A Thermostable and Acidophilic Mannanase from Bacillus mojavensis: Its Sustainable Production Using Spent Coffee Grounds, Characterization, and Application in Grape Juice Processing

Abstract

The utilization of enzymes in various industrial applications has attracted increasing attention in recent years. However, their production costs and instability in extreme environments are two significant factors that limit their use in industry. Mannanase production from Bacillus mojavensis using spent coffee grounds (SCGs), which are inexpensive and most abundant domestic industrial waste, was increased about 32-fold with optimization of submerged fermentation (SmF) medium using Plackett-Burman design (PBD) and response surface methodology-Box-Behnken design (RSM-BBD). Moreover, BmMan production in a 5-L stirred bioreactor resulted in an enzyme titer of 32.55 U/mL. BmMan was partially purified 2.28-fold with 63.01% yield using ultrafiltration and ammonium sulfate precipitation techniques. The molecular weight of partially purified mannanase (BmMan) was estimated by zymography to be 24 kDa. Biochemical characterization of BmMan showed that optimal pH and temperature for catalysis were 4.0 and 70 degrees C, respectively. BmMan showed a half-life of 40.8, 150.2, 210.2, and 387.6 min at 80, 70, 60, and 50 degrees C, respectively. Subjecting to bivalent cations and chemicals, Ag2+, Ca2+, Ni2+, Mg2+, Mn2+, Fe2+, DTT, Triton X-100, SDS, and beta-mercaptoethanol enhanced BmMan activity. Additionally, BmMan was compatible with methanol, DMSO, isopropanol, and butanol. The clarity and nutritional value of grape juice were improved after the treatment with the enzyme. Ultrafiltrate obtained by passing cell-free culture liquid through a 10-kDa molecular weight cutoff (MWCO) membrane exhibited antimicrobial and antioxidant activity without showing genotoxicity. To the best of our knowledge, this is the first report describing the thermostable and acidophilic mannanases produced by Bacillus taxa and the first study suggesting the multiple uses of SCGs.