Isolation and Characterization of a Novel Cold-Active, Halotolerant Endoxylanase from Echinicola rosea Sp. Nov. JL3085T

We cloned a xylanase gene (xynT) from marine bacterium Echinicola rosea sp. nov. JL3085T and recombinantly expressed it in Escherichia coli BL21. This gene encoded a polypeptide with 379 amino acid residues and a molecular weight of ~43 kDa. Its amino acid sequence shared 45.3% similarity with an endoxylanase from Cellvibrio mixtus that belongs to glycoside hydrolases family 10 (GH10). The XynT showed maximum activity at 40 °C and pH 7.0, and a maximum velocity of 62 μmoL min−1 mg−1. The XynT retained its maximum activity by more than 69%, 51%, and 26% at 10 °C, 5 °C, and 0 °C, respectively. It also exhibited the highest activity of 135% in the presence of 4 M NaCl and retained 76% of its activity after 24 h incubation with 4 M NaCl. This novel xylanase, XynT, is a cold-active and halotolerant enzyme that may have promising applications in drug, food, feed, and bioremediation industries.

Expression and function of an HAC1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae

To investigate the effects of transcription factor HAC1, which is involved in the unfolded protein response pathway, on the expression of xynB in Saccharomyces cerevisiae , we used overlap extension polymerase chain reaction (PCR), rDNA integration, and droplet digital PCR technology to generate a S . cerevisiae strain (S8) containing eight copies of the xylanase gene, allowing high-yield secretory expression of xylanase. HAC1 was then overexpressed in the S8 strain, and the GeXP system was used to study the effects of HAC1 overexpression on the expression of genes associated with protein folding in the endoplasmic reticulum (ER). Results confirmed the constitutive secretory expression of the multiple copies of the xylanase gene in S . cerevisiae following rDNA-based integration of the expression cassette. Specifically, recombinant S . cerevisiae strain S8, containing eight copies of the xylanase gene, showed maximum xylanase expression, with a yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL. These results confirmed that overexpression of HAC1 improves the expression of genes associated with protein folding in the ER, enhancing the protein folding and assembly functions of the ER and increasing xylanase expression.

Stimulatory Effects of Sublethal Doses of Carbendazim on the Virulence and Sclerotial Production of Botrytis cinerea

Stimulatory effects of low doses of fungicides on the virulence of phytopathogens have profound implications for applications of fungicides. The present study demonstrated that carbendazim sprayed at 0.001 to 0.03 μg/ml had stimulatory effects on the virulence of mycelia of Botrytis cinerea, and the maximum percent stimulations were 15.5 and 21.4% for isolates HB459 and HB536, respectively. Potato dextrose agar (PDA) amended with carbendazim at 0.01, 0.02, and 0.05 μg/ml inhibited mycelial growth of isolate HB536 by 0.8, 10.0, and 30.6%, respectively. However, after the inhibited mycelia were inoculated on cucumber leaves, virulence increased by 10.1, 12.9, and 10.8%, respectively. With respect to sclerotial production, carbendazim at 0.005 and 0.02 μg/ml in PDA significantly (P < 0.05) increased, while at 0.1 μg/ml significantly (P < 0.05) reduced the sclerotial number and weight of both isolates compared with nontreated controls. Conidia germination percentages slightly yet statistically significantly (P < 0.05) increased after being inoculated on PDA amended with carbendazim at 0.001 and 0.005 μg/ml. Carbendazim at 0.001∼0.02 μg/ml, either sprayed on cucumber leaves or cosuspended with conidia, exerted significantly (P < 0.05) stimulatory effects on the virulence of B. cinerea conidia. Mechanism studies showed that sublethal doses of carbendazim did not increase the expression levels of pathogenicity-related pectin methylesterase gene Bcpme1, endopolygalacturonase gene Bcpg2, cutinase gene CutA, xylanase gene Xyn11A, or NADPH oxidase gene BcnoxA.