马克斯克鲁维酵母的木糖和阿拉伯糖发酵

马克斯克鲁维酵母作为非常规酵母在燃料乙醇发酵中受到人们越来越多的关注。马克斯克鲁维具有天然的发酵戊糖的能力,但不同菌株的发酵能力存在较大差异。本研究比较了3株马克斯克鲁维菌株Kluyveromyces marxianus 9009/1911/1727(K.m 9009/1911/1727)在不同温度下的木糖和阿拉伯糖的发酵性能差异,结果发现不同发酵温度下,3株菌在耗糖速率、糖醇产率均表现出了显著的差异。菌株K.m 9009和K.m 1727在40℃下的发酵性能均优于30℃,这充分体现了马克斯克鲁维酵母的高温发酵优势。针对发酵差异,采用PCR方法获得3个不同菌株的戊糖代谢途径中的5种关键代谢酶(XR、XDH、XK、AR和LAD)的基因序列,并利用Clustalx 2.1进行了序列比对。结果显示3株菌的相关基因与文献中报道的1株克鲁维酵母的相应关键酶氨基酸编码序列相似性达98%以上,并且差异的氨基酸不在酶的关键位点处。在此基础上,通过Real-time实验,对木糖发酵差异最为明显的K.m 1727和K.m 1911的木糖代谢过程4个关键酶(XR、XDH、XK和ADH)的基因表达量进行测定,其结果显示对于耐热菌株K.m 1727,XDH和XK基因表达量低是导致木糖代谢过程中木糖醇积累、乙醇产量低的主要原因。最后,将所测得的马克斯克鲁维酵母的戊糖代谢关键酶序列与其他不同种属相比对,确定了其木糖和阿拉伯糖代谢途径,为进一步利用代谢工程方法提高戊糖发酵性能奠定了基础。     

戊型肝炎病毒样颗粒在重组汉逊酵母中的发酵表达、纯化及其免疫原性分析

为了研制戊型肝炎新型基因工程疫苗,利用汉逊酵母表达系统表达重组戊型肝炎病毒样颗粒,成功构建了重组戊型肝炎疫苗工程菌株HP/HEV2.3,对该菌株的发酵条件和纯化工艺进行了研究。先将工作种子批进行发酵培养,收集发酵后的细胞培养物;对其先后进行细胞破碎、澄清和超滤、硅胶吸附和解吸附、超滤浓缩换液、色谱纯化及除菌过滤,制得重组汉逊酵母戊型肝炎病毒样颗粒,纯化收率为33%,纯度达99%;电镜观察显示该重组汉逊酵母戊型肝炎病毒样颗粒与天然戊型肝炎病毒颗粒理论大小一致,为32 nm;基因序列与理论一致;SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质分子量大小一致,均为56 k Da,表达量占细胞总蛋白的26%,表达水平为1.0 g/L发酵液;Western blotting、ELISA活性检测及小鼠免疫接种效力试验ED_(50)结果表明,此重组汉逊酵母戊型肝炎病毒样颗粒具有良好的抗原性和免疫原性,可用于制造戊型肝炎新型基因工程疫苗。     

发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响

为研究发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响,从鼠李糖脂的结构组分、性能和应用效果等方面展开研究。薄层实验证明两种鼠李糖脂均含有单糖脂和双糖脂。液质分析发现以橄榄油作碳源时,鼠李糖脂中双糖脂(Rha-Rha-C5-C6:1和Rha-Rha-C8-C8:2)比例更大,约为73.09%。而地沟油作碳源时,单糖脂(Rha-C10-C10和Rha-C16-C16:2)的比例更高,约为76.91%。橄榄油和地沟油为碳源的鼠李糖脂的临界胶束浓度(CMC)分别为55 mg/L和80mg/L。相同投加量时,前者乳化性和乳化稳定性均优于后者。NY3菌降解含油污泥时,投加双糖脂含量高的鼠李糖脂会使C16-C30直链烷烃的去除率更高。     

Reduced levels of Cacna1c attenuate mesolimbic dopamine system function

Genetic variation in CACNA1C, which codes for the L‐type calcium channel (LTCC) Ca_v1.2, is associated with clinical diagnoses of bipolar disorder, depression and schizophrenia. Dysregulation of the mesolimbic‐dopamine (ML‐DA) system is linked to these syndromes and LTCCs are required for normal DAergic neurotransmission between the ventral tegmental area (VTA) and nucleus accumbens (NAc). It is unclear, however, how variations in CACNA1C genotype, and potential subsequent changes in expression levels in these regions, modify risk. Using constitutive and conditional knockout mice, and treatment with the LTCC antagonist nimodipine, we examined the role of Cacna1c in DA‐mediated behaviors elicited by psychomotor stimulants. Using fast‐scan cyclic voltammetry, DA release and reuptake in the NAc were measured. We find that subsecond DA release in Cacna1c haploinsufficient mice lacks normal sensitivity to inhibition of the DA transporter (DAT). Constitutive haploinsufficiency of Cacna1c led to attenuation of hyperlocomotion following acute administration of stimulants specific to DAT, and locomotor sensitization of these mice to the DAT antagonist GBR12909 did not reach the same level as wild‐type mice. The maintenance of sensitization to GBR12909 was attenuated by administration of nimodipine. Sensitization to GBR12909 was attenuated in mice with reduced Cacna1c selectively in the VTA but not in the NAc. Our findings show that Cacna1c is crucial for normal behavioral responses to DA stimulants and that its activity in the VTA is required for behavioral sensitization. Cacna1c likely exerts these effects through modifications to presynaptic ML‐DA system function.     

Screening,statistical optimized production,and application of β‐mannanase from some newly isolated fungi

Eighty‐eight fungi isolated from soil and decaying organic matter were screened for mannanolytic activity. Twenty‐eight fungi produced extracellular mannanase on locust bean gum as evidenced by zone of hydrolysis produced on mannan agar gel. Six prominent producers, including four Fusarium species namely Fusarium fusarioides NFCCI 3282, Fusarium solani NFCCI 3283, Fusarium equiseti NFCCI 3284, Fusarium moniliforme NFCCI 3287 with Cladosporium cladosporioides NFCCI 3285 and Acrophialophora levis NFCCI 3286 produced the β‐mannanase in the range of 84–140 nkat/mL. All these grew well on particulate substrates in solid‐state fermentation (SSF), producing relatively higher titers on mannan‐rich palm kernel cake (PKC) and copra meal. Two high yielding strains, F. equiseti (1747 nkat/gds) and A. levis (897 nkat/gds) were selected for statistical optimization of mannanase on PKC. Interaction of two critical solid state fermentation parameters, pH and moisture on mannanase production by these two molds was studied by response surface method. Optimized production on PKC resulted in three‐ to fourfold enhancement in enzyme yield was observed in case of F. equiseti (5945 nkat/gds) and A. levis (4726 nkat/gds). HPLC analysis of mannan hydrolysate indicated that F. equiseti and A. levis mannanase performed efficient hydrolysis of konjac gum (up to 99%) with exclusive mannooligosaccahride (DP of 4) production. A seminative SDS‐PAGE revealed that A. levis and F. solani produced three isoforms, F. moniliforme produced two isoforms while F. fusarioides, F. equiseti, and C. cladosporioides produced a single enzyme.     

Highly sensitive SERS analysis of the cyclic Arg–Gly–Asp peptide ligands of cells using nanogap antennas

The cyclic RGD (cRGD) peptide ligands of cells have become widely used for treating several cancers. We report a highly sensitive analysis of c(RGDfC) using surface enhanced Raman spectroscopy (SERS) using single dimer nanogap antennas in aqueous environment. Good agreement between characteristic peaks of the SERS and the Raman spectra of bulk c(RGDfC) with its peptide's constituents were observed. The exhibited blinking of the SERS spectra and synchronization of intensity fluctuations, suggest that the SERS spectra acquired from single dimer nanogap antennas was dominated by the spectrum of single to a few molecules. SERS spectra of c(RGDfC) could be used to detect at the nanoscale, the cells' transmembrane proteins binding to its ligand.

SERS of cyclic RGD on nanogap antenna.     

Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME)

Cyclopentyl methyl ether (CPME) was evaluated for extracting oil or triacylglycerol (TAG) from wet cells of the oleaginous yeast Lipomyces starkeyi. CPME is a greener alternative to chloroform as a potential solvent for oil recovery. A monophasic system of CPME and biphasic system of CPME:water (1:0.7) performed poorly having the lowest TAG extraction efficiency and TAG selectivity compared to other monophasic systems of hexane and chloroform and the biphasic Bligh and Dyer method (chloroform:methanol:water). Biphasic systems of CPME:water:alcohol (methanol/ethanol/1‐propanol) were tested and methanol achieved the best oil extraction efficiency compared to ethanol and 1‐propanol. Different biphasic systems of CPME:methanol:water were tested, the best TAG extraction efficiency and TAG selectivity achieved was 9.9 mg/mL and 64.6%, respectively, using a starting ratio of 1:1.7:0.6 and a final ratio of 1:1:0.8 (CPME:methanol:water). Similar results were achieved for the Bligh and Dyer method (TAG extraction efficiency of 10.2 mg/mL and TAG selectivity of 66.0%) indicating that the biphasic CPME system was comparable. The fatty acid profile remained constant across all the solvent systems tested indicating that choice of solvent was not specific for any certain fatty acid. This study was able to demonstrate that CPME could be used as an alternative solvent for the extraction of oil from the wet biomass of oleaginous yeast. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1096–1103, 2017