Characterization of an alpha-L-rhamnosidase from Aspergillus kawachii and its gene

An α-l-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on l-rhamnose as the sole carbon source. The α-l-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 °C. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 °C for 1 h. Its T ₅₀ value was determined to be 72 °C. The enzyme was able to hydrolyze α-1,2- and α-1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii α-l-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the α-l-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcusalbus

The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-β-d-glucopyranosyl-d-mannose and 4-O-β-d-galactopyranosyl-d-mannose (epilactose). Based on the sequence alignment with N-acetyl-d-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (α/α)₆ core barrel structure.     

An efficient method for N-acetyl-d-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system

N-Acetyl-d-neuraminic acid (Neu5Ac) is a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1. Two recombinant Escherichia coli strains capable of expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid aldolase were constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in Neu5Ac production. Carbon sources were optimized for Neu5Ac production, and the concentration effects of carbon sources on the production were investigated. With 2,200 mM pyruvate as carbon source and substrate, 61.9 mM (19.1 g l⁻¹) Neu5Ac was produced from 200 mM N-acetyl-d-glucosamine (GlcNAc) in 36 h by the coupled cells. Our Neu5Ac biosynthetic process is favorable compared with natural product extraction, chemical synthesis, or even many other biocatalysis processes.     

Galacto-oligosaccharide production using microbial β-galactosidase: current state and perspectives

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg⁻¹ by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80oC, and the half-life at 70oC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l⁻¹ galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l⁻¹ glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l⁻¹), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l⁻¹) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.     

Enzymatic synthesis of alkyl glucosides using <Emphasis Type="Italic">Leuconostoc</Emphasis> <Emphasis Type="Italic">mesenteroides</Emphasis> dextransucrase

Alkyl glucosides were synthesized by the reaction of Leuconostoc mesenteroides dextransucrase with sucrose and various alcohols. Alkyl α-d-glucosides were obtained with a yield of 30% (mol/mol) with primary alcohols, but secondary alcohols or tertiary alcohols gave yields below 5%. The optimal yield was 50% using 1-butyl α-d-glucoside with 0.9 M 1-butanol. The acceptor products of methanol or ethanol were confirmed as methyl α-d-glucopyranoside and ethyl α-d-glucopyranoside via MALDI-TOF MS and NMR analysis. Thus, methyl or ethyl α-d-glucoside constituted half the emulsification activities of Triton X-100 as commercially available surfactants. Young-Min Kim and Byung-Hoon Kim contributed equally to this work.     

Characterization of an acid-labile, thermostable β-glycosidase from Thermoplasma acidophilum

A recombinant putative β-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg⁻¹. The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90°C. The enzyme was unstable below pH 6.0: at pH 6 its half-life at 75°C was 28 days but at pH 4.5 was only 13 h. Catalytic efficiencies decreased as p-nitrophenyl(pNP)-β-d-fucopyranoside (1067) > pNP-β-d-glucopyranoside (381) > pNP-β-d-galactopyranoside (18) > pNP-β-d-mannopyranoside (11 s⁻¹ mM⁻¹), indicating that the enzyme was a β-glycosidase.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine production by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The l-phenylalanine (l-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved l-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N’-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in l-Phe production and a remarkable tolerance to 1 × 10¹⁰ pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced l-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH–stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on l-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ _set = 0.18 h⁻¹ in the first 20 h and a following linear varying rate feeding with F = (−0.55 × t + 18.6) ml/h, was developed to improve l-Phe production. With this two-stage feeding approach, a maximum l-Phe titer of 57.63 g/l with a high l-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential l-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).     

Comparative analysis of four <Emphasis Type="Italic">β</Emphasis>-galactosidases from <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> NCIMB41171: purification and biochemical characterisation

Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na⁺ and/or K⁺ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg⁺⁺ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺ ions exerting the most positive effect. Determination of the specificity constants (k _cat/K _m) clearly indicated that BbgI (6.11 × 10⁴ s⁻¹ M⁻¹), BbgIII (2.36 × 10⁴ s⁻¹ M⁻¹) and especially BbgIV (4.01 × 10⁵ s⁻¹ M⁻¹) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 10⁴ s⁻¹ M⁻¹) than lactose (1.48 × 10³ s⁻¹ M⁻¹). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.     

High-level Expression of an α-l-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the Production of Xylobiose from Xylan

To efficiently produce xylobiose from xylan, high-level expression of an α-l-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an α-l-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine–Dalgarno sequence. A maximum activity of 12 U mg⁻¹ was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed α-l-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and α-l-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 °C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of α-l-arabinofuranosidase gave only half this yield. Revisions requested 27 October 2005; Revisions received 5 September 2005     

Characterization of raffinose synthase from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. Nipponbare)

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45°C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-α-d-galactoside as galactosyl donors, and sucrose, lactose, 4−β-galactobiose, N-acetyl-d-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

Specific mutations within the α4–α5 loop of the bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry δ-endotoxins suggests that helices α4 and α5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the α4–α5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-β-d-thiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the α4–α5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.     

Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli

Summary A DNA fragment encoding for 598 amino acids of chitinase protein from Bacillus circulans No. 4.1 was subcloned into pQE-30 expression vector and transformed into Escherichia coli M15 (pREP4). The molecular weight of the expressed protein was approximately 66 kDa. Enzymatic activity of the recombinant protein was assayed after purification using affinity chromatography on a nickel chelating resin. The enzyme hydrolyzed N-acetylchitooligosaccharides mainly to N-acetylchitobiose, and was active toward chitin, carboxymethyl-chitin, colloidal chitin, glycol chitin and 4-methylumbelliferyl-β-d-N, N′-diacetylchitobiose. The pH and temperature optima of the chitinase enzyme were 7.0 and 45 °C, respectively. This enzyme was stable in the pH range of 5.0–9.0 and at temperatures up to 50 °C. In addition, when cleaved by a proteolytic enzyme, the 20-kDa product could retain high chitinolytic activity.     

Production of d-hydantoinase by halophilic Pseudomonas sp. NCIM 5109

About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production. The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline conditions allowed the conversion of 80 g l⁻¹ dl-5-phenylhydantoin to 82 g l⁻¹ d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%. Received: 15 September 1997 / Received revision: 5 January 1998 / Accepted: 6 January 1998     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Comparison of angiotensin converting enzyme-like activity in the Antarctic teleosts <Emphasis Type="Italic">Trematomus bernacchii</Emphasis> and <Emphasis Type="Italic">Chionodraco hamatus</Emphasis>

Biochemical parameters of the angiotensin converting enzyme-like activity (ACELA) in the gills of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii were characterized. Enzymatic activity was revealed following hydrolysis of a specific substrate of angiotensin-converting enzyme N-[3-(2-furyl)acryloyl]l-phenylalanyl-glycyl-glycine (FAPGG) and metabolites were separated by reverse phase HPLC analysis. The results showed similar Km values for the substrate FAPGG at 5°C for the two species with an increase of Km value for T. bernacchii at 25°C. The optimum pH value was 8.5 at 25°C and optimum chloride concentrations were about 300 mM. In T. bernacchii the optimum temperature for maximum enzyme activity was 50°C, while maximum activity in C. hamatus occurred at 35°C. Lisinopril was more efficient in inhibiting ACELA in C. hamatus with an I ₅₀ value of 16.83 ± 5.11 nM, compared to an I ₅₀ value of 30.66 ± 5.19 nM in T. bernacchii. In conclusion, it appears that some biochemical parameters of ACELA in C. hamatus differ from those in T. bernacchii, probably due to different ways that the enzyme adapts to the constantly cold temperatures of the animal’s environment.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.     

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.