Large-Size Subunit Catalases Are Chimeric Proteins: A H2O2 Selecting Domain with Catalase Activity Fused to a Hsp31-Derived Domain Conferring Protein Stability and Chaperone Activity.

Bacterial and fungal large-size subunit catalases (LSCs) are like small-size subunit catalases (SSCs) but have an additional C-terminal domain (CT). The catalytic domain is conserved at both primary sequence and structural levels and its amino acid composition is optimized to select H2O2 over water. The CT is structurally conserved, has an amino acid composition similar to very stable proteins, confers high stability to LSCs, and has independent molecular chaperone activity. While heat and denaturing agents increased Neurospora crassa catalase-1 (CAT-1) activity, a CAT-1 version lacking the CT (C63) was no longer activated by these agents. The addition of catalase-3 (CAT-3) CT to the CAT-1 or CAT-3 catalase domains prevented their heat denaturation in vitro. Protein structural alignments indicated CT similarity with members of the DJ-1/PfpI superfamily and the CT dimers present in LSCs constitute a new type of symmetric dimer within this superfamily. However, only the bacterial Hsp31 proteins show sequence similarity to the bacterial and fungal catalase mobile coil (MC) and are phylogenetically related to MC_CT sequences. LSCs might have originated by fusion of SSC and Hsp31 encoding genes during early bacterial diversification, conferring at the same time great stability and molecular chaperone activity to the novel catalases.

Effect of dietary crude protein degradability and corn processing on lactation performance and milk protein composition and stability.

The present study aimed to evaluate the effect of crude protein degradability and corn processing on lactation performance, milk protein composition, milk ethanol stability (MES), heat coagulation time (HCT) at 140°C, and the efficiency of N utilization for dairy cows. Twenty Holstein cows with an average of 162 ± 70 d in milk, 666 ± 7 kg of body weight, and 36 ± 7.8 kg/d of milk yield (MY) were distributed in a Latin square design with 5 contemporaneous balanced squares, 4 periods of 21 d, and 4 treatments (factorial arrangement 2 × 2). Treatment factor 1 was corn processing [ground (GC) or steam-flaked corn (SFC)] and factor 2 was crude protein (CP) degradability (high = 10.7% rumen-degradable protein and 5.1% rumen-undegradable protein; low = 9.5% rumen-degradable protein and 6.3% rumen-undegradable protein; dry matter basis). A significant interaction was observed between CP degradability and corn processing on dry matter intake (DMI). When cows were fed GC with low CP degradability, DMI increased by 1.24 kg/d compared with cows fed GC with high CP degradability; however, CP degradability did not change DMI when cows were fed SFC. Similar interactions were observed for MY, HCT, and lactose content. When cows were fed GC diets, high CP degradability reduced MY by 2.3 kg/d, as well as HCT and lactose content, compared with low CP degradability. However, no effect of CP degradability was observed on those variables when cows were fed SFC diets. The SFC diets increased dry matter and starch total-tract digestibility and reduced ß-casein (CN) content (% total milk protein) compared with GC diets. Cows fed low-CP degradability diets had higher glycosylated κ-CN content (% total κ-CN) and MES, as well as milk protein content, 3.5% fat-corrected milk, and efficiency of N for milk production, than cows fed high-CP degradability diets. Therefore, GC and high-CP degradability diets reduced milk production and protein stability. Overall, low CP degradability increased the efficiency of dietary N utilization and MES, probably due to changes in casein micelle composition, as CP degradability or corn processing did not change the milk concentration of ionic calcium. The GC diets increased ß-CN content, which could contribute to reducing HTC when cows were fed GC and high-CP degradability diets.

On the Roles of Wheat Endosperm ADP-Glucose Pyrophosphorylase Subunits.

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.

Effect of partial whey protein depletion during membrane filtration on thermal stability of milk concentrates.

Membrane filtration technologies are widespread unit operations in the dairy industry, often employed to obtain ingredients with tailored processing functionalities. The objective of this work was to better understand the effect of partial removal of whey proteins by microfiltration (MF) on the heat stability of the fresh concentrates. The micellar casein concentrates were compared with control concentrates obtained using ultrafiltration (UF). Pasteurized milk was microfiltered (80 kDa polysulfone membrane) or ultrafiltered (30 kDa cellulose membrane) without diafiltration (i.e., no addition of water) to 2× and 4× concentration, based on volume reduction. The final concentrates showed no differences in pH, casein micelle size, or mineral concentration in the serum phase. The micellar casein retentates (obtained by MF) showed a 20 and 40% decrease in whey protein concentration compared with the corresponding UF milk protein concentrates for 2× and 4× concentration, respectively. The heat coagulation time decreased with increasing protein concentration, regardless of the treatment; however, MF retentates showed a higher thermal stability than the corresponding UF controls. The average diameter for casein micelles increased after heating in UF but not MF concentrates. The turbidity (measured by light scattering) increased after heating, but to a higher extent for UF retentates than for MF retentates at the same protein concentration. It was concluded that the reduced amount of whey protein in the MF retentates caused a significant increase in the heat stability compared with the corresponding UF retentates. This difference was not due to ionic composition differences or pH, but to the type and amount of complexes formed in the serum phase.

Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase) produced by Moniliophthora perniciosa

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70 percent) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20 percent of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

A enzima glucanase de Moniliophthora perniciosa foi produzida em meio líquido e purificada a partir do sobrenadante da cultura. A metodologia de superfície de resposta (MSR) foi usada para avaliar os efeitos das variáveis, incluindo indutor (extrato de levedura) e tempo de fermentação, na atividade da glucanase de M. perniciosa detectada no meio de cultura. A enzima presente no sobrenadante foi purificada em duas etapas: precipitação com sulfato de amônio (70 por cento) e cromatografia de filtração em gel em Sephacryl S-200. A produção da enzima glucanase foi maior na concentração de 5,9 g L-1 de extrato de levedura e 13 dias de fermentação. Os resultados mostraram três diferentes isoformas (GLUI, GLUII e GLUIII) com fatores de purificação de 4,33, 1,86 e 3,03, respectivamente. O extrato enzimático parcialmente purificado mostrou um pH ótimo de 5,0 e uma temperatura ótima de 40°C. A atividade enzimática aumentou na presença de KCl em todas as concentrações estudadas. A atividade da glucanase foi maior na presença de NaCl 0,2 M. A enzima apresentou alta estabilidade térmica, perdendo apenas 10,20 por cento de sua atividade específica após 40 minutos de incubação a 90°C. Os resultados de termoestabilidade e a atividade em baixo pH mostraram que a enzima glucanase de M. perniciosa tem características promissoras para futuras aplicações industriais.