Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.
Bioreactors ; Catalysis ; Escherichia coli ; genetics ; Glucose ; Glucosyltransferases ; Industrial Microbiology ; Organisms, Genetically Modified ; Trehalose ; biosynthesis

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.
Acanthamoeba castellanii/*enzymology/genetics/metabolism ; Aldose-Ketose Isomerases/*biosynthesis ; Amebiasis/*pathology ; Benzenesulfonates ; Cell Wall/chemistry/genetics/*metabolism ; Cellulose/biosynthesis ; Down-Regulation ; Encephalitis/parasitology ; Glucosyltransferases/*biosynthesis/genetics ; Keratitis/parasitology ; Microscopy, Electron, Transmission ; RNA Interference ; RNA, Small Interfering

To investigate the mechanism of high product specificity of gamma-clodextrin glucanotransferase (CGTase) from alkalophilic Bacillus clarkii 7364, we aligned protein sequence and structure model, found out that loss of 6 amino acids at subsite -7 probably affected its product specificity. Using overlapping PCR method, we inserted 6 amino acids into subsite -7 of CGTase. The mutant CGTase gene was ligated with pET-20b (+) and expressed in Escherichia coli BL21 (DE3). The extracellular recombinant enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild CGTase, the gamma-CDs produced by mutant enzyme decreased from 76.0% to 12.5%, whereas the ratio of alpha- and beta-CDs increased from 8.7% and 15.2% to 37.5% and 50%. The possible mechanism was that, compared to alpha-, beta-CGTase, wild gamma-CGTase lacks 6 amino acids in its subsite -7. This conformation provided more space for glucose combination and was thus advantageous for forming gamma-CD. When the 6 amino acids were inserted into the subsite -7 of wild gamma-CGTase, the space to bind with glucose reduced and consequently resulted in less gamma-CD production.
Bacillus ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; biosynthesis ; genetics ; Mutant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of glucosylceramide synthase (GCS) on P-glycoprotein (P-gp) expression via extracellular signal-regulated kinase (ERK) pathways in leukemia K562/A02 cell line.

METHODSK562/A02 multidrug resistance cells were treated with GCS siRNA and U0126, respectively. Expression of multidrug resistance protein 1 (MDR1) mRNA was analyzed with qRT-PCR. Phosphorylated ERK1/2, total ERK1/2 protein and P-gp in different groups were measured with Western blotting.

RESULTSAfter treated with U0126, P-ERK1/2 was decreased along with the increased U0126 concentration. P-ERK1/2 and P-gp were apparently down-regulated by U0126 at the concentrations of 20 μmol/L, 40 μmol/L and 60 μmol/L. After being transfected with GCS siRNA, GCS mRNA was inhibited by 70.50% (58.00%-76.00%) in K562/A02 cells. Compared with the negative control, both P-ERK1/2 and P-gp were inhibited significantly after RNAi for 72 hours (P<0.01 and P<0.05, respectively.

CONCLUSIONGCS may affect the expression of P-gp by ERK signal transduction pathway in leukemia cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; metabolism ; Cell Line, Tumor ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Glucosyltransferases ; metabolism ; Humans ; K562 Cells ; Leukemia ; enzymology ; genetics ; metabolism ; MAP Kinase Signaling System

The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.
Acanthamoeba castellanii/*enzymology/genetics/growth & development ; Amebiasis/*parasitology ; Cell Wall/*metabolism ; Cellulose/*biosynthesis/genetics ; Glucosyltransferases/genetics/metabolism ; Glycogen Phosphorylase/genetics/metabolism ; Protozoan Proteins/genetics/*metabolism ; UTP-Glucose-1-Phosphate Uridylyltransferase/genetics/metabolism

Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.
Arbutin ; biosynthesis ; Catalysis ; Enzyme Stability ; Escherichia coli ; enzymology ; genetics ; Glucosyltransferases ; biosynthesis ; genetics ; metabolism ; Hydroquinones ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Sucrose ; metabolism

OBJECTIVEClone of sucrose synthase of Dendribium officinale and expression analysis, to provide the theory basis for research the relationship between polysaccharide synthesis of D. officinale and sucrose synthase activity.

METHODAccording to homologous sequence of sucrose synthase gene on GenBank, application the technology of RT-PCR and RACE, clone the full length of D. officinale. Target gene amplified with T vector was transformed into competent E. coli. BL21, IPTG induced expression, SDS-PAGE analysis.

RESULTA full length cDNA encoding sucrose synthase was isolated from the D. officinale, named DOSS1, the GenBank accession number is HQ856835, the cDNA is 2781 bp in length containing an open reading frame of 2424 bp encoding 807 amino acids with a predicted molecular mass of 92.3 x 10(3), the deduced amino acid sequence of D. officinale sucrose synthase shares 95% identity with Mokara yellow (AF530568); shares 90% identity with Oncidium goldiana (AF530567); shares more than 80% with other monocotyledonous plants.

CONCLUSIONCloned the sucrose synthase gene and induced an obvious band successfully.

3' Untranslated Regions ; genetics ; 5' Untranslated Regions ; genetics ; Cloning, Molecular ; Dendrobium ; enzymology ; genetics ; metabolism ; Escherichia coli ; genetics ; Gene Expression Regulation, Plant ; Glucosyltransferases ; genetics ; metabolism ; Phylogeny ; Polysaccharides ; biosynthesis

To enhance the thermostability and storage stability of alpha-cyclodextrin glycosyltransferase (a-CGTase), we added specific chemical additives into the preparation of alpha-CGTase, and studied the effect of additives on the storage stability of alpha-CGTase at different temperatures. Then we measured the protein structure of CGTase in the far UV (200-250 nm) and near UV (250-320 nm) ranges respectively by Circular dichroism (CD) spectra under high temperature and analyzed the relationship between thermostability and protein structure. The results indicated that the addition of selected additives (gelatin, glycerin, CaCl2 and PEG400) enhanced the thermostability of alpha-CGTase dramatically. After 45 days, the preparation of alpha-CGTase still had 100% of the enzyme activity with different additives superimposed at the optimum concentration at 40 degrees C. The CD spectra of alpha-CGTase showed that glycerin could protect the secondary and the tertiary structure of the CGTase under high temperature and therefore the enzyme maintained its high activity. Chemical additives can improve the stability of alpha-CGTase significantly and they preserve the enzyme activity by protecting its secondary structure.
Enzyme Stability ; drug effects ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; biosynthesis ; chemistry ; genetics ; Glycerol ; chemistry ; Paenibacillus ; enzymology ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

Improving stress tolerance of the microbial producers is of great importance for the process economy and efficiency of bioenergy production. Key genes influencing ethanol tolerance of brewing yeast can be revealed by studies on the molecular mechanisms which can lead to the further metabolic engineering manipulations for the improvement of ethanol tolerance and ethanol productivity. Trahalose shows protective effect on the cell viability of yeast against multiple environmental stress factors, however, further research is needed for the exploration of the underlying molecular mechanisms. In this study, the promoter region of the trehalose-6-phosphate synthase gene TPS1 was cloned from the self-flocculating yeast Saccharomyces cerevisiae flo, and a reporter plasmid based on the expression vector pYES2.0 on which the green fluorescence protein EGFP was directed by the TPS1 promoter was constructed and transformed to industrial yeast strain Saccharomyces cerevisiae ATCC4126. Analysis of the EGFP expression of the yeast transformants in presence of 7% and 10% ethanol revealed that the P(TPS1) activity was strongly induced by 7% ethanol, showing specific response to ethanol stress. The results of this study indicate that trehalose biosynthesis in self-flocculating yeast is a protective response against ethanol stress.
Base Sequence ; Cloning, Molecular ; Ethanol ; metabolism ; pharmacology ; Glucosyltransferases ; biosynthesis ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Saccharomyces cerevisiae ; enzymology ; genetics ; metabolism ; Stress, Physiological ; physiology

By constructing the genomic DNA library of Meiothermus ruber CBS-01, the genes of trehalose phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP) involved in trehalose synthesis were cloned. The genes were cloned into the plasmid pET21a, and expressed in Escherichia coli Rosetta gami (DE3). The activities of these two purified enzymes were confirmed by thin layer chromatography (TLC). Meanwhile, we tested the cellular compatible solutes of M. ruber CBS-01 under different environmental pressure, and found that under hyperosmotic pressure, this strain can accumulate trhalose-6-phosphate, but not trehalose. These results can give more insight to future research in the roles of TPS/TPP and TreS pathway.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Thermus ; enzymology ; genetics ; Trehalose ; biosynthesis