**4.3 Extraction and purification of salt-tolerant acid protease**

314 Soybean – Genetics and Novel Techniques for Yield Enhancement

protease with high proteolytic activity is used as starter to uniform the quality of the product, and shorten the ripening periods of fermentation. During the fermentation, the pH value decreased down to lower than 5 (Fukushima, 1979), thus the salt-tolerant acid

Salt-tolerant proteases were produced from halotolerant microorganisms; Halobacterium salinarium, Bacillus sp., Halobacterium halobium, Halomonas sp., Halobacillus sp., Virgibacillus sp., Oceanobacillus sp., Saccharomyces cerevisiae, Aspergillus sp.,etc (Barbosa et al., 2006; Jeong et al., 2001; Kim et al., 2004). However, few of them have been purified and characterized to be acid proteases. They are proteases produced by B. subtilis JM3 from anchovy sauce (W.J. Kim and S.M. Kim, 2005), B. megaterium KLP-98 from fermented squid (Fu et al., 2008), Rhizopus japonicus (Chung, 1984), and A. oryzae LK-101 from the

For the isolation of protiolytic bacteria, anchovy sauce fermented at 15 ± 3 °C for 3 years was inoculated on a brain–heart infusion (BHI) agar and incubated at 27.5 °C. The highest transparent colony was isolated and identified by the genetic mapping method to be *B. subtilis*. Five hundred milliliters of the medium in a 1-L wide-mouth culture flask was inoculated with 10 mL of *B. subtilis* JM-3 suspension prepared from BHI broth media cultivated at 37 °C for 3 days. The culture was incubated at 37 °C for 8 days in a shaking incubator at 150 rpm. The optimal protease production reached 1500 U/L (W.J. Kim and

Proteolytic bacteria from the fermented squid were isolated by BHI agar. After inoculating 0.1 mL of fluid of the squid with 10% NaCl concentration fermented at room temperature for 2 months, the culture media were incubated at 37 °C for 48 h. The highest transparent colony on the culture medium was isolated, identified and inoculated in BHI broth media with 10% NaCl. 500 mL of medium in a 1 L wide-mouth culture flask was inoculated with 10 mL suspension prepared from BHI broth media cultivated at 37 °C for 72 h. The culture was incubated at 37 °C for 5 days in a shaking incubator at 150 rpm. The optimal protease

Proteolytic bacterium was isolated from traditional Korean soybean paste on agar plates incubated for 2 to 4 weeks. Spores suspension was transferred to the seed culture medium (pH 3.0) containing glucose, corn steep liquor, Hyflo Super-Cel, and a defoaming agent. The bacterium was identified by rDNA sequencing method and and named as *A. oryzae* LK-101. For the production of protease, *A. oryzae* LK-101 was cultivated in a 500 mL flask containing 200 mL of 2% defatted soybean flour culture broth at 27 °C for 4 days in a shaking incubator

protease is more valuable for the production of fermented soybean products.

**4.1 Microorganisms for the production of salt-tolerant proteases** 

**4.2.1 Production of salt-tolerant acid protease by** *B. subtilis* **JM-3** 

**4.2.2 Production of salt-tolerant acid protease by** *B. megaterium* **KLP-98** 

**4.2.3 Production of salt-tolerant acid protease by** *A. oryzae* **LK-101** 

at 150 rpm. The optimal protease production reached 973 U/L (Fu et al., 2008).

traditional Korean soybean paste (Hwang et al., 2010).

**4.2 Production of salt-tolerant acid protease** 

production reached 520 U/L (Hwang et al., 2010).

S.M. Kim, 2005).

**4. Salt-tolerant acid proteases** 

Five kinds of reported salt-tolerant acid proteases were extracellular enzymes. Thus, they were extracted and purified from the culture media of each corresponding microorganism.
