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@ 國家自然科學基金

 

2015年度獲資助項目:


楊親正  國家自然科學基金面上項目《多肽修飾碳納管作為導電通道的高效生物電化學系統構建與電子傳遞機理》批準號:31570118

 摘要:提高微生物燃料電池外膜蛋白與陽極界面間電子傳遞速率是提高產電能力的核心問題。目前存在一個難題:即陽極碳納米管界面與膜蛋白活性中心接觸時如何避免蛋白構象失活。本項目參照膜細胞色素C鐵卟啉活性,來設計合成活性多肽修飾的碳納米管復合結構來解決納米管的破壞性刺入。研究不同活性多肽修飾的碳納米管分別與正常菌、OmcA缺失菌、以及純體外OmcA蛋白結合后體系導電能力的差異,構建以活性多肽修飾碳納米管作為一維導電通道的高效生物電化學系統,并闡明其電子傳遞機理。


韓 寧  國家自然基金青年基金《葡萄新型N末端截短質膜H+-ATPase活性調控及其在抗堿性鹽脅迫中的作用》批準號:31501738

 摘要:葡萄種植受到土壤鹽堿化束縛,因此提高葡萄的抗鹽堿性對產業發展非常重要。植物抗鹽堿脅迫的重要部位為根,其中根質膜H+-ATPase(質子泵)的活性對植物維持鹽堿環境中pH穩恒態至關重要。前期研究中,申請人從鹽堿抗性較強的克瑞森無核葡萄根cDNA中克隆了質膜質子泵基因 VvPMA1,酵母功能互補已證實了其強功能,隨后序列分析和試驗發現克隆的VvPMA1可能是尚未報道的剪接變體,將產生具有較高活性的N末端截短的新型質膜質子泵(VvPMA1β),利于葡萄鹽堿抗性。為了驗證推測,本項目將對VvPMA1β表達、活性及調控進行進一步分析明確堿性鹽脅迫下葡萄是否通過選擇剪接產生活性和數量不同的VvPMA1剪接變體影響根質膜質子泵總活性;VvPMA1β是否具有組織、抗性品種特異性;VvPMA1β活性如何調節。希望從基因及蛋白水平了解葡萄根質膜質子泵的表達調控方式,提高鹽堿脅迫下葡萄根質膜質子泵活性。

 

  王騰飛  國家自然基金青年基金項目《基于分子水平調控枯草芽孢桿菌芽孢表面高效穩定展示海藻糖合酶體系研究》批準號31501413

  摘要:海藻糖是一種穩定的、安全的生物制品保護劑,應用前景廣泛。利用海藻糖合酶一步轉化麥芽糖生產海藻糖的方法具有工藝簡單,符合工業化生產的現實意義。本項目擬建立一個枯草芽孢桿菌芽孢表面高效穩定展示海藻糖合酶體系,克服海藻糖合酶胞外分泌困難且穩定性差的弊端,降低因胞內表達而徒增的細胞破碎和分離成本,并減少了熱源物質的殘留。研究內容包括,通過分析麥芽糖制備海藻糖轉化體系中誘使芽孢萌發因素,結合基因敲除技術敲除與主要誘導因素相對應的關鍵受體蛋白基因,實現表面展示海藻糖合酶的芽孢特征的穩定,并探究作用機理;利用分子生物學技術克隆芽孢衣殼蛋白基因,單一或交叉融合綠色熒光蛋白展示于芽孢表面,以最大熒光強度展示組合高效展示海藻糖合酶,分析酶學特性,并利用半理性設計策略改造海藻糖合酶氨基酸組成,加強轉化體系中芽孢表面展示海藻糖合酶的耐酸性。

 

  張 松   國家自然基金青年基金《食品用SiO2、TiO2、ZnO納米材料對腸道益生菌及其菌群的影響》批準號:31501396

  摘要:納米材料應用于食品生產、加工和包裝過程中,逐漸顯現出相當的優勢,如提高食品的色澤、風味、營養利用率,并且具有抗菌或者指示食物變質的功能。但是研究表明食品用納米材料會對細胞或組織產生損傷,因此本項目研究了常用的食品用納米材料SiO2、Ti O2、ZnO對腸道益生菌及腸道菌群的生物學效應。首先從食品中分離出SiO2、TiO2、ZnO納米材料并進行表征,然后從菌體形貌觀察、體內外細胞毒性、體內外基因毒性和菌群結構變化等方面探索食品用納米材料SiO2、TiO2、ZnO對腸道益生菌及菌群的影響及其規律,在此基礎上改變納米材料SiO2、TiO2、ZnO的表面性能改善納米材料的安全性。因此本項目對于納米材料在食品領域中的應用及食品安全性具有及其重要的意義。

 

 

2014年度獲資助項目:

 

張靜  國家自然基金青年基金《我國海帶栽培品種(系)線粒體基因組遺傳分化研究》批準號:31402300

 摘要:海帶屬藻類是褐藻門關鍵的進化種和國際重要的經濟種類,本項目針對當前海帶遺傳育種研究中存在的親本鑒定缺乏有力依據及種群遺傳分化機制不明的問題,以真海帶(Saccharina japonica)、長葉海帶(Saccharina longissima)、糖海帶(Saccharina latissima)及我國海帶主要栽培品種(系)為研究對象,在線粒體基因組水平上采取比較基因組學的策略,廣泛篩選并發掘可有效用于遺傳差異研究及親本鑒定的線粒體DNA序列,并揭示我國海帶種群在長期馴化(選育)下的遺傳分化特征與機制。

 



 

高水平研究論文


Biotechnol Biofuels(SCI, 5 years IF= 7.368)

 Production of a high-efficiency cellulase complex via beta-glucosidase engineering in Penicillium oxalicum

 


Background

Trichoderma reesei is a widely used model cellulolytic fungus, supplying a highly effective cellulase production system. Recently, the biofuel industry discovered filamentous fungi from the Penicillium genus as a promising alternative to T. reesei.

Results

In our study, we present a systematic over-expression analysis of nine β-glucosidase encoding genes in the wild-type strain 114-2 of Penicillium oxalicum. We found that the over-expression of BGL1, BGL4, or BGL5 significantly enhanced both β-glucosidase activity and hydrolysis efficiency of the enzyme system on filter paper. We utilised two strategies to over-express β-glucosidase in the strain RE-10 that—although over-producing cellulase, does so at the cost of the cellulase mixture deficiency. The constitutive promoter of gene pde_02864 encoding 40S ribosomal protein S8 was used to over-express three β-glucosidases: BGL1, BGL4, and BGL5. We found that all mutants show significantly enhanced levels of β-glucosidase at transcriptional, protein, and activity levels. Furthermore, the inducible promoter from bgl2 was used to conditionally over-express the β-glucosidases BGL1 and BGL4. Surprisingly, this induced expression strategy enables significantly improved expression efficiency. The BGL1 over-expressing mutant I1-13 particularly improved the β-glucosidase activity at a factor of 65-folds, resulting in levels of up to 150 U/ml. All our BGL over-expression mutants displayed significant enhancement of cellulolytic ability on both microcrystalline cellulose and filter paper. In addition, they substantially reduced the enzyme loads in the saccharification of a natural lignocellulose material delignified corncob residue (DCCR). The mutant I4-32 with over-expression of BGL4 achieved the highest glucose yield in the saccharification of DCCR at only 25 % enzyme load compared to the parental strain RE-10.

Conclusions

In summary, genetically engineering P. oxalicum to significantly improve β-glucosidase activity is a potent strategy to substantially boost the hydrolytic efficiency of the cellulase cocktail, which will ultimately lead to a considerable reduction of cost for biomass-based biofuel.

 草酸青霉(Penicillium oxalicum)是具有纖維素酶工業生產應用價值的一種絲狀真菌,其主要特點之一是能高效表達木質纖維素酶,將難以降解的生物質水解為可發酵的寡糖和單糖。草酸青霉表達的纖維素酶系主要包括外切纖維素酶、內切纖維素酶和β-葡萄糖苷酶。這些纖維素酶組分之間合理的表達比例對于提高生物質的降解效率至關重要。在齊魯工業大學特聘教授曲音波課題組,基于草酸青霉基因組序列,預測出11個β-葡萄糖苷酶編碼基因,并構建了這些基因的突變株,篩選出β-葡萄糖苷酶比活力最高的三個β-葡萄糖苷酶編碼基因(BGL1,BGL4和BGL5)。利用篩選出的草酸青霉40S核糖體S8蛋白編碼基因PDE_02864啟動子,分別在前期構建的高產纖維素酶基因工程菌RE-10進行過表達,構建出了纖維素酶活高表達的菌株。在搖瓶發酵條件下,β-葡萄糖苷酶的酶活相比出發菌株提高65倍,達到150 U/ml。本研究論文的研究結果使草酸青霉的纖維素酶表達體系更為合理,這對進一步推廣草酸青霉在生物精煉領域的應用具有重要意義。

本論文的相關實驗研究主要在山東大學完成。山東大學曲音波教授和齊魯工業大學副教授李忠海為共同通訊作者。齊魯工業大學為第二作者單位。

  論文鏈接:http://link.springer.com/article/10.1186/s13068-016-0491-4/fulltext.html


Water Research SCI IF=5.528

 Preparation and antibacterial activity of lysozyme and layered double hydroxide nanocomposites

It is necessary to develop “green” disinfection technology which does not produce disin-fection by-products. Lysozyme-layered double hydroxide nanocomposites (LYZeLDHs) were prepared by intercalating LYZ in LDH for the first time. Their antibacterial activity was evaluated using staphylococcus aureus as a target. The bacteria removal mechanism was also studied. Characterization of LYZeLDHs by X-ray diffraction and Fourier transform infrared spectroscopy indicated that LYZ was successfully intercalated in LDH, compressed and deformed without secondary structural change. LYZeLDHs showed excellent bacte-ricidal effectiveness against staphylococcus aureus. The antibacterial performance of LYZ eLDHs was found to be affected by the LYZ/LDH ratio and the pH of the bacteria-containing water. The bacteria removal efficiency of LYZeLDHs with LYZ/LDH mass ratio of 0.8 was consistently above 94% over the pH range of 3e9. LYZeLDHs adsorbed bacteria to their surface by LDH and then killed them by the immobilized LYZ. This new material integrated the bactericidal ability of LYZ and adsorption ability of LDH. Moreover, the antibacterial ability of LYZeLDHs was persistent and not limited by the adsorption capacity.

摘要:首次通過溶菌酶插層到LDH制備出溶菌酶層狀雙氫氧化物納米復合材料(LYZ-LDHs)。選取金黃色葡萄球菌來評價其抗菌活性。LYZ-LDHs對金黃色葡萄球菌具有優良的殺菌效果。研究發現LYZ-LDHs的抗菌性能受LYZ/LDH的比值及含菌水的pH值的影響。在pH3-9的范圍內,質量比為0.8LYZ-LDHs的除菌率始終高于94%。細菌被LYZ-LDHsLDH吸附到表面,被固定化的LYZ殺死。這種新材料整合了LYZ的殺菌能力和LDH的吸附能力。

本論文的相關實驗研究主要在北京大學完成,齊魯工業大學副教授楊親正為第一作者,北京大學副教授趙華章為通訊作者。

論文鏈接http://www.sciencedirect.com/science/article/pii/S0043135413006799


Carbon SCI IF=6.196


Direct electron transfer and conformational change of glucoseoxidase on carbon nanotube-based electrodes

To gain insights into the direct electron transfer (DET) mechanism of multi-walled carbon nanotubes (MWCNTs), we investigated the conformational changes that occur in proteins when they interact with MWCNTs. We used glucose oxidase (GOD) as an example. Using cyclic voltammetry measurements, the GOD that was immobilized on the MWCNT-modified carbon paper electrode exhibited apparent direct electrochemistry compared to that on the bare electrode without MWCNTs. The structural transformation of GOD upon adsorption on the MWCNTs was characterized spectrally. GOD was not denatured, and only small shifts of the wavenumber of the β-sheet structure were observed. There was a consistent tendency for the amount of α-helix to decrease and the β-sheet to increase. The -helix content dropped from 21.2% to 19.6% as measured using Fourier transform infrared spectroscopy and from 27.1% to 25.9% as measured using circular dichroism. The reduction in the amount of α-helix led to a less shielded GOD active site and weakened the resistance of the electron transfer. These MWCNT-induced conformational changes could account for the DET between GOD and the MWCNT-modified electrode surface.

摘要:本文以葡萄糖氧化酶(GOD),采用循環伏安法論證碳紙電極經MWCNT修飾后GOD之間明顯的直接電子轉移現象。結果顯示出MWCNTs 吸附的GOD,β-折疊的紅外光譜波數發生了輕微的移位,而α-螺旋有所減少,Far-UV CD測試也得到了類似的結果,α-螺旋的含量從27.1%下降到了25.9%。密實的α-螺旋結構的減少就在一定程度上導致較為松散的外層結構,及活性中心的暴露,因而降低電子傳遞阻力,進而促進了電極界面電子轉移。

本論文的相關實驗研究主要在北京大學完成。北京大學趙華章教授和齊魯工業大學副教授楊親正為共同通訊作者。齊魯工業大學為第二作者單位。

論文鏈接http://www.sciencedirect.com/science/article/pii/S0008622309008586

Envrionmental microbiology   (SCI IF=6.78)

    Weel1 and Cdc25 control morphogenesis, virulence and multistress tolerance of Beauveria bassiana by balancing cell cycle-required cyclin-dependent kinase 1 activity

Modification of cell cycle in entomopathogenic fungi is likely crucial for host infection and environmental adaptation. Here we show that Wee1 and Cdc25 can balance cell cycle-required cyclin-dependent kinase 1 (Cdk1) activity in Beauveria bassiana. The Cdk1 phosporylation signal was strong in cdc25 but very weak in wee1 and absent in wee1 cdc25. Con-sequently, cell cycles, septation patterns and many septation-dependent gene transcripts of these mutants were reversely changed. Hyphal cells were short in wee1, slender in cdc25 and short and swollen in wee1 cdc25. Conidiation was most defective in wee1, followed by cdc25. Their conidia and yeast-like blastospores also altered antagonisti-cally in both size and complexity, accompanied with abnormally branched germlings in wee1 and wee1 cdc25. Conidial thermotolerance and UV-B resistance decreased much more in wee1 cdc25 than in wee1 but significantly increased in cdc25. The double deletion and the point mutation Cdk1T14A/P15F for inhibitory phosphorylation caused most defective virulence, followed by wee1 deletion. All the changes were restored by ectopic gene com-plementation. Virulence changes in all the mutants and control strains were highly correlated to those in blastospore size or complexity. Taken together, Wee1 and Cdc25 control cell cycle, morphogenesis, asexual development, stress tolerance and virulence of B. bassiana by balancing the Cdk1 activity.

摘要:病原真菌細胞周期調控是其侵染宿主和適應環境的關鍵。在球孢白僵菌中,調控細胞周期必須的Cdk1激酶的活性被證明是受核激酶Wee1和磷酸化酶Cdc25調控的,因為Cdk1的磷酸化信號在Dwee1減弱,在Dcdc25增強,而在雙敲除菌株Dwee1Dcdc25中基本檢測不到。因此,這些單基因和雙基因的敲除株在細胞周期、菌絲分隔方式及形態以及影響隔膜形成的若干效應基因的轉錄水平方面都發生了不同方向的改變。Dwee1菌絲細胞變短,Dcdc25的菌絲細胞細長,而Dwee1Dcdc25的菌絲細胞既粗短又腫大。產孢缺陷以Dwee1最嚴重,其次為Dcdc25。此外,突變菌株的分生孢子和芽生孢子在細胞大小和復雜度上也發生了不同的變化,其中Dwee1Dwee1Dcdc25的分生孢子萌發的芽管異常分支。分生孢子耐高溫和UV-B輻射的能力在Dwee1Dcdc25下降最多,在Dwee1中次之,而在Dcdc25中卻顯著增強。雙基因敲除和磷酸位點突變Cdk1T14A/P15F的菌株在毒力上受損最嚴重,其次為Dwee1。所有單基因敲除引起的表型變化都在回補菌株中得到很好的恢復。此外,所有供試菌株的毒力與其芽生孢子的大小和復雜度之間存在顯著的線性相關。因此,球孢白僵菌Wee1Cdc25通過平衡Cdk1的活性即其磷酸化和非磷酸化水平而調節細胞周期循環以及同生防潛能相關的多種表型。

本論文的相關實驗研究主要在浙江大學完成,齊魯工業大學邱磊為第一作者,浙江大學教授馮明光為通訊作者。

論文鏈接http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12530/full

 

 

 

 





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