枯草杆菌弹性酶的理化性质研究

对用DEAE纤维素—Sephader G—75层析所得到的4万u/g的纯酶进行分子量、等电点、最适作用条件、稳定性及水解性质等进行试验。     

竹叶青(Trimeresurus stejnegeri)蛇毒凝血酶样酶的分离纯化及其理化性质的研究

用CM—Sephadex C—25分离竹叶青粗毒,再经DEAE—Sephadex A—50和CM—Sephadex C—25纯化得到凝血酶样酶组分Ⅰ。用DEAE—Sephadex A—50分离竹叶青粗毒,再经DEAE—Sepharose CL—6B纯化得到凝血酶样酶组分Ⅱ。用聚丙烯酰胺凝胶电泳和SDS—聚丙烯酰胺凝胶电泳鉴定,组分Ⅰ在两种电泳中均为一条带; 组分Ⅱ在两朴电泳中均为二条带,经切割后鉴定证明组分Ⅱ的二条带都是凝血酶样酶。组分Ⅰ的分子量为54,500,由261个氨基酸残基组成,其中Asp和Glu含量较高,含14％中性己糖,13.1％己糖胺和14.7％唾液酸,等电点为3.5,在280nm处的消光系数为E_(1cm)~(0.1％)=0.855。组分Ⅱ的分子量分别为54,000和47,000。经凝胶电泳分离后用过碘酸—Schiff's试剂染色,证明组分Ⅰ和组分Ⅱ都是糖蛋白。     

凤丹连作对土壤理化性质和酶活性影响的研究

研究了安徽南陵凤丹园1~5年连续种植地中凤丹根际土壤的理化性质和几种酶活性的变化规律。结果表明：所有种植年限的凤丹根际土壤pH值均小于非种植土壤（p〈0.05）。全N、全P、全K、有机质等理化性质及酶活性（脲酶,硝酸还原酶,多酚氧化酶和蛋白酶）均显著高于非种植土壤（p〈0.05）。各年限根际土壤的脲酶,硝酸还原酶,多酚氧化酶和蛋白酶的R/S值均大于1,显示凤丹对4种根际土壤酶酶活性影响为正效应。连作5年凤丹和1年生凤丹根际土壤相比多酚氧化酶,脲酶和蛋白酶的活性分别下降了64.84%、52.55%、51.59%（均p〈0.05）;连作5年和2年相比,硝酸还原酶下降了23.26%（p〈0.05）,同时土壤全N、全P、全K和有机质分别下降了57.84%,36.76%,47.89%和19.84%（p〈0.05）。说明随凤丹连作,土壤中积累的根系分泌物对4种土壤酶活性表现出抑制作用,可能影响了土壤的理化性质。为揭示凤丹连作障碍的机理提供了实验依据。     

榛属群丛对土壤理化性质影响的研究

通过对相同林型下榛属丛和非榛属灌木丛下根区土壤的对比研究,发现榛属群丛下根区土壤较非榛属灌木下土壤ｐＨ值增高,有机质含量显著增加,全Ｎ和碱解氮,全磷量显著提高,有效磷降低土壤团粒结构增加,水稳性提高,表明榛属灌丛有明显地改良土壤理化性质的作用。     

昆虫来源的几丁质酶的分离纯化及酶学性质

几丁质酶在真菌和昆虫的生理和发育过程中起着关键作用,该酶本身及其酶抑制剂是获取生物农药的重要途径。本研究从蚕蛹体内提取几丁质粗酶,经硫酸铵分级沉淀和Sephadex G-150分离得到几丁质酶。用SDS-PAGE测得该酶的分子量为88kDa。水解胶体几丁质的Km值为22.3μmol/L。酶反应的最适温度为45℃,最适pH值为6.0,金属离子和有机试剂对几丁质酶活性都有影响,其中高浓度的Mn2+对酶有较强的激活作用,而Cu2+、SDS则有较强的抑制作用。研究结果为基于几丁质酶的生物农药筛选研究奠定了基础。     

沼肥对土壤理化性质的影响

目的:研究沼肥对土壤理化性质影响,为沼气的综合利用提供理论依据和技术支持.方法:通过对比栽培试验方法,研究了施用沼肥对菜园土壤密度、pH值及养分含量的影响.结果:施用沼肥使土壤密度下降9.7%、铵态氮增加60.5%、速效磷增加85%、速效钾增加225%、有机质增加30%.田间试验结果,施用沼肥组作物增产15.6%,并且产品品质也有提高,VC含量提高18.6%、硝酸盐含量降低30.3%.结论:沼肥能够有效改善土壤的营养结构,明显提高土壤的有机质含量.     

不同树种混交林及其纯林对土壤理化性质影响的研究

对针阔混交林土壤理化性质的研究表明,针阔混交林比针叶树纯林对土壤的改良作用要好,它使土壤总孔隙度增加２—１９％,水分含量增加６—３１％,枯枝落叶年凋落量增加２—２００％;土壤养分含量全Ｎ、ＮＨ４－Ｎ、代换性Ｃａ、代换性Ｍｇ和腐殖质含量分别增加４５—７５％、３３—８２％、５５—８５％、４４—８４％和３７—４６％．     

蒜氨酸酶的固定化及其酶学性质研究

为了提高蒜氨酸酶的稳定性并实现酶的反复利用,研究了影响蒜氨酸酶固定化的因素及固定化蒜氨酸酶的酶学性质。蒜氨酸酶的固定化以壳聚糖微球为载体,戊二醛为交联剂,固定化的最适条件为：戊二醛浓度4%,给酶量20.2U,交联时间2h。固定化蒜氨酸酶的最适pH值7.0,最适温度35℃,米氏常数Km 7.9 mmol/L,操作稳定性比较好,连续使用10次后酶活力损失低于10%。     

南美白对虾体壁几丁质酶的理化特性研究

报道南美白对虾体壁几丁质酶(EC3.2.1.14)的理化性质。结果表明,酶的最适pH值为5.6,最适温度为55℃。该酶在pH5.0～6.2区域较稳定,而在pH>7和pH<4.6下失活加快;在50℃以下处理1h,酶活力保持稳定,高于55℃,酶稳定性较差,很快失活。研究金属离子对酶活力影响,结果表明:Li 、Na 、K 、Mg2 和Fe2 等对该酶活力没有任何效应;Ca2 、Ba2 对酶有激活作用,Cu2 、Co2 对酶的效应先表现为激活后转为抑制作用;Ni2 、Zn2 、Mn2 、Al3 、Fe3 、Hg2 、Pb2 和Cd2 对该酶活力均具有不同程度的抑制作用,以Hg2 的抑制作用最显著,10mmol/L的Hg2 可抑制酶活力95.6%。     

海洋来源琼胶酶的分离纯化及酶学性质研究

采用Vibiro sp.ZC-1发酵制备琼胶酶,粗酶液经过中空纤维柱浓缩、硫酸铵沉淀、DEAE-阴离子交换层析,得到一个电泳纯的琼胶酶组分Aga ZC-1,其分子质量约为45k Da,比活力为114.613U/mg。对Aga ZC-1进行酶学性质分析,结果表明,其最适反应p H为7.0,在p H为5.0~9.0时保温1h仍能保持80%以上的酶活力;最适反应温度为50℃,在45℃条件下保温1h酶活力保持在60%以上。在高浓度(5mmol/L)下,Fe~(3+)、Cu~(2+)、Sn~(2+)和Zn~(2+)能完全抑制琼胶酶的活性,在低浓度(1mmol/L)下,Cu~(2+)、Ba~(2+)、Na~+、Zn~(2+)、Ag~+、Sr~(3+)、K+对琼胶酶活性具有明显抑制作用。琼胶酶的动力学参数K_m和V_(max)分别为0.538mg/ml和6.33μmol/(L·min),对琼胶底物具有高度专一性,降解产物主要为新琼四糖和新琼六糖。     

Interaction of vitronectin with Haemophilus influenzae

Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected using soluble vitronectin, suggesting that surface association of vitronectin is a prerequisite. Vitronectin binding was not likely to be mediated by fimbriae as non-fimbriated and fimbriated isogenic strains adhered equally. Adhesion could be blocked by heparin, which is also known to block vitronectin binding to Staphylococcus aureus. However, no blocking was achieved with sialic acid-rich glycoproteins such as fetuin and mucin contrasting with Helicobacter pylori for which sialic acid seems to play an important role. With Streptococcus pneumoniae binding was detected both with soluble and surface-associated vitronectin and could not be blocked by heparin. Our results suggest that H. influenzae, Streptococcus pneumoniae and Helicobacter pylori all use distinct modes to interact with vitronectin.     

Construction of antibiotic resistance cassettes with multiple paired restriction sites for insertional mutagenesis of Haemophilus influenzae

Insertional mutagenesis of cloned genes coupled with site specific recombination into the genome of the parent organism is an ideal method for characterizing gene function. In this paper we describe the production and utility of two antibiotic resistance cassettes for use in Haemophilus influenzae. The mutagenic elements encode resistance to chloramphenicol or spectinomycin. Multiple paired restriction enzyme sites bound both cassettes. Use of these constructs to create mutants in H. influenzae demonstrated that the cassettes are readily incorporated into the genome in single copy and allow easy detection of mutant constructs. The insertions are stable following repeated in vitro passage. In addition, the elements are compatible with each other and allow the construction of multiple mutations within a single strain.     

Phase variation of Haemophilus influenzae lipopolysaccharide: Characterization of lipopolysaccharide from individual colonies

Abstract The lipopolysaccharide (LPS) of Haemophilus influenzae expresses a number of core oligosaccharide epitopes on its outer surface. The expression of individual epitopes is subject to frequent (approximately 1% bacteria/generation) reversible phase variation, as determined by colony immunoblots. We have used a microtechnique for the extraction of LPS from individual colonies, whose LPS antigenic phenotype has been identified, so that the LPS can be studied by tricine sodium dodecylsulphate polyacrylamide gel electrophoresis (T-SDS-PAGE). This avoids the introduction of heterogenous phase-varying LPS which is inevitable if bacteria from colonies are grown in broth culture prior to LPS extraction and analysis. Using these techniques we have investigated the repertoire of LPS phase variation exhibited by H. influenzae strain RM7004 (a serotype b meningitis isolate). This technique will facilitate the study of bacteria in which there is variable LPS expression.     

Aberrant glycosylation of IgA from patients with IgA nephropathy

Despite the prominent role of IgA, particularly IgA₁, in the pathogenesis of IgA nephropathy (IgAN), the precise role of this molecule in the process remains unclear. Four biotin-conjugated lectins in sandwich-type enzyme-linked immunosorbent assays were devised to determine the glycosylation profiles of total IgA and its subclasses. We took advantage of differential binding properties of these lectins to sugar residues to dissect the oligosaccharide chainsO-linked to the hinge and thoseN-linked to the Fc region of total IgA and IgA subclasses in 47 patients with IgAN and an equal number of controls. The proportion of sialylated IgA₁ was higher in patients compared with controls (p<0.02), whereas IgA₂ in patients appeared less well sialylated. A reduction of galactose in pathological IgA as detected by RCA-I became significant after treatment of the molecule with neuraminidase (p<0.01). Defective galactosylation was also observed for patient IgA₁ when it was probed with ECL, a lectin that has a specificity for Gal 1,4N-acetylglucosamine groupings onN-linked oligosaccharides. The RCA and ECL results, therefore, suggest that increased sialylation on the IgA₁ is onO-linked oligosaccharides in the hinge region. This was partly confirmed by a small increase in the binding of PNA to IgA₁ from the patient group. This lectin binds preferentially to Gal 1,3N-acetylgalactosamine groups that are found onO-linked oligosaccharides.     

Catalase as a source of both X- and V-factor for Haemophilus influenzae

Haemophilus influenzae requires two growth factors, designated factor X (porphyrin) and factor V (NAD). Mammalian catalases contain both bound heme and NADPH. This study shows that catalase can supply both factors X and V to H. influenzae in vitro, thus representing a potential in vivo source of these essential growth factors.     

Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections

Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or several structural derivatives thereof protects the animals infected with a variety of deadly pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-reactive protection against meningococcal and pneumococcal infections has been demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N. meningitidis IgA1 protease and S. pneumoniae surface proteins.     

儿童肺炎链球菌和流感嗜血杆菌耐药及感染特征分析

目的 了解儿童呼吸道感染肺炎链球菌(Streptococcus pneumonia,SP)和流感嗜血杆菌(Haemophilus influenzae,Hi)的分布特征、耐药情况,及耐药菌抗生素间的相互影响,以更合理地指导临床用药.方法 对2009-2010年临床呼吸道感染患儿进行痰、咽拭子或肺泡灌洗液培养分离Hi和SP.因子需求试验鉴定Hi,头孢硝噻酚法检测β-内酰胺酶;奥普托辛和胆汁溶菌试验确认SP.两菌均采用K-B法检测常用对抗生素的耐药性.结果 收集SP 495株,Hi 515株,多见于3岁以下儿童,以呼吸科分离率最高.SP对红霉素、四环素、阿奇霉素、复方新诺明的耐药率分别为98.4％、66.1％、98％和81.6％,其对青霉素敏感性仅为9.5％.Hi有42.7％产生β-内酰胺酶,Hi对氯霉素、复方新诺明、氨苄青霉素、阿奇霉素的耐药率分别为22.1％、21.6％、36.7％和62.7％.与青霉素敏感SP和β-内酰胺酶阴性Hi相比,耐药SP和阳性Hi更易对氯霉素、四环素和复方新诺明耐药.结论 SP和Hi以婴幼儿为主,多见于呼吸科.其耐药情况严峻,青霉素耐药和产β-内酰胺酶菌株会诱导其他抗生素耐药,引发多重耐药.合理使用抗生素以及对两菌的耐药监测应引起高度重视.