Oligosaccharides from agar extends lifespan through activation of unfolded protein response via SIR-2.1 in Caenorhabditis elegans.

PURPOSE: Agaro-oligosaccharides (AGO), hydrolysis products of agarose, is known to have antioxidant and anti-inflammatory properties. Speculating that AGO is effective for preventing aging, we investigated the longevity-supporting effects of AGO and their mechanisms using Caenorhabditis elegans. METHODS: Caenorhabditis elegans were fed AGO from young adulthood. The lifespan, locomotory activity, lipofuscin accumulation, and heat stress resistance of the worms were examined. To elucidate mechanisms of AGO-mediated longevity, we conducted comprehensive expression analysis using microarrays. Moreover, we used quantitative real-time PCR (qRT-PCR) to verify the genes showing differential expression levels. Furthermore, we measured the lifespan of loss-of-function mutants to determine the genes related to AGO-mediated longevity. RESULTS: AGO extended the lifespan of C. elegans, reduced lipofuscin accumulation, and maintained vigorous locomotion. The microarray analysis revealed that the endoplasmic reticulum-unfolded protein response (ER-UPR) and insulin/insulin-like growth factor-1-mediated signaling (IIS) pathway were activated in AGO-fed worms. The qRT-PCR analysis showed that AGO treatment suppressed sir-2.1 expression, which is a negative regulator of ER-UPR. In loss-of-function mutant of sir-2.1, AGO-induced longevity and heat stress resistance were decreased or cancelled completely. Furthermore, the pro-longevity effect of AGO was decreased in loss-of-function mutants of abnormal Dauer formation (daf) -2 and daf-16, which are IIS pathway-related genes. CONCLUSION: AGO delays the C. elegans aging process and extends their lifespan through the activations of ER-UPR and the IIS pathway.

Accuracy and reliability of a smartphone application for measuring the knee joint angle.

[Purpose] Recently, a photo-based smartphone application for angle measurement-"Grid line imaging application Professional"-was developed to evaluate joint disease treatments. The aim of this study was to determine the accuracy and reliability of the application. [Participants and Methods] We measured the knee joint of a mannequin using an application and a universal goniometer. Twelve examiners measured eight knee joints of mannequins at different arbitrary angles using the application and a universal goniometer. Correlations between the application and universal goniometer measurements were examined using scatter plots and correlation coefficients. Systematic errors of the application were visually confirmed using the Bland-Altman method. Intra-class correlation coefficients were used to evaluate the inter-examiner reliability of the application. [Results] The application and universal goniometer measurements showed a good correlation (r=0.99) and no systematic error. The intra-class correlation coefficient for inter-examiner reliability was 0.999. Furthermore, to evaluate intra-examiner reliability, six examiners measured six different knee joints twice using the application on a 2-day interval. The intra-class correlation coefficient for intra-examiner reliability was 0.982. [Conclusion] The accuracy of the application was equivalent to that of a universal goniometer, and both the inter- and intra-examiner reliabilities of the application were almost perfect.

Characterization of physicochemical and digestive properties of starches from various "dainagon" adzuki beans (Vigna angularis) cultivated in Japan.

We investigated the physicochemical properties of starches extracted from Vigna angularis variants Noto-dainagon, Kyoto-dainagon, dainagon, and erimo-type adzuki beans. The particle size was larger in the Noto-dainagon starch than in the other starches, but all starches displayed a C-type crystal structure in the X-ray diffraction analysis. The onset and peak temperatures of gelatinization were approximately 7 °C lower in the Noto-dainagon and Kyoto-dainagon starches than in the dainagon and adzuki bean starches. The dainagon and adzuki bean starches also demonstrated lower swelling power at 60 °C than the other starches. The rapid visco-analyser curves of all starches showed no breakdown during the heating process, and the final viscosities of the starches increased with decreasing temperature. The apparent amylose and phosphorus contents were highest in the Noto-dainagon starch (36.6% and 95.6 ppm, respectively). The branched- chain length degree of polymerization 6-12 was lowest in the adzuki bean starch. In vitro and in vivo analyses of the digestive properties indicated that dainagon starch was more indigestible than the other starches. We considered that dainagon starch was a potentially viable health-promoting ingredient.

Comparison of the Effects of Roasted and Boiled Red Kidney Beans (Phaseolus vulgaris L.) on Glucose/Lipid Metabolism and Intestinal Immunity in a High-Fat Diet-Induced Murine Obesity Model.

Consumption of the common bean (Phaseolus vulgaris L.) is associated with beneficial effects on lipid and glucose metabolism; however, the influence of the bean processing method on these health benefits is not well understood. To investigate this, we processed red kidney beans (RKBs), a variety of the common bean, by roasting and boiling and compared the physiological effects of the two preparations in male C57BL/6N mice fed a high-fat diet (HFD). The two RKB preparations differed mainly in their resistant starch content (roasted, 24.5%; boiled, 3.1%). Four groups of mice were fed for 12 weeks on a normal diet or a HFD (45 kcal% fat) supplemented with 10% control chow (HFD control group), 10% roasted RKB, or 10% freeze-dried boiled RKB. We found that intake of roasted RKBs prevented hypercholesterolemia and increased fecal IgA and mucin content compared with the HFD control group, while intake of boiled RKBs improved glucose tolerance. Both RKB preparations suppressed the HFD-associated increase in plasma aspartate aminotransferase and alanine aminotransferase levels, which are markers of liver injury. Mice fed roasted RKBs showed significantly increased hepatic expression of cholesterol 7-alpha-monooxygenase mRNA, suggesting that cholesterol suppression may be due to enhanced bile acid biosynthesis. In contrast, mice fed boiled RKBs showed significantly increased cecal content of n-butyric acid, which may be related to the improved glucose tolerance in this group. These results indicate that the method by which RKBs are processed can profoundly affect their health benefits.

Physicochemical Properties of Starches from Lotus Rhizomes Harvested in Different Months.

We investigated the physicochemical properties of starches extracted from 8 lotus (Nelumbo nucifera Gaertn.) rhizomes harvested in different months (September 2012 to May 2013). The physicochemical properties of the lotus starches depended on the harvest date. The peak viscosity (PV) in the Rapid Visco-Analyser analysis, and the viscosity at 65 °C (V65) in the rotational viscometer analysis were significantly lower in SEP starch (extracted from the September-harvested sample) than in the other lotus starches. The Spearman's rank correlation coefficients of potassium ion (K) content vs. V65 and of K content vs. PV were 0.905 and 0.714, respectively, indicating that potassium ions are important for expressing the pasting properties of lotus starch. Principal component analysis suggested that the potassium, magnesium, calcium, and phosphorus contents are important for displaying both the pasting and gelatinization properties of the lotus starches. Meanwhile, the cluster analysis revealed that physicochemical properties of the SEP starch were different from those of the starches harvested in other months.

Dynamic viscoelasticity of protease-treated rice batters for gluten-free rice bread making.

Papain (cysteine protease), subtilisin (Protin SD-AY10, serine protease), and bacillolysin (Protin SD-NY10, metallo protease) increased the specific volume of gluten-free rice breads by 19-63% compared to untreated bread. In contrast, Newlase F (aspartyl protease) did not expand the volume of the rice bread. In a rheological analysis, the viscoelastic properties of the gluten-free rice batters also depended on the protease categories. Principal component analysis (PCA) analysis suggested that the storage and loss moduli (G' and G″, respectively) at 35 °C, and the maximum values of G' and G″, were important factors in the volume expansion. Judging from the PCA of the viscoelastic parameters of the rice batters, papain and Protin SD-AY10 improved the viscoelasticity for gluten-free rice bread making, and Protin SD-NY effectively expanded the gluten-free rice bread. The rheological properties differed between Protin SD-NY and the other protease treatments.

Application study of 1,2-α-l-fucosynthase: introduction of Fucα1-2Gal disaccharide structures on N-glycan, ganglioside, and xyloglucan oligosaccharide.

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.

Introduction of H-antigens into oligosaccharides and sugar chains of glycoproteins using highly efficient 1,2-α-l-fucosynthase.

Fucα1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via α-(1→2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-α-l-fucosidase (AfcA) into a highly efficient 1,2-α-l-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chain-containing oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced H-antigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving α-1,2-fucosyltransferases and retaining α-fucosidase.

A glycosynthase derived from an inverting chitinase with an extended binding cleft.

We created a glycosynthase from a GH19 chitinase from rye seeds (RSC-c), that has a long-extended binding cleft consisting of eight subsites; -4, -3, -2, -1, +1, +2, +3 and +4. When wild-type RSC-c was incubated with α-(GlcNAc)3-F [α-(GlcNAc)3 fluoride], (GlcNAc)3 and hydrogen fluoride were produced through the Hehre resynthesis-hydrolysis mechanism. Glu89, which acts as a catalytic base, and Ser120, which fixes a nucleophilic water molecule, were mutated to produce two single mutants, E89G and S120A, and a double mutant, E89G/S120A. E89G only produced a small amount of (GlcNAc)7 from α-(GlcNAc)3-F in the presence of (GlcNAc)4 S120A, with the highest F(-)-releasing activity, produced a larger amount of (GlcNAc)7, a fraction of which was decomposed by its own residual hydrolytic activity. However, the double mutant E89G/S120A, of which the hydrolytic activity was completely abolished while its F(-)-releasing activity was only moderately affected, produced the largest amount of (GlcNAc)7 from α-(GlcNAc)3-F and (GlcNAc)4 without decomposition. We concluded that E89G/S120A was an efficient glycosynthase, that enabled the addition of a three-sugar unit.

The crystal structure of an inverting glycoside hydrolase family 9 exo-ß-D-glucosaminidase and the design of glycosynthase.

Exo-ß-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a ß1,4-linkage at the non-reducing terminus. Asp(139), Asp(143) and Glu(555) in the active site were located near the ß-O1 hydroxy group of GlcN (D-glucosamine), with Asp(139) and Asp(143) holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp(139), we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (<0.1%), and the F(-)-release activity also decreased (<3%). Therefore the glycosynthase activity of D139E was lower than that of glycosynthases created previously from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp(139) might inevitably reduce F(-)-release activity.

Physicochemical Properties of Starches from Different Lotus Cultivars in Japan: Shinashirobana cultivar and Kanasumi-line No. 20.

We investigated the physicochemical properties of starches from rhizomes of two lotus (Nelumbo nucifera Gaertn.) cultivars, Shinashirobana cultivar and Kanasumi-line No. 20, harvested at the same farm in Japan. A rapid visco analyser analysis indicated that pasting temperatures of the starches from Shinashirobana cultivar and Kanasumi-line No. 20 were 62.1 and 66.5 °C, respectively, and the peak viscosities of the starch of both lotuses were similar 244 and 240 RVU, respectively. The starch from the Kanasumi-line No. 20 at 60 °C had low viscosity, 16.2 mPa∙s, but the starch from Shinashirobana cultivar had higher viscosity, 2,720 mPa∙s, at 60 °C. The onset and peak temperatures of the differential scanning calorimetry (DSC) curve of the starch from Shinashirobana cultivar were 4-5 °C lower than those of the starch from Kanasumi-line No. 20, although both exhibited similar ΔH values (13.4 and 11.6 J/g). The distribution patterns of the branched chain lengths of amylopectin in the two starches indicated that the rate of low-degree of polymerization (DP 5-12) of all branched chains was higher for the starch from Shinashirobana cultivar than that for the starch from Kanasumi-line No. 20. These results suggest that the increased rate of DP 5-12 in the starch from Shinashirobana cultivar is related to the higher viscosity of the starch.

Novel substrate specificities of two lacto-N-biosidases towards ß-linked galacto-N-biose-containing oligosaccharides of globo H, Gb5, and GA1.

We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galß1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of ß-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids.

Stratifying the risk of lymph node metastasis in undifferentiated-type early gastric cancer.

AIM: To study how lymph node metastasis (LNM) risk is stratified in undifferentiated-type early gastric cancer (undiff-EGC) dependent on combinations of risk factors. METHODS: Five hundred and sixty-seven cases with undiff-EGC undergoing gastrectomy with lymphadenectomy were examined retrospectively. Using clinicopathological factors of patient age, location, size, an endoscopic macroscopic tumor form, ulceration, depth, histology, lymphatic involvement (LI) and venous involvement (VI), LNM risk was examined and stratified by conventional statistical analysis and data-mining analysis. RESULTS: LNM was positive in 44 of 567 cases (7.8%). Univariate analysis revealed > 2 cm, protrusion, submucosal (sm), mixed type, LI and VI as significant prognostic factors and > 2 cm and LI-positive were independent factors by multivariate analysis. In preoperatively evaluable factors excluding LVI, sm and > 2 cm were independent factors. According to the depth and size, cases were categorized into the low-risk group [m and ≤ 2 cm, 0% (LNM incidence)], the moderate-risk group (m and > 2 cm, 5.6%; and sm and ≤ 2 cm, 6.0%), and the high-risk group (sm and > 2 cm, 19.3%). On the other hand, LNM occurred in 1.4% in all LI-negative cases, greatly lower than 28.2% in all LI-positive cases, and LNM incidence was low in LI-negative cases even in the moderate- and high-risk groups. CONCLUSION: LNM-related factors in undiff-EGC were depth and size preoperatively while those were LI and size postoperatively. Among these factors, LI was the most significantly correlated factor.

Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression.

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galß1-3GlcNAcß1-3Galß1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galß1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcß1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc), and sialyllacto-N-tetraose a (Neu5Acα2-3Galß1-3GlcNAcß1-3Galß1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-ß-lacto-N-bioside I (Galß1-3GlcNAcß-pNP) and GalNAcß1-3GlcNAcß-pNP. GalNAcß1-3GlcNAcß linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca(2+) and Mg(2+)) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Characterization of titanium particles treated with N2 plasma using a barrel-plasma-treatment system.

Metallic titanium (Ti) particles treated with N2 plasma via the barrel-plasma-treatment system were thoroughly evaluated, to investigate changes in physical and chemical properties resulting from the treatment. The color of the Ti particles changed from gray to uniform brown upon plasma-treatment, indicating good surface uniformity. Depth profiling using X-ray photoelectron spectroscopy showed that the nitrogen implanted by plasma-treatment formed Ti-N bonds at or near the surface of the particles, resulting in the formation of a stoichiometric TiN layer. It was determined from cross-sectional transmission electron microscopy and nanobeam diffraction measurements that the resulting TiN layer had two different structures. Nanoindentation data showed that the surface of a treated sample was about five times harder than that of untreated particles.

Biochemical characterization of a recombinant plant class III chitinase from the pitcher of the carnivorous plant Nepenthes alata.

A class III chitinase belonging to the GH18 family from Nepenthes alata (NaCHIT3) was expressed in Escherichia coli. The enzyme exhibited hydrolytic activity toward colloidal chitin, ethylene glycol chitin, and (GlcNAc)(n) (n=5 and 6). The enzyme hydrolyzed the fourth glycosidic linkage from the non-reducing end of (GlcNAc)(6). The anomeric form of the products indicated it was a retaining enzyme. The colloidal chitin hydrolytic reaction displayed high activity between pH 3.9 and 6.9, but the pH optimum of the (GlcNAc)(6) hydrolytic reaction was 3.9 at 37 °C. The optimal temperature for activity was 65 °C in 50 mM sodium acetate buffer (pH 3.9). The pH optima of NaCHIT3 and NaCHIT1 might be related to their roles in chitin degradation in the pitcher fluid.

A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum.

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)(n) [a ß-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)(2) fluoride] in the absence or presence of (GlcNAc)(2), (GlcNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Ser(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)(4) as a major product from α-(GlcNAc)(2)-F in the presence of (GlcNAc)(2). The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F(-) releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.

1,3-1,4-α-L-fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 chains.

α-L-fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-L-fucosynthase that specifically introduces Le(a) and Le(x) antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galß1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-L-fucosidase (EC 3.2.1.111) and α-L-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Le(a/x) epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata.

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [ß-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.

Elucidation of exo-beta-D-glucosaminidase activity of a family 9 glycoside hydrolase (PBPRA0520) from Photobacterium profundum SS9.

A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose [(GlcN)(2)] and cellobiose (CG(2)) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-ß-D-glucosaminide (GlcN-PNP) and 4-nitrophenyl-ß-D-glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent (1)H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k(cat)/K(m) of (GlcN)(2) hydrolysis was 14 times greater than that of CG(2) hydrolysis. These results suggested that the protein is an exo-ß-D-glucosaminidase (EC 3.2.1.165) rather than a glucan 1,4-ß-D-glucosidase (EC 3.2.1.74). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN)(2)-phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.