Hydrolysis Of Raffinose Research Articles

Generation of a Vibrio-based platform for efficient conversion of raffinose through Adaptive Laboratory Evolution on a solid medium

Raffinose, a trisaccharide abundantly found in soybeans, is a potential alternative carbon source for biorefineries. Nevertheless, residual intermediate di- or monosaccharides and low catabolic efficiency limit raffinose use through conventional microbial hosts. This study presents a Vibrio-based platform to convert raffinose efficiently. Vibrio sp. dhg was selected as the starting strain for the Adaptive Laboratory Evolution (ALE) strategy to leverage its significantly higher metabolic efficiency. We conducted ALE on a solid minimal medium supplemented with raffinose to prevent the enrichment of undesired phenotypes due to the shared effect of extracellular raffinose hydrolysis among multiple strains. As a result, we generated the VRA10 strain that efficiently utilizes raffinose without leaving behind degraded di- or monosaccharides, achieving a notable growth rate (0.40 h−1) and raffinose consumption rate (1.2 g/gdcw/h). Whole genome sequencing and reverse engineering identified that a missense mutation in the melB gene (encoding a melibiose/raffinose:sodium symporter) and the deletion of the two galR genes (encoding transcriptional repressors for galactose catabolism) facilitated rapid raffinose utilization. The further engineered strain produced 6.2 g/L of citramalate from 20 g/L of raffinose. This study will pave the way for the efficient utilization of diverse raffinose-rich byproducts and the expansion of alternative carbon streams in biorefinery applications.

Raffinose catabolism enhances maize waterlogging tolerance by stimulating adventitious root growth and development.

Raffinose mitigates plant heat, drought, and cold stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize raffinose synthase mutant zmrafs-1 had seedlings that lack raffinose, generated fewer and shorter adventitious roots, and were more sensitive to waterlogging stress, while overexpression of the raffinose synthase gene, ZmRAFS, increased raffinose content, stimulated adventitious root formation, and enhanced waterlogging tolerance of maize seedlings. Transcriptome analysis of null segregant seedlings compared with zmrafs-1, particularly when waterlogged, revealed that the expression of genes related to galactose metabolism and the auxin biosynthetic pathway were up-regulated by raffinose. Additionally, indole-3-acetic acid content was significantly decreased in zmrafs-1 seedlings and increased in ZmRAFS-overexpressing seedlings. Inhibition of the hydrolysis of raffinose by 1-deoxygalactonojirimycin decreased the waterlogging tolerance of maize seedlings, the expression of genes encoding proteins related to auxin transport-related genes, and the indole-3-acetic acid level in the seedlings, indicating that the hydrolysis of raffinose is necessary for maize waterlogging tolerance. These data demonstrate that raffinose catabolism stimulates adventitious root formation via the auxin signaling pathway to enhance maize waterlogging tolerance.

QTL Mapping and Genome-Wide Association Study Reveal Genetic Loci and Candidate Genes Related to Soluble Solids Content in Melon.

Melon (Cucumis melo L.) is an economically important Cucurbitaceae crop grown around the globe. The sweetness of melon is a significant factor in fruit quality and consumer appeal, and the soluble solids content (SSC) is a key index of melon sweetness. In this study, 146 recombinant inbred lines (RILs) derived from two oriental melon materials with different levels of sweetness containing 1427 bin markers, and 213 melon accessions containing 1,681,775 single nucleotide polymorphism (SNP) markers were used to identify genomic regions influencing SSC. Linkage mapping detected 10 quantitative trait loci (QTLs) distributed on six chromosomes, seven of which were overlapped with the reported QTLs. A total of 211 significant SNPs were identified by genome-wide association study (GWAS), 138 of which overlapped with the reported QTLs. Two new stable, co-localized regions on chromosome 3 were identified by QTL mapping and GWAS across multiple environments, which explained large phenotypic variance. Five candidate genes related to SSC were identified by QTL mapping, GWAS, and qRT-PCR, two of which were involved in hydrolysis of raffinose and sucrose located in the new stable loci. The other three candidate genes were involved in raffinose synthesis, sugar transport, and production of substrate for sugar synthesis. The genomic regions and candidate genes will be helpful for molecular breeding programs and elucidating the mechanisms of sugar accumulation.

Comparative affinity immobilization of α-galactosidase on chitosan functionalized with Concanavalin A and its useability for the hydrolysis of raffinose

A new enzyme immobilization strategy is bioaffinity immobilization that using biospecific affinity interactions between the enzyme and matrix. α-Galactosidase was immobilized with three different methods (Method I, II and III) on chitosan functionalized with Concanavalin A (ConA). In Method I, the chitosan was first activated with glutaraldehyde, derivatized with ConA and then utilized to immobilize the α-galactosidase. For Method II, chitosan was functionalized with ConA by adsorption at first and then used for the immobilization of the enzyme. In Method III, by using the optimized conditions of Method I affinity layers were prepared with α-galactosidase. The effect of various factors on α-galactosidase immobilization is searched to get perfect immobilization yields. Optimal immobilization conditions were determined for each method. At these conditions, α-galactosidase is immobilized with 53%, 64% and 58% activity yields by method I, II and III, respectively. α-Galactosidase immobilized with method I, II and III could hydrolyzed 52%, 94% and 30% of raffinose in 24 h at 50 °C, respectively. The biochemical characterization and stability studies confirm that these immobilized enzymes promise future in industry. Especially, the immobilized enzymes could be safely used because of their potential for the hydrolysis of raffinose and raffinose type oligosaccharides in food and feed industries.

ZmAGA1 Hydrolyzes RFOs Late during the Lag Phase of Seed Germination, Shifting Sugar Metabolism toward Seed Germination Over Seed Aging Tolerance.

Raffinose family oligosaccharides (RFOs) are accumulated during the late stage of seed development and hydrolyzed during seed germination. The process of raffinose hydrolysis during seed germination and how this process affects seed vigor remains unknown. We report here that maize alkaline α-galactosidase 1 (ZmAGA1) protein is translationally induced and is capable of hydrolyzing RFOs as well as a precursor, galactinol, during seed germination. Constitutively overexpressing ZmAGA1 in Arabidopsis decreased both RFOs and galactinol contents of mature, desiccated, and 30 hours after imbibition (HAI) seeds, yet enhanced the seed germination percentage under either salt or somewhat osmotic-stress conditions at earlier times during the time course. However, ZmAGA1 overexpression also decreased the seed aging tolerance of mature, desiccated seeds as compared with wild type (WT) or those overexpressing GFP. Compared to that of WT control seeds, the atsip2 (mutant of Arabidopsis AtSIP2 (seed imbibition protein 2, encoding alkaline α-galactosidase)) seeds have similar RFOs and galactinol contents in mature, desiccated seeds but significantly increased the amount of these metabolites at 30 HAI. This retention of RFOs and galactinol in atsip2 results in seeds that exhibit lowered seed germination percentage under either salt or osmotic stress conditions, and yet, increased seed aging tolerance relative to WT. Similarly, when maize seeds were imbibed in the presence of a specific α-galactosidase inhibitor (1-deoxygalactonojirimycin) as compared to those imbibed in water, greater amounts of raffinose and galactinol were detected; the seeds exhibited decreased seed germination percentages but increased seed aging tolerance. Taken together, these data suggest that both maize seed germination and seed aging tolerance can be simultaneously regulated through careful temporal manipulation of ZmAGA1 expression.

Whole conversion of agro-industrial wastes rich in galactose-based carbohydrates into lipid using oleaginous yeast Aureobasidium namibiae

BackgroundRaw materials composed of easily assimilated monosaccharides have been employed as carbon source for production of microbial lipids. Nevertheless, agro-industrial wastes rich in galactose-based carbohydrates have not been introduced as feedstocks for oleaginous yeasts.ResultsIn this study, Aureobasidium namibiae A12 was found to efficiently accumulate lipid from soy molasses and whey powder containing galactose-based carbohydrates, with lipid productions of 5.30 g/L and 5.23 g/L, respectively. Over 80% of the fatty acids was C16:0, C18:0, C18:1, and C18:2. All kinds of single sugar components in the two byproducts were readily converted into lipids, with yields ranging between 0.116 g/g and 0.138 g/g. Three α-galactosidases and five β-galactosidases in the strain were cloned and analyzed. Changes of transcriptional levels indicated GalB and GalC were key α-galactosidases, and GalG was key β-galactosidase. In 10 L fermentor, lipid production from SM and WP achieved 6.45 g/L and 6.13 g/L, respectively. β-galactosidase was responsible for lactose hydrolysis; sucrase and α-galactosidase both contributed to the efficient hydrolysis of raffinose and stachyose in a cooperation manner.ConclusionsThis is a new way to produce lipids from raw materials containing galactose-based carbohydrates. This finding revealed the significance of sucrase in the direct hydrolysis of galactose-based carbohydrates in raw materials for the first time and facilitated the understanding of the efficient utilization of galactose-based carbohydrates to manufacture lipid or other chemicals in bioprocess.Graphic abstract

Identification of an Invertase With High Specific Activity for Raffinose Hydrolysis and Its Application in Soymilk Treatment.

The hydrolyzation of raffinose into melibiose by using invertases under mild conditions improves the nutritional value of soybean products. However, this strategy has received little attention because a suitable invertase remains lacking. In this study, a novel invertase named InvDz13 was screened and purified from Microbacterium trichothecenolyticum and characterized. InvDz13 was one of the invertases with the highest specific activity toward raffinose. Specifically, it had a specific activity of 229 U/mg toward raffinose at pH 6.5 and 35°C. InvDz13 retained more than 80% of its maximum activity at pH 5.5–7.5 and 25–40°C and was resistant to or stimulated by most cations that presented in soymilk. In soymilk treated with InvDz13 under mild conditions, melibiose concentration increased from 3.1 ± 0.2 to 6.1 ± 0.1 mM due to raffinose hydrolyzation by InvDz13. Furthermore, the prebiotic property of InvDz13-treated soymilk was investigated via in vitro fermentation by human gut microbiota. Results showed that InvDz13 treatment increased the proportion of the beneficial bacteria Bifidobacterium and Lactobacillus by 1.6- and 3.7-fold, respectively. By contrast, the populations of Escherichia and Collinsella decreased by 1.8- and 11.7-fold, respectively. Thus, our results proved that the enzymatic hydrolysis of raffinose in soymilk with InvDz13 was practicable and might be an alternative approach to improving the nutritional value of soymilk.

Effect of Domestic Processing and Crude Extract of α-Galactosidase on Oligosaccharide Content of Red Gram (Cajanus cajan L.) Seeds

The aim of the present study was to demonstrate the effect of cooking in 1% mango powder, soaking in mixture of 1% salts and crude extract of α-galactosidase treatment on the raffinose family sugar content of red gram. Cooking of red gram seeds in 1% mango powder for 60 min resulted in a mean reduction of raffinose stachyose and verbascose by 67.26, 63.74 and 51.53% respectively. Pressure cooking of red gram seeds in 1% mango powder for 15 min led to decrease of raffinose by 69.24%, stachyose 65.13% and verbascose 47.42%. Soaking in 1% salt mixture solution led to loss of raffinose by 70.20%, stachyose 67.08% and verbascose 53.43% respectively. The crude α-galactosidase treatment led to a mean hydrolysis of raffinose by 67.18%, stachyose 65.68% and verbascose 56.55%.The domestic processing methods reduced the anti-nutrients content and in turn, improved the nutritional quality of the legume seeds.

Characterization of an acidic α-galactosidase from hemp (Cannabis sativa L.) seeds and its application in removal of raffinose family oligosaccharides (RFOs).

An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 μM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.

Bioaffinity immobilization and characterization of α-galactosidase on aminophenylboronicacid derivatized chitosan and Sepabeads EC-EA

Enzyme immobilization with affinity binding which is based on the specific affinity interactions have an important advantage as; high selectivity. In the present study, chitosan and Sepabeads EC-EA were derivatized with aminophenylboronicacid(APBA) for the affinity immobilization of α-galactosidase. The influence of various process parameters on immobilization of the enzyme is investigated to get high immobilization yields. Under optimized immobilization conditions, the chitosan and Sepabeads EC-EA immobilized enzymes exhibited activity yield of 89.5% and 72%, respectively. The maximum activities were detected at 40 °C for free and Sepabeads EC-EA immobilized enzyme and 55 °C for chitosan immobilized enzyme. The optimum pH was found as pH 5.0 for free and Sepabeads EC-EA immobilized enzyme and pH 5.5 for chitosan immobilized enzyme. Both immobilized enzymes were very stable at temperature ranged from 4 to 55 °C and also in a pH range of 2.6–7.0. The immobilized α-galactosidases were also used in the hydrolysis of raffinose. The chitosan and Sepabeads EC-EA immobilized enzymes hydrolysed 55% and 42% of raffinose in 32 h at 50 °C, respectively. The obtained results shed light for the useability of these immobilized enzymes in the hydrolysis of raffinose in food industry and make these immobilized enzymes good candidates for their various biotechnological applications.

Expression and Characteristics of an Endoglucanase from Trichoderma atroviride (TaEGII) in Saccharomyces cerevisiae.

Endoglucanase secreted by the fungus Trichoderma atroviride is a kind of cellulase. An endoglucanase gene egII was cloned from T. atroviride AS3.3013 and expressed in Saccharomyces cerevisiae INVScI. The open reading frame of the egII gene was composed of 1257bp, encoding 418 amino acids with a molecular weight of 44.23kDa plus a signal peptide of 21 amino acids. Based on sequence similarity, TaEGII belonged to the glycosyl hydrolase family 5. Expression of the egII gene in T. atroviride AS3.3013 can be induced by microcrystalline cellulose (MCC), bran, carboxymethyl cellulose (CMC), rice straw, and corn stalk but is inhibited by glucose. A highly efficient integrated expression vector (pYPIGH-B includes a sequence of the α-mating factor signal peptide (MF-α)) was constructed. The enzymatic activity of the supernatant of recombinant yeast YPIGH-B3 was 1.29 times higher than that of YES2-egII, demonstrating that the MF-α can significantly improve the expression of the recombinant EGII in S. cerevisiae. The recombinant endoglucanase TaEGII produced by S. cerevisiae showed maximum activity at pH 5.0 and temperature 60°C. Under these conditions, the Km and Kcat values for Avicel and raffinose hydrolysis were 1.22×10-2mgml-1, 9.09×10-2s-1 and 1.06×10-2mgml-1 , 9.18×10-2s-1, respectively. The enzymatic activity of recombinant TaEGII was stable when incubated from 40 to 60°C for 1h. It was stable in a wide range of pH (4.0-7.0) and sensitive to various metal ions. Transgenic yeast strain YPIGH-B3 might be applied to cellulosic ethanol production.

Hydrolysis of Oligosaccharides by a Thermostable α-Galactosidase from Termitomyces eurrhizus.

The genus of Termitomyces purchased from the market has been identified as Termitomyces eurrhizus using the Internal Transcribed Spacer (ITS) method. An α-galactosidase from T. eurrhizus (TEG), a monomeric protein with a molecular mass of 72 kDa, was purified 146 fold by employing ion exchange chromatography and gel filtration. The optimum pH and temperature was 5.0 and 60 °C, respectively. TEG was stable over pH 2–6, and also exhibited good thermostablility, retaining 100% of the original activity after incubation at 60 °C for 2 h. Inhibition of the enzyme activity by N-bromosuccinimide (NBS) constituted evidence for an essential role of tryptophan in the catalytic action of the isolated enzyme. Besides 4-nitro-phenyl α-d-galactophyranoside (pNPGal), natural substrates could also be effectively hydrolyzed by TEG. Results of thin-layer chromatography (TLC) revealed complete enzymatic hydrolysis of raffinose and stachyose to galactose at 50 °C within 6 h. These properties of TEG advocate its utilization for elevating the nutritional value of soymilk.

One‐Pot Conversions of Raffinose into Furfural Derivatives and Sugar Alcohols by Using Heterogeneous Catalysts

Inedible and/or waste biomass reserves are being strongly focused upon as a suitable new energy and chemical source. Raffinose, which is an indigestible trisaccharide composed of glucose, galactose, and fructose, is found abundantly in beet molasses, sugar cane, and seeds of many leguminous plants. Herein, we demonstrate the one-pot synthesis of furan derivatives and sugar alcohols from raffinose by using heterogeneous acid, base, and/or metal-supported catalysts. The combination of Amberlyst-15 and hydrotalcite (HT) showed a high activity (37% yield) for 5-hydroxymethyl-2-furaldehyde (HMF) through continuous hydrolysis, isomerization, and dehydration reactions. In addition, the use of a hydrotalcite-supported ruthenium catalyst (Ru/HT) successfully afforded 2,5-diformylfuran (DFF, 27% yield) from HMF produced by raffinose, directly. Moreover, the hydrogenation of hexoses obtained by raffinose hydrolysis into sugar alcohols (galactitol, mannitol, sorbitol) was also achieved in a high yield (91%) with Amberlyst-15 and Ru/HT catalysts. Thus, we suggest that raffinose has great potential for the synthesis of important industrial intermediates under mild reaction conditions.

Enzymatic preparation of melibiose and alkyl β-d-fructofuranosides by commercial lactase

Lactozyme 3000L was found to possess β-fructofuranosidase activity and catalyzed hydrolysis of raffinose to melibiose and formation of alkyl β-d-fructofuranosides from sucrose. The total chemical yield of melibiose was 95.5%, yields of alkyl β-d-fructofuranosides ranged from 5.2 to 20.2% depending on the structure of the acceptor. Alcohols larger than butanol were not glycosylated.

Purification and Characterization of Aspergillus terreus α-Galactosidases and Their Use for Hydrolysis of Soymilk Oligosaccharides

α-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4-7. Maximal activities were determined at 60, 55, and 50 °C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50 °C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg²⁺, Ag⁺, and Cu²⁺. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymes.

Production of α-Galactosidase by Aspergillus oryzae through solid-state fermentation and its application in soymilk Galactooligosaccharide hydrolysis

α-Galactosidase was produced by Aspergillus oryzae on red gram plant waste-wheat bran based media in solid-state fermentation (SSF). Optimum temperature for α-galactosidase production was 35 0C and upto 4 cm of bed height of substrate had no inhibitory effect on enzyme production. Hydrolysis of galactooligosaccharides in soymilk was carried out by α-galactosidase. Optimum temperature and pH for the hydrolysis of raffinose and stachyose of soymilk were 55(0)C and 5.2-6.2, respectively. The enzymatic treatment for 3 h completely removed the raffinose oligosaccharides in soymilk. Crude extract also showed considerable amount of invertase activity.

Extracellular α-Galactosidase fromDebaryomyces hanseniiUFV-1 and Its Use in the Hydrolysis of Raffinose Oligosaccharides

Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. ROs are hydrolyzed by alpha-galactosidases that cleave alpha-1,6-linkages of alpha-galactoside residues. The objectives of this study were the purification and characterization of extracellular alpha-galactosidase from Debaryomyces hansenii UFV-1. The enzyme purified by gel filtration and anion exchange chromatographies presented an Mr value of 60 kDa and the N-terminal amino acid sequence YENGLNLVPQMGWN. The Km values for hydrolysis of pNP alphaGal, melibiose, stachyose, and raffinose were 0.30, 2.01, 9.66, and 16 mM, respectively. The alpha-galactosidase presented absolute specificity for galactose in the alpha-position, hydrolyzing pNPGal, stachyose, raffinose, melibiose, and polymers. The enzyme was noncompetitively inhibited by galactose (Ki = 2.7 mM) and melibiose (Ki = 1.2 mM). Enzyme treatments of soy milk for 4 h at 60 degrees C reduced the amounts of stachyose and raffinose by 100%.

Metabolism of oligosaccharides and aldehydes and production of organic acids in soymilk by probiotic bifidobacteria

SummaryFour strains of Bifidobacterium were assessed for α‐galactosidase activity in MRS broth using p‐nitrophenyl‐α‐d‐galactopyranoside as substrate. Bifidobacteria were then used to ferment soymilk prepared from soy protein isolate (SPI), with and without d‐glucose and l‐cysteine supplementation. Measurements of pH and the quantification of oligosaccharides, organic acids and aldehydes in soymilk were done after 0, 12, 24, 36 and 48 h of incubation. The presence of d‐raffinose stimulated the α‐galactosidase activity of B. longum BB536 (BB536) and B. pseudolongum 20099 (BP20099). In soymilk, oligosaccharides and aldehydes were effectively metabolized by Bifidobacterium; d‐glucose and l‐cysteine supplementation enhanced the hydrolysis of raffinose and stachyose. BB536 and BP20099 degraded a significantly greater level of oligosaccharides and aldehydes than B. animalis Bb‐12 and B. longum 1941 (P < 0.05). Raffinose and stachyose were completely metabolized by BB536 after 48 h. In soymilk without any supplementation, hexanal and pentanal were not detected after 12 h of fermentation with BB536 and BP20099. BB536 was the highest producer of organic acids, with an average acetic acid/l(+)‐lactic acid ratio of 0.7.

Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular trafficking of capsular glucuronoxylomannan.

Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.

Preparation and properties of alpha-galactosidase chemically attached to activated chitin.

alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from watermelon was covalently immobilized on chitin. The immobilized alpha-galactosidase exhibited an activity of 0.61 U per g of carrier and an activity yield of 67%. The properties of free and immobilized alpha-galactosidase were also searched and compared. The results showed that, optimum conditions for activity were not affected by immobilization. The optimum pH and temperature for free and immobilized enzyme found as pH 6.0 and 65 degress C, respectively. Compared with the free enzyme, the temperature and pH stabilities of the immobilized enzyme were similar. Both the enzymes were stable between pH 2-10 and below 50 degrees C. The Km values for free and immobilized enzyme were determined using p-nitrophenyl-alpha-D-galactopyranoside (PNPG) and raffinose as substrates. Operational stability of the immobilized enzyme was investigated by using both substrates. The operational half-life (t 1/2) was calculated as 34 h for PNPG and 28 h for raffinose. The immobilized alpha-galactosidase was also utilized in the hydrolysis of raffinose. The immobilization procedure on chitin was cheap and also easy to carry out, and the immobilized enzyme had good properties that the potential for practical application is considerable.