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Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden-Meyerhof-Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Gelatin hydrolysate was reported to contain large amounts of biologically active peptides with excellent antioxidant properties. However, its inducement of antioxidant response within cells and the underlying molecular mechanism are far from clear. Here, gelatin from Nile tilapia skin was hydrolyzed by ginger protease to produce antioxidant hydrolysate, and three fish skin gelatin hydrolysate fractions (FSGHFs) were obtained by ultrafiltration. Fractionation of the hydrolysate increased the free radical scavenging capacity of the FSGHFs, particularly FSGHF3, which showed the lowest molecular weight (below 1000 Da). Furthermore, FSGHF3 treatment prior to H 2 O 2 exposure increased cell viability and membrane integrity in IPEC-J2 cells. H 2 O 2 -induced ROS production and epithelial barrier damage were suppressed by FSGHF3 pretreatment. FSGHF3 stimulated the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), along with increases in the mRNA and protein expression of catalytic and modulatory subunits of γ-glutamylcysteine ligase as well as in the level of glutathione. Silencing of Nrf2 or p62 (an upstream regulator of Nrf2) suppressed FSGHF3-induced Nrf2 activation and its protection against H 2 O 2 -induced oxidative stress. Moreover, oligopeptides in FSGHF3 may mediate the cytoprotective effect against oxidative stress, which was confirmed by the result that FSGHF3 failed to inhibit the ROS production in H 2 O 2 -exposed cells with the knockdown of Pept1 (an oligopeptide transporter). Therefore, FSGHF3 can induce glutathione synthesis and prevent oxidative stress through the Pept1-p62-Nrf2 cascade and thus may be a functional food for gastrointestinal dysfunction.

Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P 2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of <600 Da. In addition, the gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7 mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC 50 values of 79.8, 70.9, 71.7, 56.7, and 78.9 μM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes.

Superoxide dismutase (SOD) is an antioxidant enzyme protecting cells from oxidative stress. Ginger (Zingiber officinale) is known for its antioxidant properties, however, there are no data on SODs from ginger rhizomes. In this study, we purified SOD from the rhizome of Z. officinale (Zo-SOD) and determined its complete amino acid sequence using N terminal sequencing, amino acid analysis, and de novo sequencing by tandem mass spectrometry. Zo-SOD consists of 151 amino acids with two signature Cu/Zn-SOD motifs and has high similarity to other plant Cu/Zn-SODs. Multiple sequence alignment showed that Cu/Zn-binding residues and cysteines forming a disulfide bond, which are highly conserved in Cu/Zn-SODs, are also present in Zo-SOD. Phylogenetic analysis revealed that plant Cu/Zn-SODs clustered into distinct chloroplastic, cytoplasmic, and intermediate groups. Among them, only chloroplastic enzymes carried amino acid substitutions in the region functionally important for enzymatic activity, suggesting that chloroplastic SODs may have a function distinct from those of SODs localized in other subcellular compartments. The nucleotide sequence of the Zo-SOD coding region was obtained by reverse-translation, and the gene was synthesized, cloned, and expressed. The recombinant Zo-SOD demonstrated pH stability in the range of 5-10, which is similar to other reported Cu/Zn-SODs, and thermal stability in the range of 10-60 °C, which is higher than that for most plant Cu/Zn-SODs but lower compared to the enzyme from a Z. officinale relative Curcuma aromatica.

The ability of chitosan and oligochitosan to enhance ginger (Zingiber officinale) resistance to rhizome rot caused by Fusarium oxysporum in storage was investigated. Both chitosan and oligochitosan at 1 and 5g/L significantly inhibited rhizome rot, with the best control at 5g/L. Chitosan and oligochitosan applied at 5g/L also reduced weight loss, measured as a decrease in fresh weight, but did not affect soluble solids content or titratable acidity of rhizomes. The two compounds applied at 5g/L induced β-1,3-glucanase and phenylalanine ammonia-lyase enzyme activity and the transcript levels of their coding genes, as well as the total phenolic compounds in rhizome tissues. Therefore, the ability of chitosan and oligochitosan to reduce rot in stored rhizomes may be associated with their ability to induce defense responses in ginger. These results have practical implications for the application of chitosan and oligochitosan to harvested ginger rhizomes to reduce postharvest losses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

Extraction of protease from a local ginger rhizome (Zingiber officinale var. Bentong) was carried out. The effect of extraction pH (6.4, 6.8, 7.0, 7.2, 7.6, 8.0, 8.4, and 8.8) and stabilizers (0.2% ascorbic acid, 0.2% ascorbic acid and 5 mM EDTA, or 10 mM cysteine and 5 mM EDTA) on protease activity during extraction was examined. pH 7.0 potassium phosphate buffer and 10 mM cysteine in combination with 5 mM EDTA as stabilizer were found to be the most effective conditions. The extraction procedure yielded 0.73% of Bentong ginger protease (BGP) with a specific activity of 24.8±0.2 U/mg protein. Inhibitory tests with some protease inhibitors classified the enzyme as a cysteine protease. The protease showed optimum activity at 60 °C and pH 6-8, respectively. The enzyme was completely inhibited by heavy metal cations such as Cu2+, and Hg2+. SDS stimulated the activity of enzyme, while emulsifiers (Tween 80 and Tween 20) slightly reduced its activity. The kinetic analysis showed that the protease has Km and Vmax values of 0.21 mg mL-1 and 34.48 mg mL-1 min-1, respectively. The dried enzyme retained its activity for 22 months when stored at -20 °C.

Ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.), members of the Zingiberaceae, are widely used in traditional Asian cuisines and herbal medicine. Gingerols and diarylheptanoids, important compounds from these plants, appear to be produced by enzymes of the type III polyketide synthase class. Previous efforts to detect activity of such enzymes in tissues from these plants were only marginally successful in turmeric and completely unsuccessful in ginger because of very rapid hydrolysis of the hydroxycinnamoyl-CoA substrates (p-coumaroyl-CoA, feruloyl-CoA and caffeoyl-CoA) in these assays, presumably due to the presence of thioesterases in these tissues. In order to determine whether such thioesterase activities were specific and could be reduced so that the polyketide synthase activities could be better characterized, three inhibitors of the thioesterase domain of fatty acid synthase were tested in assays with leaf and rhizome crude protein extracts from these plants: orlistat, a reduced form of lipstatin, and peptide 1 and peptide 2 from hydrolysates of soybean β-conglycinin. Results of these analyses indicated that specific thioesterases do exist in these plants and that they could indeed be inhibited, with highest inhibition occurring with a mixture of these three compounds, leading for example to a reduction of caffeoyl-CoA hydrolysis in leaves and rhizomes of ginger by 40-fold and 27-fold, respectively., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

In order to search for new acetylcholinesterase inhibitors (AChEIs), 15 Zingiberaceae plants were tested for AChEI activity in rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Zingiber officinale contained a significant AChEI activity. Eighty percent saturation ammonium sulfate precipitation and diethylaminoethyl cellulose ion exchange chromatography (unbound fraction) enriched the protein to a single band on nondenaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (approximately 33.5 kDa). Gelatin-degrading zymography showed that the AChEI-containing band also contained cysteine protease activity. The AChEI activity was largely stable between -20 and 60 °C (at least over 120 min) and over a broad pH range (2-12). The AChEI activity was stimulated strongly by Mn(2+) and Cu(2+) at 1-10 mM and weakly by Ca(2+), Fe(2+), Mg(2+), and Zn(2+) at 1 mM, but was inhibited at 10 mM. In contrast, Hg(2+) and ethylenediaminetetraacetic acid were very and moderately strongly inhibitory, respectively. In-gel tryptic digestion with liquid chromatography-tandem mass spectroscopy resolution revealed two heterogeneous peptides, a 16-amino-acid-long fragment with 100 % similarity to zingipain-1, which is a cysteine protease from Z. officinale, and a 9-amino-acid-long fragment that was 100 % identical to actinidin Act 2a, suggesting that the preparation was heterogeneous. AChEI exhibited noncompetitive inhibition of AChE for the hydrolysis of acetylthiocholine iodide with a K(i) value of 9.31 mg/ml.

The effect of foliar salicylic acid (SA) applications (10⁻³ and 10⁻⁵ M) on activities of nitrate reductase, guaiacol peroxidase (POD), superoxide dismutases (SOD), catalase (CAT) and proline enzymes and physiological parameters was evaluated in two ginger varieties (Halia Bentong and Halia Bara) under greenhouse conditions. In both varieties, tested treatments generally enhanced photosynthetic rate and total dry weight. Photosynthetic rate increases were generally accompanied by increased or unchanged stomatal conductance levels, although intercellular CO₂ concentrations of treated plants were typically lower than in controls. Lower SA concentrations were generally more effective in enhancing photosynthetic rate and plant growth. Exogenous application of SA increased antioxidant enzyme activities and proline content; the greatest responses were obtained in plants sprayed with 10⁻⁵ M SA, with significant increases observed in CAT (20.1%), POD (45.2%), SOD (44.1%) and proline (43.1%) activities. Increased CAT activity in leaves is naturally expected to increase photosynthetic efficiency and thus net photosynthesis by maintaining a constant CO₂ supply. Our results support the idea that low SA concentrations (10⁻⁵ M) may induce nitrite reductase synthesis by mobilizing intracellular NO³⁻ and can provide protection to nitrite reductase degradation in vivo in the absence of NO³⁻. Observed positive correlations among proline, SOD, CAT and POD activities in the studied varieties suggest that increased SOD activity was accompanied by increases in CAT and POD activities because of the high demands of H₂O₂ quenching.

Ginger (Zingiber officinale Rosc.) is prone to photoinhibition under intense sunlight. Excessive light can be dissipated by the xanthophyll cycle, where violaxanthin de-epoxidase (VDE) plays a critical role in protecting the photosynthesis apparatus from the damage of excessive light. We isolated ~2.0 kb of ginger VDE (GVDE) gene promoter, which contained the circadian box, I-box, G-box and GT-1 motif. Histochemical staining of Arabidopsis indicated the GVDE promoter was active in almost all organs, especially green tissues. β-glucuronidase (GUS) activity driven by GVDE promoter was repressed rather than activated by high light. GUS activity was altered by hormones, growth regulators and abiotic stresses, which increased with 2,4-dichlorophenoxyacetic acid and decreased with abscisic acid, salicylic acid, zeatin, salt (sodium chloride) and polyethylene glycol. Interestingly, GUS activities with gibberellin or indole-3-acetic acid increased in the short-term (24 h) and decreased in the long-term (48 and 72 h). Analysis of 5' flank deletion found two crucial functional regions residing in -679 to -833 and -63 to -210. Northern blotting analysis found transcription to be regulated by the endogenous circadian clock. Finally, we found a region necessary for regulating the circadian rhythm and another for the basic promoter activity. Key message A novel promoter, named GVDE promoter, was first isolated and analyzed in this study. We have determined one region crucial for promoter activity and another responsible for keeping circadian rhythms.

Ginger proteases are used as milk coagulants in making a Chinese traditional milk product (Jiangzhinai or Jiangzhuangnai), suggesting their potential as a source of rennet substitute that might be applicable in the modern dairy industry. In this study, ginger proteases were extracted from fresh ginger rhizome by using phosphate buffer and subsequently purified by ion exchange chromatography. Ginger proteases, all with a molecular weight around 31 kDa, were found to exist in 3 forms with isoelectric point values around 5.58, 5.40, and 5.22, respectively. These enzymes had very similar biochemical behavior, exhibiting optimal proteolytic activity from 40 to 60 °C and maximum milk clotting activity at 70 °C. They were capable of hydrolyzing isolated α(S1)-, β-, and κ-casein, of which α(S1)-casein was most susceptible to the enzyme; κ-casein was hydrolyzed with a higher specificity than α(S1)- and β-casein. In addition, the ginger proteases exhibited a similar affinity for κ-casein and higher specificity with increasing temperature. Gel electrophoresis and mass spectra indicated that Ala90-Glu91 and His102-Leu103 of κ-casein were the preferred target bonds of ginger proteases. The milk clotting activity, affinity, and specificity toward κ-casein showed that ginger protease is a promising rennet-like protease that could be used in manufacturing cheese and oriental-style dairy foods., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60°C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg(2+) and Cu(2+). Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α(s)-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses., (© Springer-Verlag 2011)

Plant cytochrome P450s are involved in the biosynthesis of various classes of secondary metabolites. To elucidate the biosynthesis of zerumbone, a sesquiterpenoid with multiple potential anticancer properties, a family of P450 genes expressed in rhizomes of Zingiber zerumbet Smith, were cloned using a PCR-based cloning strategy. After functional expression in yeast, one of these P450s was found to convert α-humulene into 8-hydroxy-α-humulene, a proposed intermediate of zerumbone biosynthesis. This P450 has been designated CYP71BA1, a new member of the CYP71 family. CYP71BA1 transcripts were detected almost exclusively in rhizomes and showed a similar expression pattern to ZSS1 transcripts during rhizome development. Coexpression of a gene cluster encoding four enzymes of the mevalonate pathway with CYP71BA1 and ZSS1 in Escherichia coli leads to the production of 8-hydroxy-α-humulene in the presence of mevalonate, suggesting the possibility of microbial production of this zerumbone intermediate from a relatively simple carbon source by metabolic engineering.

High hydrostatic pressure (HHP) was applied to grated ginger in order to inactivate quality-degrading enzymes in a non-thermal manner. The effects of HHP treatment on the flavor and the color of the grated ginger were investigated just after treatment and during storage. After HHP treatment (400 MPa, 5 min), geraniol dehydrogenase (GeDH) was inactivated to less than 5%, but the activity of polyphenol oxidase (PPO) was reduced only to 37%. Heat treatment (100 °C, 10 min) inactivated GeDH to 43% and PPO to about 10%. In storage, the reduction of geranial, neral, and citronellal to the corresponding alcohols was observed in the untreated and the heat-treated ginger, while it was not in the HHP-treated grated ginger. In the HHP-treated sample, terpene aldehydes almost disappeared without the formation of the corresponding alcohols. Browning was not observed immediately after HHP treatment, while it was complete in the heat-treated sample. The color change during storage appeared to reflect the residual activity of PPO.

Shampoo ginger (Zingiber zerumbet Smith) has a high content and large variety of terpenoids in the essential oil of its rhizome. Here, we report on the isolation of a cDNA clone (ZSS1) encoding alpha-humulene synthase, a possible key enzyme of zerumbone biosynthesis. This clone contains an open reading frame of 1,644 bp and is predicted to encode a protein of 548 amino acids with a calculated molecular mass of 64.5 kDa. The deduced amino acid sequence shows 34-63% identity with known sesquiterpene synthases of other angiosperm species. Based on exon-intron organization, ZSS1 is classified as the terpene synthase-III (TPS-III) subfamily. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of a major product, alpha-humulene (95%) and a minor by-product, beta-caryophyllene (5%). Introduction of ZSS1 and the foreign mevalonate pathway involved in FPP synthesis into E. coli results in in vivo production of alpha-humulene. Transcript of ZSS1 was detected almost exclusively in rhizomes and was up-regulated in both leaves and rhizomes after treatment with methyl jasmonate (MeJA), suggesting its ecological function in shampoo ginger.

Two cysteine proteases, GP2 and GP3, have been isolated from ginger rhizomes (Zingiber officinale). GP2 is virtually identical to a previously identified ginger protease GPII [K.H. Choi, and R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516-1526.], and cleaves native type I collagen at multiple discrete sites, which are in the interior of the triple helical region of this molecule. In reaction with proline-containing peptides GP2 shows preference for Pro in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain. Comparison of models of GP2 and GP3 with the crystal structure of papain shows that the three enzymes have different S2 pocket structures. The S2 pocket in GP2 and GP3 is half the size of that of papain. GP2 is the only reported plant cysteine protease with a demonstrated ability to hydrolyse native collagen. The results support a role for ginger proteases as an alternative to papain, in commercial applications such as meat tenderization, where collagen is the target substrate.

Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.

Members of the Zingiberaceae such as turmeric (Curcuma longa L.) and ginger (Zingiber officinale Rosc.) accumulate at high levels in their rhizomes important pharmacologically active metabolites that appear to be derived from the phenylpropanoid pathway. In ginger, these compounds are the gingerols; in turmeric these are the curcuminoids. Despite their importance, little is known about the biosynthesis of these compounds. This investigation describes the identification of enzymes in the biosynthetic pathway leading to the production of these bioactive natural products. Assays for enzymes in the phenylpropanoid pathway identified the corresponding enzyme activities in protein crude extracts from leaf, shoot and rhizome tissues from ginger and turmeric. These enzymes included phenylalanine ammonia lyase, polyketide synthases, p-coumaroyl shikimate transferase, p-coumaroyl quinate transferase, caffeic acid O-methyltransferase, and caffeoyl-CoA O-methyltransferase, which were evaluated because of their potential roles in controlling production of certain classes of gingerols and curcuminoids. All crude extracts possessed activity for all of these enzymes, with the exception of polyketide synthases. The results of polyketide synthase assays showed detectable curcuminoid synthase activity in the extracts from turmeric with the highest activity found in extracts from leaves. However, no gingerol synthase activity could be identified. This result was explained by the identification of thioesterase activities that cleaved phenylpropanoid pathway CoA esters, and which were found to be present at high levels in all tissues, especially in ginger tissues. These activities may shunt phenylpropanoid pathway intermediates away from the production of curcuminoids and gingerols, thereby potentially playing a regulatory role in the biosynthesis of these compounds.

A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.

Geranial is one of the potent odor compounds in fresh ginger. To clarify the generation of geranial in ginger, the alcohol dehydrogenase activity was measured in a crude enzymatic system of ginger. This enzyme solution was found to contain geraniol dehydrogenase (GeDH) specifically acting on geraniol as a substrate with NADP as a coenzyme. Geranial generation and GeDH activity were investigated for different maturity stages and storage periods of ginger. Both were at maximum levels from just after harvesting to initial storage. The GeDH activity subsequently dropped, and the generation of geranial also stopped. These results suggest that the GeDH activity in ginger is related to the generation of geranial.

The induction of rooting in microshoots of Zingiber officinale cvs. Suprava, Turia local, Suruchi and V3S18 was achieved on half-strength basal Murashige and Skoog's medium supplemented with 0.5-1.0 mg/l either indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) and 2% (w/v) sucrose within 7-9 days of culture. Rooting was inhibited when the microshoots were cultured under higher concentration of auxins. The microshoots cultured on medium supplemented with NAA induced large number of thin root hairs with friable calluses within 6-7 days. Peroxidase activity was determined during root induction (0-day to the 10th day at every 2 day interval) from microshoots derived in vitro. The activity was minimum in the inductive phase (primary) and at the maximum level during the root initiative phase. These finding may be useful in monitoring the rooting behaviour in microshoots derived from different subculture and peroxidase activity as a marker for root initiation.

Three geraniol glycosides were isolated from immature fresh ginger rhizomes (Zingiber officinale Roscoe). Their structures were identified as geranyl 6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside (1) geranyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside (2) and geranyl 6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside (3) by spectrometric analyses. After incubating each glycoside with a crude enzyme solution prepared from ginger, geraniol was liberated in all of those fractions. This result indicates that the glycosides are related to the formation of geraniol-related compounds in ginger aroma.

The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine-histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is approximately 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of approximately 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1-->6(Man1-->3) (Xyl1-->2)Man1-->4GlcNAc1-->4(Fuc1-->3)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.