Cloning, expression and properties of trehalase from Pectobacterium cypripedii / 生物工程学报

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E. coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.

Distribution of archaeal community in the mud pit during strong-flavor baijiu fermentation / 生物工程学报

Multi-species solid-state fermentation in a mud pit is one of the typical features of strong-flavor baijiu, in which archaea plays important roles, however, the archaeal community distribution and diversity during fermentation are still lack of research. The biomass, composition and succession of archaea communities in fermented grains and pit mud were analyzed by high throughput sequencing. The potential interaction between archaea and bacteria was analyzed by co-occurrence network. Results demonstrate that the average biomass of archaea in pit mud was about 200 times higher than that of fermented grains. There was no significant difference in archaeal community structure between fermented grains and pit mud (r=0.017, P=0.074), but succession patterns between them showed significant correlation (r=0.30, P=0.03). Methanobacterium was the most abundant archaea in fermented grains and pit mud, and other dominant groups included Methanosarcina, Methanocorpusculum, Methanoculleus, and Methanobrevibacter. The co-occurrence network analysis showed that Methanobacterium was positively correlated with most bacteria in fermented grains and pit mud, especially with Hydrogenispora and Caproiciproducens, the dominant bacteria in pit mud. Our results revealed the temporal and spatial distribution characteristics and potential functions of the archaeal community in the mud pit of strong-flavor baijiu.

Amino acid exporters and metabolic modification of Corynebacterium glutamicum - a review / 生物工程学报

Amino acids are important compounds with a wide range of applications in the food, medicine and chemical industries. Corynebacterium glutamicum is a powerful workhorse commonly used in industrial amino acid production, with the scale of more than one million tons. In addition to its efficient anabolism, the effective exporters also ensure the high amino acid production by C. glutamicum. In this review, the research progress of amino acid exporter of C. glutamicum is summarized, to provide the foundation for further improving amino acid production by C. glutamicum via metabolic engineering.

Differences of the structure, succession and function of Clostridial communities between jiupei and pit mud during Luzhou-flavour baijiu fermentation / 生物工程学报

Clostridia inhabiting in jiupei and pit mud plays key roles in the formation of flavour during the fermentation process of Luzhou-flavour baijiu. However, the differences of Clostridial communities between jiupei and pit mud remains unclear. Here, the species assembly, succession, and metabolic capacity of Clostridial communities between jiupei and pit mud were analysed by high-throughput sequencing and pure culture approaches. The ratio of Clostridial biomass to bacterial biomass in the pit mud was relatively stable (71.5%-91.2%) throughout the fermentation process. However, it varied widely in jiupei (0.9%-36.5%). The dominant Clostridial bacteria in jiupei were Clostridium (19.9%), Sedimentibacter (8.8%), and Hydrogenispora (7.2%), while Hydrogenispora (57.2%), Sedimentibacter (5.4%), and Caproiciproducens (4.9%) dominated in the Clostridial communities in pit mud. The structures of Clostridial community in pit mud and jiupei were significantly different (P=0.001) throughout fermentation. Isolated Clostridial strains showed different metabolic capacities of volatile fatty acids in pure culture. Spatial and temporal heterogeneity of Clostridial communities existed in the baijiu fermentation pit, which was closely related to the main flavour components of Luzhou-flavour baijiu.

Effect of calcium on sporulation of Taiwanofungus camphoratus in submerged fermentation / 生物工程学报

Taiwanofungus camphoratus is a valuable and rare medicinal mushroom with various bioactivities, such as liver protection and anti-cancer. T. camphoratus can produce many arthroconidia at the end of submerged fermentation, but molecular mechanism underlying this submerged conidiation remains unknown. In this study, we found that Ca²⁺ concentration in culture medium significantly affected the arthroconidium production of T. camphoratus. Then, we identified two proteins (CaM and HSP90) involved in Ca²⁺/calmodulin signaling pathway and one protein (AbaA) involved in FluG-mediated conidiation pathway by two-dimensional electrophoresis analyses. Furthermore, we proposed a Ca²⁺/calmodulin- and FluG-mediated signaling pathway by bioinformatics analysis. By real-time quantitative PCR analyses of 23 key genes in the Ca²⁺/calmodulin- and FluG-mediated conidiation pathway, we found that expression levels of 7 genes (crz1, hsp90, flbB, brlA, abaA, wetA and fadA) showed significant responses to Ca²⁺ concentration in fermentation medium. Our research is beneficial for elucidating the underlying mechanism of submerged fermentation conidiation for T. camphoratus.

Mutation breeding of high 9α-hydroxy-androst-4-ene-3,17- dione transforming strains from phytosterols and their conversion process optimization / 生物工程学报

In order to improve transformation efficiency of phytosterols into 9α-hydroxylation of 4-androstene-3,17-dione (9α-OH-AD) by Mycobacterium sp. LY-1, we studied the strains breeding using atmospheric and room temperature plasma (ARTP) technology and optimized their conversion process. A high production strain named C33 with a good genetic stability was selected and the product molar yield reached to 15.5%, 34.8% higher than that of original strain with 15 g/L phytosterols. Furthermore, the fermentation medium was optimized through the design of orthogonal experiment. Besides, oil-water bidirectional transformation system was set up to improve the 9α-OH-AD molar yield of mutant strain C33. With adding 12 mL soybean oil to each 1 g phytosterols, the molar yield of 9α-OH-AD reached 47.0%, which increased twice than that of control (15.5%).

Application of next generation sequencing in studying food microorganisms-a review / 生物工程学报

Next generation sequencing technology has revolutionized studies in fermentation process, in particular, to explore the mechanism by which food microorganisms, including physiology, metabolic pathways, diversity and dynamic changes of microbial community. In addition, phylogenetic characteristics of different species or strains of the food microorganisms are disclosed. All these aspects will help explain how the microbes are interacting and responding to environmental factors. Bioinformatics analysis of genome and metagenome sequence data of food microorganisms could provide essential clues to improve fermentation process and function of microbes as well as control and prevention of foodborne disease outbreak. In this review, we summarized recent genomics and metagenomics studies on food microorganisms. The impact of next generation sequencing for the development and trends of food microorganism researches were discussed in details.

Optimization of hydroxylating DHEA to 7alpha,15alpha-diOH-DHEA by compound mutation and fermentation optimization / 生物工程学报

Combined with method of ketoconazole resistance screening, a 7alpha,15alpha-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7alpha,15alpha-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4 x 7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4 x 7H2O 0.015, MgSO4 x 7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7alpha,15alpha-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.

Screening, identification and culture optimization of a newly isolated aromatic nitrilase-producing bacterium--Pseudomonas putida CGMCC3830 / 生物工程学报

Microbial nitrilases have attracted increasing attention in nitrile hydrolysis for carboxylic acid production in recent years. A bacterium with nitrilase activity was isolated and identified as Pseudomonas putida CGMCC3830 based on its morphology, physiological and biochemical characteristics, as well as 16S rRNA gene sequence. The nitrilase production was optimized by varying culture conditions using the one-factor-at-a-time method and response surface methodology. Glycerol 13.54 g/L, tryptone 11.59 g/L, yeast extract 5.21 g/L, KH2PO4 1 g/L, NaCl 1 g/L, urea 1 g/L, initial pH 6.0 and culture temperature 30 degrees C were proved to be the optimal culture conditions. It resulted in the maximal nitrilase production of 36.12 U/mL from 2.02 U/mL. Investigations on substrate specificity demonstrate P. putida nitrilase preferentially hydrolyze aromatic nitriles. When applied in nicotinic acid synthesis, 2 mg/mL P. putida cells completely hydrolyzed 20.8 g/L 3-cyanopyridine into nicotinic acid in 90 min. The results indicated P. putida CGMCC3830 displayed potential for industrial production of nicotinic acid.

Breeding of high 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one transforming strains and their conversion process optimization / 生物工程学报

In order to improve transformation efficiency of dehydroepiandrosterone (DHEA) into 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one (7alpha,15alpha-diOH-DHEA) by Gibberella intermedia CA3-1, we investigated the strains breeding and their conversion process optimization. G. intermedia CA3-1 strains were treated with 0.12 mg/mL 1-methyl-3-nitro-1-nitroso-guanidin (NTG) for 30 min and chosen by 350 micromol/L minimum inhibitory concentration ketoconazole resistance marker. The high production strain named M-10 with a good genetic stability was selected and the product molar yield achieved to 70.2%, which was 20% higher than that of original strain. Under the improved conversion process with the DHEA concentration of 5 g/L, the product molar yield of the mutant M-10 reached 75.6%, which was improved by 31.3% than that of original strain.

Screening and condition optimization of a strain for efficiently biotransformation of saponins in Dioscorea zingiberensis into diosgenin / 生物工程学报

Diosgenin is an important raw material in steroid hormone and widely used in pharmaceutical industry. The traditional method for diosgenin production is acidolysis, which causes serious pollution. In order to obtain a cleaner and more efficient approach of diosgenin production, a strain of Gibberella intermedia WX12 (the sexual stage of Fusarium proliferatum) was screened from the strains deposited in our laboratory. This strain converted saponins in Dioscorea zingiberensis C.H. Wright (DZW) into diosgenin. The conversion medium was optimized by statistical experimental design. The optimized conversion medium was as follows (g/L): glucose 20.6, yeast extract 5.0, NaCl 1, K2PO4 3, ZnSO4 x 7H2O 1.5 and saponins 3. Under the optimal conditions, the yield of diosgenin achieved to (31 +/- 0.3) mg/g DZW, which was 3 times higher than that of the original medium.

Medium optimization for mycelia production of Antrodia camphorata based on artificial neural network-genetic algorithm / 生物工程学报

To illustrate the complex fermentation process of submerged culture of Antrodia camphorata ATCC 200183, we observed the morphology change of this filamentous fungus. Then we used two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) to model the fermentation process of Antrodia camphorata. By genetic algorithm (GA), we optimized the inoculum size and medium components for Antrodia camphorata production. The results show that fitness and prediction accuracy of ANN model was higher when compared to those of RSM model. Using GA, we optimized the input space of ANN model, and obtained maximum biomass of 6.2 g/L at the GA-optimized concentrations of spore (1.76x 10(5) /mL) and medium components (glucose, 29.1 g/L; peptone, 9.3 g/L; and soybean flour, 2.8 g/L). The biomass obtained using the ANN-GA designed medium was (6.1+/-0.2) g/L which was in good agreement with the predicted value. The same optimization process may be used to improve the production of mycelia and bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.

Relationship between character of atherosclerotic plaque in carotid artery and serum C reactive protein / 临床神经病学杂志

Objective To investigate the relationship of character of atherosclerotic plaque in carotid artery and serum CRP level. Methods The atherosclerotic plaques in carotid arteries were confirmed by multislice CT angiography(MSCTA),in which serum CRP level were detected in 28 patients with cerebral thrombosis,24 patients with lacunar infarction and 19 patients with TIA in internal carotid artery system in order to analysis the relationship between character of plaque and serum CRP level. Results (1)Mixed plaque and soft plaque were mainly found in cerebral thrombosis and TIA groups, while rigidity plaque was mostly found in lacunar infarction group, there were significance difference in plaques distribution among three groups(all P0.05).(3) CRP level in different groups was (4.546?2.720)mg/L in soft plaque group, (3.951?1.863)mg/L in mixed plaque group respectively,those were both higher than that of the rigidity plaque group[(2.762?1.323)mg/L],and there was significance difference between them(all P