Continuous production of sialyllactose from colominic acid using a membrane reactor

Continuous production of sialyllactose, as a typical sialylsaccharide, was examined using a membrane reactor. The synthesis of sialyllactose through the transfer reaction catalyzed by neuraminidase, has been reported to be a suitable process for industrial production, but it still has drawbacks such as a low yield, high enzyme cost, and hydrolysis by the enzyme of sialyllactose formed. We attempted to solve these problems by utilizing an appropriate membrane reactor system. We first investigated the effects of various reaction conditions on sialyllactose productivity, and found that the productivity was independent of the enzyme concentration and reaction temperature but dependent on the substrate and buffer concentrations and the hydraulic retention time (HRT). We then selected a suitable membrane that allowed sialyllactose to permeate but rejected the substrate and enzyme. Finally, we constructed a membrane reactor system with a cut-off molecular weight of 3000 and applied it to continuous sialyllactose production from colominic acid at an HRT of 80 min. Using substrate concentration of 25 g/l the system performed with a high level of productivity and gave a good yield, while maintaining high transfer ratio of 4-5% over a 160-h test period.     

In vivo formation of a stable pentameric (P2α/P1β)–P0–(P1α/P2β) ribosomal stalk complex in Saccharomyces cerevisiae

Heterodimers of acidic proteins P1α/P2β and P1β/P2α bind to P0 and are fundamental for the assembly of the ribosomal stalk. However, different inconsistencies are found in the literature regarding additional P protein heterodimer formations and their individual interactions with P0. Using the two‐hybrid approach, we have found results that help to clarify these interactions. Thus, we have found that neither P1 nor P2 directly interact with P0 unless the endogenous heterodimer partner is being expressed in the cell. In addition, a P2‐free amino end is a requisite in these heterodimers for binding to P0. With regard to the two‐hybrid interactions between P1 and P2, the known canonical P1α–P2β and P1β–P2α interactions do not depend on either a free amino end or the presence of endogenous P0, P1 or P2 proteins. Furthermore, the non‐canonical P1β–P2β pair also behaves similarly, although this interaction is weaker. Interestingly, P1α–P2α, P1α–P1β and P2α–P2β two‐hybrid interactions were also detected, although in these cases the endogenous P proteins were involved. Thus, these positive interactions are the consequence of the interaction between two canonical heterodimers. As the ribosome anchorage protein P0 is also necessary, the results suggest that, in vivo, all five P proteins form a complex, independent of the ribosome, containing the two canonical heterodimers and P0. This complex has been isolated in cells expressing a P0 protein unable to bind to the ribosome. Copyright © 2010 John Wiley & Sons, Ltd.     

MODIFIED FLUORIMETRIC FLOW-INJECTION-ANALYSIS (FIA) METHOD FOR THE DETERMINATION OF (1–3) (1–4)–B—D—GLUCAN

A simple system has been constructed for the quantitative determination of barley β-glucans. Measurements were made by the flow-injection-analysis (FIA) technique using the fluorescent dye Calcofluor as a specific reagent for (1·3) (1·4)–B—D—linkages. In this paper a single-line system is described involving only one pump samples of β-glucan being directly injected into the reagent stream. The reaction results in an increased intensity of fluorescence detected by a flow-through-cell fluorimeter and evaluated as peak heights or peak areas by a printer-plotter. The analysis is completed within 20s. The operational parameters were set during an optimization procedure.     

TOTAL AND INDIVIDUAL BARLEY (1–3), (1–4)-β-D-GLUCANASE ACTIVITIES IN SOME GREEN AND KILNED MALTS

Total β-glucanase activity was measured in extracts of malts prepared from three winter and three spring cultivars of barley. Samples were taken at intervals during modification and, after 116 hr, from malt kilned to 65°C. Good malting varieties showed highest levels of total β-glucanase activity, with a high correlation (r = 0.926) with HWE. Angora had the highest activity in the intermediate stages of malting, least loss of activity after heating extracts to 48°C for 10 mins and least loss of activity on kilning. Separation of isozymes by HPLC⁹ confirmed the greater heat stability of isozyme II, but, unlike previous studies on Australian cultivars, we found considerable activity of isozyme I after kilning, even up to 85°C. However, total β-glucanase activity was destroyed by heating extracts of all varieties to 60°C. Angora showed the highest proportion of total activity as isozyme II after kilining. Cultivar differences suggested some scope for breeding varieties with increased total activity by combining high activities of each isozyme. The high correlation of total activity with HWE suggests β-glucanase activity as a rapid test of malting quality.     

α 1,4 — α 1,6 Glucopolysaccharides Contained in Developing Maize Kernels

Corn grains with different filling times were fractionated in order to obtain their α 1,4 — α 1,6 glucopolysaccharide composition and structure. Sugary, waxy, flint and several hybrids were the varieties analyzed. There is an increase in the total content of polysaccharides, as the grain is completed. But, no differences in composition and structure were detected between the first sample analyzed (15th day after pollination) and the last one, which corresponds to the complete grain filling period. The methodology developed by us, allowed us to observe that the introduction of su character on the hybrids (F x S and S x F) has influence on the final polysaccharide structure. Also, in the F x W hybridα 1,4 — α 1,6 glucans, their structural characteristics were different from those of F or W pure.     

Enzymatic Characterisation of Novamyl®, a Thermostable α-Amylase

The thermostable -amylase Novamyl^® is used in the baking industry as an antistaling agent due to its ability to reduce retrogradation of amylopectin. We have studied its enzymatic properties at pH 5.0. We make two main conclusions: (1) Novamyl^® shows sequence homology to cycloglycosyl transferases (CGTases); like these enzymes, Novamyl^® cleaves cyclodextrins, forms transglycosylation products and is subject to product inhibition by maltose. Novamyl^® has 5 subsites in the active site and is also subject to substrate inhibition. (2) Novamyl^® is clearly different from exoglucanases like β-amylase and glucoamylase. It is able to hydrolyse a pentasaccharide with bulky substituents at both ends (INdp5) and is inhibited by the α-amylase inhibitor Trestatin A. Although Novamyl^® appears unable to hydrolyse α-1,6-linkages, it is able to degrade amylopectin to a greater extent than β-amylase as well as β-limit dextrins. Novamyl^® degrades amylose in such a manner that initially the molecular weight is drastically reduced while β-amylase does not show any detectable effect on the molecular weight of this substrate. Products of the degradation of amylopectin and amylose by Novamyl^® are maltose and oligosaccharides, whereas β-amylase and glucoamylase produce only maltose and glucose, respectively. This was shown in baking experiments as well. The new data presented here clearly show that unlike exoamylases, Novamyl^® does not require a non-reducing end and attacks amylose, Indp5 and cyclodextrins in an endo-like manner. Based on these results Novamyl^® should be reclassified.     

The (1–3, 1–4)‐β‐Glucanases in Malting Barley: Enzyme Survival and Genetic and Environmental Effects

Eighteen barley genotypes used in Brazilian malting barley breeding programs were characterized in relation to (1–3, 1–4)‐β‐glucanase activity in green and kilned malt. They were tested to determine the loss of enzyme activity during kilning in the malting process and the environmental effects on enzyme activity were measured. The genotypes analyzed showed great variation regarding the enzyme activity in both kinds of malt, in a range from 531.94 to 934.31 U/kg in green malt, and from 187.02 to 518.40 U/kg in dry malt. The mean enzyme activity loss during kilning was close to 60%, very similar to the results obtained in other studies. The loss among genotypes varied from 8.04% to 71.54%. The enzyme activity varied significantly under the different environments tested, showing existence of environmental effects on the genotypes analyzed. Embrapa 127 was the genotype that exhibited the highest enzyme activity in finished malt although it had shown a low activity in green malt, reflecting a negligible loss of activity during kilning. The data indicate promising results to malting barley breeding due to the wide variability exhibited by genotypes as to enzyme activity and levels of isoenzyme with high thermostability.     

Species identification of Wickerhamomyces anomalus and related taxa using β‐tubulin (β‐tub) DNA barcode marker

Wickerhamomyces anomalus is used in food and feed processing, although the species has been reported as an opportunistic human pathogen, predominantly in neonates. Neither phenotypic nor the most frequently applied genotypic marker (D1/D2 LSU ribosomal DNA) provide sufficient resolution for accurate identification of this yeast. In this study, the β‐tubulin gene was used for species identification by direct DNA sequencing and as marker in a species‐specific PCR assay. The results showed that all examined W. anomalus strains were clearly distinguished from the closely related species by comparative sequence analysis of the β‐tubulin gene. In addition, the species‐specific primers were also developed based on the β‐tubulin gene, which was employed for polymerase chain reaction with the template DNA of Wickerhamomyces strains. A single 218 bp species‐specific band was found only in W. anomalus. Our data indicate that the phylogenetic relationships between these strains are easily resolved by sequencing of the β‐tubulin gene and combined with species‐specific PCR assay. Copyright © 2012 John Wiley & Sons, Ltd.     

Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

α-AMYLASE IN DEVELOPING BARLEY KERNELS — A REAPPRAISAL1

The outer layers that can be dissected readily from immature barley kernels were identified by light microscopy. α-Amylase was located in the outer pericarp of developing Bonanza and Himalaya barley. Only low pl α-amylase (α-amylase 1) was detected in the pericarps but only one of the two major low pl components of α-amylase from germinated Himalaya was found in the pericarp tissue. These enzymes appeared to hydrolyze the small starch granules (2–3 μm) present in the outer pericarp of developing barley kernels. The even smaller starch granules (0.25–2μm) present in the inner pericarp were hydrolyzed at a later stage of kernel development.     

ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.     

Comparative study of enzymatic hydrolysis of α/β- and γ-gliadins

Enzymatic hydrolysis of proteins and fractionation of hydrolysates is a route of diversifying their functional properties. Chymotryptic hydrolysis of different sulphur-rich gliadins (α/β- and γ-types), major wheat storage proteins, was studied. The peptides formed in the course of digestion were characterised by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and reversed-phase high performance liquid chromatography (RP-HPLC). With reference to previous work, a general scheme of degradation was assessed for γ-gliadins. Limited hydrolysis released two types of polypeptides, comprising respectively the repetitive and the non-repetitive moieties of the protein. In spite of strong sequence homologies between the two groups of sulphur-rich gliadins, it was not possible to prepare similar peptide fractions from α/β-gliadins. They were more resistant to hydrolysis and the region where the two domains merge appeared inaccessible to chymotrypsin. Restricted accessibility of cleavage sites was attributed to the less expanded conformation of α/β-type than γ-type gliadins. A first step of scaling-up was performed. This offers opportunities to prepare functional peptides from wheat storage proteins.     

Endosperm α 1,4—α 1,6 Glucopolysaccharides Utilization During Germination of Sweet Corn and Other Maize Genotypes

The quantity variations and the structural changes of the α 1,4 —α 1,6 glucopolysaccharides contained in the endosperm of sweet corn, waxy and amylose extender maize genotypes, were studied during early germination. The α 1,4 —α 1,6 glucopolysaccharides were fractionated according to their branching degree. The results obtained showed that: the degradation pattern of the α 1,4 —α 1,6 glucopolysaccharides was similar for the different corn genotypes studied. In addition, soluble dextrins or partial degradation products were not detected, but reducing sugars were accumulated. Furthermore, the structure of the remaining polysaccharides was similar to those present in the non germinating seed. These results led us to propose that once a polysaccharide molecule was used as the substrate by degradative enzymes during germination, that molecule was totally degraded.     

Deterioration of β-carotene in certain hydrogenated fats. II.—Products of β-carotene deterioration and nature of the green pigment

The non-saponifiable fraction from a sample of β-carotene-hardened palm kernel oil which had developed a green discoloration has been examined. A number of β-carotene oxidation products were found and these included a number of β-carotene epoxide derivatives. A compound thought to be β-apo-3-carotenal was also found, but the apo-2- and apo-4-carotenals were not present in more than trace amounts. A major component was similar to, but not identical with, mutatochrome (β-carotene 5,8-epoxide). It is suggested that the green discoloration of the carotenised fat was due to oxidation of β-carotene to epoxide derivatives by traces of peroxyacids and the conversion of these epoxides into green-blue ionised forms by the action of an acidic material present in the fat.     

β-hydroxyacyl-coa-dehydrogenase (HADH) activity of unfrozen and frozen—thawed frog (Rana esculenta) legs

The β-hydroxyacyl-CoA-dehydrogenase (HADH) activity of unfrozen and thawed frog legs was investigated. The enzyme was extracted by either immersing frog legs in phosphate buffer 0.1 M, pH 6.0 at 25°C for 15 min or pressing them between trichinoscopy glasses. The enzyme activity was assayed using acetoacetyl-CoA as substrate and measured spectrophotometrically at 340 nm. It was possible by both extraction methods to distinguish between thawed and unfrozen samples although when the juice was obtained by pressing the HADH activity of the dilution was ～ 1.5 times higher than that obtained by immersion. The HADH activity was significantly higher (P≤0·001) in frozen-thawed frogs than in unfrozen legs because during freezing there is a release of HADH. No significative differences were found in the HADH activity in samples frozen in the temperature range -10 to -196°C. HADH activity was not affected by the storage time in crushed ice up to 6 days.     

The linkage of (1–3)-β-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae

Pulse-chase experiments with [¹⁴C]glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1–3)-β-glucan serves as a precursor for alkali-insoluble (1–3)-β-glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan. These cells, unable to form a glucan–chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall. Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation.