微生物转化方法生产香草酸与香草醛的初步研究

从实验室保藏的菌种中筛选到一株黑曲霉（Aspergillus niger）SW-33,能够将1g/L的阿魏酸底物转化为0.23g/L的香草酸,相应的摩尔转化率为29.35％;流加四次底物阿魏酸后,产物浓度达到1.11g/L,相应的摩尔转化率为44.9％。为了提高产物浓度,对培养基和发酵条件进行优化,使得该菌株能够将1g/l的阿魏酸底物转化为0.46g/L的香草酸,相应的摩尔转化率为57.81％。提取得到的香草酸,经HPLC测定,纯度为85.9％;提取收率为75.2％。用含香草酸的转化液,或者用提取的结晶香草酸,加入朱红密孔菌（Prcnporus cinnabarnus）SW-0203发酵培养液,可得到转化产物香草醛。     

常压甘油自催化预处理麦草浓醪发酵纤维素乙醇

目前纤维素乙醇成本偏高的根本原因在于没有达到淀粉质乙醇发酵水平的"三高"(高浓度、高转化率和高效率)指标,提高水解糖液浓度和避免发酵抑制物来实现浓醪发酵,是解决问题的关键。文中以常压甘油自催化预处理麦草为底物,尝试采用不同发酵策略,探讨其浓醪发酵产纤维素乙醇的可行性。在优化培养条件(15%底物浓度,加酶量30 FPU/g干底物,温度37℃,接种量10%)下同步糖化发酵72 h,纤维素乙醇产量为31.2 g/L,转化率为73%,发酵效率0.43 g/(L·h);采用半同步(预酶解24 h)糖化发酵72 h,纤维素乙醇浓度达到33.7 g/L,转化率为79%,发酵效率为0.47 g/(L·h),其中(半)同步糖化发酵中90%以上纤维素已被糖化水解用于发酵;采用分批补料式半同步糖化发酵,补料到基质浓度相当于30%,发酵72 h时纤维素乙醇产量达到51.2 g/L,转化率为62%,发酵效率为0.71 g/(L·h)。在所有浓醪发酵中乙酸不足3 g/L,无糠醛和羟甲基糠醛等发酵抑制物。以上结果表明,常压甘油自催化预处理木质纤维素基质适用于纤维素乙醇发酵;分批补料式半同步糖化发酵策略可用来进行浓醪纤维素乙醇发酵;未来工作中提高基质纯度和强化酶解产糖是浓醪纤维素乙醇达到"三高"指标的关键。     

酿酒酵母L610利用菊糖生产乙醇发酵条件的研究

以1株能够直接利用菊糖产乙醇的酿酒酵母L610为出发菌株,对其利用菊糖生产乙醇的发酵条件进行了一系列研究。结果表明,L610最适乙醇发酵温度为37℃,且40℃高温发酵对其产乙醇能力无显著影响;L610对酸性发酵环境有良好的耐受性,当发酵液p H值降至3.5时,其糖醇转化率及乙醇产量仍保持较高水平;以0.025~0.10 vvm的通气量通气12 h有利于L610发酵菊糖产乙醇;L610对350 g/L的高浓度菊糖有良好的转化率,乙醇浓度和生产强度分别达到129 g/L和1.35 g/(L·h);当直接以300 g/L菊芋粗粉为唯一底物进行发酵时,L610发酵产乙醇浓度达到89.6 g/L,为理论产量的78.1%。本研究所取得的成果为酿酒酵母一步法发酵菊芋生产乙醇的工业化发展提供参考。     

丁酸胁迫耦联丙酮丁醇梭菌-酿酒酵母混合培养强化丁醇发酵性能

为改善丁醇发酵性能,提出丁酸胁迫与丙酮丁醇梭菌-酿酒酵母混合培养体系协同作用的新型丁醇发酵优化控制策略.7L发酵罐中,在溶剂生产期(24 h)添加4.0 g/L-broth的丁酸浓缩液和0.2 g-DCW/L-broth的酿酒酵母进行发酵,丁醇浓度、丁醇/丙酮比和总溶剂生产效率与对照相比分别提高35％、43％和79％,达到15.74 g/L、2.83和0.52 g/L/h的最高水平.若将精馏后溶剂混合物作为高效柴油添加剂,柴油添加剂中B∶A∶E比例可达74∶17∶9(w/w)的高水平,产品质量获得显著改善.试验及分析阐明该优化控制策略可大幅诱发赖氨酸的分泌及在梭菌中的吸收/利用,提高梭菌对高丁醇浓度环境的耐受能力,促进丁醇合成;可强化梭菌对底物利用的竞争能力、提高电子往复穿梭传递系统中还原力再生速率、产生更多用于丁醇合成的NADH.两者的协同作用大幅提高了丁醇发酵的整体性能.     

稀碱预处理棕榈残渣制备纤维乙醇

以棕榈残渣(Empty fruit bunch,EFB)为原料,通过预处理、酶解、发酵等过程制备纤维乙醇.首先对比了碱、碱/过氧化氢等预处理条件对棕榈残渣组成及酶解的影响,结果表明稀碱预处理效果较好.适宜的稀碱预处理条件为:NaOH浓度为1％,固液比为1∶10,在40℃浸泡24 h后于121℃下保温30 min,在该条件下,EFB的固体回收率为74.09％,纤维素、半纤维素和木质素的含量分别为44.08％、25.74％和13.89％.对该条件下预处理后的固体样品,以底物浓度5％、酶载量30 FPU/g底物酶解72 h,纤维素和半纤维素的酶解率分别达到84.44％和89.28％.进一步考察了酶载量和底物浓度对酶解的影响以及乙醇批式同步糖化发酵,当酶载量为30 FPU/g底物,底物浓度由5％增加至25％时,利用酿酒酵母Saccharomyces cerevisiae(接种量为5％,VIV)发酵72 h后乙醇的浓度分别为9.76 g/L和35.25 g/L,可分别达到理论得率的79.09％和56.96％.     

纤维二糖发酵生产丙酮丁醇

分别考察C.acetobutylicum 810705、810706以不同浓度的麸皮和玉米粉添加物作为营养元素,纤维二糖直接进行丙酮丁醇(ABE)发酵的结果,发现2株菌对于玉米粉和麸皮的浓度变化趋势一致,C.acetobutylicum 810706转化率较高。纤维二糖ABE发酵工艺条件表明:玉米粉添加量为总糖含量的30%、底物糖质量浓度60 g/L,pH 6.5、温度35℃时,C.acetobutylicum 810706转化率达到37.38%,总溶剂质量浓度22.43 g/L,比葡萄糖、木糖ABE发酵转化率高。模拟纤维素酶水解产物配制混合糖培养基,其溶剂转化率较单独的葡萄糖、木糖发酵的转化率高,为34.95%。对比纤维素酶水解条件,C.acetobutylicum 810706具有优良的纤维素酶水解同步糖化ABE发酵能力。     

液化沙雷氏菌磷脂酶A1的克隆表达及乳糖自诱导发酵

为了利用大肠杆菌高效生产重组磷脂酶,克隆了液化沙雷氏菌磷脂酶A1的编码基因pla,分别使用pET-28a(+)和pET-20b(+)载体,实现了磷脂酶A1在大肠杆菌BL21(DE3)中的功能表达.重组菌利用载体pET-28a(+)在原始信号肽的介导下胞外PLA1酶活达40.8 U/mL,占总酶活的91％.重组菌转接至优化后的发酵诱导培养基:蛋白胨10 g/L,酵母粉5g/L,葡萄糖0.8 g/L,乳糖5 g/L,25 mmol/L Na2HPO4,25 mmol/L KH2PO4和1 mmol/L MgSO4;菌体生长6h后,添加7.5 g/L的甘氨酸,37℃恒温发酵24 h,重组菌胞外PLA1酶活达到128.7 U/mL.     

谷氨酸棒状杆菌合成5-氨基乙酰丙酸的途径构建与发酵优化

5-氨基乙酰丙酸(5-amino levulinic acid,ALA)是生物体内天然存在的一种非蛋白质氨基酸,在农业和医药等领域应用广泛。为了在谷氨酸棒状杆菌中建立ALA碳四合成途径并优化其发酵体系,首先在谷氨酸棒状杆菌中过表达沼泽红假单胞菌来源的ALA合成酶(ALAS),建立高效的ALA碳四合成途径,然后从不同发酵培养基的比较、诱导剂和底物甘氨酸的浓度以及初始接种量等不同方面对ALA摇瓶发酵工艺进行了优化。结果显示,过表达Hem A的13032/p ZWA1菌株ALA产量达到1.41 g/L,是对照菌株的67.14倍。ALA的最优摇瓶发酵条件为以酵母粉为氮源的M9培养基,采用5%的接种量0.1 mmol/L IPTG进行Hem A的诱导表达,体系中甘氨酸的浓度要控制在4 g/L,摇瓶中ALA产量可达到3.28 g/L,比优化前提高了132.62%。采用发酵优化的条件,在5 L发酵罐的放大发酵中ALA产量可达10.08 g/L,这是现有报道中谷氨酸棒状杆菌一步发酵合成ALA的最高产量。     

赭曲霉的高密度培养对坎利酮转化的影响

目的:研究赭曲霉高密度培养的发酵培养基及条件,实现坎利酮的高转化.方法:选取廉价易得的培养基成分并进行优化,同时对发酵条件进行优化,得到了最优发酵培养基配方及培养条件.结果:发酵培养基最优配方为:葡萄糖20g/L,玉米浆20g/L,酵母膏20g/L,K2HPO4 2.5g/L.种子液最佳培养时间为24h,发酵培养基初始pH 5.8,接种量为8%,装液量200mL/1000mL,摇床转速为180 r/min,28℃,底物投料时间24h,发酵结束时间72 h.结论:将该工艺在7L发酵罐中放大,菌体密度达到25.36g/L,11α羟基坎利酮的转化率为86.1%.     

代谢改造克雷伯氏菌合成D-1,2,4-丁三醇

【背景】D-1,2,4-丁三醇(D-1,2,4-butanetriol,BT)是一种重要的四碳多元醇,应用范围广,以木糖为底物的四步生化反应是目前最高效的BT生物合成路线。但大肠杆菌宿主存在严重的碳代谢抑制,限制了工程菌在木糖葡萄糖混合糖下的生长和BT合成。然而克雷伯氏菌具有生长速度更快、葡萄糖木糖混合糖利用效果好等优点。【目的】在碳代谢抑制效应较弱的克雷伯氏菌中构建以木糖为底物的BT合成途径,以提高混合糖下BT合成能力。【方法】将来源于Clostridium crescenti的木糖脱氢酶基因xdh和来源于Lactococcus lactis的2-酮异戊酸脱羧酶基因kivD及来源于Escherichia coli W3110的木糖酸脱水酶基因yjhG克隆至KlebsiellapneumoniaeZG25,得到重组菌K.pneumoniae ZG25-BT,对重组菌进行培养条件和培养基优化,进一步敲除xylA以提高BT产量。【结果】在37°C、200 r/min、接种量1%、诱导时间2 h、添加10.0 g/L CaCO3控制pH条件下,敲除xylA的重组菌在1.5倍LB培养基中以30.0 g/L木糖和10.0 g/L葡萄糖为底物,BT的产量达到4.52 g/L,摩尔转化率为0.21mol/mol,收率为15%,较优化前分别提高150%、62%和67%。【结论】实现了BT在K.pneumoniaeZG25中的发酵生产,同时通过培养条件和培养基的优化及xylA的敲除提高了BT合成能力,为进一步实验奠定了基础。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.