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.

First report of the production of a potent biosurfactant with α, ß-trehalose by Fusarium fujikuroi under optimized conditions of submerged fermentation

Abstract Biosurfactants have many advantages over synthetic surfactants but have higher production costs. Identifying microorganisms with high production capacities for these molecules and optimizing their growth conditions can reduce cost. The present work aimed to isolate and identify a fungus with high biosurfactant production capacity, optimize its growth conditions in a low cost culture medium, and characterize the chemical structure of the biosurfactant molecule. The fungal strain UFSM-BAS-01 was isolated from soil contaminated with hydrocarbons and identified as Fusarium fujikuroi. To optimize biosurfactant production, a Plackett-Burman design and a central composite rotational design were used. The variables evaluated were pH, incubation period, temperature, agitation and amount of inoculum in a liquid medium containing glucose. The partial structure of the biosurfactant molecule was identified by nuclear magnetic resonance spectrometry. F. fujikuroi reduced surface tension from 72 to 20 mN m−1 under the optimized conditions of pH 5.0, 37 °C and 7 days of incubation with 190 rpm agitation. The partial identification of the structure of the biosurfactant demonstrated the presence of an α,β-trehalose. The present study is the first report of the biosynthesis of this compound by F. fujikuroi, suggesting that the biosurfactant produced belongs to the class of trehalolipids.

Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants.

Sucrose is required for plant growth and development. The sugar status of plant cells is sensed by sensor proteins. The signal generated by signal transduction cascades, which could involve mitogen-activated protein kinases, protein phosphatases, Ca 2+ and calmodulins, results in appropriate gene expression. A variety of genes are either induced or repressed depending upon the status of soluble sugars. Abiotic stresses to plants result in major alterations in sugar status and hence affect the expression of various genes by down- and up-regulating their expression. Hexokinase-dependent and hexokinase-independent pathways are involved in sugar sensing. Sucrose also acts as a signal molecule as it affects the activity of a proton-sucrose symporter. The sucrose trans-porter acts as a sucrose sensor and is involved in phloem loading. Fructokinase may represent an additional sensor that bypasses hexokinase phosphorylation especially when sucrose synthase is dominant. Mutants isolated on the basis of response of germination and seedling growth to sugars and reporter-based screening protocols are being used to study the response of altered sugar status on gene expression. Common cis-acting elements in sugar signalling pathways have been identified. Transgenic plants with elevated levels of sugars/sugar alcohols like fructans, raffinose series oligosaccharides, trehalose and mannitol are tolerant to different stresses but have usually impaired growth. Efforts need to be made to have transgenic plants in which abiotic stress responsive genes are expressed only at the time of adverse environmental conditions instead of being constitutively synthesized.

Regulation of trehalose metabolism by Adox and AdoMet in Saccharomyces cerevisiae.

Effect of a potent methylation inhibitor oxidized adenosine (Adox), and a universal methyl group donor S-adenosyl-L-methionine (AdoMet) on trehalose metabolism was studied in two haploids of S. cerevisiae of mating types MATalpha, met3 (6460 -8D) and MATa, leu2, ura3, his4 (8534 -10A). Trehalose level decreased in presence of Adox in both strains. Both neutral trehalase (NT) and trehalose-6-phosphate (tre-6-p) synthase activities increased in presence of Adox in -8D strain. Decrease in trehalose level in -8D thus could not be explained in the light of increased tre-6-p synthase activity; however, it could be correlated with increased NT activity. In strain -10A, NT activity was reduced in presence of Adox while tre-6-p synthase activity increased. Enzyme activity profiles in -10A thus do not explain the reduced trehalose level on Adox treatment. Effect of AdoMet was not very prominent in either strain, though in -8D a small increase in trehalose level was seen on treatment. Intracellular AdoMet level of untreated cells of -10A was seen to be almost six times higher than that of -8D. Further, AdoMet treatment caused increase in its level compared to untreated cells, suggesting AdoMet uptake. No effect of either compound was seen on acid trehalase (AT) activity in any strain. The results suggest that there was a possible effect of demethylation on trehalose metabolism (particularly in the synthetic direction) in both strains, though effect of methylation was not very prominent, the reason for which is not very clear.

Aumento de la actividad panificante de levaduras comerciales por aplicación de condiciones de estrés durante su propagación / Increase of baking activity of commercial yeasts by application of stress conditions during their propagation

La actividad panificante valorada como producción de CO2 de dos cepas comerciales de Saccharomyces cerevisiae pudo ser incrementada, principalmente en amasijos azucarados, por la aplicación de un esquema de “hambreado/ pulso¼ de melaza de caña de azúcar durante su propagación bajo la forma de lote alimentado. Dicho incremento fue dependiente de la cepa utilizada. Otras características relacionadas con el comportamiento industrial de las levaduras no se vieron afectadas, con excepción de la concentración intracelular de trehalosa.Se discute la aplicabilidad del método para la producción industrial de levaduras de panificación.

Baking activity determined as CO2 production of two commercial strains of Saccharomyces cerevisiae could be increased mainly in sweet bread doughs by introducing a “starvation/ pulse feeding” schedule of sugar cane molasses during a fed-batch propagation . Such increase was strain dependent. Except for the trehalose intracellular level, other traits related to the yeast industrial performance were unaffected. Applicability of method for baker‘s yeast industrial production is discussed.

The role of trehalose in cell stress

Water is usually thought to be required for the living state, but many organisms can withstand anhydrobiosis When essentially all of their body water has been removed. The mechanisms for survival to this Kind of stress could be similar in microbes, plants and animals. One common feature is the accumulation of sugars by anhydrobiotic organisms. Trehalose, which is one of the most effective saccharides in preventing phase transition events in the lipid bilayer, is accumulated by anhydrobiotic organisms in large amounts. It lowers membrane phase transitions in dry yeast cells, thus preventing imbibitional damages when cells are rehydrated. Yeast cells have a trehalose carrier in the plasma membrane which endows them with the ability to protect both sides of the membrane. Kinetic analysis of the trehalose transport activity in Saccharomyces cerevisiae cells revealed the exoistence of a multicomponent system with a constitutive low-affinity uptake component and a high-affinity H+ - trehalose symporter regulated by glucose repression.

Trehalose metabolism: a new horizons in technological applications

Trehalose is responsible for the survival of anhydrobiotic organisms when under stress. Trehalose is a unique, non-reducing, extremely stable disaccharide which is able to protect proteins and membranes from damage caused freezing, high temperatures and dehydration. Yeasts accumulate large amounts of trehalose and constitue excellent models for studying the response of eurocaryotic cells to diverse stresses. The regulation of trehalose metabolism is reviwed and new technological applications for preservation of biological materials are discussed

Effect of dimethylsulfoxide on signal transduction in mutants of Saccharomyces cerevisiae

As the first part of a study of pesticide toxicity we report the effects of the solvent dimethylsulfoxide (DMSO) on signal transduction in mutants of Saccharomyces cerevisiae. The enzymes of trehalose metabolism, which are activated and deactivated by a "glucose signal" and by heat shock treatment, were chosen as targets for this study. DMSO was shown to be able to permeate glucose and cAMP. The effects of glucose and cAMP were enhanced by pre-incubating the cells in the presence of DMSO. No effects were observed during the heat shock, suggesting that the solvent acts on the cell membrane. The results suggest that DMSO may be used as a vehicle for small molecules which do not easily penetrate yeast cell membranes, thus providing a new tool for biochemical and toxicological studies

Fructose 2,6-bisphosphate and trehalose metabolism in Saccharomyces cerevisiae

1. A regulatory mutant of Sccharomyces (fdp) unable to activate fructose 1,6-bisphosphatase present a normal response to the glucose and fructose signals as measured by trehalase activation, indicating that the inability of the strain to grow on these sugars is caused by a defect located beyond membrane interactions. 2. In vivo experiments with a mutant strain bearing a phosphoglucoisomerase gene (pgil-delta) deletion showed that activation of trehalase and deactivation of the tehalose-6-phosphate synthase complex occurred to the same extent whether glucose or fructose was used as signal. 3. These results suggest that fructose-2,6-bisphosphate is not involved in the interconversion of forms of the enzymes of trehalose metabolism. Furthermore, when fructose-2,6-bisphosphate was assayed on trehalose synthesizing activity using cell-free extracts and partially purified preparations of the complex, no effect was observed. 4. We conclude that regulation by cAMP fulfills the requirements for control of trehalose levels in Saccharomyces