Structural Studies Of A Chemically Modified Thermostable Lipase From Geobacillus Sp. Strain T1

Alkylation has been successfully performed using propionaldehyde on four batches of T1 thermostable lipase (M1, M2, M3 and M4) with different degrees of modification (27% to 55%) to represent the different levels of hydrophobicity. Based on the crystal structure, T1 possessed 11 lysine residues, of which four of the lysine residues, Lys84, 102, 138 and 251 have scores between 53.7% and 95.8% exposure ratio, were totally exposed. Another four residues, Lys185, 329, 344 and 345 have a ratio between 20% and 50% (moderately exposed) and three of the lysine residues, Lys28, 207 and 229 are buried. The hydrolytic activity of the modified enzymes dropped drastically by 10 to 40-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and 222 nm by Far Ultra-Violet Circular Dichroism (CD) spectropolarimetry. As cooperative unfolding transitions were observed, the modified lipases were distinguished from the native state, which the former possessed a Tm in lower temperature range, 60-64 ºC whilst the latter at 68 ºC. Consequently, this has led us to the hypothesis of formation of a molten globule (MG)-like structure. Subsequent analysis of both native and modified lipases by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) study was carried out to ascertain the modifications and the location of these modifications. Four lysine residues, Lys28, 84, 207 and 329 were clearly identified from the native spectrum. As expected, Lys84 and Lys102 were clearly modified. Surprisingly, Lys185 which has a very low exposure ratio (27.5%) was also identified to be one of the modified residues. To further support the hypothesis of the formation of a molten globule, intrinsic and extrinsic fluorescence were performed. A decrease of fluorescence intensity was observed for modified lipase M1, which was modified using 0.5% of propionaldehyde. However, subsequent addition of propionaldehyde enhanced the fluorescence intensity of M2, M3 and M4, which indicated an inversion of placement for tryptophans to a more hydrophobic environment. As for extrinsic fluorescence, the alkylated lipases showed a clear enhancement of fluorescence intensity as compared to the native lipase due to the exposure of the hydrophobic interior of the enzymes

Effect of different drying methods on chemical and molecular structure of heteropolysaccharide–protein gum from durian seed.

The functional properties and biological aspects of a natural biodegradable biopolymer depend on its chemical and molecular structure. In this study, the effect of different drying processes on the chemical and molecular structure of the natural biodegradable biopolymer from durian seed was investigated. The chemical structure was analyzed by assessing the carbohydrate profile, protein, amino acid composition, moisture, and ash. Molecular weight (Mw), number average molecular weight (Mn), Mw/Mn ratio and mass recovery were assessed by using a size-exclusion chromatography coupled to multi angle laser light-scattering (SEC-MALS). The present study revealed that main monosaccharides in the chemical structure of differently dried durian seed gums were galactose (50.1–64.9%), glucose (29.4–45.7%%), arabinose (0.11–0.89%), and xylose (0.019–0.86%). The protein analysis indicated the presence of a low amount of the protein fraction (3.2–3.9%) in the chemical structure of the biopolymer from durian seed. The most abundant amino acids in the chemical structure of durian seed gum were leucine (31.78–43.02%), lysine (6.23–7.78%), aspartic acid (6.45–8.58%), glycine (6.17–7.27%), glutamic acid (5.43–6.55%), alanine (4.60–6.23%), and valine (4.49–5.52). The current study exhibited that the biodegradable biopolymer from durian seed was a heteropolysaccharideprotein complex with medium Mw ranging from 1.06 × 105 to 1.15 × 105 (g/mol)

Equilibrium headspace analysis of volatile flavor compounds extracted from soursop (Annona muricata) using solid-phase microextraction.

The influence of headspace solid-phase microextraction (HS-SPME) variables, namely, sample concentration, salt concentration and sample amount, on the equilibrium headspace analysis of the main volatile flavor compounds released from soursop was investigated. A total of 35 volatile compounds, comprising 19 esters, six alcohols, three terpenes, two acids, two aromatics, two ketones and an aldehyde, were identified. The results indicated that all response-surface models were significantly (p < 0.05) fitted for 10 target volatile flavor compounds. The results further indicated that more than 65% of the variation in the equilibrium headspace concentrations of target volatile flavor compounds could be explained by the final reduced models, with high R2 values ranging from 0.658 to 0.944. Multiple optimization results showed that extraction using a 76.6% (w/w) sample concentration, 20.2% (w/w) salt and 8.2 g of blended soursop pulp was predicted to provide the highest overall equilibrium headspace concentration for the target soursop volatile flavor compounds

Response surface modeling of processing parameters for the preparation of phytosterol Nanodispersions using an emulsification-evaporation technique.

The purpose of this study was to optimize the production parameters for water-soluble phytosterol nanodispersions. Response surface methodology (RSM) was employed to model and optimize three of the processing parameters: mixing time (t) by conventional homogenizer (1–20 min), mixing speed (v) by conventional homogenizer (1,000–9,000 rpm) and homogenization pressure (P) by high-pressure homogenizer (0.1–80 MPa). All responses [i.e., mean particle size (PS), polydispersity index (PDI) and phytosterols concentration (Phyto, mg/l)] fitted well to a reduced quadratic model by multiple regressions after manual elimination. For PS, PDI and Phyto, the coefficients of determination (R 2) were 0.9902, 0.9065 and 0.8878, respectively. The optimized processing parameters were 15.25 min mixing time, 7,000 rpm mixing speed and homogenization pressure 42.4 MPa. In the produced nanodispersions, the corresponding responses for the optimized preparation conditions were a PS of 52 nm, PDI of 0.3390 and a Phyto of 336 mg/l

Influence of soya lecithin, sorbitan and glyceryl monostearate on physicochemical properties of organogels

The objective of this study is to investigate the effects of three different organogelators, sorbitan monostearate (SMS), soya lecithin (SL) and glyceryl monostearate (GMS) prepared at different concentrations (12%, 15% and 18%, w/w) on the structural, thermal and mechanical properties of palm olein (PO)-based organogels. Polarized light microscopy analysis revealed needle-like crystals in SMS-PO, rod-shaped tubules in SL-PO and rosette-like aggregates in GMS-PO organogels. Intermolecular hydrogen bonding and van der Waals forces were the main drivers for the self-aggregation of these organogelators in PO, as observed in Fourier transform infrared (FTIR) spectroscopy. X-ray diffraction (XRD) results indicated β’-type polymorphic structure in SL-PO and GMS-PO. As the concentration of organogelators increased, there was a corresponding increase in the firmness, gel-sol transition (Tgs) and melting temperatures of the organogels. SMS-PO with amorphous structure had the lowest firmness, thus produced weaker gel with lower thermal stability. The oil binding capacity (OBC) of both SL-PO and GMS-PO were over 90%, significantly higher than that of SMS-PO organogels. These findings indicate that crystallization is the key determinant factor to the final properties of the organogel networks. This is influenced by the molecular structure and the concentration of the organogelators used

Reductive alkylation causes the formation of a molten globule-like intermediate structure in Geobacillus zalihae strain T1 thermostable lipase

A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines, in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme, as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly 15-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and 222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation temperature (T m) in lower temperature range at 61 °C while the latter at 68 °C. This was further supported by 8-anilino-1-naphthalenesulfonic acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number of lysine residues were confirmed to be alkylated

The influence of main emulsion components on the physicochemical properties of soursop beverage emulsions: a mixture design approach

The physicochemical properties of soursop beverage emulsion were investigated using mixture design. Results indicated that the regression models were significantly fitted for all response variables studied, except creaming index at 10°C. Interactions between biopolymers and oil phase had the most significant effect on creaming stability; however, modified starch played a much prominent role in maintaining the cloudiness and average droplet size. Meanwhile, WPI contributed significantly to the conductivity of the emulsions. The optimum condition resulted in desirable physicochemical properties could be achieved using 8.70% (w/w) modified starch, 1.02% (w/w) WPI, 10.11% (w/w) flavor oil, and 76.57% (w/w) water

Postharvest quality indices of different durian clones at ripening stage and their volatile organic compounds

The aim of the present work was to characterize the quality of durians at consumptions stage. Seven clones of durian namely “Musang King”, “D24″, “D88″, “IOI”, “XO”, “Red Prawn” and “Black Thorn” were characterized based on their physiochemical properties. The organic acid contents, sugar compositions and β-carotene of durian clones were measured by high-performance liquid chromatographic (HPLC), while the volatile organic compounds (VOCs) were analyzed using headspace solid phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC MS). There were significant differences on all the postharvest parameters in the selected durian clones. “Black Thorn” having orange pulp yieled the highest β-carotene content (4.55 × 10−5 kg/kg FW). The dominant sugars in the pulp of all durian clones were dominated by sucrose followed by glucose and fructose. Sulfur- and ester-containing compounds were the predominant VOCs found. Principal component analysis (PCA) allowed for the grouping of different durian clones based on VOCs

Rheological properties of modified starch-whey protein isolate stabilized soursop beverage emulsion systems

The rheological properties of soursop beverage emulsions as a function of main emulsion components, namely modified starch (5–12 % w/w), whey protein isolates (WPI) (0–2 % w/w), soursop flavor oil (5–15 % w/w), and deionized water (67.4–86.4 % w/w) were investigated using a fourcomponent with constrained extreme vertices mixture design. The apparent viscosity, flow index, yield stress, viscoelastic behavior (G′ and G′′) and consistency coefficient were evaluated. In general, analysis of variance (ANOVA) showed high coefficients of determination values (R2), ranging between 0.795 and 0.999 for the regression models, thus confirming a satisfactory adjustment of the polynomial regression models with the experimental data. Increase in both modified starch and oil phase concentration had increased the apparent viscosity of the emulsions. Contrary, higher concentrations of oil phase had negative effects on flow index and consistency coefficient, resulting in the changes of flow behavior. In addition, modified starch showed solid-like elastic properties at low concentration but behaved as liquid-like viscous as the concentration of modified starch increased. Oil phase concentration had a significant (p0.05) effect on neither the apparent viscosity nor the flow index at low concentrations but was an important element in providing elastic properties to the emulsion film

Chemical modification of lipases

Since the industrial uses of lipase is expanding, research and development on this enzyme is strategically focusing on modifying its chemical, physical and biological properties. Surface residuse of enzymes are reactive chemically and thus a potential target for chemical modification. We have carried out chemical modification via reductive alkylation on lysine residuse of T1 and Candida rugosa lipase (CRL)using propionaldehyde. Through molecular dynamics (MD) and circular dichroism (CD), intrinsic and extrinsic fluorescence studies, the altered properties of T1 lipase were examined. Reductive alkylation using propionaldehyde caused the unfolding of enzymes as observed in chemically modified Candida rugosa and T1 lipase. As the effect of reductive alkylation is not localized at the modified site, the formation of molten globule could be observed in modified T1 lipase