Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89 nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32 nmol/mg/min. The productivity of this enzyme in whole cell form is 500 mg/1 l of cultured cell. Its optimum reaction condition is 10 mM of calcium ions added to a phosphate buffer with a pH of 8.5.     

Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces Bainier

A novel ginsenoside-hydrolyzing beta-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of QSepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatographies. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and 60oC. It was highly stable within pH 3-9 and at temperatures lower than 55oC. The enzyme was specific to beta-glucoside. The order of enzyme activities against different types of beta-glucosidic linkages was beta-(1- 6)>beta-(1-2)>beta-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at 45 degrees and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->F2-->CK. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified beta-glucosidase proves to be a new protein that has not been reported before.     

Purification and characterization of ginsenoside Rb1-metabolizing beta-glucosidase from Fusobacterium K-60, a human intestinal anaerobic bacterium.

Fusobacterium K-60, a ginsenoside Rb1-metabolizing bacterium, was isolated from human intestinal feces. From this Fusodobacterium K-60, a ginsenoside Rb1-metabolizing enzyme, beta-glucosidase, has been purified. The enzyme was purified to apparent homogeneity by a combination of butyl-Toyopearl, hydroxyapatite ultragel, Q-Sepharose, and Sephacryl S-300 HR column chromatographies with a final specific activity of 1.52 micromol/min/mg. It had optimal activity at pH 7.0 and 40 degrees C. The molecular mass of this purified enzyme was 320 kDa, with 4 identical subunits (80 kDa). The purified enzyme activity was inhibited by Ba++, Fe++, and some agents that modify cysteine residues. This enzyme strongly hydrolyzed sophorose, followed by p-nitrophenyl beta-D-glucopyranoside, esculin, and ginsenoside Rb1. However, this enzyme did not change 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901) to 20(S)-protopanaxadiol, while it weakly changed ginsenoside Rb1 to IH-901. These findings suggest that the Fusobacterial beta-glucosidase is a novel enzyme transforming ginsenoside Rb1.     

Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp. 68

Pathogenic fungus Penicillium oxalicum sp. 68 was screened from soil and identified by ITS sequencing. The strain was found to be able to transform protopanaxadiol-type ginsenosides to produce a series of bioactive metabolites. Glycosidase from the culture of P. oxalicum sp. 68 was partially purified with a simple two-step procedure consisting of DEAE-cellulose chromatography and ammonium sulfate precipitation. Bioactive ginsenoside Compound K was prepared selectively and efficiently by biotransformation of ginsenosides Rb1, Rb2, Rc and Rd using the partially purified glycosidase. The optimal conditions for transforming Rb1 into Compound K were pH 4.0, 55 °C and 0.5 mg mL^?1 Rb1. The sole product is Compound K and the maximum yield reached 87.7 % (molar ratio). The transformation pathways of Rb1, Rb2, Rc and Rd are Rb1→Rd→F2→Compound K, Rb2→CO→CY→Compound K, Rc→Mb→Mc→Compound K and Rd→F2→Compound K, respectively. This biotransformation method showed great potential for preparing minor bioactive ginsenosides, especially Compound K, in the pharmaceutical industry because of its high specificity and favorable environmental compatibility.     

Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229

Ginsenoside Rb1 is the most abundant ginsenoside in Panax (ginseng). The hydrolysis of this ginsenoside produces compound K, the biologically active ginsenoside of ginseng. We previously identified a fungus Paecilomyces Bainier sp. 229 (sp. 229), which can efficiently convert ginsenoside Rb1 to compound K. In this report, the ginsenoside hydrolyzing β-glucosidases were isolated from sp. 229 and the pathway of the biotransformation of ginsenoside Rb1 to compound K by sp. 229 was investigated. Based on reverse-phase HPLC and TLC analysis, we found the main metabolic pathway is as follows: ginsenoside Rb1 → ginsenoside Rd → ginsenoside F2 → compound K. Moreover, the results showed that there were other metabolic pathways: ginsenoside Rb1 → ginsenoside XVII → ginsenoside F2 → compound K and ginsenoside Rb1 → ginsenoside Rg3 → ginsenoside Rh2. These processes would allow the specific bioconversion of ginsenoside Rb1 to various ginsenosides using an appropriate combination of specific microbial enzymes.     

Purification method improvement and characterization of a novel ginsenoside-hydrolyzing beta-glucosidase from Paecilomyces Bainier sp. 229

The purification method for a novel ginsenoside-hydrolyzing beta-glucosidase from Paecilomyces Bainier sp. 229 was successfully simplified by the application of microcrystalline cellulose (MCC) as a novel chromatographic matrix. Only two chromatographic steps, Q-Sepharose FF and MCC column in sequence, were required to purify the enzyme to apparent homogeneity. The purified enzyme, with a native molecular weight estimated to be 305 KDa, was composed of three identical subunits of approximately 102 KDa each. The optimal enzyme activity was observed at pH 3.5 at 55 degrees C. It was stable within pH 3-7 and at temperatures lower than 50 degrees C. The optimal substrate for the enzyme was p-nitrophenyl-beta-D-glucoside, followed by ginsenoside Rd, gentiobiose, and ginsenoside Rb1. It converted ginsenoside Rb1 to ginsenoside Rg3 specifically and efficiently. The hydrolyzing pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->Rg3. The specific activities against ginsenoside Rb1 and Rd were 56.7 micromol/min/mg and 129.4 micromol/min/mg respectively.     

Microbial transformation of ginsenoside Rb1 to compound K by Lactobacillus paralimentarius

In this study, the major ginsenoside Rb1 was transformed into the more pharmacologically active minor compound K by food grade Lactobacillus paralimentarius LH4, which was isolated from kimchi, a traditional Korean fermented food. The enzymatic reaction was analyzed by TLC, HPLC, and NMR. Using the cell-free enzyme of Lactobacillus paralimentarius LH4 at optimal conditions for 30 °C at pH 6.0, 1.0 mg ml^?1 ginsenoside Rb1 was transformed into 0.52 mg ml^?1 compound K within 72 h, with a corresponding molar conversion yield of 88 %. The cell-free enzyme hydrolyzed the two glucose moieties attached to the C-3 position and the outer glucose moiety attached to the C-20 position of the ginsenoside Rb1. The cell-free enzyme hydrolyzed the ginsenoside Rb1 along the following pathway: ginsenoside Rb1 → gypenoside XVII and ginsenoside Rd → ginsenoside F2 → compound K. Our results indicate that Lactobacillus paralimentarius LH4 has the potential to be applied for the preparation of compound K in the food industry.     

人参皂苷Rb3糖基水解酶的纯化及其反应特性

人参皂苷Rb3是三七茎叶皂苷的主要成分。为了充分利用廉价的三七茎叶皂苷,该研究以微生物Aspergillus sp. P90r菌为对象,综合运用生物转化的方法,经过提取、分离纯化和酶活力测定等步骤,最终以确定酶反应途径的方式得到了所产的特异性人参皂苷Rb3糖基水解酶的相关性质和动力学等反应特性。结果表明:该酶比Absidia sp. GRB3-X8r菌产酶活力高15%²5%,SDS-PAGE电泳结果测得分子量约为65.6ku,纯化后酶蛋白的含量为0.237 mg·mL^-1,蛋白比活力可达到169 U·mg^-1,纯化倍数为13.70,回收率为9.39%。人参皂苷Rb3糖基水解酶在pH=5.0的偏酸性环境下酶活力很高,最适反应条件:pH=3.0⁵.0,温度45℃,其中在pH=4.0⁶.0范围内相对稳定。该酶在20 min时进入混合级反应,酶反应米氏常数Km值为8.77 mmol·L^-1,V_max为57.44 mmol·L^-1     

Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b

F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.     

A new ether bond-splitting enzyme found in Gram-positive polyethylene glycol 6000-utilizing bacterium, <Emphasis Type="Italic">Pseudonocardia</Emphasis> sp. strain K1

Pseudonocardia sp. strain K1 is the only Gram-positive bacterium among the bacteria aerobically metabolizing polyethylene glycol (PEG). Generally, PEG is metabolized by an oxidative pathway in which a terminal alcohol group of PEG is oxidized to aldehyde and to carboxylic acid and then an ether bond is oxidatively cleaved. As the cell-free extract of Pseudonocardia sp. strain K1 has PEG dehydrogenase, PEG aldehyde dehydrogenase and diglycolic acid (DGA) dehydrogenase (DGADH) activities, all of which are constitutively formed, the strain has a metabolic pathway similar to that so far known. We purified an ether bond-splitting enzyme as DGADH. The molecular mass of the enzyme was estimated to be 55 kDa; and it consisted of two identical subunits. The enzyme oxidatively cleaved both an ether bond of PEG 3000 dicarboxylic acid and DGA. The N-terminal amino acid sequence of the purified enzyme had high homology with various superoxide dismutases and the enzyme had also superoxide dismutase activity. The atomic absorption spectrum showed that approximately one atom of Fe was included in each subunit of the enzyme. DGADH activity increased in the cells grown in a PEG medium supplemented with FeCl₃. Thus, we concluded that the enzyme purified from Pseudonocardia sp. strain K1 is a new ether bond-splitting enzyme.     

Purification and characterization of alpha-L-arabinopyranosidase and alpha-L-arabinofuranosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc

Two arabinosidases, alpha-L-arabinopyranosidase (no EC number) and alpha-L-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. alpha-L-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 micro mol/min/mg. alpha-L-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 micro mol/min/mg. The molecular mass of alpha-L-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that of alpha-L-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. alpha-L-Arabinopyranosidase and alpha-L-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40 degrees C and pH 4.5 and 45 degrees C, respectively. Both purified enzymes were potently inhibited by Cu(2+) and p-chlormercuryphenylsulfonic acid. alpha-L-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinopyranoside, followed by ginsenoside Rb2. alpha-L-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-beta-galactopyranoside or p-nitrophenyl-beta-D-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified alpha-L-arabinosidases. This is the first reported purification of alpha-L-arabinopyranosidase from an anaerobic Bifidobacterium sp.     

Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp. strain A11-2

A benzothiophene (BT) and dibenzothiophene (DBT) monooxygenase (TdsC), which catalyzes the oxidation of the sulfur atoms in BT and DBT molecules, was purified from Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme and its subunit were determined to be 200 kDa and 43 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating a tetrameric structure. The N-terminal amino acid sequence of the purified TdsC completely matched the amino acid sequence deduced from the nucleotide sequence of the tdsC gene reported previously [Ishii et al. (2000) Biophys Biochem Res Commun 270:81-88]. The optimal temperature and pH for the TdsC reaction were 65 degrees C and pH 9, respectively. TdsC required NADH, FMN and TdsD, a NADH-dependent FMN oxidoreductase, for its activity, as was observed for TdsA. FAD, lumiflavin and/or NADPH had some effect on the maintenance of TdsC activity. A comparison of the substrate specificity of TdsC and DszC, the homologous monooxygenase purified from Rhodococcus erythropolis strain KA2-5-1, demonstrated a contrasting pattern towards alkylated DBTs and BTs.     

Purification and properties of an endo-inulinase from an Arthrobacter sp.

Extracellular endo-inulinase of Arthrobacter sp. S37 was purified 63-fold, giving a single band on PAGE with activity staining. The Mr was estimated as 75 kDa by SDS-PAGE. The first 31 amino acids of the N-terminal sequence was determined. The endo-inulinase hydrolyzed inulin mainly into inulo-triose (F3), inulo-tetraose (F4) and inulo-pentaose (F5) optimally at pH 7.5 and 50°C. © Rapid Science Ltd. 1998     

Enzymatic biotransformation of ginsenoside Rb1 to 20(<Emphasis Type="Italic">S</Emphasis>)-Rg3 by recombinant β-glucosidase from <Emphasis Type="Italic">Microbacterium esteraromaticum</Emphasis>

Microbacterium esteraromaticum was isolated from ginseng field. The β-glucosidase gene (bgp1) from M. esteraromaticum was cloned and expressed in Escherichia coli BL21 (DE3). The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The recombinant β-glucosidase enzyme (Bgp1) was purified and characterized. The molecular mass of purified Bgp1 was 87.5 kDa, as determined by SDS-PAGE. Using 0.1 mg ml⁻¹ enzyme in 20 mM sodium phosphate buffer at 37°C and pH 7.0, 1.0 mg ml⁻¹ ginsenoside Rb1 was transformed into 0.444 mg ml⁻¹ ginsenoside Rg3 within 6 h. The Bgp1 sequentially hydrolyzed the outer and inner glucose attached to the C-20 position of ginsenosides Rb1. Bgp1 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 using the recombinant β-glucosidase.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

High-performance liquid chromatography coupled with tandem mass spectrometry applied for metabolic study of ginsenoside Rb1 on rat

Liquid chromatography coupled with mass spectrometry and tandem mass spectrometry has been applied to investigate the in vivo metabolism of ginsenoside Rb(1) in rat. Both positive electrospray ionization mass spectrometry and negative electrospray ionization mass spectrometry were used to identify the Rb(1) and its metabolites in rat plasma, urine, and feces samples. Oxygenation and deglycosylation were found to be the major metabolic pathways of Rb(1) in rat. A total of nine metabolites were detected in urine and feces samples collected after intravenous and oral administration. Deglycosylated metabolism of Rb(1) generated other ginsenosides as the major metabolites, such as Rd, Rg(3) or F(2), Rh(2), or C-K. This result indicates that the ginsenoside Rb(1) has many pharmacological activities and could be used as a prodrug.     

In vitro metabolism of ginsenosides by the ginseng root pathogen Pythium irregulare

The role of ginseng saponins (ginsenosides) as modulators or inhibitors of disease is vague, but our earlier work supports the existence of an allelopathic relationship between ginsenosides and soilborne microbes. Interestingly, this allelopathy appears to significantly promote the growth of the important ginseng pathogen, Pythium irregulare while inhibiting that of an antagonistic non-pathogenic fungus, Trichoderma hamatum. Herein we report on the apparent selective metabolism of 20(S)-protopanaxadiol ginsenosides by an extracellular glycosidase from P. irregulare. Thus, when P. irregulare was cultured in the presence of a purified (> 90%) ginsenoside mixture, nearly all of the 20(S)-protopanaxadiol ginsenosides (Rb1, Rb2, Rc, Rd, and to a limited extent G-XVII) were metabolized into the minor ginsenoside F2, at least half of which appears to be internalized by the organism. No metabolism of the 20(S)-protopanaxatriol ginsenosides (Rg1 and Re) was evident. By contrast, none of the ginsenosides added to the culture medium of the non-pathogenic fungus T. hamatum were metabolized. The metabolism of 20(S)-protopanaxadiol ginsenosides by P. irregulare appears to occur through the hydrolysis of terminal monosaccharide units from disaccharides present at C-3 and/or C-20 of ginsenosides Rb1, Rc, Rb2, Rd and G-XVII to yield one major product, ginsenoside F2 and one minor product (possibly G-III). A similar transformation of ginsenosides was observed using a crude protein preparation isolated from the spent medium of P. irregulare cultures.     

Isolation and characterization of a lectin from peanut roots.

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.     

Identification of a new active site for autocatalytic processing of penicillin acylase precursor in Escherichia coli ATCC11105

Penicillin acylase (PA) from Escherichia coli ATCC11105 is a periplasmic heterodimer consisting of a 24 kDa small subunit and a 65 kDa large subunit. It is synthesized as a single 96 kDa precursor and then matures to functional PA via a posttranslational processing pathway. The GST-PA fusion protein expression system was established for monitoring the precursor PA processing in vitro. The purified PA precursor was processed into mature PA the same way as in vivo, but pH dependently. From the primary sequence analysis, we identified a putative conserved lysine residue (K299) responsible for the pH dependent processing. The substitution of K299 residue by site-directed mutagenesis affected both the enzyme activity and the precursor PA processing in vivo. Furthermore, it was shown that the processing rates of wild-type and mutant precursor PAs depended on the pKa values of their side chain R group. These results demonstrated that the lysine residue (K299) was involved in the precursor processing of PA together with N-terminal serine residue (S290) of the large subunit.