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

The purposes of this study was to identify the opti mum 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 Rhodosporidium paludigenum. Protoplast fusion consists of a series of stages: protoplast isolation, protoplast fusion, pr otoplast regeneration, and analysis of hybrid fusio n results. Protoplast isolation and fusion success rate are determined by various factors, including age of the culture, med ia type, and type of lytic enzymes used. Hybrid results were analyzed us ing 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/m L of Glucanex produces the greatest number of protoplasts, 7.2 x 10 (cell/mL) for P. manshurica and 8.8 x 10 10 (cell/mL) for Rh. paludigenum. The results of analysis of hybrid fusions indicat e that the study has identified a new fusant, calle d fusant F4. Fusant F4 is capable of producing the highest i nulinase, 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.


Introduction
Microbes, especially yeasts, can produce an enzyme that degrades inulin, called inulinase (EC. 3.2.1.7). The numbers of inulinase enzymes produced by microbial products and activities are usually few, and increasing their production requires either optimizing the microbial growth environment or genetic manipulation. Naturally occurring yeast on dahlia tubers contains inulinase, the enzyme that converts inulin into fructose. Indigenous inulinolytic yeasts have been found in the wild plant Dahlia sp. in Bandungan-Ambarawa in central Java [1].
These inulinolytic yeasts are P. manshurica and Rh. paludigenum, both of which are capable of producing inulinase, albeit in small quantities [1]. Several research have created strains improved for inulinase production by manipulating growth environment or genetics [2]. One technique for improving the yeast strain is the protoplast fusion technique, which has been used intensively to improve yeast strains in a manner that overcomes the difficulties of conducting hybridization sexually. Protoplast fusion does not require cell trait or vector competence, and fusant testing is simpler and more systematic [3]. Given that inulinase enzymes produced by indigenous yeasts are few, this research seeks to increase the quality of the strain in enzyme production.

Methods
Yeast strains and media. P. manshurica and Rh. Paludigenum were obtained from laboratory at the Faculty of Mathematics and Natural Sciences, Universitas Diponegoro [1]. Both yeasts were grown at 27 o C on yeast peptone dextrose (YPD) medium containing (w/v) yeast extract 1%, peptone 2%, and glucose 2%, with a pH of 5.5-6.0 [4]. Cultures were grown in liquid media in conical flasks containing 10% by volume of the broth and agitated on a rotary shaker at 120 rpm [5].
Protoplast preparation and fusion. P. manshurica and Rh. paludigenum were grown on YPD until the log phase, when cultures were harvested and suspended in sorbitol osmotic stabilizer 1 M in 0.2 M H 2 PO 4 /Na 2 HPO 4 buffer with a pH of 5.8. [4]. After washing with the same buffer, cells were incubated at 27 o C for 90 min with Glucanex 2 mg/mL and 4 mg/mL to achieve protoplast isolation. Protoplasts were collected from both types of cultures (0.5 mL P. manshurica and 0.5 mL Rh. paludigenum), and a fusion solution containing polyethylene glycol (PEG) 6000, CaCl 2 , and glycine was prepared [6].
Assay of inulinase activity. Inulinase activity was determined through the 3.5-dinitrosalicylic acid (DNS) method [9] using inulin as a substrate. One unit of inulinase activity was defined as the amount that liberates 1 µmol of fructose equivalent from inulin per min [10].

Results and Discussion
Culture conditions and enzymatic isolation of protoplasts. In this study, P. manshurica (Pm) and Rh. paludigenum (Rhp) were grown on YPD medium and harvested after 24 h (Figure 1), in the late exponential (log) phase, when yeast cells are immature and their cell walls are easily dissolved by a lytic enzyme [4,18].
Glucanex breaks down yeast cell walls so that they become round in both P. manshurica ( Figure 2) and Rh. paludigenum ( Figure 3) compared with the cells in controls, which are oval shaped. Figure 4 shows that treatment with Glucanex 4 mg/mL (L4) rendered better results than treatment with 2 mg/mL (L2). Protoplast treatment with L4 produced 7.2 x 10 10 (cell/mL) for P. manshurica and 8.8 x 10 10 (cell/mL) for Rh. paludigenum. Treatment with L2 produced 5.2 x 10 10 and 8.2 x 10 10 , respectively. Therefore, in subsequent research, L4 will be used in the fusion process.
Protoplast fusion using PEG. This study used the fusogen PEG 6000 at concentrations of 30% and 35%. The 30% concentration of PEG 6000 formed aggregates of 2-3 ( Figure 5), while the 35% concentration formed aggregates of more than 3.  (Table 1). This MIC value was slightly lower than that of the parental P. manshurica (white), which was 300 ppm. Test of fusants F1, F4, and F7 based on inulinase production. Fusants F1, F4, and F7 were grown on ISM medium, in which inulin is used as the sole carbon   source [8]. This study observed growth patterns and inulinase production every six h for 48 h (Figures 6 and  7).
Protoplasts are cells in which the cell wall has been removed, but which have their cell membranes and internal structures intact and are able to perform normal physiological functions. Protoplast isolation must be conducted carefully and in an osmotic stabilizer solution to prevent excessive intake of water into the cells, which can lead to cell rupture [11]. Osmotic stabilizer solutions used to isolate protoplasts contain sugar and sugar alcohols [5].
In Figure 1, it is clear that P. manshurica and Rh. paludigenum have entered the late log phase at the age of 24 h without a lag phase. This can occur due to the addition of starter, which serves to negate the lag phase [12]. In this study, the starter reduced the lag phase, and the log phase was reached quickly. Cells were isolated through centrifugation. Pellets obtained were immersed in osmotic stabilizer solution containing 1 M sorbitol Glucanex 2 mg/mL (L2) and 4 mg/mL (L4). This solution served to maintain the stability of the protoplasts. Osmotic stabilizer plays a very important role in protoplast isolation, as it also increases protoplast release [6].
The lytic enzyme, containing enzyme hydrolyzed glucan, chitin, protein, and cellulose, causes protoplasts to be round. The lytic enzyme used in protoplast isolation depends on the composition of the microbial cell walls in the yeast. The main constituents of yeast cell walls are β glucan, chitin, and mannoprotein. Therefore, a suitable lytic enzyme by which to hydrolyze yeast cell walls is Glucanex, or novozim, because it contains enzymes β glucanase, cellulase, protease, and chitinase [11] to destroy yeast cell walls. When cell walls have been destroyed by Glucanex, the protoplasts appear more rounded, an effect enhanced by the osmotic stabilizer solution [19].
This result is in accordance with previous research, which found that the best concentration of PEG is 25-33% [13].
If the concentration of PEG is too high, it will be toxic to protoplasts [14]. In addition, protoplast fusion using high PEG concentrations can be toxic to recombinants [15]. However, if the concentration of PEG is too low, it will not aggregate protoplasts, and if the concentration is too high to form aggregates, there will be no fusion, only toxicity [13]. The protoplast fusion process results in cell membrane activation, causing cells to adhere to other cells. These attachments result in aggregate formation.
The addition of Ca 2+ ions during the fusion process results in an increase in fusion frequency resulting from PEG's ability to bind to Ca 2+ ions, thereby forming a bridge between the membrane and the PEG. In water, PEG (polyethylene glycol, HOCH 2 (CH 2 -O-CH 2 ) n CH 2 OH has a slightly negative charge and is able to form hydrogen bonds with the plasma membrane in protoplasts. In protoplast fusion, PEG acts as a bridge between two or more protoplasts, resulting in aggregates [16].  [6,7].
These results indicate that fusion has occurred between parentals P. manshurica and Rh. paludigenum. In other words, fusants F1, F4, and F7 were selected. Fusant F3 was not selected, because it has properties similar to the parent Rh. paludigenum. Testing of fusants F1, F4, and F7 involved continuing to select one of them based on its ability to produce inulinase.
The log phase growth pattern occurred from incubation time T 0 to T 12 ( Figure 6), during which time inulinase was produced. At 12 h, fusant F4 had produced 0.6892 IU of inulinase. During the same time period, parentals P. manshurica and Rh. paludigenum had generated 0.3263 IU and 0.557 IU, respectively. Of the four fusants, fusant F4 produced the highest amount of inulinase compared with the parentals (Figure 7). In addition, fusant F4 produced a great deal of inulinase compared with Debaryomyces hansenii DUCC-W8 (0.639 IU) [17]. Inulinase is categorized as a primary metabolite and is produced only in the log phase due to induction of inulin from the environment (in this case, from the medium). Fusant F4 had a specific growth rate (µ) of 0.3360/h and a generation time (g) of 2.0629 h. These values lie between those of the two parentals. P. manshurica had specific growth rate (µ) of 0.2793/hour and generation time (g) of 2.4815 h and Rh. paludigenum a (µ) of 0.3787/h and a (g) of 1.8304 h.

Conclusions
A Glucanex concentration of 4 mg/mL produces the greatest number of protoplasts: 7.2 x 10 10 (cell/mL) for P. manshurica and 8.8 x 10 10