Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae – to produce sorbitol from Jerusalem artichokes

The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl₂ for 30 min, the fusion frequency reached 2.64 fusants/10⁶ parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.     

Sorbitol-mediated stabilization of human IgG against thermal inactivation

Sorbitol at 30% (w/w) stabilized human IgG to thermal denaturation from 60 to 85°C, increasing the protein's half life from 25 to 266 min at 70°C. A kinetic model based on the Lumry-Eyring inactivation scheme was developed and used to estimate the apparent rate constant and activation energy. © Rapid Science. 1998     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Improvement of oxidoreductase stability of cethyltrimethylammoniumbromide permeabilized cells ofZymomonas mobilis through glutaraldehyde crosslinking

Summary Permeabilization ofZymomonas mobilis with CTAB(Cetyltrimethylammoniumbromide) was investigated in order to obtain a stable process for sorbitol production in the immobilized system. The optimum conditions for sorbitol formation were treating cells with 0.2% CTAB at 4°C for 10 min. For the immobilized system permeabilized cells were treated with glutaraldehyde to improve the system with cross-linking of enzymes. In this way, no significant loss of enzyme activity was apparent during 30 day operation in a continuous process. The productivity of the continuous process at a dilution rate 0.2 h^–1 was 6.51g/L-h for sorbitol. The CTAB-permeabilized cells could be used to produce sorbitol and gluconic acid simultaneously in the long term continuous process.     

Interaction of selected cosolvents with bovine alpha-lactalbumin

Alpha-lactalbumin constitutes about 3% of bovine milk proteins. The preferential solvent interactions between selected cosolvents (sorbitol, sucrose and glycerol) and alpha-lactalbumin at pH 7.5 was determined using precision densitimetry. The preferential interaction parameter (xi(3)) and other thermodynamic parameters were calculated at different solvent concentrations. The xi(3) parameter was maximum at 30%, 45% and 40% (w/v) concentrations with the values of -0.282g/g, -0.171g/g and -0.299g/g for sorbitol, sucrose and glycerol, respectively. Thus the principal driving energy in the system being preferential hydration and mutual exclusion of bulk solvent. There was only a marginal change in the CD spectra of the protein with these cosolvents indicating the integrity of secondary structures. The results of thermal denaturation measurements indicated an increase in thermal stability of alpha-lactalbumin with these cosolvents. In the presence of 30% sorbitol there was an increase in the apparent thermal transition temperature (apparent T(m)) from 65 to 71 degrees C. These results indicate that the selected cosolvents in this study stabilizes alpha-lactalbumin without altering the structure of the protein.     

14C-Assimilate pattern and kinetics of photosynthetic 14CO2-assimilation of the marine red alga Bostrychia scorpioides

Summary Occurrence and metabolism of dulcitol and sorbitol in the marine red alga Bostrychia scorpioides (Huds.) Mont. (Ceramiales: Rhodomelaceae) were investigated. Both hexitols are rapidly ¹⁴C-labelled during photosynthesis in a H¹⁴CO₃-seawater medium and are accumulated at comparable rates. The absolute quantity amounts to about 3.2% on a dry weight basis; the percentage of ¹⁴C-labelling after 60 min is 30% for dulcitol and 40% for sorbitol. Additionally small amounts of free [¹⁴C] glucose were found. Pulse labelling experiments and changes in specific activity provide evidence that both hexitols are rapidly available respiratory substrates, which, however, are probably not interconvertible with polymeric compounds. Some chemotaxonomic aspects are discussed.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Isomaltooligosaccharide inhibits the crystallization of sorbitol humectant and glucan formation by Streptococcus mutans

The addition of 20% (w/w) isomaltooligosaccharide (IMO) to sorbitol humectant inhibited crystallization for up to 30 days at –15°C. IMO also decreased glucan formation by Streptococcus mutans KCTC 3065 by 70%, when mixed with sorbitol humectant. Furthermore, IMO increased the moisture content and viscosity of the mixture by up to 5% and 50%, respectively. © Rapid Science Ltd. 1998     

Sensitive,selective gas chromatographic—mass spectrometric analysis with trifluoroacetyl derivatives and a stable isotope for studying tissue sorbitol-producing activity

One of the major mechanisms involved in diabetic microangiopathy is considered to be an altered polyol pathway. However, clarifying the pathophysiology is difficult due to the lack of a sensitive method for measuring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluoroacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorbitol (U-[¹³C]sorbitol, ¹³C₆H₁₄O₆, 98.7%) as an internal standard. Gas chromatography—mass spectrometry (GC—MS) using an SE-30 capillary column gave elution of TFA derivatives of sugars, polyols and U-[¹³C]sorbitol within 8 min, with clear separation of sorbitol. In the calibration study, the coefficients of correlation between the amount of sorbitol added and that determined in standard solutions containing 0.1–8.0 nmol sorbitol, erythrocyte mixture and liver cytosol mixture were r=0.999, r=0.997 and r=0.997, respectively. The precision of the GC—MS measurement of standard solution was C.V.=4.3%. Because glucose is used as a substrate, the method can clarify the polyol pathway under physiological conditions. With this method, K_m and V_max values of the reductase in erythrocytes were 115±19 mmol/l and 4.42±0.26 nmol/min/g of hemoglobin. In human liver, on the other hand, they were 75±132 mmol/l and 0.77±0.090 nmol/min/mg of protein, respectively. This difference of K_m values suggested that aldehyde reductase rather than aldose reductase is mainly responsible for reducing glucose to sorbitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentrations of sorbitol in various tissues and cells and should enable clarification of the kinetics of glucose reduction to sorbitol, which in turn can be used to evaluate the role of an altered polyol pathway in the pathophysiology of diabetic microangiopathy.     

Mechanism of solvent induced thermal stabilization of papain

In the present study an attempt is made to elucidate the effects of various cosolvents, such as sorbitol, sucrose, xylose and glycerol, on papain. The stabilizing effects of these cosolvents on the structure and function of papain is determined by the activity measurements, fluorescence spectroscopy and differential scanning calorimetry (DSC). The enzyme activity measurements indicate several fold increase in the thermal stability of the enzyme in all the cosolvents used. The thermal denaturation studies of papain in presence of various concentrations of cosolvents indicated a shift in the apparent thermal denaturation temperature (app Tm) suggesting increased thermal stability of papain in presence of cosolvents. The app Tm shifted from a control value of 83+/-1 degrees C to a value of >90+/-1 degrees C in presence of 40% sorbitol. The DSC thermogram for native papain can be clearly deconvoluted into two transitions corresponding to left and right domain and in presence of cosolvents both transitions A and B shift to higher temperature. Maximum stabilization was seen in case of 30% sorbitol where the thermal transition temperatures increased compared to control. The results from partial specific volume measurements of papain in presence of cosolvents suggest that the preferential interaction parameter (xi3) was negative in all cosolvents and maximum hydration was observed in the case of glycerol where the preferential interaction parameter was 0.165g/g. These above results suggest that there is a considerable increase in the thermal stability of papain in presence of these cosolvents as a result of preferential hydration.     

Effects of sugars on the cross-linking formation and phase separation of high-pressure induced gel of whey protein from bovine milk

The effects of sugars (xylose, arabinose, fucose, fructose, galactose, glucose, sorbitol, maltose, sucrose, and lactose; 0-20% w/v) on the properties of the pressure-induced gel from a whey protein isolate (20%, 800 MPa, 30 degrees C, 10 min) were studied. All the sugars decreased the hardness, breaking stress and water-holding capacity of the gel at the same concentration of 55.5 mM. Increasing the sugar content changed the microstructure of the gel from a honeycomb-like structure to a stranded structure, while the strand thickness was progressively reduced. These results suggest that sugars decreased the degree of intermolecular S-S bonding of proteins and non-covalent interaction, and restrained the phase separation during gelation under high pressure.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media

The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (a(w) = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89 degrees C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72 degrees C). (c) 1995 John Wiley & Sons, Inc.     

Thermal stability of <Emphasis Type="Italic">α</Emphasis>-amylase in aqueous cosolvent systems

The activity and thermal stability of α-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 ± 2°C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T _m )_app and activation energy (E _a ) of α-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T _m )_app were 20°C, 14°C, 13°C and 9°C, respectively. The E _a of thermal denaturation of α-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.     

Purification of a Bacteriocin-Like Inhibitory Substance Derived from Pediococcus acidilactici Kp10 by an Aqueous Micellar Two-Phase System

Aqueous micellar two-phase system (AMTPS) is an extractive technique of biomolecule, where it is based on the differential partitioning behavior of biomolecule between a micelle-rich and a micelle-poor phase. In this study, an AMTPS composed of a nonionic surfactant, Triton X-100 (TX-100) was used for purifying a bacteriocin-like inhibitory substance (BLIS) derived from Pediococcus acidilactici Kp10. The influences of the surfactant concentration and the effect of additives on the partitioning behavior and activity yield of the BLIS were investigated. The obtained coexistence curves showed that the mixtures of solutions composed of different surfactant concentrations (5–30% w/w) and 50% w/w crude load were able to separate into two phases at temperatures of above 60 °C. The optimum conditions for BLIS partitioning using the TX-100-based AMTPS were: TX-100 concentration of 22.5% w/w, CFCS load of 50% w/w, incubation time of 30 min at 75 °C, and back-extraction using acetone precipitation. This optimal partitioning resulted in an activity yield of 64.3% and a purification factor of 5.8. Moreover, the addition of several additives, such as sorbitol, KCl, dioctyl sulfosuccinate sodium salt, and Coomassie® Brilliant Blue, demonstrated no improvement in the BLIS separation, except for Amberlite® resin XAD-4, where the activity yield was improved to 70.3% but the purification factor was reduced to 2.3. Results from this study have demonstrated the potential and applicability of TX-100-based AMTPS as a primary recovery method for the BLIS from a complex fermentation broth of P. acidilactici Kp10. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2719, 2019     

Increased thermal stability of proteins in the presence of sugars and polyols.

Sugars and polyols stablize proteins against heat denaturation. Scanning calorimetry was used to obtain a quantitative estimate of the degree of stabilization. Solutions of ovalbumin, lysozyme, conalbumin, and alpha-chymotrypsinogen were heated at a constant rate, and the temperature of the maximum rate of denaturation was estimated (Tm). Addition of a sugar or polyol raised Tm. The magnitude of the stabilizing effect (delta Tm) depended on both the nature of the protein and the nature of the sugar or polyol, ranging from 18.5 degrees C for lysozyme at pH 3 in the presence of 50% (w/w) sorbitol to 0 degrees C for conalbumin at pH 7 in 50% glycerol solution. It is argued that this stablization is due to the effects of sugars and polyols on hydrophobic interactions. The strength of the hydrophobic interaction was measured in model systems in sucrose and glycerol solutions. Sucrose and glycerol strengthened the pairwise hydrophobic interaction between hydrophobic groups; however, they reduced the tendency for complete transfer of hydrophobic groups from an aqueous to a nonpolar environment. The extent of stabliziation by different sugars and polyols is explained by their different influences on the structure of water. The difference between the partial molar volume of the sugar or polyol and its van der Waals volume was used as a rough quantitative measure of the structure-making or structure-breaking effect. There was a linear relationship between this quantity and delta Tm.     

A simple preparation of beta-trypsin based on a calorimetric study of the thermal stabilities of alpha- and beta-trypsin

Bovine trypsin preparations contain, in addition to the single chain form of the enzyme, an active two-chain autolysis product (Schroeder, D. D., and Shaw, E., J. Biol. Chem. (1968), 243, 2943–2949). Differential scanning calorimetric (DSC) studies showed that the single chain form, β-trypsin, is more stable to thermal denaturation than the two-chain form, α-trypsin. Rate constants and activation energies for the thermal denaturation of β-trypsin are 5 × 10^?5 sec^?1 and 69 kcal/mole and of α-trypsin are 5 × 10^?3 sec^?1 and 38 kcal/mole at pH 4.4 and 48 °C. Preparation of pure β-trypsin can be greatly simplified by prior thermal denaturation of the α form. At least 75% of the α form is denatured by heating a 10–15% solution of commercial crystalline trypsin for 30–45 min at 48 °C, pH 4.4, 0.02 m Ca²⁺. The native β-trypsin is then easily isolated from the denatured α-trypsin by batchwise adsorption onto ovoinhibitor-agarose at pH 8. After elution at pH 2, dialysis, and lyophilization an average preparation contained approximately 85% β-trypsin, 10% α-trypsin, and 5% inactive material. Benzamidine was used during the isolation to decrease the rate of conversion of β- to α-trypsin. Because the separation of active β-trypsin from heat-denatured α-trypsin is relatively easy, the total preparation time has been reduced to 1 day.     

Use of polyethylene glycol in isolation and assay of stable, enzymically active starch granules from developing wheat endosperms

A procedure using polyethylene glycol (PEG), molecular weight 1000, was developed for the isolation of starch granules from wheat endosperm. Immature endosperm tissue was cut repeatedly in 300 millimolar PEG 1000 and filtered through Miracloth. Centrifugation separated a pellet from a supernatant with inhibitory activity. The pellet contained several enzyme activities, including soluble and bound components of starch synthase, starch phosphorylase, and sucrose synthase activities. The starch phosphorylase activity was unaffected by several washings with 300 millimolar PEG 1000 but was lost when the granules were washed once without PEG or washed with sucrose, glycerol, or sorbitol (up to 30%, w/v). The fraction of starch synthase, remaining on the granules after a wash without PEG (the `bound' activity) was not affected by the addition of 30% sorbitol to the wash buffer. This fraction became larger with grain development (0.2-0.7).

To obtain high activity, PEG was required not only during isolation of granules but also in the assay of both starch phosphorylase and starch synthase giving optimum activity at 225 to 255 millimolar. PEG reduced the requirement for glycogen as primer with soluble starch synthase. However, the `bound' starch synthase activity was unaffected by PEG. PEG of different size were compared by their effects in the assay of starch granules: with increase in molecular size, the same effect was obtained at ever lower polymer concentration (w/v) down to a limit.

Treatment of granules with Triton X-100 did not affect their starch synthase activity, but it removed the capacity to incorporate label from UDP [¹⁴C]G into non-starch polymers.

It is concluded that PEG, like some other active compounds (ethanol Na₃-citrate, and Ficoll) could mediate enzyme-primer interaction by exclusion.

     

Enzymatic preparation of wheat bran xylooligosaccharides and their stability during pasteurization and autoclave sterilization at low pH

Xylooligosaccharides (XOS) were prepared from wheat bran insoluble dietary fiber (WBIDF) by treatment with commercial xylanase preparation Sunzymes. XOS, with a purity of 95% (w/w) and degree of polymerization of 2-7 and the ratio of arabinose to xylose of 0.27, was obtained with a yield of approximately 31.2% of WBIDF. Their stability was evaluated by comparing with that of commercial fructooligosaccharides (FOS) during pasteurization (60–100 °C, 30 min) and autoclave sterilization (121 °C, 1 kg/cm², 10–50 min) at pH 2.0–4.0. XOS was characterized by a high thermal stability during pasteurization at pH 2.5–4.0 and sterilization at pH 3.0–4.0. Even at pH 2.0, the remaining XOS reached 97.2% (w/w) and 84.2% (w/w) during pasteurization (100 °C, 30 min) and sterilization (50 min), respectively. Compared with FOS, XOS was strongly resistant to lower acidic conditions. The results revealed that XOS was considered to be more suitable for use as functional food ingredients.     

Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii

The thermotolerant yeast strain isolated from sugarcane juice through enrichment technique was identified as a strain of Pichiakudriavzevii (Issatchenkiaorientalis) through molecular characterization. The P. kudriavzevii cells adapted to galactose medium produced about 30% more ethanol from sugarcane juice than the non-adapted cells. The recycled cells could be used for four successive cycles without a significant drop in ethanol production. Fermentation in a laboratory fermenter with galactose adapted P. kudriavzevii cells at 40 °C resulted in an ethanol concentration and productivity of 71.9 g L⁻¹ and 4.0 g L⁻¹ h⁻¹, respectively from sugarcane juice composed of about 14% (w/v) sucrose, 2% (w/v) glucose and 1% (w/v) fructose. In addition to ethanol, 3.30 g L⁻¹ arabitol and 4.19 g L⁻¹ glycerol were also produced, whereas sorbitol and xylitol were not formed during fermentation. Use of galactose adapted P. kudriavzevii cells for ethanol production from sugarcane juice holds potential for scale-up studies.