明该筛选方法简便高效,适合大规模筛选褐藻酸降解菌株。     

深渊来源Citricoccus sp.strain NyZ702的分离培养及其降解4-羟基苯甲酸的特性

【背景】深渊沉积物中存在丰富的微生物细胞和活跃的微生物碳周转,因此,分离培养微生物资源对于认识深渊中的物质循环、能量代谢具有重要意义。芳香化合物在环境中广泛存在,基于组学分析揭示了深渊中具有潜在的芳香化合物代谢菌株,然而深渊来源的芳香化合物降解微生物纯培养和相关的代谢机理研究仍然缺乏。【目的】从马里亚纳海沟沉积物样本中分离培养具有降解芳香化合物能力的微生物,对其代谢途径、中间产物和降解酶活力进行初步鉴定。【方法】以4-羟基苯甲酸为唯一碳源对马里亚纳海沟沉积物样本中的降解菌株进行分离培养,结合形态观察、16S rRNA基因扩增与序列分析对菌株进行鉴定,通过底物生长实验验证其降解能力,通过高效液相色谱和超高效液相色谱-飞行时间质谱联用仪初步鉴定全细胞生物转化中间产物,利用紫外分光光度计测定其粗酶液催化4-羟基苯甲酸的活力,进而推测菌株降解4-羟基苯甲酸的代谢途径。【结果】从深渊沉积物中分离培养获得一株好氧细菌,16SrRNA基因序列分析显示该菌株隶属于柠檬球菌属(Citricoccus),命名为Citricoccus sp. strain NyZ702。该菌株在LB固体培养基上经30°C培养4 d后呈柠檬黄色、不透明、表面光滑、边缘整齐、凸出于培养基表面、直径约为1-2 mm的圆形菌落。扫描电镜表明菌体呈球形,直径为0.4-0.6μm,无鞭毛结构。该菌株为耐盐菌,最适生长盐浓度范围为2%-8%(质量体积分数)。该菌株可利用4-羟基苯甲酸为唯一碳源进行生长,可转化4-羟基苯甲酸至中间产物原儿茶酸,推测该菌株通过原儿茶酸途径降解4-羟基苯甲酸。菌株NyZ702的粗酶液具有4-羟基苯甲酸单加氧酶活力,对4-羟基苯甲酸的催化反应需要还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)作为辅因子。【结论】从深渊沉积物样本分离得到一株4-羟基苯甲酸降解菌Citricoccus sp. strain NyZ702,该菌株以原儿茶酸为中间代谢产物降解4-羟基苯甲酸,丰富了深渊来源的微生物菌种资源,为深渊中的芳香化合物降解研究提供了一定的理论基础。     

产香细菌的筛选及产香特性分析

醇化烟叶作为巨大的微生物库,具有极其可观的研究和应用价值。本研究应用模拟烟叶生长环境的烟叶浸提物培养基筛选产香细菌,并分析所筛选菌株发酵烟叶的产香特性,用于研究其在烟叶醇化中提升烟叶香味的可行性,同时探究该微生物用于生产天然香料的可能性。实验结果表明:通过烟叶浸提物培养基筛选出一株细菌HD-40,该菌可发酵烟叶可产生明显奶香味;经生理生化和分子生物学鉴定,初步表明该菌属于甲基营养型芽孢杆菌;进一步,利用该菌株发酵处理烟叶,并将发酵液萃取后GC-MS检测,与未经菌株处理的发酵液对比,其所含的酯类、醛类、酸类等对香气有贡献的成分明显增加,特别是其中3-羟基-2-丁酮的比例可高达60%以上。HD-40可发酵烟叶产生明显的奶香味,其不仅在烟叶醇化方面,用于提高烟叶香气、改善烟叶吸味等,还可在制备天然香料香精方面均有着很好的应用前景。     

菌株DN2对烟草薄片制备液中烟碱的降解

使用O.intermedium DN2降解烟草薄片制备液中的烟碱。研究了各种工艺参数对烟碱降解的影响,单因素考察结果表明烟草薄片制备液中烟碱降解的最适条件是:添加0.1%的酵母膏,使用氨水将pH调节到7.0,接种15%种子液,培养温度30oC。在上述条件下,采用30L发酵罐对烟草薄片制备液进行3个批次的半连续发酵,烟碱的平均降解速率为140.55mg/L/h,高于其他烟碱降解菌株。该结果表明菌株DN2可以用来降低烟草薄片中的烟碱含量。     

耐盐性毒死蜱降解菌HY-1 的产酶培养基及发酵条件优化

为了明确生化处理和微生物降解的关系,通过增加耐盐菌的比例可以提高农药废水生化处理效果。从农药厂废水中分离到1株耐盐性毒死蜱降解菌——蜡状芽孢杆菌(Bacillus cereus HY-1),以从该菌中提取到的降解酶比活力为指标,进行产酶培养基和发酵条件的优化研究。通过单一因素试验和正交试验,对细菌HY-1的产酸培养基和发酵条件进行了优化。运用SPSS软件进行结果分析,所获优化培养基配方为:葡萄糖6.0 g/L,胰蛋白胨2.2 g/L,K2HPO4 2.0 g/L,KH2PO4 0.2 g/L,MgSO4.7H2O 0.1 g/L,NaCl 0.1 g/L和微量元素溶液2 mL/L。得到菌株发酵培养的最佳优化条件为:种子液培养时间为16 h,发酵培养时间为18 h,接种量为1%(V/V),发酵培养基初始pH值为7.0。氯化钠浓度为0?30 g/L时降解酶比活力不受影响,这是已报道的耐盐性最强的一株毒死蜱降解菌。     

混合菌株降解褐藻胶的筛选、鉴定及产酶条件优化

为获得仿刺参（Apostichopus japonicus）肠道菌群中可有效降解褐藻胶的混合菌株,以海藻酸钠为唯一碳源配制培养基,以透明圈法进行初筛,DNS法和紫外法复筛,从已驯化仿刺参肠道中筛选得到4株高酶活力褐藻胶降解菌株S1、S2、S10和S11,经16S rDNA序列分析、电镜观察,确定菌株S2与S11分别为微杆菌属（Microbacterium sp.）和微小杆菌属（Exiguobacterium sp.）。对该4株菌株分别进行混合培养,获得菌株S2与S11最佳配比组合,并通过单因素试验及响应面优化试验对影响混合菌株产酶条件的发酵初始pH值、NaCl质量浓度、装液量和发酵温度4个因素进行优化。得到混合菌株最佳产酶条件为pH 8,NaCl质量浓度为40 g/L,装液量80 mL,温度28 ℃。在最佳发酵条件下,混合菌株酶活力可达94.78 U/mL,相比于优化前提高了43.9%,优化后混合菌株的酶活力显著提高。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Morphology and putative function of the colon and cloaca of marine and freshwater snakes

Among tetrapods, evidence for postrenal modification of the urine by the distal digestive tract (including the colon and cloaca) is highly variable. Birds and bladderless reptiles are of interest because the colon and cloaca represent the only sites from which water and ions can be reclaimed from the urine secreted by the kidney. For animals occupying desiccating environments (e.g., deserts and marine environments), postrenal modification of the urine may directly contribute to the maintenance of hypo‐osmotic body fluids. We compared the morphology and distribution of key proteins in the colon, cloaca, and urogenital ducts of watersnakes from marine (Nerodia clarkii clarkii) and freshwater (Nerodia fasciata) habitats. Specifically, we examined the epithelia of each tissue for evidence of mucus production by examining the distribution of mucopolysaccharides, and for evidence of water/ion regulation by examining the distribution of Na⁺/K⁺‐ATPase (NKA), Na⁺/K⁺/Cl^? cotransporter (NKCC), and aquaporin 3 (AQP3). NKCC localized to the basolateral epithelium of the colon, urodeal sphincter, and proctodeum, consistent with a role in secretion of Na⁺, Cl^?, and K⁺ from the tissue, but NKA was not detected in the colon or any compartment of the cloaca. Interestingly, NKA was detected in the basolateral epithelium of the ureters, suggesting the urothelium may play a role in active ion transport. AQP3 was detected in the ureters and coprodeal complex, consistent with a role in urinary and fecal dehydration or, potentially, in the production of the watery component of the mucus secreted by the coprodeal complex. Since no differences in general cloacal morphology, production of mucus, or the distribution of ion transporters/water channels were detected between the two species, cloacal osmoregulation may either be regulated by proteins not examined in this study or may not be responsible for the differential success of N. c. clarkii and N. fasciata in marine habitats. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.