On-line optimization of glutamate production based on balanced metabolic control by RQ

In glutamate fermentations by Corynebacterium glutamicum, higher glutamate concentration could be achieved by constantly controlling dissolved oxygen concentration (DO) at a lower level; however, by-product lactate also severely accumulated. The results of analyzing activities changes of the two key enzymes, glutamate and lactate dehydrogenases involved with the fermentation, and the entire metabolic network flux analysis showed that the lactate overproduction was because the metabolic flux in TCA cycle was too low to balance the glucose glycolysis rate. As a result, the respiratory quotient (RQ) adaptive control based “balanced metabolic control” (BMC) strategy was proposed and used to regulate the TCA metabolic flux rate at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux. Compared with the best results of various DO constant controls, the BMC strategy increased the maximal glutamate concentration by about 15% and almost completely repressed the lactate accumulation with competitively high glutamate productivity.     

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. In addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.     

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.     

Effects of different traveling disturbances on the species diversity in Pinus taiwanensis communities

The effects of different traveling disturbances on the different layers of the Pinus taiwanensis community in Tianzhu Mountain National Forest Park were studied using the diversity indices of species richness index (S), Simpson index (D), Shannon-Wiener index (H) and Pielou evenness index (J). The results showed that the diversity indices of the arbor layer in low disturbance were the highest. Whereas, the diversity indices of the other layers in intermediate disturbance were the highest. The values of S, D, H, and J in low disturbance were 38, 3.0976, 0.9326, and 0.7515. The values of the indices in intermediate disturbance were 44, 3.2519, 0.9421, and 0.8594, higher than those in low disturbed forest, especially in the shrub and herb layers. In the highly disturbed forest, the diversity indices were 34, 3.0095, 0.9289, and 0.7834, lower than those in the intermediately disturbed forest. By comparing the stability of differently disturbed forests with the community dominance index (C), it was clear that the C of the intermediately disturbed forests was the lowest among the three differently disturbed forests. It is proposed that the intermediately disturbed forests can improve the species diversity, but maybe decrease the community stability.     

海洋真菌Fusariumsp.LD8岩藻多糖酶的液态发酵条件研究

通过对海洋真菌Fusariumsp.LD8产岩藻多糖酶发酵培养基组成及工艺条件的研究,得到较优培养基配方为:麸皮5%,海带粉2%,(NH4)2SO40.8%。以人工海水代替蒸馏水,接种量为3%,在恒温振荡器中以180r/min的速度振荡培养,岩藻多糖酶活可达2.10IU/mL。     

柔红霉素产生菌SIPI-1482中dnmV基因功能的阻断及恢复

dnmV基因产物为柔红霉素生物合成途径中TDP-6-脱氧己糖C4酮基还原酶,破坏该基因能阻断柔红糖胺的合成,进而阻断柔红霉素的产生。从天蓝淡红链霉菌(S. coeruleorubidus)SIPI-1482基因组DNA中经PCR扩增出dnmV及其上游dnmU基因片段,并由此构建了用于阻断dnmV基因的同源重组质粒pYG817,转化SIPI-1482菌株后成功地破坏了dnmV基因,发酵结果显示阻断突变株不再代谢产生柔红霉素,为引入新的基因来改变代谢产物的糖基结构打下了基础。通过导入dnmV基因表达质粒可重建该突变株的生物合成途径,恢复产生柔红霉素,但产量比出发菌株要低。     

A comparison of in vitro anticancerous activity and mechanism of ethanolic extracts from different Ganoderma genus

Five ethanolic extracts from the mycelia of Ganoderma lucidum, G. tsugae, G. oerstedii, G. subamboinense, and G. resinaceum were respectively studied on their anticancerous activities against leukemic HL-60 cell line in vitro. Results showed that all five extracts potently inhibited HL-60 proliferation. The extract from G. lucidum mycelia exerted the highest activity. Annexin V/PI bivariate flow cytometric analysis further revealed that the five extracts significantly induced early apoptosis in HL-60 cells. The results illustrate that not only G. lucidum but also other Ganoderma species can inhibit cancer cells, and their mechanisms are related to induction of apoptosis. __________ Translated from Journal of Shanghai Normal University (Natural Sciences), 2005, 34(2): 77–81 [译自: 上海师范大学学报 (自然科学版), 2005, 34(2): 77–81]     

Characterization of cheW genes of Leptospira interrogans and their effects in Escherichia coli

The motility and chemotaxis systems are critical for the virulence of leptospires. In this study, the phylogenetic profiles method was used to predict the interaction of chemotaxis proteins. It was shown that CheW1 links to CheA1, CheY, CheB and CheW2, CheW3 links to CheA2, MCP （LA2426）, CheB3 and CheD1; and CheW2 links only to CheW1. The similarity analysis demonstrated that CheW2 of Leptospira interrogans strain Lai had poor homology with Chew of Escherichia coli in the region of residues 30-50. In order to verify the function of these proteins, the putative cheW genes were cloned into pQE31 vector and expressed in wild-type E. coli strain RP437 or chew defective strain RP4606. The swarming results indicated that CheW1 and CheW3 could restore swarming of RP4606 while CheW2 could not. Overexpression of CheW1 and CheW3 in RP437 inhibited the swarming of RP437, whereas the inhibitory effect of CheW2 was much lower. Therefore, we presumed that CheW1 and CheW3 might have the function of CheW while CheW2 does not. The existence of multiple copies of chemotaxis homologue genes suggested that L. interrogans strain Lai might have a more complex chemosensory pathway.     

Molecular weight dependence of the poly(L-lactide)/poly(D-lactide) Stereocomplex at the air-water interface

The molecular weight dependence of poly(L-lactide)/poly(D-lactide) (PLLA/PDLA) stereocomplex behavior at the air-water interface was studied by surface pressure-area (pi-A) isotherms and atomic force microscopy (AFM). It was found that the compression-induced sterecomplexation of a PDLA/PLLA equimolar blend with high molecular weight (M(w) = 1 x 10(6) and 9.8 x 10(5), respectively) could occur at the air-water interface. This result is in marked contrast with the stereocomplexation of PDLA/PLLA blends in the bulk from the melt or in solutions, where the homocrystallites of either PLLA or PDLA rather than stereocomplex crystallites will be formed preferentially when the molecular weights of both polymers are higher than 1 x 10(5). Unexpectedly, the Langmuir-Blodgett behavior of the PDLA/PLLA blend with lower molecular weight (M(w) = 4 x 10(3) and 3.2 x 10(3), respectively), which should be favored in the stereocomplex, was distinct from that of other higher molecular weight blends. AFM images clearly disclosed for the first time the morphological changes of the equimolar blends of PLLA and PDLA at the air-water interface induced by increasing the surface pressure of the monolayer. Of particular note, the bilayer mechanism for the plateau in the isotherm was directly verified by the AFM height images.     

不同培养条件下莱茵衣藻细胞中SGAT酶活性的变化

利用模式单细胞植物莱茵衣藻,研究不同培养条件下细胞中丝氨酸：乙醛酸氨基转移酶活性的变化情况。结果表明：莱茵衣藻SGAT酶活性的最适pH介于5￣7之间,当pH高于7以后,酶活性逐渐下降;随着细胞密度增加,SGAT酶活性降低;光强可显著影响SGAT酶活性,在一定光强范围内,随着光照强度的增加,酶活性增强;乙酸作为莱茵衣藻的唯一异养碳源也会影响SGAT酶活性,两者间呈正相关;提高氧浓度,显著地提高了细胞内SGAT的酶活性;当二氧化碳浓度增加时,细胞内SGAT的酶活性也略有升高;40℃高温和15℃低温处理后,SGAT酶活性均降低。此外,提高氧浓度时细胞内Gly含量增加,Ser含量减少,Gly/Ser的比值从0.79提高到1.49。