Intergenus Protoplast Fusion between Pichia manshurica and Rhodosporidium paludigenum to Increase the Production of Inulinase

Abstract

The purpose s of this study was to identify the optimum concentration of the lytic enzyme Glucanex for protoplast isolation and to conduct fusion for the purpose of increasing inulinase production . The study performs the protoplast fusion technique using Pichia manshurica and Rh odosporidium paludigenum . Protoplast fusion consists of a series of stages: protoplast isolation , protoplast fusion , protoplast regeneration , and analysis of hybrid fusion results . Protoplast isolation and fusion success rate are determined by various factors, including age of the culture , media type, and type of lytic enzymes used . H ybrid results were analyzed using a fungicide as a marker and measuring specific growth rate ( μ) of the hybrid compared with parental growth rates. Results demonstrated that a concentration of 4 mg/ mL of Glucanex produces the greatest number of protoplasts, 7.2 x 10 10 ( c ell/mL) for P. manshurica and 8.8 x 10 10 ( c ell/mL) for Rh . paludigenum . The results of analysis of hybrid fusions indicate that the study has identified a new fusant , called fusant F4 . Fusant F4 is capable of producing the highest inulinase, 0.6892 IU, compared with parentals P. manshurica, 0557 IU, and Rh . paludigenum, 0.3263 IU. Fusant F4 has specific growth rate ( μ) of 0.3360/h and generation time ( g) of 2.0629 h .