从Staphylococcus simulans消除含有溶葡球菌酶基因质粒的研究

溶葡球菌酶是Staphylococcus simulans分泌的能分解葡萄球菌的酶,它的基因位于一个约40kb的质粒DNA上。为了探索用高拷贝质粒取代原有的质粒的可能性,本文首先进行了从该菌株中消除含有溶葡球菌酶基因质粒的实验研究,获得了相应的“消除”菌株。根据对目的菌株和原始菌株的比较分析,包括细胞蛋白质的SDS-聚丙烯酰胺凝胶电泳,Western blot分析,质粒DNA的琼脂糖胶电泳、Southern Blot分析,质粒DNA的限制性内切核酸酶酶切分析以及对溶葡球菌酶作用敏感性的分析,都表明该目的菌株确系Staphylococcus simulans的衍生菌株,只是清除了其中含有溶葡球菌酶基因的质粒。在此基础上,本文也进行了转化实验。     

溶葡球菌酶5升罐发酵研究

利用含溶葡球菌酶基因的枯草芽孢杆菌转化子,进行半合成培养基的５升自动发酵罐的中试发酵条件试验,在控制转速（４５０～５００ｒ／ｍｉｎ）和通气量（１：０．５～０．８）二个条件下,采用分批培养和补料分批培养,获得了一组在半合成培养上生产溶葡球菌酶的较优化条件,产酶高峰在８．５ｈ～９ｈ,酶产量达４６５ｍｇ／Ｌ。     

溶葡球菌酶的纯化和某些性质

1.应用本实验室构建的克隆菌株枯草杆菌0044进行了溶葡球菌酶的发酵生产,产量为150—200mg/L; 2.通过DEAE-纤维素,CM-纤维素和Sephadex G-50层析纯化了该酶;并以NaCl盐析方式,首次获得了该酶结晶; 3.测定了溶葡球菌酶的某些性质; 4.观察并讨论了溶葡球菌酶与溶菌酶等在溶菌作用上的相互加强。     

克鲁维酵母Y-85合成菊粉酶最适条件的研究

采用响应面方法（ResponseSurfaceMethod,RSM）对克鲁维酵母（Kluyveromycessp．）Y－85产菊粉酶培养基成份进行了优选,和正交试验相比,该法选出的最适培养基的酶发酵水平提高28％。用15L自控发酵罐进行产酶条件控制试验,并在1000L罐上进行5批次酶发酵中试,平均菊粉酶活性达68.9u／ml。     

热稳定β－淀粉酶高产菌株选育及发酵条件研究

从土壤中分离得到一株高温放线菌V4菌株(Thermoactinomyces sp．v4),经测定能产生热稳定β-淀粉酶。V4菌株经过热诱变获得一株具高活力β-淀粉酶的变异株A61产酶活力从400u／ml提高到1000u／ml。A61菌株产生的β-淀粉酶最适反应温度为60℃,酶的热稳定性良好,50℃保温4小时不失活,55℃保温2小时仍具有最初活力的96%。     

溶葡球菌酶前体及前体加工蛋白酶的生成控制和纯化

 为深入探讨溶葡球菌酶前体加工转化为成熟的溶葡球菌酶的机制,本文通过条件控制分别从Staphylococcus simulans的培养液中获得了溶葡球菌酶前体和它的加工蛋白酶,并分别通过HPLC和Affi-Gel 501亲和层析对它们进行了纯化,根据聚丙烯酰胺凝胶电泳和凝胶等电聚焦电泳,表明二者已基本上达到均一的程度。在此基础上,又进行了溶葡球菌酶前体的体外加工转化实验。     

溶葡球菌酶在乳酸克鲁维酵母中重组表达、诱变、优化及酶学研究

根据模仿葡萄球菌(Staphylococcus simulans)的溶葡球菌酶基因序列以及乳酸克鲁维酵母密码子偏好性设计引物扩增溶葡球菌酶基因表达片段,构建溶葡球菌酶(lysostaphin,Lys)基因表达载体(p KLAC1-Lys),转化乳酸克鲁维酵母(K.lactis GG799),实现了Lys基因的分泌表达。对重组菌株(K.lactis GG799/p KLAC1-Lys)进行NTG随机化学诱变,优化表达条件,筛选获得高表达菌株,并通过Ni-NTA亲和层析纯化蛋白并研究其酶学性质。结果表明:通过诱变重组溶葡球菌酶乳酸克鲁维菌株,Lys酶比活性提高了约5.2倍(约8 000U/L)。最适接种量为40g/L,诱导过程中每24h添加一次终浓度为20g/L的半乳糖和NH_4NO_3可提高酶比活性,最适表达p H为7.0~7.5,最适反应p H为7.0~8.0,最适反应温度为37℃。实验表明,低于40℃,p H 3~6之间时,重组溶葡球菌酶较稳定。Sr~(2+)对其酶活性有明显的促进作用,Ba~(2+)、Ca~(2+)、Zn~(2+)、Cu~(2+)、Mn~(2+)、Mg~(2+)对其有明显的抑制作用。     

脂肪酶产生菌Geofrichum sp. AS 2.1135产酶条件及酶一般性质的研究

在92株能同化油的地霉属菌株中,Geotrichum sp. AS2．1135菌株产脂肪酶活力为50—60u／ml,对其产酶条件的研究表明,不饱和长链脂肪酸和油类有利于酶的形成。在4％豆饼粉作为有机氮源的培养基中,加入0．2％尿素,酶活力显著增加,酶活达150u／ml。用聚乙二醇橄榄油乳化系统测定酶的作用最适pH为8．0,最适温度为4O一42℃。在pH4一9时5℃下存放24小时,或在pH 5和8时45℃保持15分钟,酶活力不变。     

青霉NXP25纤维素酶的产生及性质

青霉（Penicillium sp.）NXP25在5％玉米穗轴粉,3％（麦夫）,0.35％氮源10号和0.3％氯化钙组成的液体培养基（起始pH 5.0）中,10％接种量,29℃,280r/min振荡培养72h。在50℃温度下测定,发酵液内切-1,4-β-葡聚糖酶,外切-1,4-β-葡聚糖酶,β-葡糖苷酶和滤纸酶活力分别为841u/mL,13u/mL,24u/mL和46u/mL。各类型酶最适作用条件分别为pH4.8和60℃、pH5.0和50℃、pH4.     

蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证

利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓（Lumbricus rubellus）中分离的FⅠ0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae 的dbEST中获得该组分的核酸序列信息,设计特异引物, 经过RT-PCR,成功地从赤子爱胜蚓（Eisenia foetida）中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp, 编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40％,因此为蚯蚓纤溶酶中的一个新基因,GenBank 登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。     

Cloning and expression of Bacillus thuringiensis israelensis delta-endotoxin DNA in B. sphaericus

Bacillus thuringiensis israelensis delta-endotoxin genes were cloned into Bacillus sphaericus 2362, producing stable transformants reacting with antibody to the 28- and 65-kDa B. thuringiensis israelensis crystal proteins and approximately 10 times more toxic to Aedes mosquito larvae than the original host strain. The LC50 after 48 hr of exposure of Aedes larvae to the most active transformed clone was 0.19 microgram/ml, compared with an LC50 of 1.9 microgram/ml for B. sphaericus 2362 and less than 0.1 microgram/ml for B. thuringiensis israelensis. The cloning vector, plasmid pPL603E, was also effective in transforming B. subtilis 1E20 with B. thuringiensis israelensis DNA, producing highly toxic clones with less stable gene expression than the clones of B. sphaericus.     

New plasmid vector and host system for genetic engineering in Bacillus sphaericus.

A new plasmid, pNQ116, was constructed in Bacillus sphaericus by cloning a promoter fragment from B. sphaericus Ts-1 into pNQ112. The plasmid (CmrKmr, 5.23 kb) contains a restriction endonuclease polylinker used for cloning foreign genes, and its cat-86 gene is expressed at high levels from the Ts-1 promoter. This plasmid vector has been transformed into B. sphaericus AS 1.270, AS 1.465, AS 1.469, and 2362, at frequencies of 10(2)-10(3) transformants per microgram of DNA, and is maintained stably under nonselective conditions in these host strains. The presence of pNQ116 in B. sphaericus 2362 does ot interfere with the mosquito larvicidal activity of the organism.     

紫外线诱变选育碱性蛋白酶高产菌株

本实验以枯草杆菌A4－3为出发菌株,发酵产生碱性蛋白酶,其野生型菌株产酶为2412u／ml。采用孢子热处理方法处理出发菌株孢子,得到变异株As—4,产酶2732．8u／ml。对As—4菌株进行紫外线绣变处理1min,得变异菌株AX—46,产酶为3213．8u／ml,且产酶能力稳定。     

产碱性纤维素酶菌株的选育和酶合成基本特性

芽孢杆菌x－6菌株经甲基磺酸乙酯（EMS）和紫外线（UV）复会诱变,从其万古霉素（Vm）抗性突变体中选育获得一突变株EV23,所产生的碱性核甲基纤维素酶（CMCase）酶活力由原来的0．84u／ml提高到3.53u／ml。EV23菌株所产该酶基本为组成性地合成纤维素酶,酶合成明显表现出抗降解物阻遏的特点,以葡萄糖为碳源培养,4％浓度时酶合成水平最高。酶合成效率受菌体生长速率影响较大。在高浓度易代谢基质和三羧酸循环中间物存在下,酶合成将受到一定程度的阻遏。酶合成还与能量代谢有关,探讨了外源ATP、cAMP对     

Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593

A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.     

Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3, 600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Recombinant strain of Bacillus thuringiensis producing Cyt1A,Cry11B,and the Bacillus sphaericus binary toxin

A novel recombinant Bacillus thuringiensis subsp. israelensis strain that produces the B. sphaericus binary toxin, Cyt1Aa, and Cry11Ba is described. The toxicity of this strain (50% lethal concentration [LC(50)] = 1.7 ng/ml) against fourth-instar Culex quinquefasciatus was higher than that of B. thuringiensis subsp. israelensis IPS-82 (LC(50) = 7.9 ng/ml) or B. sphaericus 2362 (LC(50) = 12.6 ng/ml).     

球形芽胞杆菌C3-41磷酸果糖激酶的克隆、表达及基本生物活性

[目的]球形芽孢杆菌缺乏EMP、HMP、ED途径的关键酶,如磷酸果糖激酶等被认为是其不能以糖类物质进行生长的主要原因.杀蚊球形芽孢杆菌C3-41全基因组序列分析表明,在染色体DNA上存在的磷酸果糖激酶基因pfk,为了进一步分析球形芽孢杆菌糖酵解途径,进一步确定磷酸果糖激酶在糖酵解途径中的功能.[方法]通过pfk基因在球形芽孢杆菌菌株中的Southern-blot拷贝数鉴定,在C3-41pfk基因克隆的基础上进行pfk基因在大肠杆菌中的融合表达、序列分析和序列比对等方法进行研究.[结果]证明了球形芽孢杆菌pfk基因由960 bp核苷酸组成,表达42 kDa的PFK融合蛋白,有保守的底物结合域和ATP结合域,同时pfk基因重组表达质粒可以回复大肠杆菌pfk缺陷型菌株DFl020代谢糖的能力.[结论]杀蚊球形芽孢杆菌C3-41的pfk表达产物具有磷酸果糖激酶活性,为今后深入研究球形芽孢杆菌产能代谢机理奠定了基础.     

Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobadllus casei

The lysostaphin structural gene was cloned in Bacillus subtilis DSM402 and in Lactobacillus casei 102S. The gene was expressed in both organisms and active lysostaphin was released into the medium. Lysostaphin produced by these organisms induced lysis of growing and heat inactivated staphylococci. Expression in a protective starter organism is a prerequisite to produce lysostaphin in situ in fermenting foods and hence, to reduce the hygienical risk of staphylococcal food poisoning.     

Molecular characterization of a glucokinase with broad hexose specificity from Bacillus sphaericus strain C3-41

Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K(m) values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.