Micro-cultivation system in microbiology: frontiers and prospects / 生物工程学报

Microbial cells cultivation is not only the origin, but also the foundation of microbiology. Researches in microbiology can only be carried out when the microbial cells can be cultured. However, conventional microbial cell cultivation is not only time consuming and labour intensive, but human error is also inevitable. Recent years, automated, modularised microbial cells micro-cultivation systems with small volume, good controllability, and equipped with real-time monitoring system have attracted great attention in microbiology. This review presents the state-of-the-art micro-cultivation systems which are implemented in microbial cells cultivation. The key development, applications of various system classified based on their construction, and the prospects of micro-cultivation system are discussed and insights into them are also provided.

Interaction between orally administrated heparin and intestinal microbiota in mice / 生物工程学报

The development of orally administrated heparin drugs requires a systematic understanding of the interaction between heparin and gut flora. The in vivo distribution of fluorescein-labeled heparin that is orally administrated by mice was observed using fluorescein microscopy. In addition, the stability of heparin in simulated gastric and intestinal fluids, as well as the in vitro degradation of heparin by gut flora were detected by HPLC. The results show that orally administrated heparin was mainly distributed in the gastrointestinal tract of mice, and exerted structural stability under the condition of simulated gastric and intestinal fluids in vitro. However, heparin could be degraded by intestinal flora cultured in medium containing heparin. In order to further study the effect of orally administrated heparin on intestinal flora in mice, the fecal microbiota 16S rRNA fragment of C57BL/6J mice was tested by the Illumina Mi-Seq high-throughput sequencing technology. Compared with the gut flora of mice that orally administrated by saline, the biodiversity of gut flora in mice with orally administrated heparin was decreased. The difference of microflora structure was not significant at the phylum level, and the relative abundance of Alistipes, Parasutterella and Akkermansia was increased at the genus level, and the relative abundance of Bilophila, Enterorhabdus, Ruminiclostridium, Prevotellaceae_UCG_001, Ruminiclostridium-9, Bacteroides, Lachnoclostridium, Candidatus, Saccharimonas, Intestinimonas and Dubosiella was reduced. These findings indicate that heparin could influence the gut flora of mice. In addition, no obvious toxic and side effects were found in mice that orally administrated heparin, suggesting the safety of orally administrated heparin.

Heparinase Digestion-based Disaccharide Analysis of Clinical Heparin and Heparinoids Drug / 分析化学

Heparin and low molecular weight heparin have been widely used in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS(6S)-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are drugs prepared from heparin active pharmaceutical ingredient ( API ) that is prepared from crude heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller sizes. As a result, low molecular weight heparins are sharing the same major disaccharides but have different reducing and non-reducing ends. In current study, we focused on the disaccharide compositional analysis of clinically used heparin and heparin-based drugs. HeparinaseⅠ,II, and Ⅲ were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, Fondaparinux sodium into disaccharides. All the degraded products were analyzed by strong anion high perforance liquid chromatography ( SAX-HPLC) coupled with an UV-detector. Commercially available unsaturated disaccharide standards were then used for structral identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin, and Fondaparinux were confirmed by reversed-phase ion pair HPLC coupled with mass spectrometry. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.

Optimization of culture conditions for Clostridium cellulolyticum / 生物工程学报

Clostridium cellulolyticum, as one of obligate anaerobic bacteria capable of secreting cellulosome, has not been efficiently cultured due to its strict requirement of growing conditions. In this study, culture conditions of C. cellulolyticum were optimized using response surface methodology. Plackett-Burman design was first used to screen the dominant impact factors for the growth of C. cellulolyticum, which were determined as yeast extract concentration, cellobiose concentration and culture temperature. The steepest ascent path design was then applied to gain the suitable range close to the optimal culture conditions for obtaining high cell density. The central composite design and the response surface analysis were finally used to determine the optimal levels of the influential factors, which were 3 g/L for yeast extract concentration, 7 g/L cellobiose concentration and 34 degrees C for culture temperature. The optimized medium was used for flask culture, and OD600 of C. cellulolyticum was increased from 0.303 to 0.586. With a pH-controlled fermentor at batch mode, OD600 reached 3.432, which was 2.8 times higher than elsewhere reported. These results support further study on the high-density culture of C. cellulolyticum and its application.

Recent progress in fusion enzyme design and applications / 生物工程学报

Engineering and redesign of enzymes are important to industrial biocatalysis. Fusion enzyme technology, based on fusion protein design, is frequently used in multifunctional enzyme construction and enzyme proximity control. Here, we reviewed the recent progress in molecular design strategy and application studies of fusion enzymes. The concept and features of fusion enzymes were introduced, followed by a systematical summary of the design strategy of fusion enzymes. In particular, the effects of different linker properties on fusion enzymes and their possible mechanisms were discussed. In addition, recent studies on fusion enzyme applications were also discussed. Finally, based on our own studies on fusion enzymes and the current research progress, the key problems in fusion enzyme technology and perspectives of this field were discussed.

Operating conditions for the rapid mutation of the oleaginous yeast by atmospheric and room temperature plasmas and the characteristics of the mutants / 生物工程学报

To obtain oleaginous yeast mutants with improved lipid production and growth rates, an atmospheric and room temperature plasma (ARTP) jet was used with a 96-well plate for high throughput screening. Mutants with changes in growth rates and lipid contents were obtained. At a lethality rate of 99%, the positive mutation rate of the yeast cells was 27.2% evaluated by the growth rates of the mutants and the comparison with the wild strain. The fermentation in a medium composed of yeast extract (10 g/L), peptone (10 g/L) and D-glucose (20 g/L) resulted in the lipid yield of the mutant (C4) with 4.07% (W/W) compared with that of the wild strain (1.87%).

Heterologous expression of particulate methane monooxygenase in different host cells / 生物工程学报

Methanotrophs use methane as the sole carbon and energy source, which cause slow growth, low cell density and hinder its industrial applications. One promising solution is to heterologously express methane monooxygenase (MMO) in other host cells that can be easily cultivated at high cell density. We systematically exploited the possibility of functional expression of pMMO by choosing different promoters and different host cells. The results showed that the recombinants could oxidize methane to methanol. In particular, ethanol could also be detected in the oxidized products, but the enzyme activity was instable, implying that some changes of pMMO expressed in the host cells might have occurred. In addition, SDS-PAGE analysis showed that many recombinants could express the subunits of pMMO, but the enzyme activity could not be detected. In conclusion, correct fold of pMMO in the host cells is important for its functional expression.

Research progresses of methanotrophs and methane monooxygenases / 生物工程学报

Methanotrophs are a group of bacteria capable of utilizing methane as the sole carbon and energy source for their anabolism and catabolism. Since methanotrophs contain the unique enzymes of methane monooxygenases (MMOs), which can catalyze the oxidation of methane and short-chain alkanes and alkenes, they have potential applications in carbon recycle of nature and industrial biotechnology. Therefore, methanotrophs have been paid much more attention by the researchers in recent 20 years. In this paper, the latest progresses in studies of methanotrophs and MMOs were reviewed, including taxonomy, function and distribution of methanotrophs, and structure, function and genetic engineering of MMOs. The future research directions of methanotrophs and MMOs as well as their applications were also discussed.