Purification and characterization of a novel ß-agarase of Paenibacillus sp. SSG-1 isolated from soil.

Agar is a polysaccharide polymer material, generally extracted from seaweed. Most agar degradation strains were isolated from seawater. In order to find new species resources and novel agarase from soil, an agar-degrading bacterium Paenibacillus sp. SSG-1 was isolated from soil. Agarase SSG-1a was purified to homogeneity by 30.2 fold with a yield of 4.8% through ammonium sulfate precipitation, DEAE FF chromatography and native-PAGE separation. The tandem mass spectrometry (MS/MS) results indicated that purified SSG-1a should be a novel ß-agarase. The molecular mass of SSG-1a was estimated to be 77 kDa. The optimal temperature and pH for SSG-1a were 50°C and pH 6.0, respectively. Moreover, SSG-1a was stable in pH range of 4.0-10.0 and at temperature up to 40°C. It could hydrolyze the ß-1,4 linkage of agarose to produce neoagarohexaose (95 mol%) and neoagarooctaose (5 mol%). Metal ion Mn(2+) and reducing reagents (ß-Me and DTT) could increase its activity by 150% and 60%, respectively.

PCR-RFLP analysis of the diversity of phytate-degrading bacteria in the Tibetan Plateau.

Phytases play a very important role in increasing phytate digestion and reducing phosphorus pollution in the environment, and phytate-degrading bacteria have a ubiquitous distribution in the environment. Due to its extremely harsh environment, the Tibetan Plateau breeds possibly abundant, extreme microorganisms. In this research, 67 phytate-degrading bacteria were isolated from different habitats in the Tibetan Plateau. Among all isolates, 40.3% were screened from farmland, 25.3% from wetland, 4.5% from saline-alkaline soil, 7.5% from hot springs, and 22.4% from lawns, which showed that the distribution of the phytate-degrading bacteria varied with habitats. By the PCR-RFLP method, 16 different species were identified and named, 4 of which are reported for the first time as phytate-degrading bacteria, that is, Uncultured Enterococcus sp. GYPB01, Bacillaceae bacterium strain GYPB05, Endophytic bacterium strain GYPB16, and Shigella dysenteria strain GYPB22. Through the assay of phytase activity of 16 strains, Klebsiella sp. strain GYPB15 displayed the highest capability of phytase production. Through analysis of the optimum pH, the optimum temperature, and the thermal stability of enzyme from 16 strains, some especial phytate-degrading bacteria were obtained. Our findings clearly indicate a good relation between the composition of the soils from the different environments in the Tibetan Plateau and populations of cultivable phytate-degrading bacteria. Moreover, extreme harsh soils are logically the best soils in which to find some strains of phytate-degrading bacteria for exploiting in the fields of biotechnology and industry.

Expression of food-grade phytase in Lactococcus lactis from optimized conditions in milk broth.

The major objective of this study was to engineer lactic acid bacteria to produce the enzyme phytase from a gene native to Bacillus subtilis GYPB04. The phytase gene (phyC) of B. subtilis GYPB04 was cloned into the plasmid pMG36e for expression in Lactococcus lactis. The enzyme activity in L. lactis cultured in GM17 broth was 20.25 U/mL at 36°C. The expressed phytase was characterized as active in a pH range of 2.0-9.0 at a temperature range of 20-80°C, with an optimum pH of 5.5-6.5 and temperature of 60°C. When cultured in food-grade milk broth, the transformed L. lactis grew to an OD(600 nm) value of 1.05 and had a phytase yield of 13.58 U/mL. In same broth under optimized conditions for cell growth and phytase production, the transformant reached an OD(600 nm) value of 1.68 and a phytase yield of 42.12 U/mL, representing approximately 1.6-fold and 3.1-fold increases, respectively, compared to growth in natural milk broth. Fermentation was scaled to 5 L under optimized conditions, and product analysis revealed a final OD(600 nm) value of 1.89 and an extracellular enzyme activity of 24.23 U/mL. The results of this study may be used in the dairy fermentation industry for the development of functional, healthy yogurts and other fermented dairy foods that provide both active phytase and viable probiotics to the consumer.

A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli AppA phytase.

Despite recent advances in our understanding of the importance of protein surface properties for protein thermostability,there are seldom studies on multi-factors rational design strategy, so a more scientific, simple and effective rational strategy is urgent for protein engineering. Here, we first attempted to use a three-factors rational design strategy combining three common structural features, protein flexibility, protein surface, and salt bridges. Escherichia coli AppA phytase was used as a model enzyme to improve its thermostability. Moreover, the structure and enzyme features of the thermostable mutants designed by our strategy were analyzed roundly. For the single mutants, two (Q206E and Y311K), in five exhibited thermostable property with a higher success rate of prediction (40 %). For the multiple mutants, the themostable sites were combined with another site, I427L, we obtained by directed evolution, Q206E/I427L, Y311K/I427L, and Q206E/Y311K/I427L, all exhibited thermostable property. The Y311K/I427L doubled thermostability (61.7 %, and was compared to 30.97 % after being heated at 80 °C for 10 min) and catalytic efficiency (4.46 was compared to 2.37) improved more than the wild-type AppA phytase almost without hampering catalytic activity. These multi-factors of rational design strategy can be applied practically as a thermostabilization strategy instead of the conventional single-factor approach.

Relationship between Escherichia coli AppA phytase's thermostability and salt bridges.

In order to study on the relationship between Escherichia coli AppA phytase's thermostability and salt bridges, and indicate an effective technical route of which factor to think about and where to modify at AppA for enhancing its thermostability, a salt bridge subtraction mutant E31Q and a salt bridge addition mutant Q307D were constructed by site-directed mutagenesis. The residual activities of the wild-type AppA phytase, E31Q and Q307D were 31.42%, 17.46%, and 40.57%, respectively, after being heated at 80°C for 10 min. The salt bridge subtraction mutant E31Q showed 13.96% thermostability decreasement, and the salt bridge addition mutant Q307D showed 9.15% thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved salt bridges play a key role in E. coli AppA phytase's thermostability and the α/ß-domain of AppA may be sensitive to heat. Salt bridges and the α/ß-domain of AppA should have high priority to think about to enhance AppA's thermostability for commercial application. Besides, molecular dynamics simulation was used for salt bridges analysis.

AppA C-terminal plays an important role in its thermostability in Escherichia coli.

Due to our previous research, mainly the thermostable mutants Q307D, Y311K, and I427L, we conjectured that Escherichia coli AppA phytase's C-terminal plays an important role in its thermostability, and AppA begins to collapse from the C-terminal when at a higher temperature. So here we constructed C-lose mutant to prove it. The residual activities of the wild-type AppA phytase and C-lose were 31.42 and 70.49 %, respectively, after being heated at 80 °C for 10 min. The C-terminal deletion mutant C-lose showed 39.07 % thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved the C-lose plays a key role in E. coli AppA phytase's thermostability.