High Substrate Specificity Research Articles

Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01.

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.

Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl

The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1→6)-α-nigerosyl (CNN), a cyclic glucotetraose with alternate α-(1→6)- and α-(1→3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-α-glucosyltransferase and 3-α-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the α-(1→3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s(-1)) was much higher than that of panose (0.242 s(-1)), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the α-(1→3)-isomaltosyl moiety and effectively hydrolyzes the α-(1→3)-glucosidic linkage, it should be termed 1,3-α-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of β-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The kcat/Km for isomaltose (4.81 ± 0.18 s(-1) mm(-1)) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.

Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus.

Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60°C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus.

New Poly(Acrylic Acid)-Based Functional Nanogels as Supports of Lipase: Evaluation of Hydrolytic Activity of the Immobilized Lipase

Background: Lipases are excellent biocatalysts with dual ability of hydrolysis as well as esterification with high efficiency and substrate specificity. However, in the pristine form they have inherent limitations such as lack of reusability and limited thermal and pH stability those restrict their uses in commercial applications. These limitations can be overcome by their immobilization on the suitable supports. Methods: In the present study a new poly(acrylic acid)-based functional nanogel was prepared by a simple surfactant free emulsification method and functionalized to poly(acrylic azide) form by a simple chemical protocol. Nanogels were characterized by X-Ray diffraction (XRD), Fourier Transform Infra Red (FTIR) spectroscopy, and transmission electron microscopy (TEM) to get evidence of the network formation and post reaction. The well characterized precursor nanogel and its functional derivatives were used as supports to immobilize lipase (triacyl glycerin). The activity assay of the immobilized lipase was carried out by the standard assay method. The catalytic potential of the immobilized lipase was evaluated in hydrolytic reactions using p-nitrophenol palmitate as substrate. Results: Immobilization enhanced the lipase activity and the structure of the nanogels made significant difference in enhancing their activity as the lipase immobilized on poly(Acryl azide)-cl-divinylbenzene nanogel exhibited the higher activity than the lipase immobilized on the precursor nanogel. Also, its activity was far higher than the free lipase. The immobilized lipase exhibited thermal and pH stability, which is not possible with the free lipase. Conclusion: The lipase was immobilized by the covalent linkages. The immobilized lipase exhibited good activity, storability and reusability. These results were dependent on the nanogel structure. Hence, a suitable nanogel can be tailored to obtain the maximum gains of immobilization. The reported results have good technological potential. Keywords: Covalent immobilization, hydrolysis, lipase, nanogel, reusability.

Purification and characterization of a glycosidase with hydrolyzing multi-3-O-glycosides of spirostanol saponin activity from Gibberella intermedia

The strain Gibberella intermedia WX12 preserved in our lab showed a strong ability to degrade dioscin from Dioscorea Zingiberensis C. H. Wright into diosgenin. The glycosidase enzyme of this strain was purified by a procedure consisting of ammonium sulfate fractionation, anion-exchange and hydrophobic chromatography and was named GiGly. This enzyme was a monomer with molecular mass of approximately 45kDa. The optimal temperature and pH were 50°C and 8.0, respectively. GiGly was stable in the pH range of 5.0–8.0 and retained 80% of its original activity at pH 7.0 for 12h. In the presence of metal ions including Na+, K+ and Mg2+ slightly increased GiGly activity, and Fe2+, Fe3+, Cu2+ and Mn2+ notably inhibited the activity. Meanwhile, GiGly showed high substrate specificity for multi-3-O-glycosides of spirostanol saponins such as dioscin, trillin and polyphyllin VII, and was inactive toward the substrates with terminal groups of rhamnosyl, glucosyl, galactosyl of ginsenoside Re and saikosaponin A. GiGly from G. intermedia WX12 could only hydrolyze glycosidic bonds at the C-3 position on steroidal saponins, which have similar structure with dioscin, and can be transformed into diosgenin.

Cationic surfactant with 1,2,4-triazole- and uracil moieties as amphiphilic building blocks for supramolecular nanocontainers

Novel dicationic surfactant consisting of uracil unit bridging with 1,2,4-triazole moieties has been synthesized. Its aggregation behavior in aqueous solutions and critical micelle concentration value has been characterized by various techniques. Size and charge of aggregates have been evaluated, with agreement between different methods observed. The onset of micellization is shown to occur beyond ca. 3mM, while further structural transition is observed close to ca. 10mM. The solubilization capacity toward hydrophobic dye and micellar rate effect upon hydrolysis of ecological organophosphorus toxicants have been shown, with high substrate specificity observed.

High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles.

Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition.

Purification and Characterization of L-Arginine Deiminase from Vibrio Alginolyticus 1374

Aim : The aim of our present study was to purify and characterize l-arginine deiminase isolated from marine Vibrio alginolyticus1374. Methodology: Arginine deiminase (AD), an arginine-degrading enzyme, has been used in the treatment of tumours, sensitive to arginine deprivation, such as malignant melanoma and hepatocellular carcinoma. Proteins of microbial origin are always associated with mild to severe hypersensitivities. Moreover, proteins from terrestrial sources were found to exhibit stability problems at physiological environment. Hence considering the fact that the characteristics of the enzyme depends upon sources from which it has been isolated, we have use marine organism for the present study expecting to yield therapeutically and physiologically stable enzyme. ADI production was carried out under optimal conditions by shake flask method. The enzyme thus obtained was purified by ammonium sulphate fractionation, ion exchange and gel permeation chromatography. The purity of the enzyme was further confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was characterized to know its properties. Results: It was active at physiological pH, showed high substrate specificity towards l-arginine. It also exhibited high salt and temperature tolerance indicating good scope for its industrial and therapeutic applications. Conclusions: ADI obtained from Vibrio alginolyticus 1374 can be a good candidate for the treatment of human cancers.

Transformation of dimethyl phthalate esters (DMPEs) by a marine red yeast Rhodotorula mucilaginosa isolated from deep sea sediments of the Atlantic Ocean

A marine red yeast capable of degrading dimethyl phthalate esters (DMPEs) was isolated from deep-sea sediments of the Atlantic Ocean using enrichment culture technique. The yeast was identified as Rhodotorula mucilaginosa Mar-Y3 based on internal transcribed spacer (ITS) gene sequence analysis. The biochemical degradation pathways of three DMPE isomers, namely dimethyl phthalate (DMP), dimethyl isophthalate (DMI), and dimethyl terephthalate (DMT), were investigated using this yeast. The yeast cannot completely mineralize DMPEs, but can transform them to respective phthalate monoester or phthalic acid. R. mucilaginosa Mar-Y3 also degraded different DMPE isomers through varied metabolism pathways and biodegradation rates. The yeast performed one-step ester hydrolysis with a relatively low degradation rate to transform DMP and DMI to the respective monoester; however, monomethyl phthalate (MMP) and monomethyl isophthalate (MMI) were the dead-end products and further metabolism did not proceed. The yeast carried out sequential ester hydrolysis with a relatively high degradation rate to transform DMT to terephthalic acid (TA) via monomethyl terephthalate (MMT), but TA was resistant to further metabolism. These results suggested that phthalate esterases produced by the deep-sea yeast R. mucilaginosa Mar-Y3 had a very high substrate specificity for different DMPE isomers, and that the degradation of DMPEs by this yeast involved distinct esterases responsible for the hydrolysis of two identical carboxylic ester bonds.

Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microsporus Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source.

The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78% recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4kDa was obtained when the samples were subjected to 10% SDS-PAGE. The optimum temperature for activity was 55°C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40°C with a half-life of 115min at 60°C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72mM and V max = 94.55U/mg of protein and for p-NPP with K m = 0.04mM and V max = 106.38U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids.

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases.

Amidase activity of phosphonate analogue imprinted chymotrypsin mimics in shape-selective, substrate-specific and enantioselective amidolysis of l-phenylalanine-p-nitroanilides

Focusing on chymotrypsin mimics, highly crosslinked enzyme mimics are synthesized by molecular imprinting technique for the amidolysis of p-nitroanilide of phenylalanine, using phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate – the transition state analog of amidolysis – as the template, N-methacryloyl-l-histidine, N-methacryloyl-l-aspartic acid, and N-methacryloyl-l-serine as the functional monomers and EGDMA as the crosslinking agent. The amidase activity of the enzyme mimics follows pseudo first order kinetics. The transition state analog provides a tetrahedral geometry complementary to the transition state intermediate, which is responsible for the catalytic activity of the imprinted enzyme mimics. The enzyme mimics show stereospecificity and substrate selectivity in the amidolysis of phenylalanine p-nitroanilide. The proper orientation of the reactive functionalities in the super crosslinked macroporous polymer matrix for selective binding of the substrate through H-bonding is responsible for the high imprinting efficiency and substrate specificity of the imprinted polymer catalysts. Low cost, ease of preparation, high thermal stability, reusability and higher shelf life make the polymer catalysts better chymotrypsin mimics.

Recent Progress in the Discovery of Next Generation Inhibitors of Aromatase from the Structure-Function Perspective.

Human aromatase catalyzes the synthesis of estrogen from androgen with high substrate specificity. For the past 40 years, aromatase has been a target of intense inhibitor discovery research for the prevention and treatment of estrogen-dependent breast cancer. The so-called third generation aromatase inhibitors (AIs) letrozole, anastrozole, and the steroidal exemestane were approved in the U.S. in the late 1990s for estrogen-dependent postmenopausal breast cancer. Efforts to develop better AIs with higher selectivity and lower side effects were handicapped by the lack of an experimental structure of this unique P450. The year 2009 marked the publication of the crystal structure of aromatase purified from human placenta, revealing an androgen-specific active site. The structure has reinvigorated research activities on this fascinating enzyme and served as the catalyst for next generation AI discovery research. Here, we present an account of recent developments in the AI field from the perspective of the enzyme's structure-function relationships.

A novel L-asparaginase from Aquabacterium sp. A7-Y with self-cleavage activation.

We have identified a novel L-asparaginase, abASNase3, from Aquabacterium sp. A7-Y. abASNase3 is composed of 306 amino acids and exhibits 34% sequence homology to human asparaginase (hASNase3). Further analysis revealed that abASNase3 belongs to the N-terminal nucleophile (Ntn) family of hydrolases. Previous reports about the Ntn hydrolase family and the results of our study suggest that abASNase3 must form two subunits by self-cleavage between Gly189 and Thr190 to attain catalytic activity. The two subunits remained tightly associated to build a single functional unit. The optimum pH for abASNase3 was found to be 8.0 in Tris-HCl buffer and the enzyme was found to be stable over a broad pH range from pH 6.0 to 12.0. The optimum temperature for abASNase3 was found to be approximately 40°C, and the enzyme was stable below 65°C. abASNase3 showed high substrate specificity toward L-asparagine and had no or only slight activity toward D-asparagine, L-glutamine and D-glutamine. abASNase3 was significantly activated by Mg(2+) and was substantially inhibited by Ni(2+), Cu(2+), Mn(2+) and Co(2+). The Michaelis-Menten constant and turnover number of abASNase3 for L-asparagine were estimated to be 3.37×10(-2)M and 8.72×10(-3)s(-1), respectively. Our results indicate that abASNase3 is a novel L-asparaginase in the Ntn family of hydrolases.

Promiscuous Glucose Oxidase: Electrical Energy Conversion of Multiple Polysaccharides Spanning Starch and Dairy Milk

Enzymatic biofuel cells (EFCs) are devices that are capable of producing electrical energy from the enzymatic oxidation of simple, energy-dense fuels (such as sugars and alcohols). Glucose oxidase (GOx) is perhaps the most widely used enzyme at the bioanode of EFCs, affording devices that can be fueled by glucose. Due to its high substrate specificity, GOx was initially employed in glucose biosensor applications, which later translated into EFC technology. This high substrate specificity, however, often restricts EFC devices to operation on single fuels. Herein, we report the use of a commercial GOx with greater substrate promiscuity (broader substrate GOx, “bGOx”), which can result in the fabrication of EFCs that can utilize a single versatile catalyst for the oxidation of many mono-, di-, tri-, and polysaccharides. The EFCs presented within this study were fueled by glucose, maltose, maltotriose, cellobiose, lactose, galactose, and xylose. Additionally, the ability for the same biocatalyst to oxidize su...

Possible role of a histidine residue in the substrate specificity of yeast d-aspartate oxidase

D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids, whereas neutral and basic D-amino acids are substrates of D-amino acid oxidase (DAO). DDO of the yeast Cryptococcus humicola (ChDDO) has much higher substrate specificity to D-aspartate, but the structural features that confer this specificity have not been elucidated. A three-dimensional model of ChDDO suggested that a histidine residue (His56) in the active site might be involved in the unique substrate specificity, possibly through the interaction with the substrate side chain in the active site. His56 mutants with several different amino acid residues (H56A, H56D, H56F, H56K and H56N) exhibited no significant activity toward acidic D-amino acids, but H56A and H56N mutants gained the ability to utilize neutral D-amino acids as substrates, such as D-methionine, D-phenylalanine and D-glutamine, showing the conversion of ChDDO to DAO by these mutations. This conversion was also demonstrated by the sensitivity of these mutants to competitive inhibitors of DAO. These results and kinetic properties of the mutants show that His56 is involved in the substrate specificity of ChDDO and possibly plays a role in the higher substrate specificity toward D-aspartate.

Learning from each other: ABC transporter regulation by protein phosphorylation in plant and mammalian systems.

The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABC subfamily B (ABCB) display very high substrate specificity compared with their mammalian counterparts that are often associated with multi-drug resistance phenomena. In this review, we highlight prominent functions of plant and mammalian ABC transporters and summarize our knowledge on their post-transcriptional regulation with a focus on protein phosphorylation. A deeper comparison of regulatory events of human cystic fibrosis transmembrane conductance regulator (CFTR) and ABCB1 from the model plant Arabidopsis reveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins that chaperon both transporters to the plasma membrane in an action that seems to involve heat shock protein (Hsp)90. Further, both transporters are phosphorylated at regulatory domains that connect both nt-binding folds. Taken together, it appears that ABC transporters exhibit an evolutionary conserved but complex regulation by protein phosphorylation, which apparently is, at least in some cases, tightly connected with protein-protein interactions (PPI).

Structural analysis of enzymes used for bioindustry and bioremediation.

Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

Overexpression of Crocus carotenoid cleavage dioxygenase, CsCCD4b, in Arabidopsis imparts tolerance to dehydration, salt and oxidative stresses by modulating ROS machinery

Apocarotenoids modulate vital physiological and developmental processes in plants. These molecules are formed by the cleavage of carotenoids, a reaction catalyzed by a family of enzymes called carotenoid cleavage dioxygenases (CCDs). Apocarotenoids like β-ionone and β-cyclocitral have been reported to act as stress signal molecules during high light stress in many plant species. In Crocus sativus, these two apocarotenoids are formed by enzymatic cleavage of β-carotene at 9, 10 and 7, 8 bonds by CsCCD4 enzymes. In the present study three isoforms of CsCCD4 were subjected to molecular modeling and docking analysis to determine their substrate specificity and all the three isoforms displayed high substrate specificity for β-carotene. Further, expression of these three CsCCD4 isoforms investigated in response to various stresses revealed that CsCCD4a and CsCCD4b exhibit enhanced expression in response to dehydration, salt and methylviologen, providing a clue towards their role in mediating plant defense response. This was confirmed by overexpressing CsCCD4b in Arabidopsis. The transgenic plants developed longer roots and possessed higher number of lateral roots. Further, overexpression of CsCCD4b imparted enhanced tolerance to salt, dehydration and oxidative stresses as was evidenced by higher survival rate, increased relative root length and biomass in transgenic plants as compared to wild type. Transgenic plants also displayed higher activity and expression of reactive oxygen species (ROS) metabolizing enzymes. This indicates that β-ionone and β-cyclocitral which are enzymatic products of CsCCD4b may act as stress signals and mediate reprogramming of stress responsive genes which ultimately leads to plant defense.

Optimization of a heat-tolerant β-glucosidase production by Bacillus sp. ZJ1308 and its purification and characterization.

Response surface methodology was used to optimize the medium composition to improve the production of the heat-tolerant β-glucosidase from Bacillus sp. ZJ1308. Three significant factors were found to be corn cob, beef extract, and MnSO4 ·H2 O. The final medium compositions optimized were corn cob (51.8 g/L), beef extract (23.8 g/L), salicin (0.5 g/L), MnSO4 ·H2 O (0.363 g/L), MgSO4 ·7H2 O (0.4 g/L), and NaCl (5 g/L). Under the optimal conditions, the activity of β-glucosidase was up to 4.71 U/mL. β-Glucosidase was purified to homogeneity with a recovery rate of 5% and a specific activity of 110.47 U/mg. The optimal pH and temperature were 7.0 and 60°C, respectively. β-Glucosidase was stable within a pH range of 6.0-8.0 and showed an extremely high thermostability at 80 and 90°C, retaining 56% and 38% of its maximal activity, respectively. Ni(2+) and Ba(2+) heavily inhibited the β-glucosidase activity. The purified β-glucosidase showed a high substrate specificity. The kinetic parameters revealed that it had a high catalytic efficiency toward the substrate p-nitrophenyl-β-d-glucopyranoside (Kcat /Km = 700). It also showed a high catalytic activity toward the natural substrate salicin. This study provides a new insight into the future development and use of β-glucosidase from Bacillus sp. ZJ1308.