Screening and Mutation of Saccharomyces cerevisiae UV-20 with a High Yield of Second Generation Bioethanol and High Tolerance of Temperature, Glucose and Ethanol.

A wild-type strain was isolated from slightly rotted pears after three rounds of enrichment culture, identified as Saccharomyces cerevisiae 3308, and evaluated for its fermentation capability of second generation bioethanol and tolerance of temperature, glucose and ethanol. S. cerevisiae 3308 was mutated by using the physical and chemical mutagenesis methods, ultraviolet (UV) and diethyl sulfate (DES), respectively. Positive mutated strains were mainly generated by the treatment of UV, but numerous negative mutations emerged under the treatment of DES. A positive mutated strain, UV-20, produced ethanol from 62.33 ± 1.34 to 122.22 ± 2.80 g/L at 30-45 °C, and had a maximum yield of ethanol at 37 °C. Furthermore, UV-20 produced 121.18 ± 2.51 g/L of second generation bioethanol at 37 °C. Simultaneously, UV-20 exhibited superior tolerance to 50% of glucose and 21% of ethanol. In a conclusion, all of these results indicated that UV-20 has a potential industrial application value.

A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide.

Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher ß-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of ß-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.

Cloning, expression, sequence analysis, and partial characterization of two alkaline ß-1, 4-endoglucanases of Phaeosphaeria sp. LH21 from deep-sea mud.

Here we cloned and expressed two alkaline ß-1, 4-endoglucanases of Phaeosphaeria sp. LH21 from deep-sea mud. The two enzymes shared 71 and 63 % of identities with their known ß-1, 4-endoglucanases, respectively. According to the primary and spatial structures, the potential active sites of one of the two enzymes could be Asp122 and Asp11, while the other enzyme could be Asp16. The enzymatic properties of their recombinant enzymes from Pichia pastoris GS115 showed that they were optimally active at pH 8 and 60-65 °C, exhibited >90 % residual relative activities at pH 3-10, and obtained relative activities >75 % at pH 5-10.

Identification of Petriella setifera LH and characterization of its crude carboxymethyl cellulase for application in denim biostoning.

The phylogenetic tree of the partial elongation factor-1 alpha gene fits better than the partial 18S rDNA for generic classification. From the results of the molecular tree and analysis of morphological characters, Petriella setifera LH was identified. It can be induced to produce carboxymethyl cellulase (CMCase). The crude CMCase only shows a 44.1-kDa band by activity staining after SDS-PAGE. It is optimally active at 55°C and pH 6.0, and is stable from pH 5.0-8.0 and at 45°C or below. The crude CMCase, which is not affected by Co(2+), is strongly activated in the presence of 10 mM Na(+), K(+), Ca(2+), Mg(2+), EDTA, and Mn(2+). It is strongly inhibited by 10 mM Fe(2+), Pb(2+), Al(3+), Zn(2+), Ag(+), Fe(3+), and Cu(2+). When compared with denim treatment by Novoprime A800 (a commercial neutral cellulase), crude CMCase exhibits a similar fabric weight loss and indigo dye removal. These results indicate that crude CMCase has potential application in denim biostoning.

A comparative study of ß-1, 4-endoglucanase (possessing ß-1, 4-exoglucanase activity) from Bacillus subtilis LH expressed in Pichia pastoris GS115 and Escherichia coli Rosetta (DE3).

ß-1, 4-Endoglucanase (EG) from Bacillus subtilis LH was expressed in Escherichia coli Rosetta (DE3) and Pichia pastoris GS115, respectively. The CMCase activity of EG (EGE) from the cell lysate of DE3 reached 20,010U/ml, and that of EG (EGP) from the supernatant of GS115 was only 2008U/ml. EGE and EGP were bifunctional cellulases excluding ß-1, 4-glucosidase (BGL). The CMCases of them, optimally active at 65°C and pH 6.8, exhibited more than 80% residual activity at pH 5-10 and 60% activity at 40-70°C and pH 5-9. EGE (EGP) mixed with BGL had more than 1.5-fold higher CMCase and filter paperase activities compared to EGE (EGP). N-glycosylation protected EGP from immobilized-papain attack and accounted for 30kDa and a higher thermostability, whereas EGE was decomposed into a 33kDa active truncated EG (EGT) and two 18kDa fragments. EGE and EGP performed much better than EGT in denim biostoning.

Immunizing effects of cocultures of H22 hepatocarcinoma cells and cartilage polysaccharide on murine H22 hepatocarcinoma.

Hepatocarcinoma is a malignant cancer, which is threatening human lives. In order to disclose the immunizing effects of the cells and cartilage polysaccharide (CHCP) on liver cancer, murine H22 hepatocarcinoma model was set up. The survival time, life span, and survival rate of the CHCP group were better than model group or other groups which was immunized with cartilage short-chain polysaccharide (CPS) only or H22 cell lysate only. A series of experiments were proceeded and the results confirmed that the humoral immunity and cellular immunity were strengthened. HE staining, TUNEL assay, and Alcian staining were used to research the mechanism of the tumor specific antigen production which may be related to the apoptosis of H22 hepatocarcinoma cells induced by CPS or some new polysaccharide compound emerged. The animal experiment testified the relationship between H22 hepatocarcinoma cell apoptosis induced by CPS and the effect of murine H22 hepatocarcinoma immunoprophylaxis. Our data demonstrated that the coculture of cartilage polysaccharide and cancer cells may serve as a novel source of antihepatocarcinoma agent that may play an important role in future liver cancer immunoprophylaxis.