---

Cellulase is one of the industrial enzymes which its production and utilization is increasingly taking into consideration due to global heed to second-generation bioethanol production. Cellulase produced by different organisms such as fungi, bacteria, insects, and plants. With increase in utilization of this enzyme and need for reduction in the enzymes price for production of second-generation bioethanol, the production of recombinant enzyme has been considered noticeably.
In this study, by investigation of corn steep liquor as nitrogen source and second carbon source after glycerol, a new medium is designed based on SYN6 salt medium then biomass and endoglucanase II production by methylotrophic yeast was optimized. Experiments designed by one-factor and response surface methodology used for optimization.
Results showed that optimum conditions for biomass and endoglucanase production are 5.5% (w/v) and 6.15% (w/v) of corn steep liquor respectively. New optimized conditions increased 41.4% and 69.7% for biomass and recombinant enzyme production respectively.

---

Aims: Invertase is an enzyme that is widely used in industries. The main source of industrial production of invertase is yeast Saccharomyces cerevisiae (S. cerevisiae). Increasing thermal stability makes an important contribution to improving productivity in related production. The aim of this study was increasing thermal stability of Saccharomyces cerevisiae recombinant protein invertase by site-directed mutagenesis.
Materials and Methods: In the present experimental study, using invertase enzyme from thermophilic bacteria, Thermotoga maritima as template, it was decided to replace the threonine 345 and asparagine 349 amino acid with alanine, using site-directed mutagenesis and in Pichia pastoris, cloning was performed with the SOEing polymerase chain reaction. The activity of natural and mutant recombinant invertase enzymes at different temperatures, different pHs, stability duration, and thermal-performance stability, and Michaelis–Menten kinetics were drawn.
Findings: The thermal-structural stability of the natural and mutant invertease enzymes at 55°C showed that the mutant enzyme had a higher thermal stability at 55°C compared with the natural enzyme. Both natural and mutant enzymes exhibited a similar trend in functional stability. Reduction of K_m and increase of V_max in sucrose substrate and 5-fold increase in K_cat/K_m ratio of mutant enzyme was observed.
Conclusion: Site-directed mutagenesis has no negative effect on the amount of production as well as the secretion of recombinant protein invertase and increases enzyme activity. The mutant enzyme has a higher structural stability than the natural enzyme without altering its functional stability.