根癌农杆菌介导工业化产甘油假丝酵母的遗传转化

通过构建质粒pCAM3300-zeocin,将其电击转化到根癌农杆菌LBA4404中.将含有目的质粒pCAM3300-zeocin的根癌农杆菌和工业化产甘油假丝酵母(Candida glycerinogenes)WL2002-5共培养,利用zeocin抗性基因为筛选标记,实现了pCAM3300-zeocin对产甘油假丝酵母的转化.并根据产甘油假丝酵母的生长特性,对转化条件进行了优化,在共培养时间为24h,产甘油假丝酵母和根癌农杆菌的细胞比例为1∶(500～1000)时最高转化率达到2个转化子/104个酵母细胞.初步建立了根癌农杆菌介导转化产甘油假丝酵母的转化方法.     

流加发酵及添加L-半胱氨酸对产朊假丝酵母高密度培养合成谷胱甘肽的影响

通过7L罐考察了不同葡萄糖流加速率以及添加L-半胱氨酸对产朊假丝酵母(Candida utilis)WSH02-08高密度发酵生产谷胱甘肽(GSH)的影响.结果表明,在恒定速度(5.5g L-1h-1)流加葡萄糖30h的基础上,发酵45h后细胞干重最高达到73g L-1,此时向发酵罐中一次性添加L-半胱氨酸40mmol L-1,最终GSH产量和胞内GSH含量分别达到了1458mg L-1和2.26%.图4表1参11     

产朊假丝酵母CANDIDA UTILIS细胞壁对铜离子吸附位点的研究

目前认为所有生物大分子都对重金属离子有较强吸附性 ,但某些分子可特异地吸附重金属 ,并介导酵母细胞对重金属的抗性[1] .其中细胞表面的高分子聚合物倍受关注 ,酵母细胞壁约含 4 0种蛋白分子 ,但有关这些蛋白质在对重金属离子吸附中的作用目前还未见报道 .产朊假丝酵母Ｃａｎｄｉｄａｕｔｉｌｉｓ是重要单细胞蛋白生产菌株之一 ,因其营养条件要求不高 ,对工业废水有较强耐受和转化能力而广泛用于生产和研究 .我们观察了产朊假丝酵母细胞与分离纯化的细胞壁对铜离子的吸附能力差异 ,并对细胞壁上重金属离子吸附位点进行了研究 .1　材料与方…     

近平滑假丝酵母NAD(H)依赖型次级醇脱氢酶的分离纯化及酶学性质

从近平滑假丝酵母(Candida parapsilosis CCTCC 203011)中分离得到了NAD(H)依赖型的次级醇脱氢酶.粗酶经乙醇沉淀、Blue Sepharose Fast Flow亲和层析、DEAE Sepharose离子交换层析后在SDS-PAGE上显示为单一条带,其酶蛋白的亚基分子量(Mr)为37.5×103.该酶还原反应的最适pH为6.0,最适温度为45℃,是一个含Zn2 金属酶.该脱氢酶具有较高的底物的选择性,对2-酮具有较高的还原活力;同时,也能作用于醇类发生氧化反应,对次级醇的氧化活力最高.以β-羟基苯乙酮为底物,用纯化得到的酶催化反应后,还原产物为(R)-苯基乙二醇,因此该酶为(R)-专一性次级醇脱氢酶,是一种生产(R)-苯基乙二醇的有效的生物催化剂.经LC-MASS-MASS分析得到了酶蛋白中3个肽段的氨基酸序列,通过比对发现该酶与NCBI编号为BAA24528的次级醇脱氢酶具有较高的同源性.图4表3参17     

混合培养微生物利用甘油补料发酵生产乙醇研究

采用浸麻芽孢杆菌和红曲菌990691用甘油混合发酵生产乙醇.结果表明,分批发酵中高浓度的甘油对乙醇发酵有着较强的抑制作用,分批发酵最佳甘油浓度为0.217 mol L-1.在分批发酵的基础上补料发酵,考察了不同甘油浓度的补料液和不同补料时间对乙醇发酵的影响.最终确定乙醇补料发酵较优的工艺条件为:反应器1 L,装液量700 mL红曲发酵液,甘油初始浓度为0.217 mol L-1,以补料方式每隔60 h分5次补加0.217 mol L-1甘油浓度的红曲发酵液,每次补加100 mL,发酵培养360 h.当乙醇最高浓度达0.221 mol L-1,乙醇总产率0.628 mmol h-1,乙醇/甘油转化率达87%(mol mol-1).与分批发酵相比,补料发酵很大程度解除了高浓度甘油的抑制作用,有效地利用了甘油,提高了乙醇的产量,且乙醇产率较为稳定.     

Glycerol translocation in Condylactis gigantea

Sodium cyanide (NaCN) was used to partially uncouple respiration and photosynthesis in the symbiotic sea anemone Condylactis gigantea. NaCN significantly increased the ratio of gross photosynthesis to respiration in both intact tentacles and isolated zooxanthellae (Symbiodinium microadriaticum), increased carbon translocation from 17.7±3.5% of total fixed in controls to 43.5±5.8%, and doubled the amount of photosynthetically fixed carbon accumulating in the coelenterate host over that in controls. Only 2% of the non-particulate radioactivity recovered in the host tissue was ¹⁴C-glycerol when uninhibited symbiotic tentacles were incubated in ¹⁴C-bicarbonate for 1 h. At 10^-5 M NaCN, approximately 25% of the host nonparticulate radioactivity was recovered as ¹⁴C-glycerol, the absolute concentration of glycerol in the host tissue was three times higher than in controls, and ¹⁴C-glycerol was found in the medium. While glycerol has been proposed to play a major role in the translocation of photosynthetically fixed carbon from zooxanthellae to their coelenterate hosts, its concentration has never been measured in the animal and algal components of the symbiosis. The isolated zooxanthellae contained 3.62±0.33 mM glycerol, 26x the 0.141±0.02 mM found in the anemone. Aposymbiotic anemone tissue contained 0.169±0.06 mM glycerol. The rate of glycerol mineralization was not saturated even when exogenous glycerol levels were 70x internal concentrations. These data show that respiration and photosynthesis in symbiotic associations may be partially uncoupled by NaCN, and that this uncoupling allows the verification of the translocation and rapid catabolism of glycerol within the host.     

罗伊氏乳杆菌中甘油脱水酶的催化特性及原位再激活

对罗伊氏乳杆菌(Lactobacillus reuteri)全细胞和粗酶液催化转化甘油和1,2-丙二醇进行比较,分析其底物转化特性;使用有机溶剂制备通透细胞,外源添加辅酶B12和ATP,探索L.reuteri胞内甘油脱水酶再激活机制.L.reuteri胞内存在依赖于辅酶B12的甘油脱水酶,1,2-丙二醇不会使甘油脱水酶全酶失活,而甘油会使全酶失活;其最适温度和最适pH分别为37℃和6.2;获得了制备L.reuteri通透细胞的最佳条件;向通透细胞添加辅酶B12和ATP均能促进甘油脱水酶催化甘油转化,辅酶B12仅与甘油脱水酶单酶结合形成具催化活性的全酶,而ATP能协助失活辅酶脱离全酶和失活辅酶再激活过程.L.reuteri与Klebsiella pneumoniae类似,胞内存在甘油脱水酶再激活机制,但来源于二者的甘油脱水酶氨基酸序列同源性较低.来源于L.reuteri的甘油脱水酶是典型的依赖于辅酶B12的甘油脱水酶,尽管其序列同源性与来源于K.pneumoniae、Citrobacter freundii的甘油脱水酶差异较大,但在胞内同样存在甘油脱水酶再激活机制,可利用该机制实现胞内甘油脱水酶原位再激活,进而达到重复利用的目的.     

发酵木糖产乙醇酵母菌的选育及其发酵特性

为了提高木质纤维素水解产物中戊糖的利用率,采用划线分离法、TTC(2,3,5-氯代三苯基四氮唑)法从多年生乔木下方土壤及林中朽木中筛选出4株能利用木糖产乙醇的酵母菌。其中1株产乙醇能力较佳,其发酵条件为150 r/m in、30℃、接种量12%、初始pH值6.0时,乙醇产量达4.88g/L,木糖转化率达理论值的24.3%。经初步鉴定,该菌为假单丝酵母属(Cand ida)。研究还表明,该菌株在pH 6.5,温度40℃仍能很好地发酵木糖产乙醇。     

利用甘油脱水酶基因构建产3-羟基丙醛工程菌及其表达比较

3-羟基丙醛(3-HPA)是一种重要的化工产品,可由甘油经甘油脱水酶作用后生成.为获得产3-HPA基因工程菌,在已构建含甘油脱水酶基因及其激活因子大亚基质粒pEtac-dhaB-gdrA的基础上,构建了包含小亚基gdrB激活因子的重组质粒pEtac-dhaB-gdrA-gdrB.利用大肠杆菌通用tac启动子将该质粒在不同Escherichia coli BL21、DH5a及JM109中进行表达.阳性转化子经IPTG诱导后,提取总RNA,以cDNA为模板进行RT-PCR发现,目标基因在不同宿主都能较好转录.SDS-PAGE、酶活测定和3-HPA浓度测定结果表明,目标蛋白表达存在差异;酶活分别为4.7(±0.44)、3.5(±0.95)、8.1(±0.66)U/mg;发酵液中3-HPA的含量分别为0.012(±0.0044)、0.014(±0.003)、0.375(±0.018)g L-1,重组E.coli JM109/pEtac-dhaB-gdrA-gdrB具有较好的甘油脱水酶基因表达和产3-HPA性能.该基因工程菌与克雷伯氏菌(Klebsiella pneumoniae)相比,发酵副产物明显较少,有利于后期提取,为生产3-HPA提供了一条新思路.     

Seaweeds as a reservoir of Candida yeasts in inshore waters

The yeast populations on 9 species of seaweeds and in seawater were estimated by cultural methods over a 16-month period in Narragansett Bay, Rhode Island (USA). Maximal numbers occurred on rhodophytes and a chlorophyte, while the lower numbers on phaeophytes were attributed to the release of inhibitory polyphenolic materials. All divisions of algae showed a similar seasonal variation in yeast populations, correlated with trends in solar radiation and water temperature which would affect both the host and its microflora. The exposure of intertidal algae to unseasonable air temperatures apparently has a detectable effect on their microflora. The population changes in the water surrounding the seaweeds paralleled those on the plants. Only colorless yeasts of the genus Candida were observed, except for brief occurrences of the pink yeast Rhodotorula in late spring, when it accounted for 3 to 35% of the yeast community. Ninety five percent of the 362 representative isolates were strains of Candida. The published properties of the 84 species accepted in the genus Candida were compared with those of the marine isolates using numerical analysis. There were 7 groups: 1 solely of named species, 3 small groups with both named species and marine isolates, and 3 large groups with only one or two named species that contained 75% of the seaweed and sea-water isolates. An apparent successional sequence for three groups may be due to differences in their biochemical activity. The same three groups were preferentially enriched by 5 of the algal species. The role of this persistent yeast population as a reservoir of inshore yeasts is discussed.     

温度和发酵时间对厨余垃圾发酵产乳酸及其光学异构体的影响

通过间歇实验,探讨了温度和发酵时间对非灭菌的厨余垃圾发酵产乳酸及其光学异构体的影响.结果表明,控制温度在35℃时,发酵液中乳酸浓度远高于45℃和55℃,发酵120h后,总乳酸的产率和产量最大,分别达到0.59gL-1d-1和0.62gg(VS)-1,以有机碳表示的乳酸占发酵液中总有机碳的78%;控制发酵时间小于120h,L-乳酸在35℃、45℃和55℃三个温度条件下都是主要的异构体形式;35℃和55℃时,发酵72h后,随着发酵时间的延长,L-乳酸浓度在总乳酸中的比例呈下降趋势;45℃时,L-乳酸浓度随着发酵时间的延长逐渐增加,L-乳酸在总乳酸中的比例在发酵144h后达到97%.图4表1参21