Abstract
Protoplasts are useful for applications such as cell fusion and genetic engineering. Here, we sought to find a proteinase inhibitor that can suppress proteinase activity in the protoplast preparation system based on preparation of a cell wall lytic enzyme from Trichoderma harzianum. Of the inhibitors tested, only the microbial alkaline proteinase inhibitor (MAPI), which is a specific inhibitor of serine-thiol proteinases, effectively reduced proteinase activity in the Trichoderma enzyme preparation. The number of protoplasts in the lytic reaction mixture with the Trichoderma enzyme reached a maximum after 2-3 h of incubation and gradually decreased thereafter. By contrast, the lytic reaction with MAPI incubation produced about 90% that of the lytic enzyme system without MAPI by 2 h of incubation and the number of protoplasts was still increasing after 4h. Furthermore, the protoplasts produced by the lytic reaction with MAPI reverted in regeneration medium at about 2.2times higher than the rate of those prepared by only the original Trichoderma enzyme. This rate was about 10% higher than that observed for protoplasts produced in the presence of bentonite.
