大肠杆菌Zn2+敏感突变株构建及其功能验证

【背景】Zn²⁺在细胞解毒及许多生理过程中发挥着关键作用,Zn²⁺转运蛋白已逐渐引起人们的重视。在大肠杆菌中,zntA和zitB是2个外排Zn²⁺的关键基因。【目的】构建大肠杆菌Zn²⁺敏感突变株,并对其功能进行验证。【方法】以Escherichia coli DH5α为出发菌株,利用λ Red重组系统,通过携带卡那霉素抗性基因的同源重组片段敲除zntA基因。在单基因敲除菌株基础上,利用携带庆大霉素抗性基因的同源重组片段敲除zitB基因,获得一株敲除了zntA和zitB的双基因敲除菌株KZAB04。通过功能互补实验检测基因敲除菌株及对照菌株对不同浓度Zn²⁺的敏感程度。【结果】基因敲除菌株KZAB04比出发菌株E.coli DH5α具有更高的Zn²⁺敏感性。【结论】大肠杆菌Zn²⁺敏感突变株构建成功。该菌株的构建为zntA和zitB基因功能的研究提供了必要条件,同时也为其他Zn²⁺转运蛋白基因的功能鉴定与分析奠定了基础。     

木聚糖酶生产菌Saccharothrix variisporea YJ的筛选、发酵条件及酶学性质研究

以Azo-xylan为底物,利用双层平板法从堆肥中筛选到可降解木聚糖的菌株,16S rRNA测序分析显示该菌株与糖丝菌属（Saccharothrix variisporea）的同源性最高（99.33%）,命名为S. variisporea YJ。研究发现以酵母提取物或(NH₄)₂SO₄作为氮源、甘蔗叶作为碳源、初始pH值 7.0、发酵温度40 ℃、发酵时间5 d时,发酵液中木聚糖酶的酶活性最高。酶学性质研究表明该木聚糖酶的最适反应温度及pH值分别为55 ℃和8.0,在55 ℃以下及pH值 4.0~10.0的范围内保持较高稳定性。Na⁺能有效提高木聚糖酶活性,Mg²⁺和Mn²⁺没有明显影响,Cu²⁺则严重抑制木聚糖酶活性。此外,发酵液还可以直接对天然底物玉米芯进行降解。     

甘蔗ScNRAMP基因家族的鉴定与生物信息学分析

NRAMP蛋白(natural resistance-associated macrophage proteins)家族在植物响应重金属胁迫时具有重要作用,能够转运Fe²⁺、Mn²⁺、Zn²⁺和Cd²⁺等重金属离子。为探究甘蔗ScNRAMP基因家族的特征,该文基于甘蔗割手密基因组鉴定了ScNRAMP基因家族,并进行了理化特性、基因结构、顺式作用元件、保守基序、结构域和进化关系等分析。结果表明:甘蔗ScNRAMP基因家族含有29个成员,不均匀分布在19条染色体上; 编码蛋白均为不稳定蛋白,无信号肽,亚细胞均定位在质膜上; 各成员保守基序有6～10个不等,跨膜数有6～12个不等,二级结构主要构成元件为α-螺旋和无规则卷曲; 顺式作用元件分析表明甘蔗ScNRAMP基因家族可能通过激素代谢参与逆境胁迫和生长发育等生物过程; 利用割手密的RNA-seq转录组表达数据进行的组织特异性分析发现,ScNRAMP在甘蔗不同发育阶段的叶和茎中具有时空表达特性; 进化树分析将甘蔗ScNRAMP家族成员分为3个亚家族(I、Ⅱ和Ⅲ)。该研究在全基因组水平上系统地鉴定了现代栽培甘蔗祖先种之一割手密NRAMP基因家族,既为进一步了解甘蔗NRAMP基因家族提供了基础,也为后续甘蔗重金属研究提供了重要候选基因。     

粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育所得的漆酶高产菌株,Active-PAGE分析表明SAH-12在高氮低碳无机盐培养液（LM3）中至少分泌3种漆酶同工酶（Lac1、Lac2、Lac3）。采用硫酸铵盐析、透析和Sephadex-G75分子筛层析从其培养液中分离纯化得到电泳纯的Lac1,纯化倍数6.54,酶活性回收59.7%。Lac1经SDS-PAGE验证为一条带,其表观分子量为61.5kDa。Lac1为一种糖蛋白,含糖量11.6%,等电点pI 4.40,催化氧化底物ABTS的最适反应温度为60℃,最适pH为2.6,Km值为25μmol/L。Lac1在40℃（pH4.0）以下和pH1.5～5.0（28℃）范围内稳定。金属离子Fe²⁺、Ag⁺、Hg²⁺和Cr³⁺与抑制剂DTT、SDS、EDTA和DMSO对Lac1有抑制作用,其中Fe²⁺和DTT完全抑制酶活,而Cu²⁺对酶有明显激活作用,Mn²⁺、Zn²⁺对酶活影响不大。Lac1不仅可使一些合成染料明显脱色,而且对苹果汁多酚祛除也有较好效果。40℃用该酶（1U/mL）处理苹果汁5h,其多酚含量可降低40%。     

粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育所得的漆酶高产菌株,Active-PAGE分析表明SAH-12在高氮低碳无机盐培养液（LM3）中至少分泌3种漆酶同工酶（Lac1、Lac2、Lac3）。采用硫酸铵盐析、透析和Sephadex-G75分子筛层析从其培养液中分离纯化得到电泳纯的Lac1,纯化倍数6.54,酶活性回收59.7%。Lac1经SDS-PAGE验证为一条带,其表观分子量为61.5kDa。Lac1为一种糖蛋白,含糖量11.6%,等电点pI 4.40,催化氧化底物ABTS的最适反应温度为60℃,最适pH为2.6,Km值为25μmol/L。Lac1在40℃（pH4.0）以下和pH1.5～5.0（28℃）范围内稳定。金属离子Fe²⁺、Ag⁺、Hg²⁺和Cr³⁺与抑制剂DTT、SDS、EDTA和DMSO对Lac1有抑制作用,其中Fe²⁺和DTT完全抑制酶活,而Cu²⁺对酶有明显激活作用,Mn²⁺、Zn²⁺对酶活影响不大。Lac1不仅可使一些合成染料明显脱色,而且对苹果汁多酚祛除也有较好效果。40℃用该酶（1U/mL）处理苹果汁5h,其多酚含量可降低40%。     

六种重金属离子胁迫诱导鱼类细胞凋亡的研究

以草鱼（Ctenopharyngodon idellus）ZC7901细胞为研究模型,选用六种重金属离子进行胁迫诱导鱼类细胞凋亡试验.结果显示,在Cd²⁺、Cr⁶⁺、Hg²⁺、Cu²⁺、As⁵⁺、Pb²⁺的胁迫作用下,ZC7901细胞均出现了明显的染色质凝集、趋边化、形成凋亡小体等凋亡形态特征,核酸电泳显示DNA发生特异降解而呈现电泳阶梯,用末端脱氧核糖核酸转移酶介导的dUTP切口末端标记（TUNEL）法,检测到DNA的3′-OH断端均被原位特异标记,表明六种重金属离子均能诱导鱼类细胞发生典型的凋亡.     

NtMTP1基因过表达对增强烟草Zn胁迫耐受性的研究

该研究采用实时荧光定量PCR（qRT PCR）技术,对烟草金属耐受蛋白1（MTP1）基因（NtMTP1）在烟草不同组织以及不同质量浓度ZnSO4处理下的表达进行了分析;利用农杆菌介导法,将NtMTP1基因植物过表达载体pBI121 35S∶∶MTP1转化野生型烟草,筛选得到NtMTP1基因过表达的转基因烟草植株,并进行不同质量浓度ZnSO₄处理,检测NtMTP1基因过表达对烟草Zn胁迫耐受性的影响。结果表明：NtMTP1基因在烟草中呈现组织特异性表达,主要在花与叶中表达;NtMTP1基因的表达受到Zn²⁺诱导,在400 μmol/L ZnSO₄处理后,表达量达到最高,为对照组的3.81倍;3株转基因烟草植株中NtMTP1基因表达量分别为野生型的10.42、7.61和11.84倍,与野生型相比,过表达植株对Zn胁迫的耐受性显著增强。研究结果为阐明NtMTP1基因在烟草体内Zn²⁺转运过程中的生物学功能提供了重要依据。     

直链烷基苯磺酸钠(LAS)降解菌的筛选及其降解特性的初步研究*

从洗衣粉生产废水中分离到一株高效降解 LAS（Linear Sodium Alkylbenzenesulfonate）菌GZ6,经初步鉴定其为杰氏棒杆菌（Corynebaerium jeikeium GZ6）。该菌株最高可以降解700mg/L左右的LAS,降解LAS的最适pH值和温度分别为7.0和30℃,最适LAS浓度为400mg/L,所需时间为24h,降解率可达98.7％。实验还表明一些重金属离子如Hg²⁺、Co²⁺、Cd2+等对该菌     

海藻糖酶产生菌的选育、鉴定及其酶学特性初探

海藻糖酶可特异性将1分子海藻糖分解为2分子葡萄糖,在乙醇工业、食品等行业中具有广阔的应用前景。从环境土壤中筛选到1株海藻糖酶产生菌C2,根据形态学分析和分子生物学鉴定将其命名为大黄欧文氏菌（Erwinia rhapontici）。该菌株产海藻糖酶的最适温度为40 ℃,最适pH为5.0,在酸性及低于35 ℃条件下,该酶具有较高的稳定性,Ca²⁺、Zn²⁺、Ni²⁺、Mn²⁺、Cu²⁺、K⁺、Na⁺和Fe²⁺对海藻糖酶酶活具有促进作用,DMSO和DTT对酶活具有一定的提升作用,而Triton X-114、SDS和PMSF则对酶活具有抑制作用。     

Bacillus sp.ZJS3基因组测序及砷胁迫下相关基因表达分析

【背景】环境中高毒性As³⁺的微生物氧化在砷的生物地球化学循环中起重要作用,具有潜在的应用价值。【目的】Bacillus sp.ZJS3菌株是本实验室前期分离鉴定的一株As³⁺耐受菌株,而且对多种重金属具有耐受性,期望进一步明确该菌株在As³⁺胁迫下菌体形态变化及应对砷胁迫的遗传基础,为As³⁺耐受细菌的研究提供基础数据。【方法】使用单分子实时测序（single-molecule real-time sequencing,SMRT）及Illumina测序技术对Bacillus sp.ZJS3菌株进行全基因组测序,对其基因进行功能注释和生物信息学分析,并结合绝对定量PCR技术对砷抗性及砷代谢相关基因进行分析。【结果】Bacillus sp.ZJS3菌株基因组大小为5.82 Mb,GC含量为35.9%,包含染色体1个、质粒3个、CDS数量为5 981个、tRNA 104个、sRNA 136个、rRNA 42个、串联重复序列173个、基因岛13个、转运蛋白1 023个、跨膜蛋白1 717个和双组分调控基因160个。NR、Swiss-Prot、Pfam、COG、GO和KEGG数据库分别可注释Bacillus sp.ZJS3菌株基因组中97.66%、69.30%、78.52%、65.49%、67.65%和43.87%的基因。绝对定量PCR结果表明,arsC基因在砷处理条件下显著高于对照组,而arsB基因在砷处理条件下显著低于对照组。【结论】Bacillus sp.ZJS3菌株在As³⁺胁迫下可能导致细胞分裂无法正常进行,进而影响细胞形态。基因组中aqpZ、arsA、arsB、arsC等基因的存在表明该菌株具有As³⁺外排和还原As⁵⁺的能力,phoUpstBACS的存在表明菌株可以吸收As⁵⁺,但菌株受到外界环境As³⁺胁迫时arsB表达水平降低。     

Characteristics of extracellular proteases produced by Bacillus laterosporus and Flavobacterium sp. isolated from gelatinfactory effluents

Forty bacterial isolates from the effluents of a gelatin factory (Jabalpur, India) were screened for protease activity and the two most potent producers were identified as Bacillus laterosporus and a Flavobacterium sp. The enzymes of both isolates were optimal at pH 8 and 60°C, with maximum activity after 90 min. The enzyme activity of B. laterosporus was suppressed by Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ ions but was enhanced by Ba²⁺ and Ca²⁺. That of Flavobacterium sp. was suppressed by Mg²⁺ and Mn²⁺ ions but enhanced by Ba²⁺, Ca²⁺ and Fe²⁺. The enzyme activity of the former was strongly inhibited by KCN, whereas that of the latter was only slightly inhibited by 8-hydroxyquinoline.     

Evidence of metal binding activities of pentadecapeptide from Panax ginseng

A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg²⁺. Mobility was also affected in the presence of La³⁺, Mn²⁺, Ca²⁺ and Zn²⁺ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca²⁺-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg²⁺ and Mn²⁺ ions.     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

Determination of the growth and solubilization capabilities of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> T1

The growth capability of Trichoderma harzianum Rifaii Tl was tested on Malt Extract and Czapeks Dox agar containing different concentrations of Cu²⁺, Zn²⁺, Mn²⁺, Fe²⁺ and Ca²⁺. The T. harzianum Tl isolate was observed to produce mycelia and spores in various mineral-containing media. It showed the lowest tolerance to Ca²⁺ and the highest tolerance to Fe²⁺. Solubilization capability of T. harzianum Tl for some insoluble minerals via acidification of medium has been tested on MnO₂, CuO, Fe₂O₃ and metallic Zn. T. harzianum Tl was able to solubilize MnO₂ and metallic Zn in a liquid medium.     

Production of melanin bleaching enzyme of fungal origin and its application in cosmetics

We screened wild fungal isolates for melanolytic activity and found thatSporotrichum pruinosum was the most promising of the very limited number of fungi that decolourised synthetic melanin. We used a submerged aerobic process to produce a skin depigmentation enzyme by this strain, and found that in the medium the presence of Mn²⁺ ions was necessary, the limitation of carbon source was beneficial, and Zn²⁺ ions were inhibitory. Cultivation in a stirred bioreactor required immobilization of mycelium and use of low stirring velocity. A partially purified enzyme was prepared and tested for depigmentation of human skin corneocytes and whole epidermis of phototypes III and V. This is the first study demonstrating the effective enzymatic degradation of the skin melanin rather than inhibition of its synthesis. This opens the possibility of using melanolytic enzymes in cosmetic skin lightening.     

In Solution Cation-Induced Secondary and Tertiary Structure Alterations of Human Calprotectin

Calprotectin (CP) is widely considered to have diverse roles including growth inhibitory and apoptosis induction in a number of tumor cell lines and antimicrobial activities. As CP has been proposed to bind metal ions with high affinity, we have studied its functional and primarily its structural behavior upon Zn²⁺ and Mn²⁺ chelation solely and along with Ca²⁺. We employed fluorescence spectroscopy and circular dichroism to determine the resulting modifications. Based upon our findings it is clear that treating CP with ions effectively weakened its natural growth inhibitory activity. Moreover, structural analysis of Zn²⁺ and Mn²⁺-treated CPs indicated remarkable alterations in the regular secondary structures in favor of irregular structures while Zn²⁺ and Mn²⁺ treatment of CP after incubation with Ca²⁺ displayed no remarkable shifts. Tertiary structure investigation using fluorescence spectroscopy showed that CP undergoes conformational changes upon Zn²⁺ and Mn²⁺ treatment whereby Trp residues of protein is slightly exposed to the hydrophilic environment, compactness of CP is compromised, whereas in Ca²⁺-treated CP, the tertiary structure integrity is intact upon Zn²⁺ and Mn²⁺ chelation. Interestingly, CP structural modifications upon Zn²⁺ and Mn²⁺ treatment was significantly comparable, probably due to similar radii and charges of ions. Taken all together, we have concluded that CP maintains its normal nature in Ca²⁺-loaded state when treated with Zn²⁺ and Mn²⁺ ions. It can be suggested that Ca²⁺ not only stabilize CP structure but also helps CP to keep its structure upon metal ions chelation which is involved in host organism defense system.     

Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions

Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30 °C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH₂PO₄ (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60 °C while Phy II was 4.0 and 60 °C, respectively. Phy I was stable in the pH range 1.5–3.5 while Phy II was stable in the wider pH range, 2.0–7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg²⁺, Mn²⁺, Ca²⁺ and Fe³⁺ ions and inhibited by Zn²⁺ and Cd²⁺ ions while Phy II activity was moderately stimulated by Fe³⁺ ions and was inhibited by Hg²⁺, Mn²⁺ and Zn²⁺ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 μmols/min/mg protein, respectively.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

Effects of divalent metal cations on lovastatin biosynthesis from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> in chemically defined medium

Effects of six divalent metal cations: Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺, Cu²⁺and Mn²⁺ on fungal cell growth and lovastatin biosynthesis were investigated by submerged cultivation of Aspergillus terreus in a modified chemically defined medium. The influences of different initial concentrations of the above six metal cations were also examined at 1, 2, and 5 mM, respectively. Cu²⁺ apparently inhibited the cell growth, but had no influence on biosynthesis of lovastatin. All of Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺ and Mn²⁺ promoted the cell growth and lovastatin biosynthesis in different extents. The highest biomass of 13.8 ± 0.5 g l⁻¹ and specific lovastatin titres of 49.2 ± 1.4 mg gDCW⁻¹ were obtained at the level of 2 and 5 mM in the presence of Zn²⁺, respectively. The values were improved double and 14.4-fold. Excess Zn²⁺ inhibited the cell growth, but enhanced lovastatin biosynthesis with an increment of 17.6 mg l⁻¹ per mM. The interactions of all metal cations slightly inhibited the lovastatin production comparing with the existence of Zn²⁺, Fe²⁺ and Mg²⁺ solely, yet remarkably improved the cell growth. These results suggest that the divalent metal ions Zn²⁺ or Fe²⁺ influence the production by regulating the action of key enzymes such as LovD or LovF in lovastatin biosynthesis.