Effect of Vegetable Protein Sources on Extracellular Protease Activity of Bacillus velezensis Strains

다국어 초록 (Multilingual Abstract)

Bacillus is an important microorganism employed in the food fermentation industry. From Bacillus spp., the mixture of various extracellular proteases, which is directly secreted into the medium, is produced during the late exponential phase to early stationary phase of cell growth. Among the factors influencing protease synthesis in Bacillus, nitrogen source has been considered to either promote or inhibit enzyme production. Therefore, extensive studies have been conducted to date, utilizing various nitrogen sources for large-scale production of Bacillus proteases. In this study, the effect of soy, rice, and pea proteins on the production of Bacillus proteases was investigated using casein and skim milk as control substrates. First, the aprE gene encoding one of the major proteases (BsAprE) from B. subtilis ATCC6051 type strain was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. BsAprE showed its highest activity at pH 10.0 and 50°C, and exhibited high activity in the following order: casein, pea, soybean, and rice proteins. Two Bacillus strains exhibiting high protease activity were isolated from fermented soybean products. They were identified and designated as Bacillus velezensis SMB164 (BvSMB164) and B. velezensis SMB201 (BvSMB201), based on their whole genome sequences. Both strains showed the highest protease activity after 24 hours of cultivation in Tryptic Soy Broth (TSB) medium. When 1% of single protein source was added to TSB medium and cultured for 36-48 hours, the order of high protease productivity was observed as follows: skim milk, pea protein, soy protein, rice protein, wheat gluten, and casein. Especially, the addition of protein sources resulted in a delay in the time to reach maximum protease activity along with an increase in protease productivity. Subsequently, the effects of complex protein sources on protease productivity were examined. In experiments where skim milk and one of plant-based protein sources were added to TSB medium, additional increases in protease activity were observed. As a result, BvSMB164 exhibited the highest activity of 1220 U/mL TSB containing 1% skim milk and 1% pea protein #2, while BvSMB201 showed an increase to 1223 U/mL TSB with skim milk and soy protein #2, representing increases of 3.9-fold and 4.4-fold, respectively, compared to TSB medium without any additional protein source. In conclusion, it was confirmed that the addition of various protein sources selectively increases the production of extracellular protease from Bacillus. Particularly, a combination of skim milk and plant-based protein sources allowed for more efficient protease production. Future research combining these results with further transcriptome analysis to decipher the gene expression regulation mechanisms could contribute to the development of industrial-scale production processes for Bacillus proteases.

목차 (Table of Contents)

• I. Introduction 1
• 1. Proteases 1
• 1.1. Classification of proteases 1
• 1.1.1. endo- and exo-peptidases 1
• 1.1.2. Intracellular and extracellular proteases 5
• 1.2. Microbial enzymes in protein fermentation 9
• 1.2.1. Bacillus proteases 12
• 1.2.2. Expression mechanism of major proteases in Bacillus 17
• 1.3. Factors affecting protease production 18
• 1.4. Nitrogen source for protease production 21
• 2. Genome sequencing of Bacillus spp. 25
• 3. Transcriptome analysis of Bacillus spp. 25
• 4. Research objectives 28
• II. Materials and methods 29
• 1. Materials 29
• 1.1. Bacterial strains and DNAs 29
• 1.2. Enzymes and reagents 29
• 1.3. Oligonucleotides & sequencing 31
• 1.4. Protein sources 31
• 2. Methods 31
• 2.1. Bioinformatic analysis of proteases from Bacillus spp. 31
• 2.2. Transformation into Escherichia coli 31
• 2.3. Isolation of plasmid DNA 34
• 2.4. Whole genome sequencing 35
• 2.4.1. Isolation of genomic DNAs 35
• 2.4.2. Hybrid assembly 35
• 2.5. Polymerase Chain Reaction (PCR) 35
• 2.6. Gene cloning 36
• 2.7. Gene expression and enzyme purification 36
• 2.8. Protein analysis 37
• 2.8.1. Determination of protein concentrations 37
• 2.8.2. SDS-PAGE analysis 38
• 2.9. Protease activity assay 39
• 2.9.1. Effect of pH on protease activity 40
• 2.9.2. Effect of temperature on protease activity 40
• 2.10. Isolation of novel Bacillus strains 40
• 2.11. Protease production induced by protein sources 41
• III. Results and discussion 42
• 1. Prediction of extracellular protease genes in B. subtilis type strain 42
• 2. Gene cloning of protease genes in B. subtilis type strain 42
• 2.1. Gene cloning of BsAprE 42
• 2.2. Gene expression and enzyme purification from E. coli 48
• 2.3. Functional characterization of BsAprE 48
• 2.4. Gene cloning and expression of BsNprE and BsNprB from E. coli 50
• 3. Isolation of Bacillus strains with high protease activity 53
• 3.1. Agar plate assay 53
• 3.2. Whole genome sequencing of B. velezensis SMB164 56
• 4. Effect of vegetable protein sources on protease activity 65
• 4.1. Effect of culture media 65
• 4.2. Cell growth and protease activity of Bacillus spp. 69
• 4.3. Effect of single protein source on protease production 72
• 4.4. Effect of complex protein sources on protease production 75
• Ⅳ. Conclusion 80
• Ⅴ. Abstract in Korean 82
• VI. References 84
• VII. Appendix 94