Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

The Role of Phyllosphere Bacteria in Improving Cotton Growth and Yield Under Drought Conditions

Cotton is a valuable fiber and cash crop in Telangana, India. This study examines how crop growth and fiber yield are affected by the uneven distribution of rainfall. Cotton phyllosphere bacterial isolates were gathered from the Rangareddy and Warangal Districts of Telangana, and in total, 31 phyllosphere bacterial isolates were obtained. These isolates were screened for drought tolerance and it was found that fifteen suitable isolates grew at −1.25 Mpa stress level with copious amounts of exopolysaccharides production. These isolates were further screened for ACC deaminase production and we observed 0.13–0.40 mM of α-ketobutyrate per milligram of cellular protein per hour. Five efficient bacterial isolates, namely Pseudomonas stutzeri, Acinetobacter sp., Bacillus mojavensis, Pseudomonas chlororaphis, and Enterobacter asburiae were found to produce ACC deaminase and were able to grow at −1.25 Mpa stress level. The cotton variety ADB-542 (drought susceptible) was treated with drought-tolerant five isolates. Acinetobacter sp. treated seeds had the highest seed germination and seedling vigor of 76.67 and 45.81%, respectively. The nutrient status of inoculated plots was considerably improved. The root length, fresh weight, proline content, and the number of bolls were increased by 28.52, 41.9, 28.78, and 12.99%, respectively, with the inoculation of Acinetobacter sp., to plants at −0.75 Mpa water potential. Overall the performance of cotton was improved significantly with the inoculation of phyllosphere bacteria to seeds; hence they can be recommended for the application of field crops as bio-inoculants.

Characterization of a Novel Keratinase With Surfactant Stability in a Wide Range of pH Activity From a Native Isolate, Bacillus Mojavensis FUM125

Keratinases are enzymes with the most diverse sources and applications. Different forms of keratinase have been applied in environment and variety of industries, highlighting the necessity for novel potential keratinases, which could be applicable in variety of industries. Accordingly, the present study aimed to identify and characterize a novel keratinase producing bacterium with high potential in variety of industries. In the present study, the native isolate of Bacillus sp. FUM125 was isolated, identified and optimized for the keratinolytic activity. The keratinase was purified and characterized using biochemical assays. The Bacillus sp. FUM125 isolate was identified as Bacillus mojavensis R-OH-1 with 99.8% similarity. The isolate showed the maximum keratinolytic activity at pH of 8.5 after 24-hour incubation at 37°C (2.1-fold enzyme production). According to the biochemical analysis, the keratinase belonged to a serine protease family, whit 33.5 kDa molecular weight and was stable in a wide range of pH and temperature with maximum keratinolytic activity at 60°C and pH 8. Among the metal ions, K+, Ca2+, Na2+ and Mg2+ increased the enzyme activity. The activity was increased by the reducing agents of DTT and beta-mercaptoethanol. Based on the substrate profile findings, the enzyme was active in various soluble and insoluble substrates. The enzyme showed a half-life of 98 min in the optimal temperature and the ratio of keratinolytic:caseinolytic to be 0.95. Our enzyme with higher temperature and pH stability compared to existing commercial enzymes can be considered as a potential candidate for use in various industries.

Diversity, Enzyme Production and Antibacterial Activity of Bacillus Resource in Sesame-Flavored Liquor Daqu

Daqu provides enzymes and precursors for liquor fermentation, and is the core of liquor fermentation. In this study, 11 strains of Bacillus were isolated from sesame-flavored liquor Daqu, which can not only produce protease and amylase, but also have antagonistic effects on common pathogens Escherichia coli and Staphylococcus aureus. According to the gyrA gene phylogeny analysis, these 11 Bacillus strains belong to three species, B1, Y14, Y15, and YPDW9 belong to Bacillus mojavensis, W7, W13, YPDW6, and YPDW12 belong to Bacillus subtilis, W14, Y5 and YPDW1 belong to Bacillus velezensis. According to the results of random amplified polymorphic DNA (RAPD) typing, these 11 Bacillus strains are completely different. The specific primers were used to randomly amplify the biological control genes expressing lipopeptide antibiotics (bioA, bmyB, ituc, fend, srfAA, srfAB, yngG and yndJ), and they all expressed different expressions in these 11 Bacillus strains. This research provides new ideas for strengthening Daqu and lays a foundation for improving the quality of liquor.

Quality of Immature and Mature Pepper (Capsicum annuum L.) Seeds in Relation to Bio-Priming with Endophytic Pseudomonas and Bacillus spp.

Fruit maturity for seed production can occur at various times because of the continual flowering of pepper plants. Accordingly, seeds with different maturity are acquired as the fruits are collected in a single harvest. Immature seeds obtained in this harvest may lead to a decrease in the quality of seed lots. Therefore, this research aimed to evaluate the influence of four different endophytic bacteria strains (Pseudomonas fluorescens strain L5b, Pseudomonas gessardii strain L13, Bacillus subtilis strain Bs1 and Bacillus mojavensis strain ApBm) on germination and seedling vigor of immature and mature bell pepper seeds. To obtain seeds with different maturity levels, fruits were collected 45–49 days after flowering for immature seeds and 65–69 days for mature seeds. The effectiveness of these bacteria strains was examined by coating seeds with four different endophytic bacteria strains separately. Additionally, to see the activity of endophytic bacteria more clearly, a mock treatment with sterile water was added to the experiment as a control (+) group. Bio-priming (especially strain Bs1 and L13) improved germination and seedling emergence characteristics of both immature and mature seed lots compared to control groups (p < 0.05). The results demonstrate that bio-priming with beneficial endophytic bacteria can be used to stimulate the quality of both immature and mature seeds from the pepper.