Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions.

BSA stabilizes Streptococcus thermophilus beta-galactosidase against thermal inactivation and binds to the active enzyme subunits formed on heating. The mechanism of interaction and stabilization, however, is unknown, and it was investigated using different proteins. The results show that several proteins increased the enzyme half-life (t 1/2) at 64 degrees C in the presence of the substrate lactose. The best stabilizers were BSA (9-fold) and casein (6-fold). There was a significant correlation between enzyme half-life (t 1/2) and surface hydrophobicity of the proteins (So), of the form t 1/2 varies; is directly proportional to S0.5o. The surface hydrophobicity of the enzyme increased upon heating, while that of BSA declined. Heating enzyme and BSA together caused a net loss in surface hydrophobicity, indicating hydrophobic interactions, but there was no change in the absence of heating. Stabilization of the enzyme by BSA was markedly affected by chaotropic and kosmotropic salts. Stabilization was increased by 1 M Na2SO4 and reduced by 1 M NaBr; 1 M NaCl had little effect. None of the three salts increased the stability of the enzyme itself, indicating that the effect was on the enzyme-protein interaction. The results indicate that BSA stabilized the enzyme by hydrophobic interactions with the heated enzyme and that surface hydrophobicity is a major determinant of the extent of stabilization by a protein.

Purification and thermostability of beta-galactosidase (lactase) from an autolytic strain of Streptococcus salivarius subsp. thermophilus.

beta-Galactosidase from an autolytic strain of Streptococcus salivarius subsp. thermophilus was purified 109-fold to near homogeneity. The yield of purified enzyme was 41% and the specific activity was 592 o-nitrophenyl beta-D-galactopyranoside U/mg at 37 degrees C. Two isozymes were present, but only one subunit was detected, having a mol. wt of 116,000. Enzyme stability was 37-83 times greater in milk than in buffer in the range 60-65 degrees C. At 60 degrees C the half-life in milk was 146 min. Denaturation in buffer was first-order, but in milk the overall reaction order with respect to enzyme concentration was approximately 0.5. The activation energy for denaturation was 453 kJ/mol in milk and 372 kJ/mol in buffer. In milk the activation energy for lactose hydrolysis was 35.1 kJ/mol.

Effect of guar gum, lignin and pectin on proteolytic enzyme levels in the gastrointestinal tract of the rat: a time-based study.

The effects of dietary pectin (P), guar gum (G) and lignin (L) on stomach emptying and potential levels of pepsin, trypsin and chymotrypsin during a 2-h period after force-feeding were investigated in growing rats. All of the fibers delayed stomach emptying by 21-26 min. Total potential pepsin activity over 2 h decreased for P (57%), G (44%) and L (20%). In the intestine, total potential trypsin activity over 2 h increased for L (16%) but decreased for P (21%). Total potential chymotrypsin activity over 2 h increased for L (54%) and G (39%). Sixteen to 21% of the variability in intestinal activity over time was statistically attributable to variation in the weight of intestinal contents. The results indicate that fiber components altered proteolytic enzyme levels in the gastrointestinal tract, but the decreased protein utilization previously observed with these fibers is probably not due to reduced levels of intestinal proteases.

Effect of dietary supplements of guar gum and cellulose on intestinal cell proliferation, enzyme levels and sugar transport in the rat.

Male Wistar rats (approximately 200 g) were given fibre-free semi-synthetic diets containing either sucrose (S) or a sucrose-starch mixture (SS) as the carbohydrate component, or a diet similar to SS containing 40 g guar gum/kg (G), or 100 g cellulose/kg (C). The animals remained healthy, and weight gain after 30 d was similar in all groups. The small intestines of the animals given diet G were significantly longer than those of the other groups, and showed signs of increased mitotic activity and mucosal growth. No significant differences in mucosal enzyme activity were detected between the two fibre-free control groups. Lactase (EC 3.2.1.23) and alkaline phosphatase (EC 3.1.3.1) activities were significantly lower than controls in group G, but were higher in group C. Kinetic analysis of 3-O-methyl glucose uptake by isolated intestine indicated that the maximum transport rate (Vmax) of tissue from group G tended to be lower than from the fibre-free group SS and group C. It is concluded that materials which are classed as dietary fibre but which differ markedly in their physical properties may also differ in the functional changes to which they give rise in the small intestine. These changes may be at least partially mediated by effects on mucosal cell proliferation.

Effect of dietary fiber components on fecal nitrogen excretion and protein utilization in growing rats.

The effects of purified fiber components and wheat bran on several indices of protein utilization were determined in growing rats. A control diet containing 10% casein was diluted with either cellulose (C), pectin (P), lignin (L), guar gum (G), or wheat bran (W) at fiber levels ranging from 3% to 20%. All fibers except C caused a decrease in net protein ratio (NPR) as compared to the control casein diet. This depression in NPR increased as the dietary fiber level increased. Apparent and true nitrogen digestibilities also decreased with all fibers at all levels. At the highest level of fiber (20%) the depression was greater for G and W and was least for C. NPR when divided by digestibility (analogous to biological value) decreased with P,L, W (all levels) and G (20% level) but not with C. When rats were fed fiber without protein, there was increased excretion of endogenous fecal nitrogen with all fibers at all levels. The results demonstrate that fiber(s) affected protein utilization as measured by NPR, digestibility and endogenous fecal nitrogen excretion and that the negative effect increased with the level of fiber consumed.

Purification and characterization of beta-galactosidase from a strain of Bacillus coagulans.

beta-Galactosidase from B. coagulans strain L4 is produced constitutively, has a mol. wt. of 4.3 x 10(5) and an optimal temperature of 55 degrees C. The optimal pH at 30 degrees C is 6.0 whereas at 55 degrees C it is 6.5. The energy of activation of enzyme activity is 41.9 kJ/mol (10 kcal/mol). No cations are required. The Km with ONPG as substrate is 4.2-5.6 mM and with lactose is 50 mM. The Ki for inhibition by galactose is 11.7-13.4 mM and for dextrose is 50 mM. Galactose inhibited competitively while dextrose inhibited noncompetitively. The purified and unprotected enzyme is 70% destroyed in 30 min at 55 degrees C whereas in the presence of 2 mg/ml of BSA 42% of the activity is destroyed in 30 min at 55 degrees C. An overall purification of 75.3-fold was achieved.

Selection of strain, growth conditions, and extraction procedures for optimum production of lactase from Kluyveromyces fragilis.

Forty-one strains of Kluyveromyces fragilis (Jörgensen) van der Walt 1909 varied 60-fold in ability to produce lactase (beta-galactosidase). The four best strains were UCD No. 55-31 (Northern Regional Research Center NRRL Y-1196), UCD No. C21(-), UCD No. 72-297(-), and UCD No. 55-61 (NRRL Y-1109). Biosynthesis of lactase during the growth of K. fragilis strain UCD No. 55-61 was followed on both lactose and sweet whey media. Maximum enzyme yield was obtained at the beginning of the stationary phase of growth. Bets lactase yields from K. fragilis UCD No. 55-61 were obtained with 15% lactose and an aeration rate of at least .2 mmol oxygen/liter per min. Supplementary growth factors were unneccessary for good lactase yeilds when yeast was grown on whey media. Best extraction of lactase from fresh yeast cells was obtained by toluene autolysis (2% vol/vol) at 37 C in .1 M potassium phosphate buffer, pH 7.0, containing .1 mM manganese chloride and .5 mM magnesium sulfate. The enzyme was concentrated and purified partially by acetone precipitation. At least 95% of the enzyme activity of the concentrated solution was retained after storage for 7 days at 22 C, for 3 wk at 4 C, and for 6 wk at -20 C.