曲霉型豆豉用高产蛋白酶菌种的选育

从曲霉型阳江豆豉曲中筛选出5株菌落形态不同的曲霉(Aspergillus spp.),通过纯种发酵制备豆豉曲,测定了豆豉曲中碱性蛋白酶和中性蛋白酶的活性,从中筛选出1株蛋白酶活性较高的菌种,并对该曲霉进行ITS r DNA序列分析,发现该曲霉与Aspergillus oryzae strain P6B2(JX429926)的同源性最高,故鉴定为米曲霉(Aspergillus oryzae).以该米曲霉为出发菌株,通过紫外线诱变和硫酸二乙酯化学复合诱变处理,选育得到优良高产蛋白酶活性的突变株,其中性蛋白酶酶     

三氯生与镉单一及复合污染对土壤呼吸和酶活性的影响

为了解典型药品和个人护理品(PPCPs)——三氯生与镉复合污染的生态效应,采用室内培养实验和联合毒性预测模型,首次评价了三氯生与镉单一及复合污染对土壤呼吸及参与土壤碳氮循环的相关酶活性的生态毒性。结果表明:三氯生与镉单一及复合污染对土壤呼吸呈现激活—抑制—激活的生态效应;刺激了蛋白酶活性,激活率先降低后升高,56 d时达到最大。整个实验期间均抑制了蔗糖酶活性,镉(10.0 mg kg-1)单一污染培养14 d抑制率达到最大值(81%),三氯生单一胁迫呈现负的剂量效应关系,两者复合污染无显著的剂量效应关系。联合效应评价模型预测表明,相比三氯生或镉单一污染,两者的复合胁迫对土壤呼吸呈现随时间变化的拮抗—协同—加和效应,对土壤蛋白酶呈现协同—加和—协同的联合效应,而对土壤蔗糖酶活性则主要为协同效应。     

Response of broiler chicks to non-steam- or steam-pelleted diets containing raw,full-fat soybean meal

The objective of this study was to evaluate the effects of steam pelleting of diets containing graded levels of raw, full-fat soybean meal (RSBM) on the chemical properties and feeding values of the diets. Samples of diets with steam- or non-steam-pelleted as well as the mash containing varying levels of RSBM were subjected to detailed chemical analysis. As a result of this study, trypsin inhibitor (TI) concentrations in the diets ranged between 4,153 and 10,484 TIU/g. Amino acid concentrations were higher in the non-steam-pelleted and mash diets than the steam-pelleted diets. A 4 × 2 factorial arrangement (RSBM: zero, 10, 20 or 30%, equivalent to zero, 30, 60, and 90 g/kg of diet, respectively, and non-steam- or steam-pelleted diets) was used while feeding broiler chicks (zero to 14 d of age). Each treatment was replicated 6 times with 8 birds per replicate. As a result of this study, there was no difference (P > 0.05) in mortality of birds among the groups. Feed intake (FI) (P < 0.05) and body weight gain (BWG) (P < 0.001) decreased with increasing levels of RSBM. Birds fed on steam-pelleted diets gained less (P < 0.001) weight than birds on the non-steam-pelleted diets, but the FI was not significantly (P > 0.05) different. The FCR was negatively affected (P < 0.05) by increasing levels of RSBM. There was no interaction effect between RSBM and pelleting method on the FI, BWG, or FCR of birds. The weight of the pancreas (P < 0.001) and duodenum (P < 0.01) increased with a rise in the level of RSBM in diets. Non-steam pelleting increased (P < 0.05) the pancreatic protein content, whereas the activity of chymotrypsin was reduced (P < 0.01) when the RSBM level was increased. Birds fed with RSBM-free diets had thicker muscle, longer villi, wider villus surface area, and higher villus to crypt depth ratios than birds on the other diets, but these differences were not significant. It can be concluded that steam pelleting of diets containing RSBM is inadequate to reduce the adverse impact of TI on chicks.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

Optimization of 0-8 Aspergillus oryzae Solid State Fermentation Conditions and the Effect of Fermented Product on Nutrient Digestion of Broiler

The high-yield protease was obtained by screening high-yield protease Aspergillus oryzae and optimizing its solid-state fermentation conditions, and then the effect of fermented product on nutrient digestion in broiler was studied. After the screening and re-screening of 8 different Aspergillus oryzae spores, a new Aspergillus oryzae strain 0-8 with high acid protease was obtained. The fermentation conditions of acid protease were optimized by single factor and orthogonal test experiment. The results showed that the optimum temperature was 30℃, the most suitable fermentation time was 66 h, the initial water content was 47.40%, the inoculation quantity was 1.5×10⁷ spores/mL, the content of bran was 80%, C/N was 1/3, the optimized vigor of acid protease reached 14 416.64 U/g, which was 157.02% higher than that before optimization. 500 mg/kg Aspergillus oryzae 0-8 fermented product was added in the broiler diet, and the mutrient digestibility of broiler were measured after 10 d of adaptation and 5 d of test period. The results of the nutrient digestion of broilers showed that feeding Aspergillus oryzae 0-8 fermentation increased the CP and energy digestibility of broiler in a certain degree compared to control group, but there's no significant difference (P>0.05). Dietary supplementation of Aspergillus oryzae fermentation was beneficial to the production of broilers, but its additive dose or feeding pattern should be further studied.     

Furanoditerpene ester and thiocarbonyldioxy derivatives inhibit photosynthesis

6α,7β-Dihydroxyvouacapan-17β-oic acid (1) and methyl 6α,7β-dihydroxyvouacapan-17β-oate (8) were isolated from Pterodon polygalaeflorus Benth. 1 was modified to obtain 6α-hydroxyvouacapan-7-β,17β lactone (2). Then, 6-oxovouacapan-7β,17β lactone (3) was obtained from 2. The furanoditerpene ester derivatives: propyl 7β-hydroxy-6-oxovouacapan-17β-oate (4), butyl 7β-hydroxy-6-oxovouacapan-17β-oate (5), 2-methoxyethyl 7β-hydroxy-6-oxovouacapan-17β-oate (6) and 3-methylbut-2-enyl 7β-hydroxy-6-oxovouacapan-17β-oate (7) were synthesized from (3) and methyl 6α,7β-thiocarbonyldioxyvouacapan-17β-oate (9) was obtained from (8). In this work, the lactone ester derivatives 4-7 and 9 were tested on photosynthetic activities in an attempt to search for new compounds as potential herbicide agents that affect photosynthesis. All compounds inhibited ATP synthesis and electron flow from water to MV, therefore, they act as Hill reaction inhibitors, being 4- to 9-fold more potent than 2 and 3 as inhibitors of ATP synthesis. Their interaction site was located at PSII in a similar way to diuron. Furthermore, furanoditerpene esters 6 and 7 act as uncouplers, and were corroborated by enhancement of the light-activated Mg²⁺-ATPase, while 5 act as an energy transfer inhibitor. Finally 5-7 behave as herbicides, since they inhibit the biomass production of weeds assay.     

纤细齿梗孢丝氨酸蛋白酶cDNA克隆及其序列分析和表达

纤细齿梗孢Olpitrichum tenellum是一种寄生在轮枝镰孢菌Fusarium verticillioides上的活体营养菌寄生真菌。丝氨酸蛋白酶在菌寄生过程可能起到重要作用。根据丝状真菌丝氨酸蛋白酶的同源保守序列设计简并引物,通过RT-PCR及RACE的方法,克隆得到1845bp的全长cDNA片段。其可读框为1554bp（GenBank登录号为EU368754）,编码517个氨基酸的蛋白质。比对分析发现该蛋白是一种分泌型的丝氨酸蛋白酶,属于丝氨酸蛋白酶S8家族的枯草杆菌蛋白酶subtilisin,具有典型的信号肽-前肽-成熟肽的结构。将该基因可读框除去信号肽插入酵母表达载体pPIC9K,转化毕赤酵母Pichia pastoris GS115,在甲醇诱导下成功分泌出具有生物活性的重组蛋白酶,诱导120h后酶活性可达153U/ml。重组蛋白酶经DEAE-Sepharose层析进行纯化,SDS-PAGE分析其分子量为39kD。其反应的最适温度为60℃,最适pH为8。     

蚕蛹蛋白高效分解菌的分离筛选及部分酶学性质的研究(英文)

[目的]获得蚕蛹蛋白高效分解菌,探索其有关酶学性质。[方法]从腐败蚕蛹中采用稀释平板分离蛋白高效分解菌。先进行菌株的初筛:取采集的样品2g,分别加入装有LB培养基的摇瓶中,置摇床上180r/min,37℃培养4～5d。将发酵液稀释一定倍数,涂布法进行分离,根据酪素培养基中单菌落透明圈和菌落直径比值(H/C)大小,选取H/C比值较大的菌株进行复筛。将初筛得到的待测菌株接种于发酵培养基中,32℃摇床培养60h。发酵液离心(4000r/min,5min)后取上清,测定酶活力,并比较不同菌株发酵液蛋白酶pH、温度作用范围。对筛选出的菌株所产中性蛋白酶再进行pH值条件下酶活力稳定性测验,酶活力热稳定性试验,及不同金属离子对中性蛋白酶活力的影响试验。蚕蛹粉采用筛选出的菌株固体发酵后,于60℃烘箱烘干,测定其中小分子蛋白含量。[结果]经筛选得到1株产中性蛋白酶的KB细菌,在摇瓶发酵条件下其酶活达到589.08U/ml,发酵蚕蛹粉后中小分子蛋白含量达到15.09%,比未发酵的对照和实验室现用菌株发酵分别提高了95.72%和15.67%,这表明KB菌株在蚕蛹粉发酵中优于实验室现用菌株。KB菌株所产中性蛋白酶在pH值6.5～9.0条件较稳定,pH值8.0为其最适pH值,在pH值8.0、温度55℃条件下处理0～30min时的酶活较稳定,残余酶活力在90.0%以上。[结论]初步获得蚕蛹蛋白高效分解菌。     

温和气单胞菌TL97528株丝氨酸蛋白酶基因片段克隆与序列分析

中华鳖(Trionyx sinensis)细菌性疾病主要由气单胞菌感染引起的,通过脱脂奶平板检测及酶活性试验得出温和气单胞菌TL97528株分泌的胞外蛋白酶为丝氨酸蛋白酶,而丝氨酸蛋白酶是气单胞菌的主要毒力因子。根据已发表的嗜水气单胞菌的丝氨酸蛋白酶基因序列保守区域设计引物,PCR扩增出一长度为809 bp大小的特异片段,该序列在Genbank上的登录号为FJ357446。对该序列进行分析发现,已发表的温和气单胞菌(Aeromonas sobria)丝氨酸蛋白酶基因(GenBank登录号为AF253471)同源性为98%、与其他几株已发表的嗜水气单胞菌(Aeromonas hydrophila)丝氨酸蛋白酶基因(GenBank登录号分别为AF126213、AY841795、DQ127822、CP000462、CP000644、AF159142)同源性分别为98%、82%、82%、82%、81%、81%,与杀鲑气单胞菌(Aeromonas salmonicida)丝氨酸蛋白酶基因(GenBank登录号为X67043)同源性为80%。用DNAstar软件分析,与鳖源温和气单胞菌TK961010株的同源性为98%,...