The plant surface, which is representative of the phylloplane and rhizoplane, is a characteristic habitat for microorganisms. In this review, the ecological roles of phytoepiphytic bacteria will be described. The phylloplane and rhizoplane, which are adjacent to the atmosphere and soil sphere respectively, accumulate topically and/or selectively release secondary metabolites that are specific to the plant genera and species which reside within these regions. Some epiphytes have abilities to decarboxylate xenobiotic phenolic acids that have accumulated in the plant tissues and surfaces as a majority of such secondary metabolites. In physicochemically stressed soil, rhizosphere microflora often remedy such microenvironments within the rhizosphere in order to assist in the survival of the host, and some of the microfloral compositions behave as if they were symbionts. Specifically, some Sphingomonas spp., which are frequently isolated from the rhizosphere of acidic soil-tolerant plants in tropical zones, make possible the development of a rhizo-biocomplex. In this review, the possibility of rhizosphere regulation utilizing such a rhizo-biocomplex is discussed.
Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.
The lectin-antibody enzyme immunoassay of the alphafetoprotein-L3 carbohydrate chain, a tumor marker of liver cancer, has not been automated. We improved the technique of the assay for automation. Consequently, alphafetoprotein-L3 and total alphafetoprotein were detected with two lectins using an automatic paramagnetic bead handling robot. This indicates that the improved method is potentially applicable to the automated enzyme immunoassay robot.
Epichloe typhina is an endophytic fungus, while Cladosporium phlei is a pathogenic fungus of the timothy plant (Phleum pretense L.). We found two activities in the culture filtrate of E. typhina: one stimulated the pathogenic fungus, C. phlei, to produce phleichrome and the other inhibited its growth. The active ingredients that stimulated the production of phleichrome and inhibited the growth of C. phlei were isolated and characterized. The isolated compounds were identified as cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were stimulatory compounds, and p-hydroxybenzaldehyde, which was the growth inhibitory compound, based on an analysis of their spectral data. Of the two stimulatory compounds, cyclo-(L-Pro-L-Phe) showed higher activity. However, when 500 μg of cyclo-(L-Pro-L-Phe) was spotted on the TLC plate for bio-autography, a growth inhibitory zone was identified in the central red region, which contained phleichrome. On the other hand, phleichrome showed antifungal activity against E. typhina in the light, so it is assumed that there might be antagonism between the endophytic fungus, E. typhina, and the pathogenic fungus, C. phlei.
The benzyl-protected disaccharide building blocks of core 8 O-glycan (15a/15b) for glycopeptide were stereoselectively synthesized by two glycosidation reactions with the glycosyl fluoride method. The building blocks were utilized in the solid-phase synthesis of a glycopeptide carrying two O-glycans with the consensus sequence of the tandem-repeat domain of MUC5AC. The synthetic glycopeptide was detached from the resin with reagent K, and subsequent debenzylation under conditions of low-acidity TfOH afforded glycopeptide 2. The synthetic sample will be used as a suitable standard in studies of the physicochemical or immunochemical characterization of mucin glycoforms.
One novel labdane-type diterpene, named ptychantin O, and two known diterpenes, ptychantin G and F, were isolated from cultured cells of the Phychanthus striatus liverwort, and their structures were determined by NMR spectroscopic analyses.
A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10−4 M, using the culture of single-node segments of potato stems in vitro.
The major sex pheromone components of Drosophila ananassae and D. pallidosa, (Z,Z)-5,25-hentriacontadiene and (Z,Z)-5,27-tritriacontadiene, respectively, were synthesized by using the Wittig olefination and sulfone coupling reactions as the C–C bond-forming steps.
In the course of isolating hyaluronan-degradation inhibitors from the Tricholoma orirubens mushroom, new phenones, orirubenones D to G, were isolated. Although none of the compounds had the inhibitory activity, this result and the result in the previous paper allow us to conclude that the catechol moiety in the molecules was indispensable for exhibiting inhibitory activity.
Lipid vesicles are potentially useful as microcapsules for drug and/or gene delivery. We developed cationic lipid vesicles consisting mainly of sorbitan monooleate (Span 80) and cationic peptide lipid (CPL), and evaluated the CPL vesicles as gene transfection vectors. The optimum CPL concentration for gene transfection into HeLa cells was found to be 20 wt % of total lipid, and such CPL vesicles did not exhibit significant cytotoxicity. Co-culture of Poly-L-lysine and plasmids prior to making CPL vesicle-plasmid complexes was effective. Lipofection using LipofectAMINE was suppressed in 10% serum-supplemented medium. The transfection efficiency of 20 wt % CPL vesicles, however, was not affected by serum in the medium when plasmids were treated with poly-L-lysine.
A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to α-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl α-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.
Random mutagenesis of the gene encoding family 11 xylanase was used to obtain alkalophilic mutants. The catalytic domain of the chimeric enzyme Stx15, which was constructed from Streptomyces lividans xylanase B and Thermobifida fusca xylanase A, was mutated using error-prone PCR and screened for halo formation on dye-linked xylan plates and activity toward soluble xylan. A positive mutant, M1011, was isolated, and it was found that mutation A49V was responsible for the alkalophilicity of the mutant. Mutation A49V increased the specific activity at pH 9.1 and the stability of mutant A49V was not significantly different from that of Stx15 at 60 °C. Both enzymes retained more than 90% of their relative activity from pH 4.7 to 9.1 after 1 h of incubation at 60 °C. Analysis of the kinetic parameters at various pH values showed that the A49V mutation reduced the Km in the alkaline pH range, resulting in the higher specific activity of the A49V mutant enzyme.
Five full-length cDNA encoding gibberellin 2-oxidases, VaGA2oxA1, VaGA2oxA2, VaGA2oxB1, VaGA2oxB2, and VaGA2oxB3, were cloned from etiolated adzuki bean (Vigna angularis cv. Dainagon) seedlings, and their enzymatic characteristics were examined using recombinant enzymes fused with glutathione S-transferase (GST). Recombinant VaGA2oxA1 (rVaGA2oxA1) and rVaGA2oxA2 showed 2β-hydroxylation activity by converting GA1, GA4, GA9, GA20, GA4-methyl ester, and 16,17-dihydro-GA4 to the corresponding 2β-hydroxylated gibberellins, which were identified by GC/MS. rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3 showed similar activity by converting [3H4]-16,17-dihydro-GA4 to a metabolite showing an Rf value of 16,17-dihydro-GA34. RNA-blot analysis showed that VaGA2oxA1 and VaGA2oxA2 were the major ones expressed in etiolated hypocotyls. The addition of Co2+ instead of Fe2+ to the assay medium apparently reduced the enzymatic activity, but increased the binding of [3H4]-16,17-dihydro-GA4 to rVaGA2oxA1, indicating the possibility that VaGA2oxs can be detected as gibberellin-binding proteins under certain conditions.
Gibberellin-binding proteins (GBPs) were purified ca. 230,000 fold. The characteristics of adzuki GBP were examined and compared with those of a recombinant gibberellin 2-oxidase (rVaGA2oxA1) that was fused with glutathione S-transferase (GST). VaGA2oxA1 was most abundantly expressed in etiolated adzuki bean seedlings, and VaGA2oxA1 and GBPs from adzuki bean seedlings showed gibberellin-binding activity when incubated with 2-oxoglutarate and Co2+. Both rVaGA2oxA1 and partially purified GBPs from adzuki bean seedlings showed very similar selectivity to gibberellins in binding assays, where biologically active gibberellins such as GA4, GA3, GA7, and GA1 showed higher binding affinity than biologically inactive gibberellins such as GA8, GA34, and 3-epi-GA4. The polyclonal antibody raised against rVaGA2oxA1 cross-reacted with all rVaGA2oxs (rVaGA2oxA1, rVaGA2oxA2, rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3) whose cDNAs were cloned from adzuki bean seedlings. Treated with the antibody, the recombinants that originally showed gibberellin-binding activity lost both binding activity and enzymatic activity. In contrast to the recombinants, the gibberellin-binding activity of GBPs from adzuki bean seedlings was hardly affected by the antibody treatment. The GBPs showed very weak gibberellin 2-oxidase-like activity, and it was not affected by the antibody treatment either. These observations suggest that a major component that showed GA-binding activity was apparently different from any gibberellin 2-oxidase cloned from the seedlings.
Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxo-acids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.
The spc operon of Escherichia coli encodes 11 ribosomal proteins and SecY. The secY gene and downstream rpmJ encoding a ribosomal protein, L36, are located distal to the promoter of the spc operon. It has been suggested that the stability of SecY mRNA depends on rpmJ unless a ρ-independent terminator is inserted immediately downstream of secY. Moreover, it has been suggested that RpmJ is dispensable for E. coli. We constructed rpmJ null strains, AY101 (ΔrpmJ::tetA) and AY201 (ΔrpmJ::cat), by replacing rpmJ with tetA, which encodes a membrane protein responsible for tetracycline-resistance, and cat, which encodes a cytoplasmic chloramphenicol acetyltransferase, respectively. Depletion of RpmJ did not inhibit protein synthesis, whereas the growth of AY101 was defective at high temperatures. The level of SecY mRNA decreased significantly in both disruptants even though the ρ-independent terminator was inserted immediately downstream of secY. Some periplasmic proteins were missing in the disruptants with a concomitant increase in the amount of phage shock protein in the inner membrane. These phenotypes caused by the rpmJ null mutation were corrected by a plasmid carrying secY, but not by one carrying rpmJ.
We discovered that luteolin, a typical flavonoid contained in various kinds of plants, inhibits the secretion and gene expression of endothelin-1 (ET-1), a potent vasoconstrictor regulating blood pressure, in porcine aortic endothelial cells. Its ED50 was about 10 μM. In addition, the inhibition of ET-1 by a glycoside compound of luteolin (luteolin-6-C-glucoside) was weak.
A monoclonal antibody to lactophorin (LP) was prepared by creating hybridoma from mouse myeloma cells and spleen cells from mice immunized with PAS-4 concentrated fraction from bovine milk fat globule membrane. The prepared antibody recognized a polypeptide moiety of LP27, the major component constituting LP, but not a carbohydrate moiety. Immunoblot analysis showed that all polypeptides (LP17, LP20, LP27, LP40, and LP50) constituting LP were recognized by the antibody. The identities of LP20, LP40, and LP50 were verified by N-terminal and internal amino acid sequencing. LP20 contains hydrolysate of LP27 besides LP27 without the O-glycosyl sugar chain. These results suggest that LP40 and LP50 are homo- or heterodimers of LP20 and LP27. This is the first report to the effect that LP was constructed from several forms of polypeptides, derived from LP27.
The influence of saccharides on the formation of acrylamide (AcA) was investigated. The reducing saccharides reacted with asaparagine to form AcA, but the non-reducing saccharides, except sucrose, gave no AcA. AcA formation from a mixture containing glucose and asaparagaine was suppressed by the non-reducing saccharides, especially trehalose (76% suppression) and neotrehalose (75% suppression). Glucose is heat-degraded into pyruvaldehyde and 5-hydroxymethyl-2-furfural in the water system. The degradation products react with asparagines to generate AcA. Trehalose appears to inhibit not only the formation of these intermediates and asparagines for AcA, but also the AcA formation from these intermediates.
Mispyric acid is a novel natural triterpene dicarboxylic acid which has inhibitory activity against DNA polymerase β (pol β) isolated from the plant, Mischocarpus pyriformis. In this report, we examine the selectivity of the inhibitory activity against mammalian pols and the mode of inhibition in vitro. Natural mispyric acid (compound 1) inhibited the activities of all the mammalian pols tested (pol α, β, γ, δ and ε) with an IC50 value in the range of 3.6–44.5 μM. The inhibition was strongest for pol γ among these five pols. The enantiomer of mispyric acid (compound 2, ent-mispyric acid) had similar effects to those of the natural compound. However, derivatives of compounds 1 and 2 with hydroxyl groups instead of carboxyl groups (i.e., compounds 3 and 4, respectively) exhibited no inhibitory effect on mammalian pols. The moiety of two carboxylic acids in mispyric acid was important for the inhibition of pols, and the stereoisomers of mispyric acid had no inhibitory effect.
Linoleic acid (LA) incubated with a homogenate of Lentinula edodes or Tricholoma matsutake mushroom significantly increased the amount of (R)-1-octen-3-ol. The alcohol was identified as (S)-10-HODE with 90–87% and >99% enantiomeric excess (ee), respectively. During the incubation of LA with these homogenates in the presence of glutathione–glutathione peroxidase (GSH–GPx), which can reduce hydroperoxy fatty acids to the corresponding hydroxy acids, the formation of (R)-1-octen-3-ol was significantly inhibited, whereas the amount of 10-hydroxy-(8E,12Z)-8,12-octadecadienoic acid (10-HODE) was significantly increased. The acid was identified as (S)-10-HODE with 92–88% ee and >99% ee, respectively. The decrease in the amount of alcohol was approximately the same as the increase in amount of HODE in both mushrooms. These results indicate a stereochemical correlation between (R)-1-octen-3-ol and (S)-10-hydroperoxy-(8E,12Z)-8,12-octadecadienoic acid [(S)-10-HPODE] in both mushrooms.
Alkylhydroperoxides in oxidized oil are undesirable components because they become alkylperoxyl radicals (ROO•) in the presence of heme, hemoglobin, or myoglobin in red meat. ROO• are biochemically reactive and damage nucleic acids and proteins, thereby harming living cells. We isolated a component, a highly potent ROO• scavenger, from crude canola oil (rapeseed), which we designated canolol, and identified its chemical structure, 4-vinyl-2,6-dimethoxyphenol. The canolol content of crude canola oil greatly increased after the seed was roasted as compared with that from unroasted seed, but it decreased in highly purified oil. This anti-ROO• activity was highest in crude oil, deceased after each refining step, and was lowest in highly purified refined oil. Canolol was thus generated during roasting. As shown previously, canolol is one of the most potent anti-ROO• components in crude canola oil and its potency is much greater than that of well-known antioxidants, including α-tocopherol, vitamin C, β-carotene, rutin, and quercetin.
D-Mannitol, one of the main phytochemicals of the edible Tamogi-take mushroom (Pleurotus cornucopiae), was found to inhibit an angiotensin I converting enzyme (ACE). The antihypertensive effect of D-mannitol and a hot water extract of Tamogi-take mushroom was demonstrated in spontaneously hypertensive rats (SHR) by oral administration.
Gly-Leu-Phe (GLF), an immunostimulating peptide derived from α-lactalbumin, prevented alopecia induced by an anticancer agent etoposide in a neonatal rat model after intraperitoneal injection at a dose of 100 mg/kg for 4 d or oral administration at a dose of 300 mg/kg for 6 d. By microscopic analysis of skin sections, GLF proved to inhibit etoposide-induced loss of hair, thickening of the epidermis, and thinning of the adipocyte layer. The anti-alopecia effect of GLF was inhibited by pyrilamine, a histamine H1 receptor antagonist, suggesting that the anti-alopecia effect is mediated by histamine release.
Previously we reported that a mutant of Corynebacterium glutamicum ATCC14067 with reduced H+-ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 μg/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534–540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 μg/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 μg/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the H+-ATPase-defective mutant of C. glutamicum is useful in glutamic acid production.
Four Magnetospirillum strains degrading toluene, phenol, benzoate, and other aromatic compounds under anaerobic conditions were isolated from denitrifying enrichment cultures. One of the isolates, toluene-degrading strain TS-6, contained genes that are homologous to those encoding benzylsuccinate synthase (Bss) and benzoyl-CoA reductase (Bcr), two key enzymes of anaerobic toluene and benzoate degradation respectively in known denitrifying bacteria. Transcription of the genes was confirmed. It was controlled by growth substrates and oxygen conditions, but bcr genes were unexpectedly expressed in aerobic cells grown on benzoate. It was confirmed that the genus Magnetospirillum represents the third genus of denitrifying bacteria capable of degrading aromatic compounds under anaerobic conditions, besides the genera Thauera and Azoarcus.
The methylotrophic yeast Pichia pastoris can degrade peroxisomes selectively though two distinct pexophagic pathways, viz., micropexophagy and macropexophagy. These micro- and macropexophagy pathways are induced by adaptation of methanol-grown cells to glucose-containing and ethanol-containing media respectively. However, our understanding of the molecular signal(s) that determine which pathway is activated or repressed in response to environmental changes is limited. In this study, the determinant for these pathways was sought using cells undergoing pexophagy under a variety of conditions. Micropexophagy and macropexophagy were distinguished in living cells by fluorescence microscopy. Our results indicate that glucose and ethanol were not specific inducers of micro- and macropexophagy respectively. Micropexophagy was found to be more sensitive to ATP-depletion than macropexophagy, suggesting that the micropexophagic process requires a higher level of ATP than the macropexophagic process. From these and other results, we postulate that intracellular ATP levels play an important role in determining which pexophagic pathway is activated.
Hydroxycitric acid (HCA) is a major acid component of the tropical plants Garcinia cambogia and Hibiscus subdariffa. (2S,3S)-HCA from G. cambogia was shown to be a potent inhibitor of ATP citrate lyase (EC22.214.171.124), which catalyzes the extramitochondrial cleavage of citrate to oxaloacetate and acetyl-CoA. (2S,3R)-HCA from H. subdariffa inhibits α-amylase and α-glucosidase, leading to reduction of carbohydrate metabolism. The availability of HCA is limited by the restricted habitat of the plants as well as the difficulty of stereoselective organic synthesis. Hence, we screened microorganisms producing HCA to find an alternative source of optically pure bulk HCA. Two strains, Streptomyces sp. U121 and Bacillus megaterium G45C, were screened by HPLC analysis. Particular metabolites were purified from their culture broths and compared with authentic HCA from plants. NMR studies indicated that the products are identical to Hibiscus-type HCA. This is the first report showing isolation of microorganisms producing HCA.
Clostridium stercorarium Xyn10B having hydrolytic activities on xylan and β-1,3-1,4-gllucan is a modular enzyme composed of two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of the glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. We investigated the function of family-9 and family-22 CBMs in a modular enzyme by comparing the enzymatic properties of a truncated enzyme composed of two family-22 CBMs and the catalytic module (rCBM22-CM), an enzyme composed of the catalytic module and family-9 CBM (rCM-CBM9), an enzyme composed of two family-22 CBMs, the catalytic module, and family-9 CBM (rCBM22-CM-CBM9), and the catalytic module polypeptide (rCM). Although the addition of family-9 CBM to rCM and rCBM22-CM did not significantly change catalytic activity toward xylan and β-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially β-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 °C to 75 °C, but that of family-9 CBM to rCM and rCBM22-CM did not affect the optimum temperature. These facts suggest that the enzyme properties of Xyn10B were mainly dependent on the presence of the family-22 CBMs but not family-9 CBM.
The sulfite resistance gene, SSU1-R, is widely distributed in wine yeasts. This gene has an upstream region distinct from that of the allelic gene, SSU1 and SSU1-R is expressed at a much higher level than SSU1. We characterized the promoters of both of these genes by analysis of their activity using the LacZ gene as a reporter. FZF1, the activator gene of SSU1, was shown to regulate SSU1-R expression indirectly. SSU1-R expression was activated under microaerobic conditions, and four 76-bp repeats, present within the SSU1-R promoter region, was essential for high expression. These results indicate that SSU1-R expression is regulated in different manner from that of SSU1. By deletion analysis of the SSU1-R promoter region, we found that at least two of the 76-bp repeats are necessary for promoter activity, and that the number of 76-bp repeats influences the activity. Hence, it was suggested that the number of 76-bp repeats increases in wine yeasts that require strong sulfite resistance.
Streptomyces subtilisin inhibitors (SSIs) are produced by a wide variety of Streptomyces species. Streptomyces coelicolor A3(2) contains two genes, SCO0762 and SCO4010, encoding an SSI-like protein. Of these two genes, SCO0762 was transcribed actively throughout growth. Gene disruption of SCO0762 (mutant ΔSCO0762) resulted in overproduction of extracellular protease activity, showing that SCO0762 serves as a modulator of extracellular protease activities. Mutant ΔSCO0762 showed no apparent phenotypic changes in morphological differentiation, forming aerial hyphae and spores in the same time course as the parental strain. SCO4010 appeared to be a pseudogene, because mutant ΔSCO4010 showed the same protease activity as the parental strain, probably due to amino acid replacement of one (Arg-60) of the important residues for SSI activity, and because the transcription of this gene was extremely low.