Protein Expression Autoinduction in a Cold-Shock Expression System in Escherichia coli.

The cold-shock expression system in Escherichia coli was developed on a manual induction approach using optical density at 600 nm (OD600) measurements and isopropyl ß-D-1-thiogalactopyranoside (IPTG) addition. In this study, we show that cold-shock expression performs equally well using an autoinduction approach wherein OD600 measurements and IPTG addition may be eliminated. We further demonstrate that cold-shock expression with autoinduction can better facilitate high-throughput experiments.

Enzymatic synthesis of ß-d-fructofuranosyl α-d-glucopyranosyl-(1→2)-α-d-glucopyranoside using Escherichia coli glycoside phosphorylase YcjT.

YcjT is a kojibiose phosphorylase found in Escherichia coli. We found that sucrose was a good acceptor of YcjT in reverse phosphorolysis using ß-d-glucose 1-phosphate as a donor. The product was identified as ß-d-fructofuranosyl α-d-glucopyranosyl-(1→2)-α-d-glucopyranoside. This sugar was also synthesized from sucrose and maltose using YcjT and maltose phosphorylase and promoted the growth of the probiotic Bifidobacterium breve.

Identification and biochemical characterization of a novel N-acetylglucosamine kinase in Saccharomyces cerevisiae.

N-acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae, a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The Km values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.

Functional characterization of a novel GH94 glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase, and implication of the metabolic pathway of acidic carbohydrates in Paenibacillus borealis.

Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of ß-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl ß-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-ß-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl ß-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be ß-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-ß-d-glucopyranosyl ß-d-glucuronide structure released from bacterial and plant acidic carbohydrates.

Validation of a phenol-sulfuric acid method in a microplate format for the quantification of soluble sugars in ruminant feeds.

Soluble sugars in feeds are important for ruminant production; however, performing numerous sugar analyses within a short period is a laborious task. Here, we developed a phenol-sulfuric acid (PSA) assay in a microplate format to quantify soluble sugars in ruminant feeds. This method is easy and quick and requires only a small quantity of harmful reagents. We found that assay measurements were not affected by the representative organic acids and sugar alcohol contained in feeds. The treatment of activated charcoal with ethanol extract prior to the PSA assay was effective in removing interfering compounds for a more accurate determination of soluble sugars in certain feeds. Furthermore, the inter-day and intra-day repeatability of the present method was acceptable. Hence, we conclude that the method developed in this study is suitable for routine analysis of soluble sugars content in ruminant feeds.

The Emi2 Protein of Saccharomyces cerevisiae is a Hexokinase Expressed under Glucose Limitation.

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg 2+ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.

Purification and characterization of 1,3-ß-D-glucan phosphorylase from Ochromonas danica.

1,3-ß-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-ß-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-ß-glucans.

A wild and tolerant yeast suitable for ethanol fermentation from lignocellulose.

Ethanol fermentation from food wastes containing mainly starch without carrying out sterilization was investigated by using wild and tolerant yeast, Issatchenkia orientalis MF-121. The MF-121 strain is not a suitable choice for ethanol fermentation from lignocellulosic biomass because it is only capable of fermenting hexoses of glucose, mannose, and fructose to ethanol. Therefore, we first isolated acid- and salt-tolerant yeast that are capable of fermenting various monosaccharides to ethanol, and the isolated yeast that showed the ability to ferment ethanol from glucose, mannose, galactose, fructose, and xylose, was identified as Zygoascus hellenicus LK-5G on the basis of the 26S rRNA sequence analysis.

A comparative study of ethanol production by Issatchenkia orientalis strains under stress conditions.

The ability of 13 strains of multi-stress-tolerant Issatchenkia orientalis yeast to produce ethanol was examined under different stress conditions, including conditions of elevated Na2SO4 and Na2SO4 concentrations and increased heat. The MF-121 strain produced a significant amount of ethanol after the incubation in acidic media containing high concentrations of salt, e.g., 50 g/l Na2SO4 at pH 2.0, or at high temperatures, e.g., 43°C, when compared with other strains.

Construction of a beta-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis.

We demonstrate the value of the thermotolerant yeast Issatchenkia orientalis as a candidate microorganism for bioethanol production from lignocellulosic biomass with the goal of consolidated bioprocessing. The I. orientalis MF-121 strain is acid tolerant, ethanol tolerant, and thermotolerant, and is thus a multistress-tolerant yeast. To express heterologous proteins in I. orientalis, we constructed a transformation system for the MF-121 strain and then isolated the promoters of TDH1 and PGK1, two genes that were found to be strongly expressed during ethanol fermentation. As a result, expression of beta-glucosidase from Aspergillus aculeatus could be achieved with I. orientalis, demonstrating successful heterologous gene expression in I. orientalis for the first time. The transformant could convert cellobiose to ethanol under acidic conditions and at high temperature. Simultaneous saccharification and fermentation (SSF) was performed with the transformant, which produced 29 g l(-1) of ethanol in 72 h at 40 degrees C even without addition of beta-glucosidase when SSF was carried out in medium containing 100 g l(-1) of microcrystalline cellulose and a commercial cellulase preparation. These results suggest that using a genetically engineered thermotolerant yeast such as I. orientalis in SSF could lead to cost reduction because less saccharification enzymes are required.

Relationship of granule size distribution and amylopectin structure with pasting, thermal, and retrogradation properties in wheat starch.

Starches separated from 18 Indian wheat varieties were evaluated to see relationship of granule size distribution and amylopectin structure with pasting, thermal, and retrogradation properties. Average diameter of A-, B-, and C-granules among different starches varied between 23.0 and 28.5, 10.0 and 12.0, and 2.3 and 2.7 mum, respectively. Amylopectin chain length distribution varied significantly, short length chains (DP 6-12) and long length chains (DP > 24) ranged between 44.5 and 52.4% and 3.7 and 6.5%, respectively, whereas amylose content ranged between 18.2 and 28.8%. Short length chains of amylopectin had inverse relationship with starch gelatinization temperatures T(o), T(p), and T(c). Starches with higher crystallinity had higher enthalpy of gelatinization and lower swelling power. Paste characteristics were mainly dependent upon granule type and all pasting parameters except pasting temperature, showed significant positive correlations with A-granules and negative with the proportion of B- and C-granule.

Diversity in amylopectin structure, thermal and pasting properties of starches from wheat varieties/lines.

Structural, thermal and pasting diversity of starches from Indian and exotic lines of wheat was studied. Majority of the starches showed amylose content ranging between 22% and 28%. Endotherm temperatures (T(o), T(p) and T(c)) of the starches showed a range between 56-57, 60 -61 and 65.5-66.5 degrees C, respectively. Exotherms with T(p) between 87.0 and 88.2 degrees C were observed during cooling of heated starches, indicating the presence of amylose-lipid complexes. Exotherm temperatures were negatively correlated to swelling power. Amylopectin unit chains with different degree of polymerization (DP) were observed to be associated with pasting temperature, setback and thermal (endothermic T(o), T(p), and T(c)) parameters. Amylopectin unit chains of DP 13-24 showed positive relationship with endothermic T(o), T(p) and T(c). Pasting temperature showed positive correlation with short chains (DP 6-12) while negative correlation with medium chain (DP 13-24) amylopectins. Setback was positively correlated to DP 16-18 and negatively to DSC amylose-lipid parameters.

Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.).

In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule-bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N-terminal noncatalytic region (N-domain), and a C-terminal catalytic region (C-domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N-domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N-domain, and recombinant kidney bean SSIII C-domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high k(cat) values, and that the K(m) values of recombinant kidney bean SSIII C-domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C-domain had similar chain-length specificities for the extension of glucan chains, indicating that the N-domain of kidney bean SSIII does not affect the chain-length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N-domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N-domain of kidney bean SSIII as a carbohydrate-binding module.

Structure of lintnerized starch is related to X-ray diffraction pattern and susceptibility to acid and enzyme hydrolysis of starch granules.

Acid-resistant residues (lintnerized starches, Ls) were prepared from starches showing A-, B- and C- X-ray diffraction patterns. Ls retained the same X-ray crystalline type as their native counterparts with an improvement in diffraction intensity. Fluorophore-assisted capillary electrophoresis (FACE) study indicated that structural characteristics of Ls were associated with X-ray diffraction patterns. Double helices originated from linear chains with an approximate average degree of polymerisation (DP) 14, 16, and 15 would span the entire length of crystalline lamellae of A-, B-, and C-type starches, respectively. The proportion of singly branched materials (SB) with DP 25 protected in Ls was higher for A-type Ls (10-17%) than for B-type Ls (4-6%) and C-type Ls (8%). The structures of SB were similar in which branched chain (DP 13-15) was longer than main chain (DP 10-12). The structural characteristics of Ls are discussed in relation to acid and enzymatic degradations of starch granules.

Characterization of starch synthase I and II expressed in early developing seeds of kidney bean (Phaseolus vulgaris L.).

Plant starch synthase (SS) contributes to the elongation of glucan chains during starch biosynthesis and hence plays an essential role in determining the fine structure of amylopectin. To elucidate the role of SS activity in the formation of amylopectin in kidney bean (Phaseolus vulgaris L.), a study was undertaken to isolate cDNA clones for SS and to characterize the enzymatic properties of the coded recombinant enzymes. Two SS cDNAs, designated pvss1 and pvss21, which were isolated from early developing seeds, encoded SSI and SSII (designated PvSSI and PvSSII-1) that displayed significant identity (more than 65%) with other SSI and SSII members, respectively. RNA gel blot analysis indicated that both transcripts accumulate in leaves and developing seeds at the early stage. Immunoblot analysis with antisera raised against both recombinant proteins (rPvSSI and rPvSSII-1) showed that the accumulation of both proteins parallels the gene expression profiles, although both were detectable only in starch-granule fractions. Recombinant enzymes expressed by Escherichia coli cells showed distinct chain-length specificities for the extension of glucan chains. Our results suggest that these SS isozymes for synthesis of transitory starch are also responsible for synthesis of storage starch in early developing seeds of kidney bean.

Enzymatic synthesis of two novel non-reducing oligosaccharides using transfructosylation activity with beta-fructofuranosidase from arthrobacter globiformis.

Two novel non-reducing oligosaccharides together with tri- and tetra-saccharides were synthesized by transfructosylation activity from sucrose as a donor and cellobiose or cellotriose as an acceptor with a purified beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and these oligosaccharides were identified as O-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside and O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside by spectrometric analyses. Both oligosaccharides were stable under condition at 100 degrees C for 30 min, and showed no degradation at pH 2.

Cloning and heterologous expression of a beta-fructofuranosidase gene from Arthrobacter globiformis IFO 3062, and site-directed mutagenesis of the essential aspartic acid and glutamic acid of the active site.

We have cloned the gene encoding a beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and subsequently, the gene was heterologously expressed in Escherichia coli. This beta-fructofuranosidase gene encodes a protein of 548 amino acid residues with a calculated molecular mass of 60,519 Da. We have examined the roles of three residues of A. globiformis IFO 3062 beta-fructofuranosidase by site-directed mutagenesis, and found that aspartic acid 130 and glutamic acid 392, which are two of the apparent catalytic residues, are essential for hydrolase activity. This study provides the first experimental evidence showing that these two amino acid residues of beta-fructofuranosidase play a critical role in hydrolyzing sucrose.

Differential characteristics and subcellular localization of two starch-branching enzyme isoforms encoded by a single gene in Phaseolus vulgaris L.

Starch-branching enzymes (SBE) have a dominant role for amylopectin structure as they define chain length and frequency of branch points. We have previously shown that one of the SBE isoforms of kidney bean (Phaseolus vulgaris L.), designated PvSBE2, has a molecular mass (82 kDa) significantly smaller than those reported for isologous SBEs from pea (SBEI), maize (BEIIb), and rice (RBE3). Additionally, in contrast to the dual location of the pea SBEI in both the soluble and starch granule fractions, PvSBE2 was found only in the soluble fraction during seed development. Analysis of a pvsbe2 cDNA suggested that PvSBE2 is generated from a larger precursor with a putative plastid targeting sequence of 156 residues. Here we describe the occurrence of a larger 100-kDa form (LF-PvSBE2) of PvSBE2 found both in the soluble and starch granule fractions of the developing seeds. The determined N-terminal sequence, VKSSHDSD, of LF-PvSBE2 corresponded to a peptide sequence located 111 amino acids upstream from the N terminus of purified PvSBE2, suggesting that LF-PvSBE2 and PvSBE2 are products of the same gene. Analysis of the products by 5'-RACE (rapid amplification of cDNA ends) and reverse transcription PCR indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing. Recombinant LF-PvSBE2 (rLF-PvSBE2) was purified from Escherichia coli and the kinetic properties were compared with those of recombinant PvSBE2 (rPvSBE2). rLF-PvSBE2 had much higher affinity for amylopectin (K(m) = 4.4 mg/ml) than rPvSBE2 (18.4 mg/ml), whereas the V(max) of rLF-PvSBE2 (135 units/mg) for this substrate was much lower than that of rPvSBE2 (561 units/mg). These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for localization in starch granules by altering its enzymatic properties.