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1.
Planta ; 258(6): 116, 2023 Nov 09.
Article in English | MEDLINE | ID: mdl-37946063

ABSTRACT

MAIN CONCLUSION: Each ß-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of ß-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and ß-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal ß-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall ß-glucan, it was compared with ß-1,3-glucanase with different substrate specificities. We obtained recombinant ß-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I ß-1,3-glucanase) and CcGluA (GH64 ß-1,3-glucanase).


Subject(s)
Ficus , beta-Glucans , Antifungal Agents/pharmacology , Antifungal Agents/metabolism , Saccharomyces cerevisiae/metabolism , beta-Glucans/metabolism , Ficus/metabolism , Latex/metabolism , Glycoside Hydrolases/genetics , Glycoside Hydrolases/analysis , Glycoside Hydrolases/metabolism , Fungi/metabolism , Bacteria/metabolism , Cell Wall/metabolism
2.
Appl Environ Microbiol ; 88(12): e0065222, 2022 06 28.
Article in English | MEDLINE | ID: mdl-35652665

ABSTRACT

Chitin is a biopolymer of N-acetyl-d-glucosamine with ß-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the ß-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme's thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCE We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9°C higher than wild type (WT) and a half-life at 60°C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.


Subject(s)
Antifungal Agents , Chitinases , Antifungal Agents/chemistry , Chitin/chemistry , Chitinases/chemistry , Disulfides , Enzyme Stability , Ficus/enzymology , Fungi , Latex , Proline
3.
Appl Environ Microbiol ; 87(21): e0114421, 2021 10 14.
Article in English | MEDLINE | ID: mdl-34406826

ABSTRACT

Certain Aspergillus and Penicillium spp. produce the fungal cell wall component nigeran, an unbranched d-glucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation. The mechanism underlying nigeran biosynthesis and the physiological role of nigeran in fungal survival are not clear. We used RNA sequencing (RNA-seq) to identify genes involved in nigeran synthesis in the filamentous fungus Aspergillus luchuensis when grown under nitrogen-free conditions. agsB, which encodes a putative α-1,3-glucan synthase, and two adjacent genes (agtC and gnsA) were upregulated under conditions of nitrogen starvation. Disruption of agsB in A. luchuensis (ΔagsB) resulted in the complete loss of nigeran synthesis. Furthermore, the overexpression of agsB in an Aspergillus oryzae strain that cannot produce nigeran resulted in nigeran synthesis. These results indicated that agsB encodes a nigeran synthase. Therefore, we have renamed the A. luchuensis agsB gene the nigeran synthase gene (nisA). Nigeran synthesis in an agtC mutant (ΔagtC) increased to 121%; conversely, those in the ΔgnsA and ΔagtC ΔgnsA strains decreased to 64% and 63%, respectively, compared to that in the wild-type strain. Our results revealed that AgtC and GnsA play an important role in regulating not only the quantity of nigeran but also its polymerization. Collectively, our results demonstrated that nisA (agsB) is essential for nigeran synthesis in A. luchuensis, whereas agtC and gnsA contribute to the regulation of nigeran synthesis and its polymerization. This research provides insights into fungal cell wall biosynthesis, specifically the molecular evolution of fungal α-glucan synthase genes and the potential utilization of nigeran as a novel biopolymer. IMPORTANCE The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3/1,4-glucosidic linkages. The mechanisms regulating the biosynthesis and function of nigeran are unknown. Here, we performed RNA sequencing of Aspergillus luchuensis cultured under nitrogen-free or low-nitrogen conditions. A putative α-1,3-glucan synthase gene, whose transcriptional level was upregulated under nitrogen-free conditions, was demonstrated to encode nigeran synthase. Furthermore, two genes encoding an α-glucanotransferase and a hypothetical protein were shown to be involved in controlling the nigeran content and molecular weight. This study reveals genes involved in the synthesis of nigeran, a potential biopolymer, and provides a deeper understanding of fungal cell wall biosynthesis.


Subject(s)
Aspergillus , Cell Wall/genetics , Glucans/biosynthesis , Glucosyltransferases/genetics , Aspergillus/enzymology , Aspergillus/genetics , Fungal Proteins/genetics , Nitrogen , Polymerization , RNA-Seq
4.
Planta ; 253(6): 120, 2021 May 13.
Article in English | MEDLINE | ID: mdl-33987712

ABSTRACT

MAIN CONCLUSION: A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.


Subject(s)
Chitinases , Ficus , Antifungal Agents/pharmacology , Chitin , Chitinases/genetics , Cloning, Molecular , DNA, Complementary , Hypocreales , Latex
5.
J Biosci Bioeng ; 131(4): 348-355, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33281068

ABSTRACT

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC50 value of 0.81 µM.


Subject(s)
Antifungal Agents/pharmacology , Chitinases/metabolism , Lysobacter/enzymology , Chitinases/genetics , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Lysobacter/genetics , Schizophyllum/drug effects , Trichoderma/drug effects
6.
J Biosci Bioeng ; 130(4): 352-359, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32522405

ABSTRACT

Aspergillus luchuensis has been used to produce awamori, a distilled liquor, in Okinawa, Japan. Vanillin, derived from ferulic acid (FA) in rice grains, is one of the characteristic flavors in aged and matured awamori, known as kusu. Decarboxylation of FA leads to the production of 4-vinylguaiacol (4-VG), which is converted to vanillin by natural oxidization. However, the mechanism underlying FA conversion to 4-VG has remained unknown in awamori brewing. In our previous studies, we showed that phenolic acid decarboxylase from A. luchuensis (AlPAD) could catalyze the conversion of FA to 4-VG, and that AlPAD is functionally expressed during koji making (Maeda et al., J. Biosci. Bioeng., 126, 162-168, 2018). In this study, to understand the contribution of AlPAD to 4-VG production in awamori brewing, we created an alpad disruptant (Δalpad) and compared its 4-VG productivity to that of the wild-type strain. The amount of 4-VG in the distillate of moromi prepared with the wild-type strain showed a significant increase, proportional to the time required for koji making. In the Δalpad strain, the amount of 4-VG was very small and remained unchanged during the koji making. In an awamori brewing test using koji harvested 42-66 h after inoculation, the contribution of AlPAD to 4-VG production was in the range of 88-94 %. These results indicate that AlPAD plays a key role in 4-VG production during awamori brewing.


Subject(s)
Alcoholic Beverages/microbiology , Aspergillus/enzymology , Carboxy-Lyases/metabolism , Guaiacol/analogs & derivatives , Aspergillus/metabolism , Biocatalysis , Guaiacol/metabolism
7.
Appl Biochem Biotechnol ; 192(2): 616-631, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32500428

ABSTRACT

Talaromyces cellulolyticus is a promising fungus for providing a cellulase preparation suitable for the hydrolysis of lignocellulosic material, although its mannan-degrading activities are insufficient. In the present study, three core mannanolytic enzymes, including glycosyl hydrolase family 5-7 (GH5-7) ß-mannanase (Man5A), GH27 α-galactosidase, and GH2 ß-mannosidase, were purified from a culture supernatant of T. cellulolyticus grown with glucomannan, and the corresponding genes were identified based on their genomic sequences. Transcriptional analysis revealed that these genes were specifically induced by glucomannan. Two types of Man5A products, Man5A1 and Man5A2, were found as major proteins in the mannanolytic system. Man5A1 was devoid of a family 1 carbohydrate-binding module (CBM1) at the N-terminus, whereas Man5A2 was devoid of both CBM1 and Ser/Thr-rich linker region. The physicochemical and catalytic properties of both Man5A1 and Man5A2 were identical to those of recombinant Man5A (rMan5A) possessing CBM1, except for the cellulose-binding ability. Man5A CBM1 had little effect on mannan hydrolysis of pretreated Hinoki cypress. The results suggest that an improvement in Man5A CBM1 along with the augmentation of identified mannanolytic enzyme components would aid in efficient hydrolysis of softwood using T. cellulolyticus cellulase preparation.


Subject(s)
Mannans/metabolism , Talaromyces/enzymology , beta-Mannosidase/metabolism , Hydrolysis , Talaromyces/genetics , Talaromyces/metabolism , Temperature , Transcription, Genetic
8.
Int J Biol Macromol ; 154: 1295-1302, 2020 Jul 01.
Article in English | MEDLINE | ID: mdl-31733252

ABSTRACT

PrChiA is an antifungal chitinase obtained from Pteris ryukyuensis, a fern plant. It consists of two N-terminal lysin motif (LysM) domains and a C-terminal catalytic domain of glycoside hydrolase family 18. Previous studies have shown that the deletion of LysM domains or loss of hydrolytic activity causes the loss of the antifungal activity of chitinases. In this study, we produced LysM-domain multimers (LysMn, n = 2-5) and the respective multimer fusion chitinases (LysMn-Cat, n = 1-4), and characterized their enzymatic and antifungal properties. LysMn and LysMn-Cat showed a higher affinity to insoluble chitin than single LysM domain and single catalytic domain alone, respectively. LysMn-Cat hydrolyzed insoluble chitin more efficiently than the catalytic domain alone. Surprisingly, LysMn showed antifungal activity without chitinolytic activity. Further, LysMn-Cat exhibited a stronger antifungal activity than LysMn. Microscopic observation revealed that LysMn attacked only the tips of the fungal hyphae; LysMn-Cat attacked not only the tips, but also the lateral walls around the septa of the fungal hyphae. It is suggested that the LysMn act on the growing point of the hyphal tip through their chitin-binding ability and that the LysMn-Cat act on not only the hyphal tips, but also on the lateral walls through their chitin-hydrolyzing and -binding activities.


Subject(s)
Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Chitinases/chemistry , Chitinases/pharmacology , Protein Multimerization , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacology , Antifungal Agents/metabolism , Catalytic Domain , Chitin/metabolism , Chitinases/metabolism , Hydrolysis , Models, Molecular , Protein Structure, Quaternary , Pteris/enzymology , Recombinant Fusion Proteins/metabolism
9.
Biosci Biotechnol Biochem ; 82(10): 1742-1752, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29966504

ABSTRACT

Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc)4 by SdChiA showed that SdChiA specifically recognizes the ß-anomer in the + 2 subsite of the substrate (GlcNAc)4 and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains. ABBREVIATIONS: AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.


Subject(s)
Chitinases/genetics , DNA, Complementary/genetics , Selaginellaceae/enzymology , Selaginellaceae/genetics , Amino Acid Sequence , Chitin/metabolism , Chitinases/chemistry , Chitinases/metabolism , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA, Plant/genetics , Electrophoresis, Polyacrylamide Gel , Open Reading Frames , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Substrate Specificity
10.
J Biosci Bioeng ; 126(2): 162-168, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29519654

ABSTRACT

Awamori is a traditional distilled liquor in the Ryukyu Islands, made from steamed rice by the action of the black-koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae. One of the specific flavors in aged awamori kusu is vanillin, which is derived from ferulic acid (FA) in rice grains. FA is released from the cell wall material in the rice grain by ferulic acid esterase produced by A. luchuensis. Through decarboxylation of FA, 4-vinylguaiacol (4-VG) is produced, which is transferred to the distilled liquor, and converted to vanillin by natural oxidization during the aging process. However, the actual mechanism for conversion of FA to 4-VG in the awamori brewing process is unknown. A genetic sequence having homology to the phenolic acid decarboxylase (PAD)-encoding region from bacteria and the yeast Candida guilliermondii has been identified in A. luchuensis mut. kawachii. In the present study, recombinant PAD from A. luchuensis, designated as AlPAD, expressed as a homodimer, catalyzed the conversion of FA to 4-VG, displayed optimal catalytic activity at pH 5.7 and 40°C, and was stable up to 50°C. Both rice bran and FA could induce the bioconversion of FA to 4-VG and the expression of AlPAD in A. luchuensis. The amount of AlPAD determined using western blotting correlated with the level of FA decarboxylase activity during koji production. In awamori brewing process, AlPAD might be responsible for a part of the conversion of FA to 4-VG.


Subject(s)
Aspergillus/genetics , Carboxy-Lyases/biosynthesis , Carboxy-Lyases/genetics , Alcoholic Beverages , Aspergillus/enzymology , Benzaldehydes/metabolism , Candida/metabolism , Carboxy-Lyases/isolation & purification , Carboxy-Lyases/metabolism , Carboxylic Ester Hydrolases/metabolism , Cloning, Molecular , Coumaric Acids/metabolism , Edible Grain , Enzyme Induction , Guaiacol/analogs & derivatives , Guaiacol/metabolism , Oryza/enzymology , Oryza/genetics , Oryza/metabolism , Saccharomyces cerevisiae
11.
Appl Biochem Biotechnol ; 183(1): 155-170, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28236189

ABSTRACT

An amylolytic lactic acid bacterium isolate K-1 was isolated from the wastewater of a cassava starch manufacturing factory and identified as Entercoccus faecium based on 16S rRNA gene sequence analysis. An extracellular α-amylase was purified to homogeneity and the molecular weight of the purified enzyme was approximately 112 kDa with optimal pH value and temperature measured of 7.0 and 40 °C, respectively. It was stable at a pH range of 6.0-7.0, but was markedly sensitive to high temperatures and low pH conditions, even at a pH value of 5. Ba2+, Al3+, and Co2+ activated enzyme activity. This bacterium was capable of producing 99.2% high optically pure L-lactic acid of 4.3 and 8.2 g/L under uncontrolled and controlled pH at 6.5 conditions, respectively, in the MRS broth containing 10 g/L cassava starch as the sole carbon source when cultivated at 37 °C for 48 h. A control pH condition of 6.5 improved and stabilized the yield of L-lactic acid production directly from starch even at a high concentration of starch at up to 150 g/L. This paper is the first report describing the properties of purified α-amylase from E. faecium. Additionally, pullulanase and cyclodextrinase activities were also firstly recorded from E. faecium K-1.


Subject(s)
Bacterial Proteins , Enterococcus faecium/enzymology , Lactic Acid/biosynthesis , Manihot/chemistry , Starch/chemistry , alpha-Amylases , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Enterococcus faecium/growth & development , Hydrogen-Ion Concentration , alpha-Amylases/chemistry , alpha-Amylases/isolation & purification , alpha-Amylases/metabolism
12.
Biochem Biophys Res Commun ; 477(3): 383-7, 2016 08 26.
Article in English | MEDLINE | ID: mdl-27329813

ABSTRACT

We previously reported the crystal structure of an acetyl esterase (TcAE206) belonging to carbohydrate esterase family 3 from Talaromyces cellulolyticus. In this study, we solved the crystal structure of an S10A mutant of TcAE206 complexed with an acetate ion. The acetate ion was stabilized by three hydrogen bonds in the oxyanion hole instead of a water molecule as in the structure of wild-type TcAE206. Furthermore, the catalytic triad residue His182 moved 0.8 Å toward the acetate ion upon substrate entering the active site, suggesting that this movement is necessary for completion of the catalytic reaction.


Subject(s)
Acetates/chemistry , Acetylesterase/chemistry , Amino Acid Sequence , Catalysis , Crystallography, X-Ray , Sequence Homology, Amino Acid , Substrate Specificity , Talaromyces/enzymology
13.
J Biosci Bioeng ; 121(1): 1-6, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26031195

ABSTRACT

We biosynthesized 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose, which rarely exist in nature, from L-fucose by coupling and sequential enzymatic reactions. The first product, 6-deoxy-L-talose, was directly produced from L-fucose by the coupling reactions of immobilized D-arabinose isomerase and immobilized L-rhamnose isomerase. In one-pot reactions, the equilibrium ratio of L-fucose, L-fuculose, and 6-deoxy-L-talose was 80:9:11. In contrast, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were produced from L-fucose by sequential enzymatic reactions. D-Arabinose isomerase converted L-fucose into L-fuculose with a ratio of 88:12. Purified L-fuculose was further epimerized into 6-deoxy-L-sorbose by D-allulose 3-epimerase with a ratio of 40:60. Finally, purified 6-deoxy-L-sorbose was isomerized into both 6-deoxy-L-gulose with an equilibrium ratio of 40:60 by L-ribose isomerase, and 6-deoxy-L-idose with an equilibrium ratio of 73:27 by D-glucose isomerase. Based on the amount of L-fucose used, the production yields of 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were 7.1%, 14%, 2%, and 2.4%, respectively.


Subject(s)
Aldose-Ketose Isomerases/metabolism , Deoxy Sugars/biosynthesis , Fucose/metabolism , Hexoses/biosynthesis , Monosaccharides/biosynthesis , Carbohydrate Epimerases/metabolism , Fructose/metabolism , Hexoses/metabolism , Sorbose/analogs & derivatives , Sorbose/biosynthesis
14.
Biosci Biotechnol Biochem ; 79(10): 1725-9, 2015.
Article in English | MEDLINE | ID: mdl-26020767

ABSTRACT

L-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert L-psicose and D-tagatose to L-allose and D-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce L-allose and D-talose. Conversion reaction was performed with the reaction mixture containing 10% L-psicose or D-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of L-allose and D-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert L-psicose to L-allose without remarkable decrease in the enzyme activity over 7 times use and D-tagatose to D-talose over 37 times use. After separation and concentration, the mixture solution of L-allose and D-talose was concentrated up to 70% and crystallized by keeping at 4 °C. L-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% L-allose and 7.30% D-talose that were obtained from L-psicose and D-tagatose, respectively.


Subject(s)
Aldose-Ketose Isomerases/chemistry , Bacterial Proteins/chemistry , Cellulomonas/chemistry , Fructose/metabolism , Glucose/biosynthesis , Hexoses/metabolism , Lactones/metabolism , Aldose-Ketose Isomerases/metabolism , Bacterial Proteins/metabolism , Cellulomonas/enzymology , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Fructose/chemistry , Gene Expression , Glucose/chemistry , Glucose/isolation & purification , Hexoses/chemistry , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Kinetics , Lactones/chemistry , Lactones/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribose/chemistry , Ribose/metabolism
15.
Appl Microbiol Biotechnol ; 99(15): 6303-13, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25661811

ABSTRACT

L-Ribose isomerase from Cellulomonas parahominis MB426 (CpL-RI) can catalyze the isomerization between L-ribose and L-ribulose, which are non-abundant in nature and called rare sugars. CpL-RI has a broad substrate specificity and can catalyze the isomerization between D-lyxose and D-xylulose, D-talose and D-tagatose, L-allose and L-psicose, L-gulose and L-sorbose, and D-mannose and D-fructose. To elucidate the molecular basis underlying the substrate recognition mechanism of CpL-RI, the crystal structures of CpL-RI alone and in complexes with L-ribose, L-allose, and L-psicose were determined. The structure of CpL-RI was very similar to that of L-ribose isomerase from Acinetobacter sp. strain DL-28, previously determined by us. CpL-RI had a cupin-type ß-barrel structure, and the catalytic site was detected between two large ß-sheets with a bound metal ion. The bound substrates coordinated to the metal ion, and Glu113 and Glu204 were shown to act as acid/base catalysts in the catalytic reaction via a cis-enediol intermediate. Glu211 and Arg243 were found to be responsible for the recognition of substrates with various configurations at 4- and 5-positions of sugar. CpL-RI formed a homo-tetramer in crystals, and the catalytic site independently consisted of residues within a subunit, suggesting that the catalytic site acted independently. Crystal structure and site-direct mutagenesis analyses showed that the tetramer structure is essential for the enzyme activity and that each subunit of CpL-RI could be structurally stabilized by intermolecular contacts with other subunits. The results of growth complementation assays suggest that CpL-RI is involved in a novel metabolic pathway using L-ribose as a carbon source.


Subject(s)
Aldose-Ketose Isomerases/metabolism , Cellulomonas/enzymology , Pentoses/metabolism , Protein Multimerization , Ribose/metabolism , Aldose-Ketose Isomerases/chemistry , Aldose-Ketose Isomerases/genetics , Crystallography, X-Ray , Mutagenesis, Site-Directed , Substrate Specificity
16.
Biosci Biotechnol Biochem ; 78(2): 317-25, 2014.
Article in English | MEDLINE | ID: mdl-25036688

ABSTRACT

6-Deoxy-L-glucose, 6-deoxy-L-altrose, and 6-deoxy-L-allose were produced from L-rhamnose with an immobilized enzyme that was partially purified (IE) and an immobilized Escherichia coli recombinant treated with toluene (TT). 6-Deoxy-L-psicose was produced from L-rhamnose by a combination of L-rhamnose isomerase (TT-PsLRhI) and D-tagatose 3-epimerase (TT-PcDTE). The purified 6-deoxy-L-psicose was isomerized to 6-deoxy-L-altrose and 6-deoxy-L-allose with L-arabinose isomerase (TT-EaLAI) and L-ribose isomerase (TT-AcLRI), respectively, and then was epimerized to L-rhamnulose with immobilized D-tagatose 3-epimerase (IE-PcDTE). Following purification, L-rhamnulose was converted to 6-deoxy-L-glucose with D-arabinose isomerase (TT-BpDAI). The equilibrium ratios of 6-deoxy-L-psicose:6-deoxy-L-altrose, 6-deoxy-L-psicose:6-deoxy-L-allose, and L-rhamnulose:6-deoxy-L-glucose were 60:40, 40:60, and 27:73, respectively. The production yields of 6-deoxy-L-glucose, 6-deoxy-L-altrose, and 6-deoxy-L-allose from L-rhamnose were 5.4, 14.6, and 25.1%, respectively. These results indicate that the aldose isomerases used in this study acted on 6-deoxy aldohexoses.


Subject(s)
Deoxy Sugars/metabolism , Hexoses/metabolism , Intramolecular Oxidoreductases/metabolism , Rhamnose/metabolism , Bacteria/enzymology , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Intramolecular Oxidoreductases/chemistry
17.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 7): 890-5, 2014 Jul.
Article in English | MEDLINE | ID: mdl-25005083

ABSTRACT

The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase from Pseudomonas cichorii and the D-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit-subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV.


Subject(s)
Archaeal Proteins/chemistry , Carbohydrate Epimerases/chemistry , Methanocaldococcus/chemistry , Agrobacterium tumefaciens/chemistry , Agrobacterium tumefaciens/enzymology , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Clostridium cellulolyticum/chemistry , Clostridium cellulolyticum/enzymology , Crystallography, X-Ray , Deoxyribonuclease IV (Phage T4-Induced)/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hot Temperature , Methanocaldococcus/enzymology , Models, Molecular , Protein Folding , Protein Multimerization , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structural Homology, Protein
18.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 12): 2330-9, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24311575

ABSTRACT

L-Ribulose 3-epimerase (L-RE) from Mesorhizobium loti has been identified as the first ketose 3-epimerase that shows the highest observed activity towards ketopentoses. In the present study, the crystal structure of the enzyme was determined to 2.7 Šresolution. The asymmetric unit contained two homotetramers with the monomer folded into an (α/ß)8-barrel carrying four additional short α-helices. The overall structure of M. loti L-RE showed significant similarity to the structures of ketose 3-epimerases from Pseudomonas cichorii, Agrobacterium tumefaciens and Clostridium cellulolyticum, which use ketohexoses as preferred substrates. However, the size of the C-terminal helix (α8) was much larger in M. loti L-RE than the corresponding helices in the other enzymes. In M. loti L-RE the α8 helix and the following C-terminal tail possessed a unique subunit-subunit interface which promoted the formation of additional intermolecular interactions and strengthened the enzyme stability. Structural comparisons revealed that the relatively small hydrophobic pocket of the enzyme around the substrate was likely to be the main factor responsible for the marked specificity for ketopentoses shown by M. loti L-RE.


Subject(s)
Carbohydrate Epimerases/chemistry , Mesorhizobium/enzymology , Amino Acid Sequence , Carbohydrate Epimerases/metabolism , Catalytic Domain , Enzyme Stability , Mesorhizobium/chemistry , Mesorhizobium/metabolism , Molecular Sequence Data , Pentoses/metabolism , Protein Conformation , Sequence Alignment , Substrate Specificity , Temperature
19.
Biosci Biotechnol Biochem ; 77(3): 511-5, 2013.
Article in English | MEDLINE | ID: mdl-23470755

ABSTRACT

A gene encoding L-ribulose 3-epimerase (L-RE) from Mesorhizobium loti, an important enzyme for rare sugar production by the Izumoring strategy, was cloned and overexpressed. The enzyme showed highest activity toward L-ribulose (230 U/mg) among keto-pentoses and keto-hexoses. This is the first report on a ketose 3-epimerase showing highest activity toward keto-pentose. The optimum enzyme reaction conditions for L-RE were determined to be sodium phosphate buffer (pH 8.0) at 60 °C. The enzyme showed of higher maximum reaction a rate (416 U/mg) and catalytic efficiency (43 M(-1) min(-1)) for L-ribulose than other known ketose 3-epimerases. It was able to produce L-xylulose efficiently from ribitol in two-step reactions. In the end, 7.2 g of L-xylulose was obtained from 20 g of ribitol via L-ribulose at a yield of 36%.


Subject(s)
Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Mesorhizobium/enzymology , Mesorhizobium/genetics , Xylulose/chemistry , Amino Acid Sequence , Carbohydrate Epimerases/chemistry , Cloning, Molecular , Enzyme Stability/drug effects , Hydrogen-Ion Concentration , Kinetics , Metals/pharmacology , Molecular Sequence Data , Pentoses/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribitol/chemistry , Sequence Analysis , Substrate Specificity , Temperature
20.
Carbohydr Polym ; 92(2): 2135-40, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23399268

ABSTRACT

A novel α-glucan substituted rare 6-deoxy-D-altropyranose was isolated from edible fruiting bodies of a mushroom (Lactarius lividatus) grown in Okinawa, Japan. The polysaccharide consists of D-glucose, D-galactose and 6-deoxy-D-altrose in a molar ratio of 3.0:1.0:1.0. The specific rotation [α](589) was estimated as +64.3° (0.2% in water) at 25 °C. Based on results of IR, NMR ((1)H, (13)C, 2D-COSY, 2D-HMQC, 2D-ROESY and 2D-HMBC), and methylation analyses, the structure of the polysaccharide was determined as [formula, see text] This work is the first demonstration of rare 6-deoxy-D-altropyranose moiety on polysaccharides.


Subject(s)
Agaricales/chemistry , Deoxy Sugars/chemistry , Glucans/chemistry , Hexoses/chemistry , Deoxy Sugars/isolation & purification , Hexoses/isolation & purification , Methylation
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