Properties of theβ-glucosidase fromCellulomonas flavigena and fromEscherichia coli harboring the recombinant plasmid pJS3

Summary Plasmid-coded -glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The -glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the -glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of -glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheK _m values for cellobiose and PNPG indicated that the -glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the -glucosidase fromE. coli pJS3 showed higher affinity for PNPG.     

Enhanced Thermotolerance of <Emphasis Type="Italic">E. coli</Emphasis> by Expressed OsHsp90 from Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A gene encoding the rice (Oryza sativa L.) 90-kDa heat shock protein (OsHsp90) was introduced into Escherichia coli using the pGEX-6p-3 expression vector with a glutathione-S-transferase (GST) tag to analyze the possible function of this protein under heat stress for the first time. We compared the survivability of E. coli (BL21) cells transformed with a recombinant plasmid containing GST-OsHsp90 fusion protein with control E. coli cells transformed with the plasmid containing GST and the wild type BL21 under heat shock after isopropyl β-d-thiogalactopyranoside induction. Cells expressing GST-OsHsp90 demonstrated thermotolerance at 42, 50, and 70°C, treatments that were more harmful to cells expressing GST and the wild type. Further studies were carried out to analyze the heat-induced characteristics of OsHsp90 at 42, 50, and 70°C in vitro. When cell lysates from E. coli transformants were heated at these heat stresses, expressed GST-OsHsp90 prevented the denaturation of bacterial proteins treated with 42°C heat shocks, and partially prevented that of proteins treated at 50 and 70°C; meanwhile, cells expressing GST-OsHsp90 withstood the duration at 50°C. These results indicate that OsHsp90 functioned as a chaperone, binding to a subset of substrates, and maintained E. coli growth well at high temperatures.     

Molecular cloning and expression of the pyranose 2-oxidase cDNA from<Emphasis Type="Italic"> Trametes ochracea</Emphasis> MB49 in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28°C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg^–1). The effect of isopropyl -d-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28°C. The synthesis of P2O at 28°C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg^–1). At 25°C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg^–1. The soluble enzyme represented 70% of the total amount of P2O.     

A thermostable β-glucosidase isolated from a bacterial species of the genus Thermus

Summary An extremely thermophilic aerobic bacterium which produced -glucosidase was isolated from soil collected at the Yudanaka hot spring in Japan. It was identified as belonging to the genus Thermus. Production of -glucosidase by this bacterium was stimulated by the addition of cellobiose or laminaribiose to the medium. The optimum pH and temperature of the enzyme were 4.5–6.5 and 85° C respectively. The enzyme was stable in the pH range of 4.5–7.0 at 70° C for 2 h and the half-life at 75° C was 5 days. The K _m value of the enzyme for p-nitrophenyl--d-glucopyranoside, determined at 70° C in 0.1 M sodium phosphate buffer (pH 6.5), was 0.28 mM while the K _m was 2.0 mM for cellobiose. The enzyme effectively hydrolysed cellobiose at 70° C and the conversion yields of cellobiose to glucose were 95%, 93% and 90% at substrate concentrations of 5%, 10% and 15%, respectively.     

Cloning and high expression of glutaryl 7-aminocephalosporanic acid acylase gene from Pseudomonas diminuta

The gene coding for the glutaryl 7-aminocephalosporanic acid (GL 7-ACA) acylase from Pseudomonas diminuta KAC-1 was cloned and expressed in Escherichia coli. The acylase gene was composed of 2160 base pairs and encoded a polypeptide of 720 amino acid residues. The E. coli BL21 carrying pET2, the plasmid construct for high expression of GL 7-ACA acylase gene, produced this enzyme at approx. 30% of the total proteins with 3.2 units activity mg protein^–1. Growth at temperature below 31 °C and deletion of signal peptide increased the processing of precursor acylase to active enzyme in the recombinant E. coli cells.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Production of l-alanine from ammonium fumarate using two immobilized microorganisms

Summary For the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate -decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.     

Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts

The potential of 89 culturable cold-adapted isolates from uncontaminated habitats, including 61 bacterial and 28 yeast strains, to utilize representative fractions of petroleum hydrocarbons (n-alkanes, monoaromatic and polycyclic aromatic hydrocarbons) for growth and to produce various enzymes at 10°C was investigated. The efficiency of bacterial and yeast strains was compared. The growth temperature range of the yeast strains was significantly smaller than that of the bacterial strains. Sixty percent of the yeasts but only 8% of the bacteria could be classified as true psychrophiles, showing no growth above 20°C. A high percentage (89%) of the yeast strains showed lipase activity. More than one-third of the 61 bacterial strains produced amylase, -lactamase, -galactosidase or lipase; more than two-thirds were protease producers. Only 6% of the bacterial strains but 79% of the yeast strains utilized n-hexadecane for growth; 13% of the bacterial strains and 21–32% of the yeast strains utilized phenol, phenanthrene or anthracene for growth. Only four yeast strains but none of the bacterial strains could grow with all hydrocarbons tested. The biodegradation of phenol was investigated in fed-batch cultures at 10°C. Three yeast strains degraded phenol concentrations as high as 10 mm (one strain) or 12.5 mm (two strains). Of eight bacterial strains, two strains degraded up to 10 mm phenol. The optimum temperature for phenol degradation was 20°C for all eight bacterial strains and for two yeast strains. Biodegradation by five yeast strains was optimal at 10°C and faster at 1°C than at 20°C. All phenol-degrading strains produced catechol 1,2 dioxygenase activity.Communicated by K. Horikoshi     

Isolation of a new cellulase gene from a thermophilic anaerobe and its expression inEscherichia coli

Summary A new cellulase gene was cloned and expressed inEscherichia coli from a thermophilic anaerobe, strain NA10. A 7.4 kbEcoRI fragment of NA10 DNA encoded the cellulase which hydrolyzed carboxymethyl cellulose, lichenan, andp-nitrophenyl--d-cellobioside, but could not digest laminarin andp-nitrophenyl--d-glucoside. The cloned enzyme could digest cellooligosaccharides and release cellobiose as a main product from cellotetraose but could not digest cellobiose. It was distinct from the endoglucanase which was cloned by us previously from NA10 strain in terms ofp-nitrophenyl--d-cellobioside degradation activity and the location of restriction enzyme sites. The enzyme produced byE. coli transformant was extremely heat-stable and the optimum temperature for the enzymatic reaction was 80°C. Fifty three percent of the cloned enzyme was detected in the periplasm and the remaining activity existed in the cellular fraction in theE. coli transformant.     

Using native hydantoinase promoter to induce d-carbamoylase soluble expression in Escherichia coli

By use of PCR, the genes encoding d-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coli BL21 (DE3) to overexpress d-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (P_Hase) whose optimal length was confirmed to 209 bp. Furthermore, the RBS region in the downstream of P_Hase was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E. coli strain Top10F′. The enzyme activity of Top10F′/pGEMT-R-DCB grown at 37 °C was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 °C.     

Production and properties ofβ-glucosidase byNeurospora sitophila

Growth at 25°C and pH 5.50 favour the production of-glucosidase. De-fatted oilseed flour and Tween 80 enhanced the production of-glucosidase, Lactose, gentibiose, gentibiose-acetate, laminarabiose and xylobiose induced-glucosidase activity. Precipitation of the culture filtrate with (NH₄)₂SO₄ resulted in 26-fold purification with 67% recovery. The optimum pH and temperature for activity were 5.0 to 5.4 and 55°C respectively. The enzyme was stable at 40°C with half-life at 12 h at 50°C. TheK _m andV _max for the hydrolysis ofp-nitrophenyl--d-glucoside at 40°C H 5.0 are 0.28mm and 0.60 U/mg protein, respectively.     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin     

d-Xylanase produced by Schizophyllum radiatum

Summary d-Xylanase (1,4--xylan xylanohydrolase, EC 3.2.1.8) was obtained from mycelial submerged culture of the mushroom Schizophyllum radiatum, grown on wheat straw pretreated with steam explosion as the substrate. The enzyme was purified 192-fold (specific activity 455 IU mg^-1 protein), with 37% yield with respect to total d-xylanase activity. Polyacrylamide electrophoresis of the d-xylanase peak showed a single band of protein whose molecular weight, calculated by electrophoretic mobility, was 25 700. The enzyme exhibited maximum activity at pH 4.9 and 55°C. d-Xylanase was stable from pH 5.0 to 7.5; its half-life was 12 h at 45°C. The Michaelis constant was 9.5 mg ml^-1 and V _max 0.37 mole min^-1. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylobiose, d-xylotriose, d-xylotetraose, and d-xylopentose showing the mode of action of an endo-type enzyme.     

Overexpression of the gene forN-acylamino acid racemase fromAmycolatopsis sp. TS-1-60 inEscherichia coli and continuous production of optically active methionine by a bioreactor

A DNA sequence encodingN-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c. The recombinant plasmid was introduced intoEscherichia coli MM194 lysogenized with a bacteriophage having a T7 RNA polymerase gene. The amount of AAR produced by theE. coli transformant was 1100-fold more than that produced byAmycolatopsis sp. TS-1-60, the DNA donor strain. The AAR was purified to homogeneity from the crude extract of theE. coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography. Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR andd- orl-aminoacylase.d- orl-methionine was continuously produced with a high yield fromN-acetyl-dc-methionine by the bioreactor.     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

Purification and characterization of a Bacillus sp. SAM1606 thermostable α-glucosidase with transglucosylation activity

We purified a novel -glucosidase to homogeneity from an Escherichia coli recombinant transformed with the -glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known -glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O--d-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k _O) and the Michaelis constant (K _m) values at 55°C and pH 6.0 for sucrose were 54.6 s^–1 and 5.3 mm, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75°C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 m sucrose at 60°C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65°C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.     

Metabolism of d(+)-carnitine by Escherichia coli

Summary Escherichia coli 044 K74 grown under anaerobic conditions in the presence of l(–)-carnitine is able to convert d(+)-carnitine into the l(–)-enantiomer. This activity is repressed by electron acceptors such as oxygen and nitrate as well as by glucose. d(+)-Carnitine shows no effect on the induction or repression of the corresponding enzyme or enzyme system. Resting cells of E. coli 044 K74 were used for the formation of l(–)-carnitine from d(+)-carnitine. The maximum obtained yield was 50%. -Butyrobetaine was formed as a by-product. Offprint requests to: H. Jung     

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004     

Characterization of a major xylanase purified from Lentinula edodes cultures grown on a commercial solid lignocellulosic substrate

Summary The white-rot basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler is the dominant edible mushroom cultivated on wood. The major xylanase detected in cultures grown on a commercial oak wood medium was extracted, purified, and characterized. The enzyme was a non-debranching endo--d-xylanase (1,4--d-xylan xylanohydrolase; E.C.3.2.1.8) highly specific for xylans, with a molecular weight of 41 000 (on sodium dodecyl sulfate gels) and an isoelectric point of 3.6. With aspen glucuronoxylan as substrate, the enzyme showed optimal activity at pH 4.5–5.0 and 60° C, with a K _m of 0.66 mg/ml and specific activity of 310 units/mg protein at 40° C. It was capable of hydrolyzing (forming reducing sugars from) 40%–50% of the hydrolyzable linkages in either glucuronoxylan or arabinoxylan. The enzyme produced xylose and major identifiable products in the xylobiose or xylotriose (and presumably larger) size range including xylobiose and xylooligosaccharides, but neither glucuronic acid nor arabinose. Products were also produced from arabinoxylan that appeared to be arabinoxylobiose and arabinoxylotriose.Research carried out while a visiting scientist from the National Chemistry Laboratory, Pune, India 411008The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by US Government employees on official time, and it is therefore in the public domain and not subject to copyright Offprint requests to: G. F. Leatham