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.
Trehalase/metabolism* ; Trehalose/metabolism* ; Escherichia coli/metabolism* ; Ethanol/metabolism* ; Cloning, Molecular

Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are aerobic microorganisms that can remove nitrogen under high-salt conditions, but their performance in practical applications are not satisfactory. As a compatible solute, trehalose helps microorganisms to cope with high salt stress by participating in the regulation of cellular osmotic pressure, and plays an important role in promoting the nitrogen removal efficiency of microbial populations in the high-salt environment. We investigated the mechanism of exogenous-trehalose-enhanced metabolism of HN-AD community under high-salt stress by starting up a membrane aerobic biofilm reactor (MABR) to enrich HN-AD bacteria, and designed a C₁₅₀ experimental group with 150 μmol/L trehalose addition and a C₀ control group without trehalose. The reactor performance and the community structure showed that NH₄⁺-N, total nitrogen (TN) and chemical oxygen demand (COD) removal efficiency were increased by 29.7%, 28.0% and 29.1%, respectively. The total relative abundance of salt-tolerant HN-AD bacteria (with Acinetobacter and Pseudofulvimonas as the dominant genus) in the C₁₅₀ group reached 66.8%, an 18.2% increase compared with that of the C₀ group. This demonstrated that trehalose addition promoted the enrichment of salt-tolerant HN-AD bacteria in the high-salt environment to enhance the nitrogen removal performance of the system. In-depth metabolomics analysis showed that the exogenous trehalose was utilized by microorganisms to improve proline synthesis to increase resistance to high-salt stress. By regulating the activity of cell proliferation signaling pathways (cGMP-PKG, PI3K-Akt), phospholipid metabolism pathway and aminoacyl-tRNA synthesis pathway, the abundances of phosphoethanolamine, which was one of the glycerophospholipid metabolites, and purine and pyrimidine were up-regulated to stimulate bacterial aggregation and cell proliferation to promote the growth of HN-AD bacteria in the high-salt environment. Meanwhile, the addition of trehalose accelerated the tricarboxylic acid (TCA) cycle, which might provide more electron donors and energy to the carbon and nitrogen metabolisms of HN-AD bacteria and promote the nitrogen removal performance of the system. These results may facilitate using HN-AD bacteria in the treatment of high-salt and high-nitrogen wastewater.
Nitrification ; Denitrification ; Trehalose ; Phosphatidylinositol 3-Kinases/metabolism* ; Heterotrophic Processes ; Salt Stress ; Nitrogen/metabolism* ; Aerobiosis ; Bioreactors/microbiology*

This study was aimed to evaluate the effect of benzyl alcohol on trehalose-loading red blood cells (RBCs). The RBCs were incubated in 10, 30, 50 and 100 mmol/L concentrations of benzyl alcohol-trehaloe solution at 4 degrees C for 24 hours. The hemolysis rate of loaded RBCs was detected by using cyanohemoglobin kit, the intracellular trehalose level were assayed by sulfate anthrone method. The results showed that the intracellular trehalose concentration in group with 100 mmol/L benzyl alcohol was 72 +/- 12.98 mmol/L, compared with that in groups of 10, 30 and 50 mmol/L, the statistical difference were significant (p = 0.000); the hemolysis rate of loaded RBCs in group with 100 mmol/L of benzyl alcohol was 17.99 +/- 3.75%, as compared with groups of 10, 30 and 50 mmol/L, the statistical difference was significant (p = 0.000). It is concluded that benzyl alcohol can enhance the intracellular trehalose concentration. As concentration of benzyl alcohol ascends, the intracellular trehalose concentration increases. 100 mmol/L benzyl alcohol may be proper for loading RBCs.
Benzyl Alcohol ; pharmacology ; Blood Preservation ; methods ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; drug effects ; metabolism ; Freeze Drying ; methods ; Humans ; Trehalose ; metabolism ; pharmacology

Lyophilization of human red blood cells has important significance in clinical application. Some sugars, especially trehalose, can be more tolerant of some organism or cells to dry environments, But, how to bring sugars into cells is a challenge. This study was aimed to investigate the regularity of sugar-uptake in human red blood cells. The absorption rate of trehalose and glucose in red blood cells, free hemoglobin level and erythrocyte deformation index were determined at different incubation temperature (4, 25 and 37 degrees C), different sugar concentration (0, 0.2, 0.4, 0.6, 0.8 and 1 mol/L) and different incubation time (1, 3, 5, 7 and 9 hours). The results showed that with increase of temperature and extracellular sugar concentration, the uptake of sugar in red blood cells also increased, the intracellular trehalose and glucose concentrations were over 30 mmol/L and 40 mmol/L respectively. The effects of incubation time on uptake of trehalose and glucose were different. With prolonging of incubation time, the uptake of trehalose showed firstly increase and then decrease, however, the uptake of glucose showed a constant increase. But the loading process had side-effect on free hemoglobin and maximum deformation index (MAXDI) of red blood cells, especially for trehalose, which mainly come from high osmotic pressure. It is concluded that the uptake of sugars in red blood cells is closely dependent on incubation temperature, extracellular sugar concentration and incubation time. In certain condition, the efficiency of sugar uptake is very high, but this process also damages red blood cells so as to affect the application of sugars in lyophilization of red blood cells. The research in the future should focus on how to deal with the relation between cell injury and uptake efficiency of sugar in red blood cells.
Blood Preservation ; adverse effects ; Cryoprotective Agents ; pharmacokinetics ; Erythrocyte Membrane ; drug effects ; Erythrocytes ; metabolism ; Freeze Drying ; Glucose ; pharmacokinetics ; Humans ; Trehalose ; pharmacokinetics

Lyophilization is the best method for preservation of blood cells at present. Lyophilized blood cells could be stored at room temperature for long periods of time, while maintaining a high degree of viability. Lyophilized blood cells facilitates transportation and the costs are low. However, the membrane of blood cells is damaged and viability of blood cells is decreased in lyophilization. Trehalose has been shown to protect membrane, proteins and nucleic acids during freezing and desiccation. Now, researchers present a methor for loading blood cells with trehalose. In this paper, damage effect of lyophilization on blood cells, the mechanism of trehalose protection and the experimental studies on trehalose were reviewed.
Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; drug effects ; Freeze Drying ; methods ; Humans ; Trehalose ; pharmacology

The aim of this research was to study the technology and methods of loading lyoprotectant-trehalose into cytoplasm of human platelets before lyophilization, to optimize experimental conditions of loading trehalose, to investigate the changes of platelets response to agonists and activation after incubation of platelets for 4 hours at 37 degrees C in the presence of lyoprotectant-trehalose, to protract the figures of loading efficiency and intracellular trehalose concentration versus incubation time, temperature and external trehalose concentration, to optimize loading parameters. The response of platelets to different agonists--thrombin, ADP, collagen and ristocetin were measured respectively by APACT2 aggregometer before and after loading trehalose into platelets; the expressions of CD62p and PAC-1 on platelet membranes in the presence and absence of reversible platelets activation inhibitors were measured by flow cytometry respectively before and after loading trehalose into cytoplasm of platelets. The results showed that the loading efficiency was linear to incubation time (2 hours later) and incubation temperature (rang from 30 degrees C to 40 degrees C), respectively. The loading efficiency almost reached 60% when the platelets were incubated at 37 degrees C for 4 hours. The intracellular trehalose concentration was higher with the increase of the extracellular trehalose concentration (< 50 mmol/L). Compared to untreated groups, the values of MPV and aggregation to different agonists in treated groups showed no significant difference, respectively (P > 0.01). After incubation of platelets for 4 hours, the expression of CD62p increased to some extent, however, the expression of CD62p decreased again when the reversible platelets activation inhibitor PGE-1 and adenosine were added to the incubation buffer. It is concluded that 37 degrees C, 4 hours and the extracellular trehalose concentration < 50 mmol/L are the optimal conditions for loading with trehalose. The processing of loading with trehalose before platelet lyophilization has no significant effects on response of platelets to agonists and activation.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cell Survival ; Cryopreservation ; methods ; Freeze Drying ; Humans ; Trehalose ; blood ; pharmacology

The aim of this study was to search a procedure of platelet lyophilization and find a way of long-term storage of human platelets at normal temperature with smaller size and lighter weight, to be convenient to transport at long distance thus to meet the demands in accidents and war time. Human platelets were pretreated by freezing, the first and the second desiccation, and were added with reversible activation-inhibitors of platelets, DMSO and trehalose, then were rehydrated. At the same time, the recovery rate of platelets, platelet maximal aggregation induced by thrombin, coagulation of platelets, CD62p expression and PAC-1 expression were assayed. The results indicated that the recovery rate of the platelets was 56.29%. The platelet maximal aggregation induced by thrombin had no significant difference between lyophilized platelets and the fresh platelet-rich plasma (FPRP), but the aggregation of platelets induced by ADP or propyl gallate was decreased by 49.34% and 26.25%. Coagulation of the lyophilized platelets was not significantly different from FPRP. CD62p expression of the lyophilized platelets (42.36%) was higher than that in FPRP while PAC-1 expression was 2.12%. CD62p re-expression rate induced by thrombin was 50.88% and PAC-1 re-expression was 54.55%. It is concluded that the ability of recovered lyophilized platelets added with reversible activation-inhibitors, DMSO and trehalose to aggregate and coagulate has showed no significant difference as compared with FPRP. The reversible activation-inhibitors can decrease CD62p expression of lyophilized platelets, and may enhance their survival ability and prolongate survival time. Therefore the efficiency of lyophilizing platelets can be improved based on this freeze-drying procedure.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cell Survival ; Freeze Drying ; methods ; Humans ; Trehalose ; pharmacology

This study was aimed to further optimize trehalose loading technique including loading temperature, loading time, loading solution and loading concentration of trehalose, based on the established parameters. Loading efficiency in plasma was compared with that in buffer at 37 degrees C; the curves of intracellular trehalose concentration versus loading time at 37 degrees C and 16 degrees C were measured; curves of mean platelet volume (MPV) versus loading time and loading concentration were investigated and compared. According to results obtained, the loaing time, loading temperature, loading solution and trehalose concentration were ascertained for high loading efficiency of trehalose into human platelet. The results showed that the loading efficiency in plasma was markedly higher than that in buffer at 37 degrees C, the loading efficiency in plasma at 37 degrees C was significantly higher than that at 16 degrees C and reached 19.51% after loading for 4 hours, but 6.16% at 16 degrees C. MPV at 16 degrees C was increased by 43.2% than that at 37 degrees C, but had no distinct changes with loading time and loading concentration. In loading at 37 degrees C, MPV increased with loading time and loading concentration positively. Loading time and loading concentration displayed synergetic effect on MPV. MPV increased with loading time and concentration while trehalose loading concentration was above 50 mmol/L. It is concluded that the optimization parameters of trehalose loading technique are 37 degrees C (temperature), 4 hours (leading time), plasma (loading solution), 50 mmol/L (feasible trehalose concentration). The trehalose concentration can be adjusted to meet the requirement of lyophilization.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Freeze Drying ; Humans ; Trehalose ; metabolism ; pharmacology

This study was aimed to investigate the feasibility of cryopreserving red blood cells (RBCs) by loading with trehalose and to evaluate the effect of trehalose on lyophilized RBCs. Based on the thermal properties of RBC plasma membrane, the RBCs were incubated in 0.8 mol/L trehalose solution at 37 degrees C for 7 hours, and RBCs incubated in phosphate-buffered saline were used as control. The morphology of RBCs was observed by light and scanning electron microscopy, the hemolysis rate of loaded RBCs was detected by using cyanohemoglobin kit, the intracellular trehalose levels were assayed by sulfate anthrone method, the intracellular ATP and 2, 3-DPG levels were determined by bioluminescence assay and 2, 3-DPG kit respectively, meanwhile the deformation and osmotic fragility of RBCs were measured. The results showed that the intracellular trehalose concentration was 36.56 +/- 7.95 mmol/L, the electronical microscopic images of trehalose-loaded RBCs showed the membrane integrity, the hemolysis rate in trehalose-loaded RBCs was 15.663 +/- 3.848%, while hemolysis rate in controlled RBC was 5.03 +/- 1.85% (P < 0.05). Maximum index of deformation in trehalose-loaded RBC was 0.0289 +/- 0.00738, while maximum index of deformation in control group was 0.1200 +/- 0.0121 (P < 0.05), The level of ATP in trehalose-loaded RBC was 2.67 +/- 0.54 micromol/gHb, while the level of ATP in control group was 5.22 +/- 1.10 micromol/gHb (P > 0.05). Osmotic fragility data showed that trehalose exerted osmotic protection on RBC. During loading period the level of 2, 3-DPG in trehalose-loaded RBC was maintained close to the level in control. When trehalose-loaded RBCs were lyophilized and rehydrated, the recovery rate of hemoglobin was about 46.44 +/- 4.14% and that in control was 8.33 +/- 2.34% (P < 0.001). The recovery rate of hemoglobin of trehalose-loaded RBC was higher than that of control. It is concluded that trehalose can be integrated in the membrane of RBC in lyophilization state, maintain the integrity of RBC membrane, and significantly enhance the recovery rate of hemoglobin of cryopreserved RBCs. Cryopreserving RBCs by loading with trehalose is feasible.
Blood Preservation ; methods ; Cells, Cultured ; Cryoprotective Agents ; pharmacology ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; metabolism ; Freeze Drying ; methods ; Hemolysis ; drug effects ; Humans ; Trehalose ; pharmacology

Ethanol tolerance of self-flocculating yeast SPSC01 was studied in a 3-L bioreactor under fed-batch culture. Yeast floc populations with the average sizes around 100, 200, 300, and 400 microm were obtained by adjusting the mechanical stirring rates of the fermentation system. When subjected to 20% (V/V) ethanol shock for 6 h at 30 degrees C, the remained cell viability was 3.5%, 26.7%, 48.8% and 37.6% for the aforementioned four floc populations, respectively. The highest ethanol yield 85.5% was achieved for the 300 microm flocs, 7.2% higher than that of the 100 microm flocs. The amounts of trehalose and ergosterol (including free ergosterol and total ergosterol) were positively correlated with the average size distributions from 100 to 300 microm. However, in the 400 microm flocs, the content of trehalose and ergosterol decreased, which coincided with its reduced ethanol tolerance compared to that of the 300 microm flocs. Furthermore, when subjected to 15% (V/V) ethanol shock at 30 degrees C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient(P') of the 300 microm flocs accounted for only 43% and 52% respectively of those of the 100 microm and 200 microm populations. The effect of floc size distribution on the ethanol tolerance of the self-flocculating yeast strain SPSC01 was closely related to plasma membrane permeability. An optimal floc size distribution with the highest ethanol tolerance and ethanol production level could be obtained by controlling mechanical stirring speed of the bioreactor, which provides basis for the process optimization of fuel ethanol production using this self-flocculating strain.
Bioreactors ; microbiology ; Drug Tolerance ; Ergosterol ; biosynthesis ; Ethanol ; metabolism ; pharmacology ; Fermentation ; Flocculation ; Industrial Microbiology ; methods ; Particle Size ; Trehalose ; biosynthesis ; Yeasts ; drug effects ; growth & development ; metabolism