To study the changes in secondary, tertiary and quaternary structures and the alteration in protein–protein interactions during the protein aggregation upon heating, bovine serum albumin (BSA) was incubated over a range of temperature, and its structure was monitored by various biophysical techniques including circular dichroism (CD), second-derivative UV absorption spectroscopy, dynamic light scattering (DLS) and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS). The experimental data demonstrated that the size of BSA was relatively stable up to 60 1C. Above 65 1C, however, BSA underwent the obvious heat-induced aggregation (a dramatic growth of hydrodynamic radius Rh), accompanied with great unfolding of its secondary structure (a sigmoidal reduction of a-helix with the transition midpoint at 67.6 1C), with significant expansion of its tertiary structure (an increased solvent exposure of some aromatic residues with the transition midpoint at 65.5 1C), with distinct screening of electrostatic repulsion (a great reduction of second virial coefficient A2 above 65 1C) and with remarkable formation of aggregates (an evident increase of molecular weight above 65 1C).

The effect of pancreatic hydrolysis on the aggregate size and molecular weight distribution (MWD) of bovine casein was investigated by size exclusion chromatography (SEC), coupled with multiangle laser light scattering (MALLS) and dynamic light scattering (DLS). The concentrations of both casein aggregates and monomers decreased as the reaction proceeded and almost disappeared after a 10min hydrolysis. The ratio of aggregates to monomers was maintained at 1:(2-2.5) during the initial 10min hydrolysis, which shows that the caseins in the solution are in a dynamic equilibrium system, between aggregates and monomers. Upon limited hydrolysis, casein aggregates displayed a continuous growth in their sizes and molecular weights, together with a decrease in the intermolecular repulsion. This result was further supported by on-line DLS measurements, which showed the increase in the hydrodynamic radii and the decrease in light scattering intensities of casein hydrolysates. Moreover, the release kinetics of peptide fractions with different molecular weights were also followed. It was suggested that the increase in the hydrophobic attraction and the reduction in the protein repulsion might promote casein aggregation, during the limited pancreatic hydrolysis.

Bovine casein was digested with pancreatin at pH 8.0 in a batched stirred tank reactor. In total, 52 casein phosphopeptides (CPPs) in the time-course samples were separated and identified using liquid chromatography-tandem mass spectrometry by means of neutral loss scanning and database searching. Of these peptides, 23, 7, 8, 10 and 4 are singly, doubly, triply, quadruply and quintuply phosphorylated, respectively. Furthermore, 14 peptides contained the cluster sequence, S(P)S(P)S(P)EE, providing the mineral binding sites; these peptides were all formed after 10 min of hydrolysis, but none of them survived after 6 h of pancreatic digestion. A study of the release kinetics of CPPs allowed determination of the degrees of hydrolysis for the preparation of target peptides with high yields. The discrimination of pancreatic attack on different phosphorylated regions of bovine casein was also analyzed in terms of the release of CPPs.-2006 Elsevier Ltd. All rights reserved.

Bovine serum albumin was hydrolyzed with trypsin in a batch stirred tank reactor. The influence of operating parameters on the degree of hydrolysis was investigated by pH-stat method. Molecular weight distribution of hydrolysates was analyzed by high performance sizeexclusive chromatography. The kinetic model of hydrolysis reaction was studied by a lumping technique. The hydrolysates were combined into several lumps according to their molecular weights. Reaction of the lumps was described with intrinsic kinetic equations, and the lumping kinetic model for bovine serum albumin tryptic hydrolysis reaction was established. The rate constant of each reaction was estimated by Marquardt method in two steps from lower level to upper level. The reliability of the model was tested by comparing the computed values with the experimental values.

In the enzymatic membrane reactor for separating casein hydrolysatem backflushing technology has been used to decrease the fouling of the membrance. Predication of the backflushing efficiency poses a complex non-linear problem as the system integrates enzymatic hydrolysis, membrane separation and periodic backflushing together. In this paper an alternative artificaial neural network approach is developed to predict the vackflushing efficiency as a function of duration and interval. A contour plot of backflushing performance is presented to model these effects, and the backflushin conditions have been optimized as duration of 10s and interval of 10 min using this neural network, Also, simpleneural networks are established to predict the time evolution of flux before and after backflushing. The results predicted by the modelass are in good agreement with the experimental data, and the average deviations for all te cases are well within +-5% The neural network approach is found to be capable of modeling the backflushing with confidence.

The kinetics of casein tryptic hydrolysis to prepare active peptides was inveestigated. Taking into account the reaction mechanism including single substrale hydrolysism irreversible enzyme inactivation, and substrate inhibition, a set of exponential equations was established to characterize the enzymatic hydrolysis curves The verification was carried out by a series of experimental results and indicated that the average regrcssive error was less than 5% According to the proposed kinetic model, the kinetic constants and thermodynamic constants of the reaction system were also calculated.

To obtain high concentration of ethanol from cellulose, corncob was pretreated with acid and alkali to remove non-cellulose components, and then subjected to simultaneous saccharification and fermentation (SSF). An ethanol concentration as high as 69.2g/L was achieved with 19% dry matter (DM) using batch SSF, resulting in an 81.2% overall ethanol yield. A fed-batch process using a high solid concentration was also investigated. Fresh substrate was pretreated with dilute sulfuric acid-sodium hydroxide, and then added at different amounts during the first 24h, to yield a final dry matter content of 25% (w/v). SSF conditions with cellulose loading of 22.8 FPU/g glucan, dry yeast (Saccharomyces cerevisiae) loading of 5g/L and substrate supplementation every 4 h yielded the highest ethanol concentration of 84.7g/L after 96 h. This corresponded to a 79% overall ethanol yield.

Assembly and fibrillation of amyloid proteins are believed to play a key role in the etiology of various human diseases, including Alzheimer’s, Parkinson’s, Huntington’s and type II diabetes. Insights into conformational changes and formation processes during amyloid fibrillation are essential for the clinical diagnosis and drug discovery. To study the changes in secondary, tertiary, quaternary structures, and the alteration in the collective vibrational mode density of states during the amyloid fibrillation, bovine insulin in 20% acetic acid was incubated at 60℃, and its multi-level structures were followed by various biophysical techniques, including circular dichroism (CD), thioflavin T fluorescence (ThT), dynamic light scattering (DLS), electron microscopy, and terahertz (THz) absorption spectroscopy. The experimental data demonstrated a transformation of a-helix into b-sheet starting at 26 h. This was followed by the aggregation of insulin, as shown by ThT binding, with a transition midpoint at 41 h, and by the bulk formation of mature aggregates after about 71 h. THz is a quick and non-invasive technique, which has the advantage of allowing the study of the conformational state of biomolecules and tissues. We first applied THz spectroscopy to study the amyloid fibrillation. At the terahertz frequency range of 0.2–2.0 THz, there was an apparent increase in both the absorbance and refractive index in THz spectra. Thus, THz is expected to provide a new way of looking into amyloid fibrillation.

zMicrocrystalline cellulose Avicel was subjected to an ionic liquid (IL) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) pretreatment step to obtain a high conversion of soluble sugars in the enzymatic hydrolysis of cellulose. Avicel was dissolved completely in [BMIM]Cl, followed by precipitating it into deionized water to obtain the regenerated cellulose. The high performance liquid chromatography (HPLC) analysis results indicated that after 24 h, the sugar released from IL-treated Avicel was 94.65%, much higher than that from the original Avicel (48.57%). Structure, crystallinity and morphology of the IL-treated Avicel were also investigated as follows: the hydrogen bonding, detected by FTIR, decreased; the crystallinity, monitored by XRD, changed significantly from crystalline to amorphous pattern; the morphology, examined by SEM, demonstrated that the orderly structure was destroyed remarkably with some pores and holes appearing in the cellulose substrates. These changes of Avicel pretreated with [BMIM]Cl led to the increase of sugar conversion during cellulose enzymatic hydrolysis.

以微晶纤维素为研究对象，设计了离子液体1-丁基-3-甲基咪唑氯盐（1-butyl-3-methylimidazoliumchloride，[BMIM]Cl）预处理微晶纤维素Avicel的实验方法以实现纤维素的高效酶解糖化.在[BMIM]Cl中Avicel完全溶解，经水洗沉淀得到再生纤维素，回收后的离子液体可重复利用.预处理后底物酶解的可溶性糖转化率在24h时高达94.65%，较之同样条件下未经预处理底物的酶解糖转化率（48.57%）有飞跃性提升。进一步考察了离子液体预处理对纤维素结构及形态的影响，结果表明：[BMIM]Cl预处理后Avicel氢键减弱；结晶度明显下降，结晶型态由纤维素I型转变为纤维素II型；由规整的平行排布转变为疏松有孔的无序形貌。正是离子液体预处理引起的纤维素微观与宏观结构性质的显著改变使得再生后纤维素酶解的可溶性糖转化率大幅提高。