Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

α‐(1,4)‐Amylase,but not α‐ and β‐(1,3)‐glucanases,may be responsible for the impaired growth and morphogenesis of Paracoccidioides brasiliensis induced by N‐glycosylation inhibition

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N‐glycosylation of proteins such as N‐acetyl‐β‐d ‐glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)‐mediated inhibition of N‐linked glycosylation on α‐ and β‐(1,3)‐glucanases and on α‐(1,4)‐amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α‐ or β‐(1,3)‐glucanase production and secretion. Moreover, incubation with TM did not alter α‐ and β‐(1,3)‐glucanase activity in yeast and mycelium cell extracts. In contrast, α‐(1,4)‐amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N‐glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N‐glycans were essential for α‐(1,4)‐amylase activity involved in the production of malto‐oligosaccharides that act as primer molecules for the biosynthesis of α‐(1,3)‐glucan. Our results suggest that reduced fungal α‐(1,4)‐amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells. © 2013 The Authors. Yeast published by John Wiley & Sons Ltd.     

Optimised methodology for carboxymethylation of (1→3)‐β‐d‐glucan from Yeast (Saccharomyces cerevisiae) and promotion of mechanical activation

With a view to utilise yeast (1→3)‐β‐d ‐glucan as biological response modifiers with better water solubility, carboxymethylation was carried out by a two‐step alkalisation and etherification with monochloroacetic acid. Four technological parameters of carboxymethylation were investigated by orthogonal experiments for obtaining the maximum degree of substitution (DS), apparent viscosity (η) and solubility of carboxymethyl derivatives. In view of the orthogonal analysis, the optimal technological parameters were reaction temperature 50 °C, total reaction time 5 h, 3 mL of 50% sodium hydroxide as the second alkali dosage and 15 mL of 4 m chloroacetic acid. In addition, it was found that ball milling pretreatment for original (1→3)‐β‐d ‐glucan can be an advantage for carboxymethylation. By contrast, DS, η and solubility of carboxymethyl product increased 24%, 6% and 22%, respectively, suggesting the effect of ball milling pretreatment could not be neglected on improvement of DS, η and solubility for carboxymethyl products.     

Effect of d‐Psicose Used as Sucrose Replacer on the Characteristics of Meringue

Excessive intake of sugar‐rich foods leads to metabolic syndrome. d ‐Psicose (Psi) not commonly found in nature, is noncalorie sweetener with a suppressive effect on the blood glucose level. Thus, Psi has the potential to be utilized as a sucrose (Suc) replacer in sugar‐rich foods, including meringue‐based confectionery (MBC). In this study, we investigated the effect of Psi on the physical and chemical properties of meringue. Meringue was made by whipping egg white and Suc (at a weight ratio of 1:1) and baking at 93 °C for 2 h. Thirty percent of the total weight of Suc was replaced with d ‐ketohexoses such as Psi, d ‐fructose, d ‐tagatose, and d ‐sorbose. The meringues containing d ‐ketohexoses had higher specific volume than the meringue not containing d ‐ketohexoses (Ct‐meringue). Baking of meringue caused differences between Psi and the other d ‐ketohexose meringues. Meringue containing Psi (P30‐meringue) had the highest breaking stress (7.00 × 10⁵ N/m²) and breaking strain (4.40%), resulting in the crunchiest texture. In addition, P30‐meringue also had the highest antioxidant activity (491.84 μM TE/mg‐meringue determined by ABTS method) and was the brownest due to a Maillard reaction occurring during baking. The replacement of Suc with Psi improved the characteristics of baked meringue. Thus, Psi was found to be useful in modifying the physical and chemical properties of MBC.     

Investigations on the conversion of onion aroma precursors S‐alk(en)yl‐L‐cysteine sulphoxides in onion juice production

Onion flavour develops when the cells are disrupted, allowing the enzyme alliinase (EC 4.4.1.4) to act upon the aroma precursors S‐alk(en)yl‐L ‐cysteine sulphoxides (ACSOs). For maximum flavour release, optimal cell disintegration and complete enzymatic conversion of ACSOs are desired. To assess an industrial scale onion juice extraction in regard to the extent of cell disintegration and the rate of enzymatic ACSO conversion, samples from all mass flows (ie onion, macerate, juice and pomace) were subjected to ACSO analysis. Applying a modified HPLC method, which allows baseline separation of the aroma precursors, three ACSOs, namely methyl‐, propyl‐ and 1‐propenyl‐L ‐cysteine sulphoxides (MCSO, PCSO and 1‐PeCSO), were determined. Initial ACSO ratios changed in the course of increasing degree of comminution from 17:83 to 100:0 (MCSO/1‐PeCSO). Only negligible amounts of PCSO were detected (<9.2 nmol g^?1 fresh weight (fw)), contributing less than 0.2% to the total ACSO content. For evaluation of the disintegration efficiency a chart was established consisting of an abscissa representing a percentage scale of ruptured cells and ordinates for the development of the marker parameters ACSOs and reaction product pyruvate relative to their initial contents. From this correlation a cell disintegration percentage after grinding of about 70% was deduced according to the determined ACSO contents. On a molar basis, macerate, juice and pomace showed 15, 0.6–2.4 and 0.7–1.0% of initial ACSO contents respectively. Only trace amounts of 1‐PeCSO were found in the juice (<16 nmol g^?1 fw). MCSO was converted to almost 90%, indicating that enzymatic transformation, in a practical sense, goes to completion. Copyright © 2004 Society of Chemical Industry     

Flavan‐3‐ol C‐glycosides – Preparation and model experiments mimicking their human intestinal transit

In order to study the human intestinal transit of flavan‐3‐ol C‐glycosides, several C‐glycosyl derivatives were prepared by non‐enzymatic reaction of (+)‐catechin with α‐D ‐glucose, α‐D ‐galactose and α‐D ‐rhamnose, respectively. In contrast to literature data, we propose that the reaction mechanism proceeds in analogy to the rearrangement of flavan‐3‐ols during epimerization under alkaline conditions. Four of the 12 synthesized flavan‐3‐ol C‐glycosides were incubated under aerobic conditions at 37°C using saliva (2 min) and simulated gastric juice (3 h). To simulate human intestine, the C‐glycosides were also incubated under anaerobic conditions at 37°C both in human ileostomy fluid (10 h) and colostomy fluid (24 h), respectively. The flavan‐3‐ol C‐glycosides under study, i.e. (+)‐epicatechin 8‐C‐β‐D ‐glucopyranoside (1a), (+)‐epicatechin 6‐C‐β‐D ‐glucopyranoside (1d), (+)‐catechin 6‐C‐β‐D ‐galactopyranoside (2b), (+)‐catechin 6‐C‐β‐D ‐rhamnopyranoside (3b) were analyzed in the incubation samples by HPLC‐DAD and HPLC‐DAD‐MS/MS. They were found to be stable in the course of incubation in saliva, simulated gastric juice and ileostomy fluid and underwent degradation in colostomy fluid. While the 6‐C‐β‐D ‐glucopyranoside 1d was completely metabolized between 2 and 4 h, decomposition of the 6‐C‐β‐D ‐galactopyranoside 2b reached only 16±2% within 4 h of incubation. Linear degradation rates of 1d and 2b in colostomy fluid differed significantly. As microbial metabolism of flavan‐3‐ols is known not to be influenced by the stereochemistry of the aglycon, varying degradation rates are ascribed to the effect of the sugar moiety. Based on these results we assume that flavan‐3‐ol C‐glycosides pass through the upper gastrointestinal tract (oral cavity, stomach and small intestine) unmodified and are then metabolized by the colonic microflora.     

Formation of 4(5)‐Methylimidazole in Aqueous d‐Glucose‐Amino Acids Model System

The International Agency for Research on Cancer (IRAC) has classified 4(5)‐methylimidazole (4‐MeI) as a group 2B possible human carcinogen. Thus, how 4‐MeI forms in a d ‐glucose (Glu) amino acids (AA) model system is important, as it is how browning is affected. An aqueous solution of Glu was mixed individually in equimolar amounts at 3 concentrations (0.05, 0.1, and 0.15 M) with aqueous solutions of l ‐Alanine (Ala), l ‐Arginine (Arg), Glycine (Gly), l ‐Lysine (Lys), and l ‐Serine (Ser). The Glu‐AA mixtures were reacted at 60, 120, and 160 °C for 1 h. The 4‐MeI levels were measured by gas chromatography‐mass spectrometry after derivatization with isobutylchloroformate. No 4‐MeI was formed at 60 °C for any treatment combination; however, at 120 °C and 0.05 M, Glu‐Arg and Glu‐Lys produced 0.13 and 0.14 mg/kg of 4‐MeI. At 160 °C and 0.05 M all treatment combinations formed 4‐MeI. At 160 °C and 0.15 M, the observed levels of Glu‐Ala, Glu‐Arg, Glu‐Gly, Glu‐Lys, and Glu‐Ser were 0.21, 1.00, 0.15, 0.22, and 0.16 mg/kg. The AA type, reactant concentrations, and temperature significantly affected (P < 0.001) formation of 4‐MeI as well as browning. Glu‐Lys treatment in all combinations produced the most browning, but Glu‐Arg produced the most 4‐MeI. This method showed that foods processed using low temperatures may have reduced levels of 4‐MeI.     

Effects of oxidised linoleic acid on the formation of Nε‐carboxymethyl‐lysine and Nε‐carboxyethyl‐lysine in Maillard reaction system

This study investigated the effects of oxidised linoleic acid (18:2) on N^ε‐carboxymethyl‐lysine (CML) and N^ε‐carboxyethyl‐lysine (CEL) formation in Maillard reaction systems. Model systems of lysine/glucose (L/G), lysine/18:2 (L/18:2), lysine/18:2/glucose (L/18:2/G), myofibrillar protein/glucose (MFP/G), MFP/18:2 and MFP/18:2/G were maintained at 37 °C for 6 weeks. The results showed that CML/CEL contents in L/G (6.99 and 0.96 mmol mol^?1 lysine, respectively) were significantly higher than those in L/18:2/G (1.43 and 0.41 mmol mol^?1 lysine, respectively), and there is a small amount of CML/CEL generation in L/18:2. However, the CML/CEL levels in MFP/G (197.2 and 83.8 ng mg^?1 protein, respectively) were markedly lower than those in MFP/18:2/G (283.2 and 118.5 ng mg^?1 protein, respectively). 18:2 favours the formation of CML/CEL in MFP/18:2/G, not in L/18:2/G. All these findings indicated that the role of 18:2 on CML/CEL formation in Maillard reaction system was complex, and depended on CML/CEL formation rate and substrate types (lysine or lysine residue in protein).     

Simultaneous Analysis of Nε‐(Carboxymethyl)Lysine and Nε‐(Carboxyethyl)Lysine in Foods by Ultra‐Performance Liquid Chromatography‐Mass Spectrometry with Derivatization by 9‐Fluorenylmethyl Chloroformate

Isotope dilution ultra‐performance liquid chromatography–electrospray tandem mass spectrometry with derivatization by 9‐fluorenylmethyl chloroformate was successfully applied to quantify N^ε‐(carboxymethyl)lysine (CML) and N^ε‐(carboxyethyl)lysine (CEL) in processed foods. We demonstrate that this analytical method is well validated for the determination of CML and CEL contents in processed foods. Relative standard deviations (RSD) indicate repeatability (RSD < 6% for CML and CEL) and reproducibility (RSD < 6% for CML and < 7% for CEL) in this method. Percent recovery is also good. We obtain recoveries of 102% to 112% for CML and 86% to 114% for CEL. CML levels detected in the samples vary from 2.29 to 480 mg/kg food, whereas CEL is detected in significantly lower concentrations ranging from 0.56 to 107 mg/kg food. These data could help consumers make better food choices by monitoring intake of advanced glycation end‐products, which may pose a risk to human health.     

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N‐acetyl‐β‐D‐glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa, which is purified by affinity with immobilized N‐acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels of TNFα and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the β‐1,4‐homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin‐affinity purification of paracoccin. This procedure provided higher yields than those achieved by means of the technique based on the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS–PAGE and Western blot analysis revealed similarities between the N‐acetylglucosamine‐ and chitin‐bound fractions, confirmed by MALDI–TOF–MS of trypsinic peptides. Western blot of two‐dimensional gel electrophoresis of the yeast extract showed a major spot with M_r 70 000 and pI approximately 5.63. Morevover, an N‐acetyl‐β‐D ‐glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright © 2009 John Wiley & Sons, Ltd.     

The yeasts of the genus Spathaspora: potential candidates for second‐generation biofuel production

Yeasts of the Spathaspora clade have the ability to convert d ‐xylose to ethanol and/or xylitol. This is an important trait, as these yeasts may be used to produce bioethanol from lignocellulosic biomass or as a source of new d ‐xylose metabolism genes for recombinant industrial strains of Saccharomyces cerevisiae. The core group of the genus Spathaspora has 22 species, both formally described and not yet described. Other species, such as Sp. allomyrinae, Candida alai, C. insectamans, C. lyxosophila, C. sake, Sp. boniae and C. subhashii are weakly associated with this clade, based on LSU rRNA gene D1/D2 sequence analyses. Spathaspora passalidarum, Sp. arborariae, Sp. gorwiae and Sp. hagerdaliae produce mostly ethanol from d ‐xylose, whereas the remaining species within the Spathaspora clade already tested for this property may be considered xylitol producers. Among the d ‐xylose‐fermenting Spathaspora species, Sp. passalidarum is the best ethanol producer, displaying high ethanol yields and productivities when cultured in media supplemented with this pentose under oxygen‐limited or anaerobic conditions. The species also exhibits rapid d ‐xylose consumption and the ability to ferment glucose, xylose and cellobiose simultaneously. These characteristics suggest that Sp. passalidarum is a potential candidate for domestication and use in the fermentation of lignocellulosic materials. Copyright © 2017 John Wiley & Sons, Ltd.     

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on l‐arabinose and d‐xylose

Genes encoding l ‐arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on l ‐arabinose was dependent on a functioning l ‐arabinose transporter, or by screening a differential display library, respectively. These transporters also transport d ‐xylose and were designated KmAXT1 (arabinose–xylose transporter) and PgAXT1, respectively. Transport assays using l ‐arabinose showed that KmAxt1p has K_m 263 mm and V_max 57 nm /mg/min, and PgAxt1p has K_m 0.13 mm and V_max 18 nm /mg/min. Glucose, galactose and xylose significantly inhibit l ‐arabinose transport by both transporters. Transport assays using d ‐xylose showed that KmAxt1p has K_m 27 mm and V_max 3.8 nm /mg/min, and PgAxt1p has K_m 65 mm and V_max 8.7 nm /mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K_m 371 mm and V_max 341 nm /mg/min for l ‐arabinose, and K_m 25 mm and V_max 76 nm /mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both l ‐arabinose and d ‐xylose, one scenario for the complete usage of biomass‐derived pentose sugars would require only the low‐affinity, high‐throughput transporter Gal2p and one additional high‐affinity general pentose transporter, rather than dedicated d ‐xylose or l ‐arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040 Copyright © 2015 John Wiley & Sons, Ltd.     

N,N‐Bis(2‐hydroxyethyl)formamide as a New Plasticizer for Thermoplastic Starch

N,N‐Bis(2‐hydroxyethyl)formamide (BHF) was synthesized efficiently and used as a new plasticizer for corn starch to prepare thermoplastic starch (TPS). The hydrogen bond interaction between BHF and starch was proven by Fourier‐transform infrared (FT‐IR) spectroscopy. As detected by scanning electron microscopy (SEM), starch granules were completely disrupted and a continuous phase was obtained. The crystallinity of corn starch and BHF‐plasticized TPS (BTPS) was characterized by X‐ray diffraction (XRD). The thermal behavior of glycerol‐plasticized TPS (GTPS) and BTPS was investigated by differential scanning calorimetry (DSC). The water resistance of BTPS was better than that of GTPS. Generally, at low relative humidity (RH), the tensile strength of BTPS was higher than that of GTPS. At high RH, the elongation at break of BTPS was higher than that of GTPS.     

Artificial simulated gastrointestinal digestion of four carbohydrates containing beta‐d‐1 → 4 linkages and new GC‐TQ/MS‐MS method for characterising released monosaccharides

Four types of carbohydrates, including Dendrobium officinale polysaccharide, Dendrobium aphyllum polysaccharide and β‐glucans from yeast and barley, were examined, and their structures were found to mainly contain 1,4‐linked‐β‐d ‐Glcp. Artificially simulated gastrointestinal digestion was conducted to characterise the changes of molecular weight, reducing sugars and released free monosaccharides by high‐performance liquid chromatography, kits and the newly developed gas chromatography (GC)‐mass spectrometry (MS)/MS analysis, which indicated that high molecular weight and complex spatial structures contributed to delayed monosaccharide release following exposure to digestive solution. The spatial structures of carbohydrates were changed during gastric digestion, but their primary structures were destroyed during intestinal digestion. Additionally, for the developed 7890A/7000 GC‐TQ/MS‐MS, the new analytical method was successfully used to analyse very low concentrations of monosaccharides in the simulated gastrointestinal digestive system.     

In vitro culture of Pandanus amaryllifolius and enhancement of 2‐acetyl‐1‐pyrroline,the major flavouring compound of aromatic rice,by precursor feeding of L‐proline

Shoots, plantlets and semi‐differentiated callus (SDC) cultures of Pandanus amaryllifolius capable of producing high levels of basmati rice flavour were established in vitro using Murashige and Skoog nutrient medium. A total of 10% of the initial explants responded to produce shoot cultures in the presence of benzylamino purine (BAP) (0.5 mg L^?1) and glutamine (100 mg L^?1). Leaf explants and basal portions of shoots produced SDC whereas elongated in vitro shoots could be continuously multiplied, using BAP (1.5 mg L^?1) and kinetin (Kn) (1.0 mg L^?1), and rooted in half‐strength medium for ex vitro cultivation leading to a process of micropropagation. Steam‐distillation extraction (SDE) followed by gas chromatography‐mass spectrometry (GC‐MS) analysis of various cultured organs and spent liquid medium used for SDC revealed the presence of 2‐acetyl‐1‐pyrroline (2‐AP) to various extents. This 2‐AP compound has been identified as the major flavouring compound of scented basmati and other scented rice varieties. 2‐AP was found to be highest, on a fresh weight basis, in SDC (19.7 mg kg^?1) on the 40th day, whereas in vitro roots, shoots and field leaves (of one‐year‐old plant) had lower levels of 15, 6.8 and 14 mg kg^?1, respectively. Further enhancement of 2‐AP in SDC using precursor was possible by feeding into medium 1 mmol L^?1 of L ‐proline where a highest level of 21.67 ppm of 2‐AP accumulated on the seventh day whereas a higher level of 2 mmol L^?1 of L ‐proline suppressed 2‐AP levels. The present report is the first on the tissue culture studies of P. amaryllifolius where continuous production of plantlets as well as synthesis of high levels of 2‐AP has been documented. Copyright © 2005 Society of Chemical Industry     

Reduction of fumonisin B₁ in corn grits by twin-screw extrusion

Abstract: This study was designed to investigate the fate of fumonisins in flaking corn grits during twin‐screw extrusion by measuring fumonisin B₁ (FB₁) and its analogs with a mass balance approach. Food grade corn grits and 2 batches of grits contaminated with FB₁ at 10 and 50 μg/g by Fusarium verticillioides M‐2552 were processed with or without glucose supplementation (10%, w/w) with a twin‐screw extruder. Extrusion reduced FB₁ in contaminated grits by 64% to 72% without glucose and 89% to 94% with added glucose. In addition, extrusion alone resulted in 26% to 73% reduction in the levels of fumonisin B₂ and fumonisin B₃, while levels of both mycotoxins were reduced by >89% in extruded corn grits containing 10% glucose. Mass balance analysis showed that 38% to 46% of the FB₁ species detected in corn extruded with glucose was N‐(deoxy‐D‐fructos‐1‐yl)‐FB₁, while 23% to 37% of FB₁ species detected in extruded corn grits with and without added glucose was bound to the matrix. It was also found that the hydrolyzed form of FB₁ was a minor species in extruded corn grits with or without added glucose, representing <15% of the total FB₁ species present. Less than 46% of FB₁ originally present in corn grits could be detected in the fumonisin analogues measured in this study. Research is needed to identify the reaction products resulting from extrusion processing of fumonisin‐contaminated corn products. Practical Application: Twin‐screw extrusion is widely used in food industry for its versatility. This technology may reduce the level of fumonisins in corn particularly with added glucose.     

Preparation and Use of Poly(hydroxyethyl methacrylate) Cryogels Containing L‐Histidine for β‐Casein Adsorption

The objective of this study was to determine β‐casein adsorption by using supermacroporous poly(2‐hydroxyethyl methacrylate‐N‐methacryloyl‐(l) ‐histidine methyl ester) [p(HEMA‐MAH)] cryogel. β‐Casein adsorption properties of p(HEMA‐MAH) cryogel were studied for the application of β‐casein purification. The cryogel was produced by free radical polymerization initiated by N,N,N’,N’‐tetramethylene diamine and ammonium persulfate pairs in an ice bath. P(HEMA‐MAH) cryogel was characterized by swelling tests, Fourier transform infrared spectroscopy, and scanning electron microscopy. The effects of the flow rate, pH, temperature, initial β‐casein concentration, and ionic strength on the adsorption efficiency of cryogel were studied. The equilibrium swelling degree of the p(HEMA‐MAH) cryogel was 6.73 g H₂O/g cryogel. β‐Casein adsorption capacity of p(HEMA‐MAH) cryogel from aqueous solution was estimated as 31.17 mg/g cryogel. It was also observed that β‐casein could be repeatedly adsorbed and desorbed with p(HEMA‐MAH) cryogel without significant loss in the adsorption capacity.     

Functional Expression of the Raw Starch‐Binding Domain of Bacillus sp. Strain TS‐23 α‐Amylase in Recombinant Escherichia coli

Two DNA fragments encoding the starch‐binding domain (SBD) of Bacillus sp. strain TS‐23 α‐amylase were prepared by polymerase chain reaction and cloned into the Escherichia coli expression vector, pQE‐30, to generate pQE‐N428/C607 and pQE‐N465/ C607. In isopropyl‐β‐D ‐thiogalactopyranoside (IPTG)‐induced E. coli strain M15 harboring these expression plasmids, the recombinant SBDs (N428/C607 and N465/C607) could comprise up to 20% of the total soluble proteins. The His‐tag/SBD fusion proteins were purified to homogeneity with a His‐bind affinity column and had molecular masses of approximately 22.6 and 16.5 kDa, respectively. Starch‐binding assays revealed that about 7.1 and 8.3 μg, respectively, of N428/C607 and N465/C607 were bound by 1 mg of raw corn starch, indicating that the SBD of Bacillus sp. strain TS‐23 α‐amylase retain sufficient function in the absence of a catalytic center.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.