Safety evaluation of a high-lipid algal biomass from Chlorella protothecoides.

Chlorella are traditionally freshwater green algae that have been evaluated for dietary purposes because of their nutritional value. This study investigates the safety of Chlorella protothecoides in a 28-day study. Sprague-Dawley rats were administered 0 (control), 2.5, 5.0, or 10% of their diet for 28days using an FDA Redbook protocol. The average daily dietary intake of algal biomass was determined to be 0, 1794, 3667, and 7557 mg/kg body weight for males and 0, 1867, 3918, and 8068 mg/kg body weight for females. Hematological and biochemical analyses were conducted, and upon completion, gross and microscopic evaluations were performed. No signs of toxicity were observed. Although statistically significant alterations were noted in several parameters among males and females, these changes were deemed to be of no toxicological significance due to the lack of dose-response relationships, the fact that they occurred in only one sex, and the lack of any supporting gross or microscopic alterations. The no-observed-adverse-effect level for the algal biomass under the conditions of this study was considered to be 10% in the diet, the highest dose tested.

Red algal cellular biomass lowers circulating cholesterol concentrations in Syrian golden hamsters consuming hypercholesterolaemic diets.

Preliminary evidence suggests that consumption of Porphyridium cruentum (PC) biomass results in hypocholesterolaemic effects; however, mechanisms responsible have not been elucidated. The aim of the present study was to determine whether PC biomass lowers circulating cholesterol concentrations, dose dependently, in hamsters fed hypercholesterolaemic diets for 28 d and determine whether cholesterol biosynthesis is affected. Biomass added to diets at 2.5, 5 and 10% resulted in 14, 38 and 53% reductions (P < 0.001) in total plasma cholesterol, respectively, compared with a control diet. Similarly, non-HDL-cholesterol concentrations in the 5 and 10% PC groups were reduced (P < 0.001) 28 and 45%, respectively, v. controls. These effects were unrelated to cholesterol fractional synthesis rate (FSR), as this did not differ between either treatment or control animals. PC consumption had no effect on food intake, plasma glucose concentrations or energy expenditure, but percentage of body fat was lower (P < 0.001) in the 5 and 10% PC groups compared with controls. These data show that PC reduces total plasma cholesterol and non-HDL-cholesterol when incorporated into the diet at levels as low as 2.5%. The mechanism of action for this reduction may be related to increased excretion since food intakes and cholesterol FSR were not reduced in the animals receiving the PC. In conclusion, the use of PC biomass reduces circulating cholesterol, dose dependently, in hypercholesterolaemic hamsters but not via reductions in cholesterol FSR. There is potential for the use of this biomass as a functional ingredient to aid in the management of blood cholesterol concentrations.

A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor.

A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.). The strong aprE promoter was used to drive the transcription of the fusion gene and the AprE signal sequence was fused to the mature BCE103 cellulase for efficient secretion of the fusion protein into the culture medium. It was necessary to use a B. subtilis strain deficient in nine protease genes in order to reduce the proteolytic degradation of the fusion protein during growth. The fusion protein was produced in shake flasks at concentrations >1g/L. After growth, the sBBI was activated by treatment with 2-mercaptoethanol to allow the disulfide bonds to form correctly. An economical and scalable purification process was developed to purify sBBI based on acid precipitation of the fusion protein followed by acid/heat cleavage of the fusion protein at labile Asp-Pro bonds in the CBD linker. If necessary, non-native amino acids at the N- and C-termini were trimmed off using glutamyl endopeptidase I. After purification, an average of 72 mg of active sBBI were obtained from 1L of culture broth representing an overall yield of 21% based on the amount of sBBI activated before purification.

The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase.

Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 microM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.