Solid state fermentation with Trametes versicolor was carried out on agricultural wastes containing bisphenol A (BPA). It was found that BPA degradation was along with the occurrence of laccase production, and wheat bran and corn straw were identified as suitable mixed substrates for laccase production. In the process of BPA degradation with T. versicolor, laccase activity increased rapidly at the 6th–10th day after inoculation. Moreover, BPA can enhance the production of laccase. After 10 days of fermentation, degradation rate of BPA exceeded 90% without the usage of mediators ABTS and acetosyringone at pH 4.0–8.0. In addition, metal ions did not affect the BPA degradation with T. versicolor. In vitro, the optimum pH range of BPA degradation with laccase was in the acidic region with the optimal performance of pH 5.0. Metal ions Cu2+, Zn2+, and Co2+ showed little effect on BPA degradation. However, Fe3+ and Fe2+ substantially inhibited the BPA degradation. Natural mediator acetosyringone showed optimum enhancement on BPA degradation. Greater than 90% of the estrogenic activity of BPA was removed by T. versicolor and its laccase. Compared to in vitro degradation with laccase, this study shows that the process of simultaneous laccase production and BPA degradation with T. versicolor was more advantageous since BPA can enhance the laccase production, mediators were unnecessary, degradation rate was not affected by metal ions, and the applicable pH range was broader. This study concludes that T. versicolor and laccase have great potential to treat industrial wastewater containing BPA.

CBH1 (cellobiohydrolase) comprises the majority of secreted proteins by Trichoderma reesei. For expression of Talaromyces thermophilus lipase gene in T. reesei, a self-designed CBH1 promoter was applied to drive the lipase gene expression cassette which was bracketed by flanking sequences of cbh1 gene for homologous recombination. Protoplast and Agrobacterium-mediated plasmid transformations were performed and compared, resultantly, transformation mediated by Agrobacterium was overall proved to be more efficient. Stable integration of lipase gene into chromosomal DNA of T. reesei transformants was verified by PCR. After shaking flask fermentation, lipase activity of transformant reached 375 IU mL−1, whereas no cellobiohydrolase activity was detected. SDS-PAGE analysis further showed an obvious protein band about 39 kDa and no CBH1 band in fermentation broth, implying lipase gene was successfully extracellularly expressed in T. reesei via homologous recombination at cbh1 locus. This study herein would benefit genetic engineering of filamentous fungi and industrial application of thermo-alkaline lipase like in paper making and detergents addition.

To heterologously express a Talaromyces thermophilus lipase gene in Trichoderma reesei, an efficient binary vector pChph-pCBH1sigpro-ttl which includes a newly designed cbh1 promoter and hygromycin-resistant marker was constructed. This plasmid was then transformed into T. reesei via improved Agrobacterium EHA 105-mediated transformation. After modification of co-culture conditions and enzymolysis treatment of conidia, 258 transformants were produced. A two-step screening method based on antibiotic resistance and capacity to utilize lactose and tributyrin was introduced to further select promising candidates, which would be additionally verified by PCR analysis, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and lipase activity assay. Lipase production was carried out in shaking flasks, and the activity reached 241 IU/mL (7415.4 IU/mg) after 84-h fermentation. It was found that this lipase performed high alkali and thermostable tolerance with the optimal pH 9.5 and temperature 60 °C, and it could retain more than 70 % activity after being disposed in pH 11 or 70 °C for 1 h. This study herein would benefit the genetic engineering of T. reesei and the industrial application of this important fungal lipase.

The neutral endo-β-glucanase gene cel5A from Humicola insolens was cloned and connected with the cellobiohydrolase 1 promoter from Trichoderma reesei to construct a recombinant plasmid pCB-hEG with the hygromycin B resistance marker. The plasmid was introduced into conidia of T. reesei using the Agrobacterium tumefaciens mediated transformation method. Eight transformants were obtained on screening plates with sodium carboxymethyl cellulose as the sole carbon source. Stable integration of the cel5A gene into the chromosomal DNA of T. reesei was confirmed by PCR. An obvious protein band (approximately 52 kDa) was detected by SDS-PAGE from fermentation broth, which showed that the cel5A gene in recombinant T. reesei successfully fulfilled efficient expression and extracellular secretion. After 96 h shaking-flask fermentation, the endo-β-glucanase activity at pH 6.5 from recombinant T. reesei reached 3,068 U/ml, which was 11 times higher than that of the host strain. In a 2 m3 fermenter, the endo-β-glucanase activity could be further increased to 8,012 U/ml after 96 h fermentation. The results showed a good prospect for application of neutral endo-β-glucanase in the textile industry.

The codon modified neutral endo-β-1,4-glucanase gene celEn, originating from the anaerobic fungus Orpinomyces sp. strain PC-2, was inserted between the strong promoter Pcel7A and the terminator Tcel7A from Trichoderma reesei. The resulting expression cassette was ligated to the pCAMBIA1300 Agrobacterium binary vector to construct pCB-hE that also contains a hygromycin B resistance marker. pCB-hE was introduced into T. reesei ZU-02 through an Agrobacterium tumefaciens–mediated transformation procedure that has been modified with an improved transformation efficiency of 12,500 transformants per 107 conidia. Stable integration of the celEn gene into the chromosomal DNA of T. reesei ZU-02 was confirmed by PCR. After 48 h fermentation in shaking flasks, the endo-β-1,4-glucanase activities increased to 55–70 IU ml−1 in transgenic strains, which were about 6–7 times higher than that of the original ZU-02 strain (9.5 IU ml−1). When the avicel was added in fermentation medium, the endo-β-1,4-glucanase activity in the transgenic strains could be further increased to 193.6 IU ml−1 after 84 h fermentation. Transgenic T. reesei strains with high neutral endo-β-1,4-glucanase activity will be particularly suitable for certain applications in textile industry. The improved procedures for overproduction and secretion of heterologous proteins in transgenic T. reesei can also be used to generate similar recombinant proteins for research or industrial purposes.

Three corn stover hydrolysates, enzymatic hydrolysates prepared from acid and alkaline pretreatments separately and hemicellulosic hydrolysate prepared from acid pretreatment, were evaluated in composition and fermentability. For enzymatic hydrolysate from alkaline pretreatment, ethanol yield on fermentable sugars and fermentation efficiency reached highest among the three hydrolysates; meanwhile, ethanol yield on dry corn stover reached 0.175 g/g, higher than the sum of those of two hydrolysates from acid pretreatment. Fermentation process of the enzymatic hydrolysate from alkaline pretreatment was further investigated using free and immobilized cells of recombinant Saccharomyces cerevisiae ZU-10. Concentrated hydrolysate containing 66.9 g/L glucose and 32.1 g/L xylose was utilized. In the fermentation with free cells, 41.2 g/L ethanol was obtained within 72 h with an ethanol yield on fermentable sugars of 0.416 g/g. Immobilized cells greatly enhanced the ethanol productivity, while the ethanol yield on fermentable sugars of 0.411 g/g could still be reached. Repeated batch fermentation with immobilized cells was further attempted up to six batches. The ethanol yield on fermentable sugars maintained above 0.403 g/g with all glucose and more than 92.83% xylose utilized in each batch. These results demonstrate the feasibility and efficiency of ethanol production from corn stover hydrolysates.

Four commonly used chemical pretreatment processes based on dilute acid, lime, aqueous ammonia steeping followed by dilute acid hydrolysis, and sodium hydroxide, were evaluated to provide comparative performance data. An obverse correlation between lignin removal and enzymatic digestibility of pretreated corn stover was observed. Compared with other three pretreatments, pretreatment of corn stover with 2% NaOH substantially increased the lignin removal and enhanced the accessibility and digestibility of cellulose. The hydrolysis yield of NaOH-pretreated corn stover reached 81.2% by 48 h at 8.0% substrate concentration and cellulase dosage of 20 FPU g−1 substrate. Chemical analysis showed that the enzymatic hydrolysate from NaOH-pretreated corn stover contained higher content of fermentable sugars and less inhibitors, which is suitable for subsequent fermentation process to produce ethanol. The research results are meaningful in bioconversion and utilization of renewable lignocellulosic biomass.

•A fungal lipase from Talaromyces thermophilus was secretorily expressed by Pichia pastoris.•Lipase production of recombinant Pichia pastoris increased by 2.6 times through optimization of fermentation condition.•Recombinant lipase worked excellently in alkaline and high temperature conditions.•Strong tolerance of recombinant lipase toward Ca2+, K+ and Na + was observed.Expression of a thermo-alkaline lipase gene from Talaromyces thermophilus in Pichia pastoris was researched to enhance its production. The lipase gene (TTL) was genetically optimized and inserted into the downstream of AOX1 promoter and α-factor to construct recombinant plasmid pPIC9K-TTL, which was then transformed into P. pastoris via electroporation, producing 220 positive transformants. Stable integration of lipase gene into chromosomal DNA of transformants was confirmed through PCR analysis. Lipase production was performed at lab scale, and an obvious protein band about 39kDa was detected using SDS-PAGE, which suggested lipase gene in recombinant P. pastoris was successfully extracellularly expressed. This lipase worked efficiently at pH range from 8.9 to 10.5, and showed the maximum activity at pH 9.5. After treating at pH 11 for one hour, 75% of its activity could be remained. It was active above 40 °C up to 70 °C, and the optimal temperature for reaction was 60 °C. High thermostability was observed, and more than 70% activity could be kept after one hour treatment at temperatures up to 80 °C. Lipase activity was promoted by Ca2+ and inhibited by Zn2+ and Cu2+. This research showed a bright prospect for industrial application of thermo- and alkaline-stable lipase.

•Isoproturon can be completely degraded by laccase-mediator systems.•Synthetic mediators are more effective for the isoproturon degradation.•Mediators can affect laccase stability in the degradation of isoproturon.•PEG can reduce the HBT dosage and enhance the degradation rate of isoproturon.•A higher degradation rate of isoproturon was observed in real wastewater.This study investigated the degradation of the herbicide isoproturon using Trametes versicolor laccase and its laccase-mediator systems. Isoproturon was poorly degraded with laccase alone, due to the presence of the relatively strong withdrawing electron group (NHCON(CH3)2) in the chemical structure of isoproturon. This study showed that laccase-mediator systems can effectively enhance the degradation rate of isoproturon. Within 24 h, isoproturon was completely degraded in the presence of 0.3 U/mL laccase and 1 mM 1-hydroxybenzotriazole (HBT). Compared with natural mediators, synthetic mediators are more effective in the laccase-mediated degradation of isoproturon. However, laccase activity rapidly declined in the presence of the synthetic mediator HBT. Degradation occurred at an acidic pH and optimum temperature was 50 °C. A high concentration (10 mM) of metal ions Cu2+, Zn2+ and Cd2+ positively enhanced isoproturon degradation with the laccase-HBT system. Polyethylene glycol (PEG) can reduce the HBT dosage in isoproturon degradation with the laccase-HBT system and enhance the degradation rate of isoproturon by increasing laccase stability. Moreover, a higher degradation rate of isoproturon was observed when incubating isoproturon with the laccase-HBT system in real wastewater compared to that in sodium citrate buffer. Finally, transformation products showed much lower ecotoxicity to green algae than the original isoproturon. This study concludes that laccase-mediator systems have great potential to treat industrial wastewater containing the herbicide isoproturon.