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.

The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence

BACKGROUND Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

Screening and bioinformatic analysis of trehalase in Thelazia callipaeda / 中国血吸虫病防治杂志

Objective To characterize the trehalase gene in Thelazia callipaeda through screening the annotated data of the T. callipaeda genome, and to investigate the biological characteristics of the trehalase gene-coding protein. Methods The trehalase gene was screened from the T. callipaeda genome and subjected to validation by using a PCR assay. The structural features of the coding protein were analyzed with bioinformatics tools, including hydrophobicity, transmembrane region, signal peptides, conserved domains, as well as the secondary and tertiary structures and the antigen epitope. Homology analysis of the amino acid sequences was performed, and the phylogenetic tree was built by the MEGA X software. In addition, the protein-protein interaction network was deduced from the STRING database. Results The sequence of the trehalase gene with the complete CDS region was obtained from T. callipaeda genome, which had a length of 1 638 bp and encoded 545 amino acids. The encoded protein was predicted to have a molecular weight of 63 478.48 ku and be a secretory protein. The 5′ domain of the encoded protein contained a signal peptide without transmembrane regions, and was predicted to contain 7 antigen epitopes. Based on the protein-protein interaction network of nematodes in the STRING database, the protein-protein interaction network of the trehalase gene of T. callipaeda was deduced, and 27 interactions covering 10 genes were identified. Conclusions A trehalase gene is successfully identified in T. callipaeda genome and its coding protein receives a bioinformatics analysis, which provides insights into the research on the biological functions of the protein and the screening of vaccine candidates for thelaziasis callipaeda.

Effects of low temperature on trehalose and trehalase contents in Culex pipiens pallens / 中国血吸虫病防治杂志

Objective To examine the effect of low temperature on trehalose and trehalase levels in Culex pipiens pallens. Methods The fourth instar larvae and female adult mosquitoes of Cx. pipiens pallens were exposed at 4 ℃ for 0, 1, 3, 6, 12, 18, 24 h and 0, 1, 3, 6, 12, 18, 24, 36, 48, 72 h, respectively. Then, the trehalose and trehalase contents were detected by enzyme-linked immunosorbent assay (ELISA) in mosquitoes. Results The contents of trehalose and trehalase significantly increased in the larval and female adult mosquitoes post-exposure to low temperature. The changing trend of trehalose levels was consistent in the larval and female adult mosquitoes, and the highest levels were (2.458 8 ± 0.379 2) mg/g and (2.825 7 ± 0.211 1) mg/g 3 h post-exposure to low temperature, respectively. The trehalose and trehalase levels fluctuated greatly within the first 6 h post-exposure to low temperature. Following adaptation for a period of time, the trehalose and trehalase levels remained at a relatively high level. Conclusion Low temperature may induce the production of trehalose and trehalase in Cx. pipiens pallens, and the trehalose and trehalase may play an important role in the improvement of the cold resistance.

Effects of low temperature on trehalose and trehalase contents in Culex pipiens pallens / 中国血吸虫病防治杂志

Objective To examine the effect of low temperature on trehalose and trehalase levels in Culex pipiens pallens. Methods The fourth instar larvae and female adult mosquitoes of Cx. pipiens pallens were exposed at 4 ℃ for 0, 1, 3, 6, 12, 18, 24 h and 0, 1, 3, 6, 12, 18, 24, 36, 48, 72 h, respectively. Then, the trehalose and trehalase contents were detected by enzyme-linked immunosorbent assay (ELISA) in mosquitoes. Results The contents of trehalose and trehalase significantly increased in the larval and female adult mosquitoes post-exposure to low temperature. The changing trend of trehalose levels was consistent in the larval and female adult mosquitoes, and the highest levels were (2.458 8 ± 0.379 2) mg/g and (2.825 7 ± 0.211 1) mg/g 3 h post-exposure to low temperature, respectively. The trehalose and trehalase levels fluctuated greatly within the first 6 h post-exposure to low temperature. Following adaptation for a period of time, the trehalose and trehalase levels remained at a relatively high level. Conclusion Low temperature may induce the production of trehalose and trehalase in Cx. pipiens pallens, and the trehalose and trehalase may play an important role in the improvement of the cold resistance.

Evaluation urinary trehalase in patients with renal proximal tubular damage / 中华检验医学杂志

Objective To investigate the value of urinary trehalase (T) in patients with renal proximal tubular damage.Methods 134 patients with kidney disease (male:66 female:68 age:18-59 ; 31cases with acute renal failure,30 cases with chronic renal failure,20 cases with drug-induced renal impairment,21 cases with renal transplantation and 32 cases with nephritic syndrome) and 101 healthy controls (58 males and 43 females) were selected.Urinary T,N-acetyl-D-glucosaminidase (NAG),β2-MG,gamma-glutamyl transferase (GGT) were detected.Data were analyzed by SPSS 11.5,including nonparametric rank test,ROC analysis.Results The level of urinary trehalase in control group was normally distributed (7.1 ± 4.1) μmol/h · g Cr (0-25 μmol/h · g Cr).There was no significant difference between men and women (t =0.63,P =0.53).Urinary T levels were significant higher in all kidney disease groups than in control group (Z =6.80,5.90,5.23,6.00,8.04,P ＜0.01).According to ROC curve,the area of urinary T under the ROC curve (AUC) in 134 patients was 0.9,significantly different with NAG,β2-MG,GGT area (P ＜ 0.01),the AUCs of T were 0.94,0.85,0.90,0.90,0.91 in acute and chronic renal failure group,drug-induced renal impairment group,renal transplantation group and nephritic syndrome group,respectively; Youden index were 0.85,0.65,0.77,0.66,0.72 respectively.Corresponding to the Youden index,sensitivity and specificity were 90.3％ and 95.1％,73.3％ and 92.1％,85.0％ and 92.1％,81.0％ and 85.2％,87.5％ and 84.2％ respectively.Conclusions The Urinary trehalase is better than other markers in the diagnosis of the proximal renal tubular damage.It was better to evaluate the proximal tubular function early in time.The diagnostic value of urinary trehalase played a key role in diagnosis,treatment and prognosis of kidney diseases.

Two forms of trehalase in rabbit enterocyte: Purification and chemical modification.

Trehalase found to be associated with the brush border membrane vesicles and the Ca2+ aggregated basolateral membrane vesicles were purified to homogeneity. They were found to differ in their molecular weight, subunit structure, heal stability, N-terminal residues, amino acid composition and also the active site residues. Chemical modification showed the presence of a histidine and tyrosine at the active site of brush border membrane vesicle trehalase and two histidines at the active site of basolateral membrane vesicle.

Purification and properties of trehalase from monkey small intestine.

Brush border membrane trehalase was purified from monkey small intestine by a procedure which includes solubilisation by Triton X-100, ammonium sulphate fractionation, and chromatography on DE-52 and hydroxyapatite. The purified enzyme had a specific activity of 11 units/mg protein and was purified 140-fold. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis. It had a Km value of 17·4 mM for trehalose and a Vmax of 1·33 units. Sucrose and Tris acted as competitive inhibitors of the enzyme.