Coix lacryma-jobi chymotrypsin inhibitor displays antifungal activity.

A novel chymotrypsin inhibitor, named ClCI, was purified from coix seed (Coix lacryma-jobi L.) by aqueous two-phase extraction, chymotrypsin-Sepharose 4B affinity chromatography and centrifugal ultrafiltration. ClCI was a 7.9 kDa competitive inhibitor with pI 6.54. The inhibition constants (Ki) for bovine pancreatic chymotrypsin and bacterial subtilisin were 1.27 × 10-10 M and 1.57 × 10-9 M respectively. ClCI had no inhibitory activity against bovine trypsin and porcine elastase. ClCI had wide pH stability and good heat resistance. It can maintain >90% inhibition activity against chymotrypsin at 20-80 °C for 1 h. The primary structure of ClCI was highly similar (57%-92%) to those of several inhibitors belonging to the Gramineae crop potato protease inhibitor- I superfamily and showed the typical sequence motif of the protease inhibitor of the seed storage protein group. ClCI (12.5 mg) inhibited mycelial growth of the phytopathogenic fungi Mycosphaerella melonis, Helminthosporium turcicum, Alternaria solani, Phytophthora capsici, Isariopsis griseola, and Colletotrichum gloeosporioides, and caused 89% inhibition of the proteases from spore germination of plant-pathogenic fungi. The results of the present study indicate that ClCI had biotechnological potential as an alternative agent to combat the important phytopathogenic fungi.

Optimized culturing conditions for an algicidal bacterium Pseudoalteromonas sp. SP48 on harmful algal blooms caused by Alexandrium tamarense.

Bacteria play an important role in preventing algal blooms and reducing their harm to the environment. To improve the algicidal activity of Pseudoalteromonas SP48 which had an inhibition effect on dinoflagellate Alexandrium tamarense, its growth medium and fermentation conditions were optimized for this bacterium. In this study, we used two steps to establish the optimum conditions. First, the proper proportion of medium was selected based on an orthogonal design. Then, the fermentation conditions were further optimized through uniform design in an enlarged 5L bioreactor. To test the algicidal ability of Pseudoalteromonas SP48 under the optimum conditions, algal cell morphology was observed by transmission electron microscopy (TEM). After the orthogonal design, we found that the optimum medium was [0.7% (m/v) tryptone, 0.2% (m/v) soluble starch, 0.2% (m/v) sucrose, 0.1% (m/v) FeSO4 , and 1.2% (m/v) K2 HPO4 ] for Pseudoalteromonas SP48 growth. Based on these results, optimum fermentation conditions were further explored in a 5L fermentation cylinder using a uniform design; the influence of variables such as incubation time, carbon type, and rotation speed were tested. The optimal fermentation conditions were fermentation time (42 hr), tryptone (1.1%), seeding volume (1.4 × 1013  cells), and rotation speed (250 r/min). Under these established optimum conditions, the biomass of strain SP48 increased by 79.2% and its lethal dose 50% (LD50 ) decreased by 54.0%, respectively. The TEM results showed that compared with the control group, the cell wall and cell membrane of A. tamarense were significantly damaged, and the structure and shape of the organelles were destroyed by algicidal bacteria of Pseudoalteromonas SP48. Overall, our results demonstrate that the optimized culture conditions could significantly enhance the algicidal activity of Pseudoalteromonas SP48 against a harmful dinoflagellate, such as A. tamarense. It will effectively provide a scientific foundation for both production of algicidal substances and HABs control.

[16S rDNA clone library analysis of microbial diversity associated with the PSP-producing dinoflagellate Alexandrium tamarense].

The composition of bacterial community in the Alexandrium tamarense culture was determined by analyzing the 16S rDNA clone libraries. 16S rRNA gene was amplified by using the total DNA extracted from the A. tamarense culture at three growth stages as templates. 34 different restriction fragment length polymorphism (RFLP) patterns of the clones were obtained from the three libraries. Clones representing each RFLP patterns were sequenced and phylogenetic analysis revealed that the Proteobacteria and the Bacteroidetes group can be the dominant component in the phycosphere microflora. Results showed that in the lag phase, alpha-Proteobacteria (36.4%), gamma-Proteobacteria (27.3%) and Bacteroidetes (27.3%) were the dominant group; in the late-exponential phase, the clones belonged to alpha-Proteobacteria (53.3%), beta-Proteobacteria (13.3%), gamma-Proteobacteria (6.7%), and Bacteroidetes (26.7%); and in the stationary phase, the community was comprised of alpha-Proteobacteria (47.8%), beta-Proteobacteria (8.7%), gamma-Proteobacteria (21.7%), delta-Proteobacteria (4.3%) and Bacteroidetes (17.4%). The results also suggest that a part of the bacteria associated with A. tamarense are uncultured and novel species. And these bacteria may play a major role in regulating the processes of algal bloom initiation, maintenance and decline. So these results may provide us great academic and practical importance in controlling the algal blooms by microbes.