Improving the Yield of Glucoamylase and α-amylase in Solid-state Co-culture

Steamed rice inoculated with Aspergillus oryzae, called Koji, is an essential ingredient for making amazake or brewing sake. However, A. oryzae usually offers low enzyme yield, especially in case of glucoamylase. Co-culture of Aspergillus and Rhizopus strains in Koji increased glucoamylase and α-amylase activities. The ratio of initial spore counts of A. oryzae and R. oryzae influenced the activity of amylolytic enzymes. When this ratio was 1:1, α-amylase showed maximum activity (573 U/g-substrate, 95 h), and when this ratio was 200:1, glucoamylase showed maximum activity (180 U/g-substrate, 95 h).


Introduction
Amylases are among the most important enzymes, with great significance in biotechnological applications, constituting around 25% of the enzyme industry [1]. The amylase family has two major classes: glucoamylase and α-amylase.
Amazake, a traditional non-alcoholic beverage in Japan, is produced from a mixture of rice Koji and boiled rice that is incubated at 55 °C to 60 °C for 8 h 4 . During this process, the glucoamylase and α-amylase produced by Aspergillus oryzae, a fungus found in the rice Koji, hydrolyze starch to dextrin and glucose; the chief sweet component in amazake is glucose [4,7,8].
The surface of rice grains contains many proteins; the proteolytic enzymes from A. oryzae hydrolyze these proteins to generate large amounts of amino acids and peptides [7].
Therefore, glucoamylase and α-amylase production by A. oryzae is important for the production of amazake.
However, the production of glucoamylase by the Aspergillus species is lower than that of α-amylase.
Many fungal species are capable of producing glucoamylase under different conditions and techniques of fermentation. Among them, Rhizopus oryzae, assigned the "generally regarded as safe" (GRAS) status, is commonly used in the industrial production of glucoamylase [9]. Moreover, much work has been undertaken to enhance the activity of the glucoamylase isolated from this fungus, such as optimization of the culture conditions and identification of the genes coding for glucoamylase.
Mertens et al. [10] reported that the glucoamylase produced by Rhizopus can be divided into two different taxonomic groups, namely, Type-I and Type-II, on the basis of the differences in phenotype (e.g., acid production), rDNA internal transcribed spacer regions, and genomic structure.
Glucoamylase-I can absorb raw starch and has high raw starch-digesting activity, while glucoamylase-II cannot absorb raw starch and has extremely weak raw starch-digesting activity. Glucoamylase-I contains starchbinding sites in the N-terminal region, whereas glucoamylase-II is formed by protease digestion of the Nterminal region of the glucoamylase-I and has reduced ability to digest starch [11,12].
Solid-state fermentation is mostly undertaken with mixed cultures. Mixed culture fermentations are those in which the inoculum consists of two or more organisms and are widely used in many processes, including the production of antibiotics, enzymes, and several types of fermented food, composting, dairy fermentation, and bioconversion of apple distillery and domestic wastewater sludge [13]. The mixed culture may offer internal regulation of growth and product formation, which might be difficult to achieve otherwise. Several reports describe the co-culture of two strains for enhanced enzyme production [14,15]. For example, the synergistic interaction of Trichoderma reesei RUT-C30 and Aspergillus niger LMA in a submerged fermentation system allowed more efficient cellulose degradation than in monoculture with either T. reesei or A. niger, owing to the complementary interactions between the cellulases from T. reesei and the glucosidases from A. niger that allowed complete cellulose hydrolysis [16]. In addition, Yun-wei and Yan [17] reported that acid protease activity was enhanced by the co-culture of A. oryzae AS3042 and A. niger SL-09. The inoculation of A. niger SL-09 significantly enhanced enzyme production, primarily because of the synergetic interaction between the strains.
Building upon these previous findings, we developed a solid-state co-culture involving two fungi for glucoamylase and α-amylase production. A. oryzae and R. oryzae were used for α-amylase and glucoamylase production, respectively.

Rice Koji preparation
Material rice (Yumetukushi) was obtained from Fukuoka (Japan) and polished to 90%. Steamed rice was inoculated with the spore suspension (final concentration, 2.5 × 10 4 spores/g-substrate; initial water content, 38%) and cultured for 20, 45, 70, 95, or 120 h. This system was incubated at 31 °C at 90% moisture for the initial 20 h. Then, it was moved to 38 °C at 85% moisture.

Enzyme extraction from rice Koji
The crude enzyme was extracted from 10 g of rice Koji by using 50 mL of sterile water for 3 h at room temperature. It was filtered through the filter paper (No. 2; diameter, 125 mm; Tokyo Roshi Kaisya, Ltd., Tokyo, Japan) to remove the Koji; the resulting filtrate was used as the crude enzyme solution.

Co-culture system with pre-culture time
The co-culture system was set up for a controlled culture time. For pre-culture, the spores (final concentration, ~1.25 × 104 spores/g-substrate) of A. oryzae or R. oryzae were added to steamed rice, and cultured for 20 h (initial water content, 38%). The inoculated rice was incubated at 31 °C at 90% moisture for the initial 20 h. The pre-culture Koji was inoculated with the spore suspension (final concentration, 1.25 × 10 4 spores/g-substrate) of other strains and cultured for 20, 40, 65, 90, 115, and 140 h. This system was incubated at 31 °C at 90% moisture for 20 h. It was subsequently moved to 38 °C at 85% moisture.

Co-culture system with controlled spore count
The co-culture system was set up with controlled number of spores. R. oryzae and A. oryzae (from the slants) were separately added to 10 mL of sterile water. Spore suspensions containing different ratios of R. oryzae and A. oryzae spores (1:200, 1:50, 1:10, 1:1, 10:1, 50:1, or 200:1) were prepared, and the total spore count was adjusted to 2.5 × 10 4 spores per gram of substrate.

Determination of glucoamylase, α-amylase, and acidic protease activities
Glucoamylase activity was measured using the method described by Morita et al. [18]. The reaction mixture for glucoamylase activity assay was composed of 1 mL of 2% (w/v) soluble starch (Nacalai Tesque Co., Kyoto, Japan) solution in 0.1M acetate buffer (pH 4.5) and 1 mL of the enzyme solution. The reaction progressed at 40 °C for 20 min and it was stopped by heating the test tubes in a boiling water bath for 10 min. The amount of liberated glucose was determined using the glucose oxidase method [19], by using a commercially available glucose test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). One unit of glucoamylase activity was defined as 1 microgram of glucose liberated from soluble starch per minute per milliliter of the enzyme solution.
A modification of the Anson hemoglobin method [20] was used for determining acidic protease activity. Briefly, 2.5 mL of hemoglobin solution (0.6% in 0.05M acetate buffer, pH 4.5) and 1 mL of the enzyme solution were mixed and incubated at 40 °C for 30 min. The reaction was stopped by adding 5 mL of 5% trichloroacetic acid. After 60-min incubation at 40 °C, the precipitated unhydrolyzed hemoglobin was removed by filtration, and the absorbance of the solution was measured at 280 nm. One unit of the acidic protease was defined as one microgram of tyrosine per gram of solidsubstrate per minute.  Figure 1 shows the glucoamylase, α-amylase, and acidic protease activity in A. oryzae NBRC 5238 pure culture, R. oryzae NBRC 4716 pure culture, and co-culture. The highest glucoamylase activity was 79 U/gsubstrate for 70 h and the highest α-amylase activity was 573 U/g-substrate for 95 h in co-culture. The glucoamylase and α-amylase activities were extremely low in R. oryzae pure culture. The acidic protease activity in A. oryzae NBRC 5238 pure culture, R. oryzae NBRC 4716 pure culture, and co-culture was not significantly different ( Fig. 1 C). These results indicated that the highest glucoamylase activity in the co-culture system was lower than that observed in A. oryzae NBRC 5238 pure culture (167 U/g-substrate, 95 h). This can be attributed to the fact that it might be difficult to grow Rhizopus species on steamed polished rice cultures, because the enzyme produced by Rhizopus might not act on the heat-denatured proteins contained in rice. In particular, Rhizopus strains possessing low acidic carboxypeptidase activity are incapable of breaking down heat-denatured proteins. Therefore, the growth of Rhizopus species is delayed because of the lack of nitrogen sources [21,22]. Moreover, Harayama et al. [23] reported that R. oryzae IFO 5418 and Rhizopus oligosporus NRRL 2710 showed extremely weak αamylase and glucoamylase activities in rice and soybean Koji. These results were in agreement with those of our study. : Co-culture involving equal number of R. oryzae and A. oryzae spores (1:1), ▲: A. oryzae pure culture, ■: R. oryzae pure culture

Effects of a co-culture system with preculture time on glucoamylase and α-amylase production
Previous studies showed that the glucoamylase activity in the co-culture system was lower than that in the A. oryzae NBRC 5238 pure culture system. Therefore, we adopted the co-culture system involving pre-culture time to enhance glucoamylase activity.

Figure 2
Influence of co-culture system with pre-culture time on glucoamylase and α-amylase production A. Glucoamylase (GA) activity (U/g-substrate) and B. α-Amylase (α-A) activity (U/g-substrate ) : pre-culture of A. oryzae, : pre-culture of R. oryzae, : co-culture start point  Figure 2 shows the glucoamylase and α-amylase activities of A. oryzae or R. oryzae during the pre-culture time. High α-amylase production (545 U/g-substrate) was observed at 90 h (Fig. 2B), but glucoamylase production was reduced ( Fig. 2A) in the co-culture system with preculture time. According to some reports, enzyme production by A. oryzae depended on the temperature [24,25]. Acid protease is well produced at temperatures below 35 °C, but temperatures above 38 °C can decrease the production. The expression of pepA (acid proteaseencoding gene) is induced at low temperatures [24]. Hisada et al. [25] reported that glaB (glucoamylaseencoding gene) promoter is induced by exposure to high temperature (for example, growth at 42 °C).
In this experiment, the temperature was maintained at 31 °C for 40 h during the early stages of cultivation. Thus, we assumed that glucoamylase production by A. oryzae was inhibited at low temperature.We found that the coculture system with pre-culture time was not the optimal culture method.

Effects of a co-culture system with controlled spore count on glucoamylase and αamylase productions
The above-described results showed that the glucoamylase activity in the co-culture system with preculture time was extremely low. Additionally, α-amylase production by the seed culture of Rhizopus in the coculture system was extremely low. Mikai et al. [2] have reported the co-culture of A. oryzae and Rhizopus arrhizus. Therefore, we tried another co-culture system with controlled spore count.
The graphs in Fig. 3 and 4 show glucoamylase and αamylase activities in the presence of controlled spore count.
The optimal initial spore count of A. oryzae and R. oryzae was in the ratio of 200:1 for glucoamylase production (180 U/g-substrate, 95 h, Fig. 3). The highest glucoamylase activity in the co-culture system (spores of A. oryzae and R. oryzae, 200:1) was higher than that observed in the A. oryzae NBRC 5238 pure culture (167 U/g-substrate, 120 h). The optimal initial spore count of A. oryzae and R. oryzae for α-amylase production (573 U/g-substrate, 95 h) was in the ratio of 1:1 (Fig. 4).
Glucoamylase and α-amylase activities improved on equalizing or increasing the initial spore ratio of A. oryzae and R. oryzae (Fig. 3A, 4A) and it reduced on increasing the initial spore ratio of R. oryzae and A. oryzae (Fig. 3B, 4B). Taken together, these data showed that the optimal conditions for the production of both glucoamylase and α-amylase in a co-culture system involve equal spore count of A. oryzae and R. oryzae (1:1 ratio). Under these conditions, glucoamylase activity (167 U/g-substrate) reached ~47% of that observed in A. oryzae pure culture, and α-amylase activity (491 U/gsubstrate) reached ~117% of that observed in A. oryzae pure culture.
Thus, these results suggest that the production of glucoamylase and α-amylase can be controlled by adjusting the numbers of A. oryzae and R. oryzae spores.
Furthermore, these data imply that increased production of glucoamylase and α-amylase can be obtained by coculturing A. oryzae and R. oryzae.

Conclusions
In this study, we investigated the production of glucoamylase and α-amylase by A. oryzae and R. oryzae in solid-state co-culture.
Glucoamylase activity of co-culture Koji was optimized 180 U/g-substrate when the ratio of initial spore counts was 200:1 and α-amylase of co-culture Koji was optimized 573 U/g-substrate when the ratio of initial spore counts was 1:1. These results indicated that amylolytic enzymes can be regulated from adjusting the numbers of A. oryzae and R. oryzae spores.
However, from a perspective in productivity of glucoamylase and α-amylase, our results suggested that the co-culture system with simultaneous inoculation with equal number of spores of A. oryzae and R. oryzae represents optimal conditions for amylase production.
Further investigations are necessary to improve the production of glucoamylase and α-amylase under optimal conditions (e.g., moisture, temperature) required for the co-culture system.