Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan

In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5' and 3' rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme.     

Structural studies by stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan

Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides and have a pectin-like structure. The results of a structural analysis of SSPS by using polygalacturonase (PGase) and rhamnogalacturonase (RGase) clarified that the main backbone consisted of galacturonan (GN) and rhamnogalacturonan (RG), which were composed of the diglycosyl repeating unit, -4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-. The side chains of beta-1,4-galactans, branched with fucose and arabinose residues, were linked to the C-4 side of rhamnose residues in the RG regions. The degree of polymerization (dps) of GN, which linked the RG regions together, was estimated to be about 4-10 residues, and some were modified with xylose residues on the C-3 side of the galacturonates. The dps of GN at the reducing end of SSPS was estimated to be about 7-9 residues. Moreover, the fragment of the basic structure of the RG region, -[4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-]2-, some of which had long-chain beta-1,4-galactans branched on the C-4 side of rhamnose residues, were liberated from SSPS by the RGase treatment. The dps of the galactan side chain was estimated to be about 43-47 residues by an analysis of the digestion products from the beta-galactosidase treatment.     

Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate

A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site.     

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by ¹H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg⁻¹. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.     

海藻糖合酶在毕赤酵母表面的展示

海藻糖合酶能够利用麦芽糖一步法转化生产海藻糖,其底物专一性较高,该酶体系生产工艺简单,不受底物麦芽糖浓度的影响,是工业生产海藻糖的首选。为获得具有生产海藻糖合酶能力的毕赤酵母表面展示载体,实验以筛选的Pseudomonas putide P06海藻糖合酶基因为模板,PCR扩增得到海藻糖合酶基因(tres,2064 bp),连接至pPICZαA质粒中,获得重组质粒pPICZαA-tres。以来自酿酒酵母的共价连接细胞壁的Pir系列蛋白的Pir1p成熟肽蛋白作为毕赤酵母表面展示的锚定蛋白,利用PCR技术扩增得到pir1p(847 bp),连接至重组质粒pPICZαA-tres中,获得重组质粒pPICZαA-tres-pir1p。将重组质粒电击转入毕赤酵母GS115中,利用α-factor信号肽将蛋白引导分泌至细胞壁展示于毕赤酵母表面。通过Zeocin抗性筛选,挑选出阳性克隆子并摇瓶发酵。发酵产物经离心、破碎并使用昆布多糖酶水解,洗脱,结果显示,SDS-聚丙烯酰胺凝胶电泳分析可见明显融合蛋白条带,表明海藻糖合酶已成功地锚定在毕赤酵母。将重组毕赤酵母使用pH 7.5的缓冲液清洗并重悬,与底物浓度为30%的麦芽糖在30℃~60℃水浴条件下作用2 h,反应产物利用HPLC检测,能够检测到酶学活性。在优化后的条件pH 7.5,50℃,表面展示海藻糖合酶酶活达到300.65 U/g。40℃~50℃酶活较稳定,保温60 min,残留酶活相对活力达75%以上;最适反应pH值为7.5,并在碱性环境下稳定。     

Rhamnogalacturonan alpha-L-rhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin.

Two alpha-L-rhamnohydrolases with different substrate specificities were isolated from a commercial preparation produced by Aspergillus aculeatus. The first rhamnohydrolase was active toward p-nitrophenyl-alpha-L- rhamnopyranoside, naringin, and hesperidin and was termed p-nitrophenyl-alpha-L-rhamnopyranohydrolase (pnp-rhamnohydrolase). From the data collected, the enzyme seemed specific for the alpha-1,2- or alpha-1,6-linkage to beta-D-glucose. The pnp-rhamnohydrolase had a molecular mass of 87 kD (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH optimum of 5.5 to 6, a temperature optimum of 60 degrees C, and a specific activity toward pnp-alpha-L-rhamnopyranoside (pnp-Rha) of 13 units mg-1 protein. The second rhamnohydrolase, on the contrary, was active toward rhamnogalacturonan (RG) fragments, releasing Rha, and was therefore termed RG-rhamnohydrolase. The RG-rhamnohydrolase had a molecular mass of 84 kD, a pH optimum of 4, a temperature optimum of 60 degrees C, and a specific activity toward RG oligomers of 60 units mg-1 protein. The RG-rhamnohydrolase liberated Rha from the nonreducing end of the RG chain and appeared specific for the alpha-1,4-linkage to alpha-D-galacturonic acid. The enzyme was hindered when this terminal Rha residue was substituted at the 4-position by a beta-D-galactose. The results so far obtained did not indicate particular preference of the enzyme for low or high molecular mass RG fragments. From the results it can be concluded that a new enzyme, an RG alpha-L-rhamnopyranohydrolase, has been isolated with high specificity toward RG regions of pectin.     

棉铃虫组织蛋白酶B酶原在毕赤酵母中的表达

棉铃虫组织蛋白酶B（ Helicoverpa armigera Cathepsin B ,HCB）属于半胱氨酸蛋白酶类,参与胚胎发育中卵黄蛋白水解供给胚胎发育的氨基酸。本研究将HCB基因克隆到pPIC9K载体并转化毕赤酵母KM71菌株,经甲醇诱导,HCB表达并分泌到培养上清中。表达产物经SDS-PAGE测定分子量为38 kD, 与HCB基因编码的蛋白质分子量一致。用HCB的特异性抗体检测表明重组表达产物为棉铃虫组织蛋白酶B,原位水解实验显示重组表达的蛋白酶具有蛋白水解活性,表明在毕赤酵母中表达了有活性的棉铃虫组织蛋白酶B, 可用于组织蛋白酶B酶原活化机理研究及开发新蛋白酶产品。     

Production and characterization of recombinant Phanerochaete chrysosporium beta-glucosidase in the methylotrophic yeast Pichia pastoris

The extracellular beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium was expressed heterologously in the methylotrophic yeast Pichia pastoris. After 7 days' cultivation in an induction medium containing 1% (v/v) methanol, the expression level of the recombinant enzyme was 28,500 U/l, 38 times that of the wild-type enzyme. The specific activity of the crude recombinant enzyme for p-nitrophenyl-beta-D-glucoside was 52 U/mg, 37 times that of the wild-type enzyme; this difference made the purification of the enzyme simple. On a SDS-PAGE, the molecular mass of the recombinant enzyme was 133 kDa, and that of the wild-type enzyme was 116 kDa, but the difference had no effect on the hydrolysis of cellobiose or p-nitrophenyl-beta-D-glucoside. We concluded that the recombinant enzyme produced by Pichia pastoris retains the catalytic properties of the wild-type enzyme from Phanerochaete chrysosporium.     

Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate

Bacillus subtilis strain 168 YteR has been identified as a novel enzyme "unsaturated rhamnogalacturonyl hydrolase" classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid (DeltaGalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9A resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in DeltaGalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of DeltaGalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of DeltaGalA.     

Overexpression, purification, and characterization of a barley alpha-glucosidase secreted by Pichia pastoris

alpha-Glucosidases (EC 3.2.1.20) are recognized as important in starch degradation during cereal seed germination. A barley (Hordeum vulgare) alpha-glucosidase expressed in Pichia pastoris was cultured in flasks; however, the yield was low necessitating the use of multiple batches. Problems arose because of significant variation between batches. We solved these problems by switching to a fermentation system producing a sufficient quantity of a uniform sample. Here we present the expression and purification of a recombinant alpha-glucosidase grown under fermentation conditions. We also present the results of experiments to characterize the thermostability, pH optimum, and substrate specificity of the recombinant enzyme. The optimal pH for the hydrolysis of maltose by recombinant alpha-glucosidase is between 3.5 and 4.5. The thermostability of recombinant alpha-glucosidase was determined at pH 4, where activity is optimal, and at pH 5 and 6, which better mimic the conditions used to convert barley starch to fermentable sugars during industrial processing. The results indicate the enzyme is most thermolabile at pH 4. However, the enzyme is protected from heat inactivation at pH 4 by high concentrations of sucrose. The purified enzyme hydrolyzed maltose three times more rapidly than nigerose and 20 times more rapidly than trehalose and isomaltose. Concentrations of maltose greater than 20 mM inhibited maltose hydrolysis. This is the first report of substrate inhibition for any alpha-glucosidase. The results indicate that the only significant difference between the recombinant enzyme and the previously characterized barley isoforms was the V(max) for maltose hydrolysis.     

Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase : An Enzyme That Specifically Removes the Terminal Nonreducing Galacturonosyl Residue in Rhamnogalacturonan Regions of Pectin1

A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg⁻¹ was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h).     

High-level production of recombinant fungal endo-beta-1,4-xylanase in the methylotrophic yeast Pichia pastoris

Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.     

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.     

Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.     

Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis

Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase. known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized. The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/microg protein) and only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/microg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells.     

Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris

Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.     

Production and characterization of the agarase ofCytophaga flevensis

Cytophaga flevensis produced an inducible agarase which was extracellular under most conditions tested. The effect of cultural conditions on the production of enzyme was studied in batch and continuous culture. In batch culture, production was optimal whenCytophaga flevensis was incubated at 20C in a mineral medium with agar as the sole carbon source and ammonium nitrate as the nitrogen source at an initial pH of 6.6–7.0. The enzyme appeared to be subject to catabolite repression, since its synthesis was repressed when glucose was added to the medium in batch culture. Furthermore, in continuous culture, enzyme production decreased with increasing growth rate. Extracellular agarase was partially purified and the enzyme preparation obtained was very stable. The enzyme has a molecular weight of 26000 daltons. It is a β-agarase which is highly specific for polysaccharides containing neoagarobiose units. The final products of hydrolysis of agarose by the endo-acting enzyme were neoagarotetraose and neoagarobiose. Optimal conditions for its activity were pH 6.3 and 30C. When agarose was used as a substrate, an apparent temperature optimum of 35C was found, due to gelling of the substrate during the assay procedure.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Multiple attack in porcine pancreatic alpha-amylase-catalyzed hydrolysis of amylose studied with a fluorescence probe.

1. A large fluorescence enhancement of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) observed in the presence of amylose was utilized to monitor quantitatively the time course of porcine pancreatic alpha-amylase [EC 3.2.1.1] (PPA)-catalyzed hydrolysis of amylose with a number-average degree of polymerization of 16.8. 2. The slope of the plot of decrease in the relative fluorescence intensity of the TNS-amylose system (termed as the fluorescence value) versus the number of linkages hydrolyzed (reducing value) (Kondo, H. et al. (1977) Agric. Biol. Chem. 41, 631-634) in the course of PPA-catalyzed hydrolysis was shown to be useful to describe the degree of "multiple attack," which is defined by the number of reattacks on a long chain substrate molecular per one encounter of the enzyme and the substrate. A parameter gamma was defined as the ratio of the reciprocal of the slopes obtained at each pH to that at pH 10.5, where the multiple attack is not operating. 3. The gamma versus pH profile gave an apparent pK value of about 9, indicating that some ionizable groups participate in the multiple attack mechanism. 4. Based on a reaction scheme involving a "sliding" of the substrate molecule on the enzyme, which may contribute to the multiple attack mechanism, besides binding, dissociation, and cleavage steps of the substrate, and on the assumption of the steady state for the enzyme-substrate complex, rate equations were obtained to describe the time course of hydrolysis of a linear substrate. The product distribution with the progress of the reaction can be calculated theoretically, and is dependent on the number of multiple attack and the mode of sliding. The number of multiple attack can be estimated from this distribution, and the fluorescence value can be calculated theoretically by combining the product distribution with the relative efficiency of fluorescence intensity of each maltooligosaccharide (Nakatani, H. et. al. (1977) Biopolymers 16, 2363-2370). By comparing the experimental data with the theoretical ones, it was suggested that the multiple attack occurs through the sliding by maltose unit of the retained fragment on the enzyme, which is one of the fragments produced by the initial cleavage of the substrate molecule. 5. It was found that anions (chloride, bromide, and nitrate ions) which critically affect the enzyme activity have no effect on the degree of multiple attack.     

Enantioselective resolution of racemic styrene oxide at high concentration using recombinant Pichia pastoris expressing epoxide hydrolase of Rhodotorula glutinis in the presence of surfactant and glycerol

The reaction medium was optimized to accomplish epoxide hydrolase-catalyzed, batch enantioselective hydrolysis of racemic styrene oxide at high initial substrate concentrations. The recombinant Pichia pastoris containing the epoxide hydrolase gene of Rhodotorula glutinis was used as the biocatalyst. Enantiopure (S)-styrene oxide with 98% ee was obtained with 41% yield (maximum yield = 50%) from 1.8 M racemic styrene oxide at pH 8.0, 4 degrees C in the presence of 40% (v/v) Tween 20 and 5% (v/v) glycerol.