吡咯喹啉醌合成及生产工艺研究进展

吡咯喹啉醌(pyrroloquinoline quinone, PQQ)是继烟酰胺和核黄素之后发现的第三类氧化还原酶辅因子,普遍存在于生物体中参与呼吸链电子传递,具有促进线粒体产生、清除自由基、增强细胞代谢和预防心肌损伤等生理功能,在医药、食品和农业领域具有广泛的应用前景。微生物发酵法是PQQ生产的主要方式,解析PQQ生物合成途径及其调控机制,通过代谢工程选育短周期、高产量的生产菌是PQQ工业化的研究方向之一。本文综述了PQQ的合成途径、高产菌株选育以及微生物发酵生产与分离纯化的研发工作,为深入阐释PQQ的生物合成机制和工业化生产菌株的选育提供参考。     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b₅ at the N-terminal of interferon (b₅-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b₅ at the C-terminal of interferon (Met-IFN-b₅-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b₅-chimera), resolved this problem and gave enhanced biological activity.     

重组人心房利钠肽 (ANP) 的原核表达、纯化及鉴定

为提高重组人心房利钠肽(Atrial natriuretic peptide,ANP)的表达量,将3个ANP通过赖氨酸(Lysine,K)串联,并构建相对应的重组表达载体p ET28a(+)/ANP3。转染大肠杆菌进行诱导表达,目的蛋白约占菌体总蛋白的60%。经过包涵体变复性,赖氨酸酶(Lys-C)和羧肽酶(CPB)水解,以及一系列层析纯化,每升培养液可获得约16 mg的ANP蛋白。最终,纯化后的ANP经UPLC及Tricine SDS-PAGE鉴定,纯度大于90%,LC-MS鉴定显示其分子量为3 080 Da,且为二硫键正确形成的ANP单体,通过ELISA试剂盒检测,其具有和参比品一致活性。本研究为ANP的大规模制备打下了基础。同时,所采用的串联表达技术也为其他多肽类药物的重组表达提供了新的思路。     

Environmental DNA metabarcoding of lake fish communities reflects long‐term data from established survey methods

Organisms continuously release DNA into their environments via shed cells, excreta, gametes and decaying material. Analysis of this ‘environmental DNA’ (eDNA) is revolutionizing biodiversity monitoring. eDNA outperforms many established survey methods for targeted detection of single species, but few studies have investigated how well eDNA reflects whole communities of organisms in natural environments. We investigated whether eDNA can recover accurate qualitative and quantitative information about fish communities in large lakes, by comparison to the most comprehensive long‐term gill‐net data set available in the UK. Seventy‐eight 2L water samples were collected along depth profile transects, gill‐net sites and from the shoreline in three large, deep lakes (Windermere, Bassenthwaite Lake and Derwent Water) in the English Lake District. Water samples were assayed by eDNA metabarcoding of the mitochondrial 12S and cytochrome b regions. Fourteen of the 16 species historically recorded in Windermere were detected using eDNA, compared to four species in the most recent gill‐net survey, demonstrating eDNA is extremely sensitive for detecting species. A key question for biodiversity monitoring is whether eDNA can accurately estimate abundance. To test this, we used the number of sequence reads per species and the proportion of sampling sites in which a species was detected with eDNA (i.e. site occupancy) as proxies for abundance. eDNA abundance data consistently correlated with rank abundance estimates from established surveys. These results demonstrate that eDNA metabarcoding can describe fish communities in large lakes, both qualitatively and quantitatively, and has great potential as a complementary tool to established monitoring methods.     

Optimization,purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn²⁺ and strongly inhibited by Ba²⁺. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.     

A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris

As an eco-friendly biocatalyst for alginate hydrolysis, bacteria-derived alginate lyase (AlgL) has been widely used in research and industries to produce oligosaccharides. However, the cost of AlgL enzyme production remains high due to the low expression and difficulty in purification from bacterial cells. In this study we report an effective method to overexpress the Pseudomonas aeruginosa AlgL (paAlgL) enzyme in Pichia pastoris. Fused with a secretory peptide, the recombinant paAlgL was expressed extracellularly and purified from the culture supernatant through a simple process. The purified recombinant enzyme is highly specific for alginate sodium with a maximal activity of 2,440 U/mg. The enzymatic activity remained stable below 45°C and at pH between 4 and 10. The recombinant paAlgL was inhibited by Zn²⁺, Cu²⁺, and Fe²⁺ and promoted by Co²⁺ and Ca²⁺. Interestingly, we also found that the recombinant paAlgL significantly enhanced the antimicrobial activity of antibiotics ampicillin and kanamycin against Pseudomonas aeruginosa. Our results introduce a method for efficient AlgL production, the characterization, and a new feature of the recombinant paAlgL as an enhancer of antibiotics against Pseudomonas aeruginosa.