Overproduction of beta-1,6-glucanase in Trichoderma harzianum is controlled by extracellular acidic proteases and pH

To produce high amounts of extracellular endo-beta-1,6-glucanase, we overexpressed the gene bgn16.2 from Trichoderma harzianum under the control of the pyruvate kinase gene promoter (pki) of T. reesei. Transcription of bgn16.2 gene increased under most conditions but not extracellular beta-1,6-glucanase levels. Relationship of extracellular BGN16.2 protein and presence of proteases was studied in order to maximize production. After changing the carbon and nitrogen sources and buffering the culture media at different pHs, four major proteases, the acidic ones being pH-regulated, were detected. Overexpression of BGN16.2 at low pH resulted in BGN16.2 degradation, due to the induction of aspartyl proteases and to instability at pH below 3. Maximal overproduction of BGN16.2 albeit pure was achieved in buffered medium, where pH-induced aspartyl proteases were absent or when some nitrogen sources, such as yeast extract, peptone or casein were substrate for these proteases.