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Acta Biochimica et Biophysica Sinica 2007年第2期39卷 101-107页

作者： Yunhua HOU~(1,2) Tianhong WANG~1 Hao LONG~1 and Huiynan ZHU~(1,2) ~1state key laboratory of microbial technology,shandong university,jinan 250100,china ~2Department of Food and Biologic Engineering,shandong Institute of Light Industry,jinan 250323,china state key laboratory of microbial technology Shandong University Jinan 250100 China Department of Food and Biologic Engineering Shandong Institute of Light Industry Jinan 250323 China

A cellobiohydrolase 1 gene (cbhl) was cloned from Penicillium chrysogenum FS010 by amodified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR).DNA sequencing showsthat cbhl has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid *** amino acid sequence revealed that CBHI has a modular structure with a predicted molecular massof 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain bya threonine rich linker *** putative gene product is homologous to fungal cellobiohydrolases inFamily 7 of the glycosyl hydrolases.A novel cbhl promoter (1.3 kb) was also cloned and sequenced,whichcontains seven putative binding sites (5'-SYGGRG-3') for the carbon catabolite repressor *** ofvarious carbon sources to the cbhl transcription of *** was examined by Northern analysis,suggesting that the expression of cbhl is regulated at transcriptional *** cbhl gene in cold-adaptivefungus *** was expressed as an active enzyme in Saccharomyces cerevisiae *** recombinantCBHI accumulated intracellularly and could not be secreted into the medium.

关键词： Penicillum chrysogenum cellobiohydrolase TAIL-PCR promoter of cbh1

Effect of physiological heterogeneity of E. coli population on antibiotic susceptivity test

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Science china(Life Sciences) 2007年第6期50卷 808-813页

作者： LIU YuQing1,3, ZHANG HuaiQiang2, SHEN JianZhong3 & GAO PeiJi2 1 Institute of Animal Science and Veterinary Medicine, shandong Academy of Agricultural Science, jinan 250100, china 2 state key laboratory of microbial technology, shandong university, jinan 250100, china 3 College of Veterinary Medicine, china Agricultural university, Beijing 100094, china Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Science Jinan China College of Veterinary Medicine China Agricultural University Beijing China state key laboratory of microbial technology Shandong University Jinan China

According to the instantaneous growth rate (dN/dt) of E. coli CVCC249 growing in batch culture, the entire growth progress was distinguished into four phases: accelerating growth phase, constant growth phase, decelerating growth phase; declining phase, in each of which obvious variation in physiological; biochemical properties was detected, including total DNA, total protein,; MTT-dehydrogenase activity, etc., that led to difference in their antibiotic susceptivity. Antibiotic susceptivity of the population sampled from each phase was tested by Concentration-killing Curve (CKC) approach following the formula N=N 0/{1+exp[r·(x-BC 50)]}, showing as normal distribution at the individual cell level for an internal population, in which the median bactericidal concentration BC 50 represents the mean level of susceptivity, while the bactericidal span BC 199=(2lnN 0)/r indicates the variation degree of the antibiotic susceptivity. Furthermore, tested by CKC approach, the antibiotic susceptivity of E. coli CVCC249 population in each physiological phase to gentamicin or enoxacin was various: susceptivity of the population in the constant growth phase; declining phase all increased compared with that in the accelerating growth phase for gentamicin but declined for enoxacin. The primary investigations revealed that the physiological phase should be taken into account in the context of antibiotic susceptivity; research into antimicrobial mechanism. However there are few reports concerned with this study. Further research using different kinds of antibiotics with synchronized continuous culture of different bacterial strains is required.

关键词： instantaneous growth rate, batch culture, physiological heterogeneity, concentration-killing curve, antibiotic susceptivity test

Isolation and diversity analysis of heterotrophic bacteria associated with sea anemones

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Acta Oceanologica Sinica 2010年第2期29卷 62-69页

作者： DU Zongjun ZHANG Wanyi XIA Hongjie LU Guoqiang CHEN Guanjun state key laboratory of microbial technology Shandong University Jinan 250100 China College of Marine Science Shandong University at Weihai Weihai 264209 China

A study was undertaken to investigate the heterotrophic bacterial flora associated with the sea anemones. Samples of the sea anemones Anthopleura midori were collected from the coast of Weihai and bacteria were isolated from these samples. Additionally, high numbers of viable bacteria were obtained from the celom wall and surface of anemone, the community of cultivable bacteria was very diverse. As a result of this isolation, 60 strains were obtained, 56 of them were selected for identification and characterization by 16S rRNA gene sequence analysis and limited phenotypic testing. Among these isolates, 16 strains were phylogenetically related to members of the genus Pseudoalteromonas and neighboring taxa. Other isolates included members of the genera Colwcllia, Vibrio, Acinetobactcr, Pseudomonas, Endozoicomonas, Roseovarius, Paraeoceus, Loktanella, Leisingcra, Sulfitobacter, Bacillus, Staphylococcus, Plantibacter, Microbaeterium, Micro- coccus, Joostella, Psychroserpens, Cellulophaga, Krokinobaeter, Polaribacter and Psychrobaeter. Seven potential novel species were found. Among 60 strains, 17 of them can produce proteolytic exoenzyme, 20 can produce lipolytic exoenzyme. Strain NQ8 has strong antagonistic effects on some Vibrio strains. This study demonstrates that the culturable fraction of bacteria from the sea anemones Anthopleura midori is diverse and appears to possess much potential as a source for the discovery of novel bioactive materials.

关键词： Anthopleura midori Bacterial diversity 16S rRNA gene Phylogeny Identification

木酮糖激酶表达水平对酿酒酵母木糖代谢产物流向的影响

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生物化学与生物物理进展 2004年第8期31卷 746-751页

作者：沈煜郑华军王颖鲍晓明曲音波白凤武山东大学微生物技术国家重点实验室济南250100 大连理工大学环境与生命学院大连116023

在酿酒酵母中分别引入真菌和细菌的木糖代谢关键酶 ,木糖还原酶基因XYL1、木糖醇脱氢酶基因XYL2和木糖异构酶基因xylA .并在此基础上以共转化策略超表达下游关键酶木酮糖激酶基因XKS1.与亲本菌株相比 ,用pMA91和YEp2 4质粒表达XKS1的重... 详细信息

在酿酒酵母中分别引入真菌和细菌的木糖代谢关键酶 ,木糖还原酶基因XYL1、木糖醇脱氢酶基因XYL2和木糖异构酶基因xylA .并在此基础上以共转化策略超表达下游关键酶木酮糖激酶基因XKS1.与亲本菌株相比 ,用pMA91和YEp2 4质粒表达XKS1的重组菌株 ,木酮糖激酶 (xylulokinase,XK)活性分别提高了 14和6 7倍 .在限氧条件下 ,重组菌株对木糖和葡萄糖的共发酵结果显示 ,表达XYL1,XYL2以及XKS1的重组菌株HSXY 2 5 1木糖消耗为 12 4 g/L ,提高了 12 0 9% ,乙醇产量达到 9 4 g/L ,提高了 36 % ,副产物木糖醇产量为 0 7g/L ,下降了 84 9% .

关键词：木糖木酮糖激酶酿酒酵母代谢产物乙醇

粗糙脉孢菌(Neurospora crassa)木糖发酵的研究

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微生物学报 2003年第4期43卷 466-472页

作者：张潇朱冬青王丹林建强曲音波余世袁山东大学微生物技术国家重点实验室济南250100 南京林业大学化工学院南京210037

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌 (Neurosporacrassa)AS 3 1 60 2木糖发酵的影响。结果表明 ,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行 ,... 详细信息

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌 (Neurosporacrassa)AS 3 1 60 2木糖发酵的影响。结果表明 ,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行 ,此时乙醇的转化率达到63 2 %。木糖醇发酵适合在微好氧的条件下进行 ,转化率达到 31 8%。木糖醇是在培养基中乙醇达到一定浓度后才开始积累。培养基的初始pH对木糖发酵产物有较大的影响 ,乙醇产生最适pH5 0 ,木糖醇产生最适pH4 0。在培养基pH为碱性条件时 ,木糖发酵受到很大的抑制。初始木糖浓度对产物乙醇及木糖醇的产率有很大的影响。葡萄糖的存在会抑制木糖的利用 ,对乙醇和木糖醇的产生也有很大的影响。

关键词：粗糙脉孢菌木糖发酵乙醇木糖醇

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麦草生物化机浆的木聚糖酶漂白

纤维素科学与技术 2004年第1期12卷 14-18页

作者：于红秦梦华卢雪梅曲音波高培基山东轻工业学院制浆造纸工程省级重点学科山东济南250100 山东大学微生物技术国家重点实验室山东济南250100

探讨了在由PhanerochaetechrysosporiumME446麦草上固态发酵制得的木聚糖粗酶对麦草生物化机浆漂白效果的改善作用,并对其预处理工艺进行了优化。实验结果表明,当木聚糖酶用量为15IU/g,浆浓15%,处理3h后,麦草生物化机浆的白度可达到66.1... 详细信息

探讨了在由PhanerochaetechrysosporiumME446麦草上固态发酵制得的木聚糖粗酶对麦草生物化机浆漂白效果的改善作用,并对其预处理工艺进行了优化。实验结果表明,当木聚糖酶用量为15IU/g,浆浓15%,处理3h后,麦草生物化机浆的白度可达到66.1%ISO。文章还对木聚糖酶处理前后的麦草化机浆纤维进行了扫描电镜的观察。

关键词：麦草生物漂白木聚糖酶生物化机浆预处理工艺生物技术造纸业

Estimation of Cellobiohydrolase I Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy

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Acta Biochimica et Biophysica Sinica 2006年第6期38卷 372-378页

作者： Bin WU Yue ZHAO Pei-Ji GAO The state key laboratory of microbial technology,shandong university,jinan 250100,china The state key laboratory of microbial technology Shandong UniversityJinan 250100 China

1,4-β-D-glucan cellobiohydrolase I （CBH I）,p-nitrophenyl β-D-cellobioside,p-nitrophenol andcellobiose show distinct ultraviolet spectra,allowing the design of an assay to track the dynamic process ofp-nitrophenyl β-D-cellobioside hydrolysis by CBH *** on the linear relationship between p-nitrophenolformation in the hydrolysate and its first derivative absorption Curve of AUC340400nm（area under the curve）,a new sensitive assay for the determination of CBH I activity was *** dynamic parameters ofcatalysis reaction,such as Vm and kcat,can all be derived from this *** influence of β-glucosidase andendoglucanase in crude enzyme sample on the assay was discussed in *** approach is useful foraccurate determination of the activity of CBHs.

关键词： cellobiohydrolase dynamic spectrum area under the curve(AUC)

重组人β干扰素在DHFR~--CHO细胞中的高效表达

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中国生化药物杂志 2006年第3期27卷 129-133页

作者：史艳秋陈畅顾黎邹思湘宋静王鹏南京农业大学动物医学院山东大学微生物技术国家重点实验室山东大学微生物技术国家重点实验室江苏南京210095 山东济南250100

目的由二氢叶酸还原酶缺陷型的中国仓鼠卵巢(DHFR--CHO)细胞高效表达重组人β干扰素(rhIFN-β),比较来源于***、Yeast和CHO细胞的IFN-β抗增殖活性。方法采用PCR技术,从pLG104R载体中扩增目的基因,经XhoⅠ和SmaⅠ双酶切后克隆到真核表... 详细信息

目的由二氢叶酸还原酶缺陷型的中国仓鼠卵巢(DHFR--CHO)细胞高效表达重组人β干扰素(rhIFN-β),比较来源于***、Yeast和CHO细胞的IFN-β抗增殖活性。方法采用PCR技术,从pLG104R载体中扩增目的基因,经XhoⅠ和SmaⅠ双酶切后克隆到真核表达载体pCI-dhfr中,采用阳离子脂质体介导方法,将构建的表达载体转染到CHO细胞中。通过MTX的加压筛选,以病毒诱导的细胞病变抑制(CPEI)法进行活性定量检测,获得稳定、高效表达rhIFN-β的CHO细胞株,并扩大培养。分离纯化后,定量并鉴定表达产物。采用四甲基偶氮唑盐(MTT)法比较***、Yeast和CHO细胞表达体系所生产的rhIFN-β的抗增殖活性。结果得到由CHO细胞株表达的rhIFN-β纯品,且抗增殖活性显著高于其他2个表达体系。结论CHO细胞可大规模生产rhIFN-β,且活性高于其他表达体系,显示了以CHO细胞为生产平台的优越性。

关键词：中国仓鼠卵巢细胞 β干扰素真核表达抗增殖活性

Influence of Substrate Conformation on the Deglycosylation of Ribonuclease B by Recombinant Yeast Peptide:N-glycanase

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Acta Biochimica et Biophysica Sinica 2007年第1期39卷 8-14页

作者： Shengjun WANG Peng George WANG and Qingsheng QI state key laboratory of microbial technology,Life Science Schooll,shandong university,jinan 250100,china state key laboratory of microbial technology Life Science School Shandong UniversityJinan 250100 China

Peptide:N-glycanase has been thought to be responsible for proteasome-dependent degradationof misfolded glycoproteins translocated from the endoplasmic reticulum (ER) to the ***,theenzyme was supposed to be able to distinguish between native and non-native *** the presentstudy,a recombinant,yeast peptide:N-glycanase,Png lp, was expressed in Escherichia coli as inclusionbodies and was purified,refolded and *** results showed that the recombinant enzymehas a broad pH range adaptation,from pH 4.0 to pH 10.0,and has an optimum temperature of 30 ℃.This enzyme is a zinc *** activity was abolished with the addition of EDTA and notrestored by adding metal ***,the deglycosylation efficiency of recombinant Pnglpfrom *** was investigated with respect to the substrate conformation in *** ribonuclease B(RNase B) was denatured at 60-65 ℃ or by 40-60 mM dithiothreitol, indicated by its obvious structuralchange and sharpest activity change,its deglycosylation by Pnglp was most *** deglycosylationefficiency of RNase B by Pnglp was found to be related to its structural conformation and enzymaticactivity.

关键词： glycanase ribonuclease B glycoprotein deglycosylation circular dichroism

粗糙脉孢菌(Neurospora crassa)木聚糖酶的合成及性质研究

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纤维素科学与技术 2002年第4期10卷 20-26页

作者：张潇朱冬青王丹林建强曲音波余世袁山东大学微生物技术国家重点实验室济南250100 南京林业大学化工学院南京210037

粗糙脉孢菌1602(Neurosporacrassa)可以产生木聚糖酶。以不同的植物纤维物料及纯木聚糖作为底物都可诱导产生较高的木聚糖酶活,其中玉米芯粉的诱导能力最强,酶活可达25.02IU/mL。纤维素与木聚糖混合对木聚糖酶合成具有促进作用。葡萄糖... 详细信息

粗糙脉孢菌1602(Neurosporacrassa)可以产生木聚糖酶。以不同的植物纤维物料及纯木聚糖作为底物都可诱导产生较高的木聚糖酶活,其中玉米芯粉的诱导能力最强,酶活可达25.02IU/mL。纤维素与木聚糖混合对木聚糖酶合成具有促进作用。葡萄糖、木糖对木聚糖酶的合成有很强的阻遏作用,葡萄糖的阻遏作用大于木糖的阻遏作用。其木聚糖酶最适作用pH值为5.4,最适作用温度为60℃。

关键词：粗糙脉孢菌木聚糖酶阻遏