Statistical optimization of a cellulase from Aspergillus glaucus CCHA for hydrolyzing corn and rice straw by RSM to enhance yield of reducing sugar.

OBJECTIVE: The unique GH5 cellulase, AgCMCase, from Aspergillus glaucus CCHA was identified and characterized as having high cellulose and straw hydrolysis activities that were thermostable, pH stable and salt-tolerant. Therefore, it is a potential straw-degradation enzyme that can release reducing sugars in industrial applications. To increase the efficiency of the AgCMCase' hydrolysis of straw to release simple sugars, response surface methodology (RSM) was introduced to optimize hydrolysis parameters such as pH, temperature, reaction time and enzyme dose. RESULTS: The enzyme showed only one major protein band from the fermentation broth by the Pichia pastoris GS115 expression. The crude enzyme (without purification) showed a satisfactory capability to hydrolyze CMC-Na after 4 days of production. Here, the crude AgCMCase also showed cellulose and straw hydrolysis capabilities as assessed by scanning electron microscopic and Fourier-transform infrared spectroscopic analyses. A high-performance liquid chromatographic analysis demonstrated that the degradation of corn and rice straw by crude AgCMCase mainly produced glucose and cellobiose. Temperature, reaction time and enzyme dose were the significant variables affecting corn and rice straw degradation. After the optimization of RSM, a model was proposed to predict 1.48% reducing sugar yield with the optimum temperature (51.45 °C) and reaction time (3.84 h) from the straw degradation. The reaction of crude AgCMCase and rice straw in the optimized condition resulted in reducing sugar production of 1.61% that agrees the prediction. CONCLUSION: Our findings suggest that the crude AgCMCase is suitable to be used in straw conversion.

Abiostress resistance and cellulose degradation abilities of haloalkaliphilic fungi: applications for saline-alkaline remediation.

Soda saline-alkaline lands are significantly harmful to agriculture; thus, effective strategies to remediate such soil are urgently needed. Multiple negative factors exist in the community structure of saline-alkaline fields, among which the lack of fungal species diversity remains the most prominent problem. The haloalkaliphilic fungi are a unique group of extremophiles that grow optimally under conditions of extreme salinity and alkalinity; these fungi, which buffer salinity and alkalinity by absorbing and/or constraining salt ions, produce organic acids and/or macromolecules, secrete macromolecules such as cellulose degradation enzymes, and provide biomass that is beneficial for soil health. Considering that haloalkaliphilic fungi are a valuable genetic resource of resistance and degradation genes, these fungi are expected to be applied in biotechnology. Aspergillus glaucus exhibits high resistance to a variety of stressors and the ability to degrade crop straw; and it is a practical genetic tool that can be used to identify and validate genes involved in abiotic stress resistance and cellulose decomposition genes. This review will focus on the following aspects: isolation of extreme haloalkaliphilic fungi, fungal genes involved in salt and alkalinity resistance, macromolecule degrading enzymes, applications for genetic improvement of haloalkaliphilic fungi, and application of haloalkaliphilic fungi in saline-alkali soil mycoremediation.

The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications.

In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.