A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Purification and Biochemical Characterization of Digestive Lipase in Whiteleg Shrimp

Penaeus vannamei lipase was purified from midgut gland of whiteleg shrimp. Pure lipase (E.C. 3.1.1.3) was obtained after Superdex 200 gel filtration and Resource Q anionic exchange. The pure lipase, which is a glycosylated molecule, is a monomer having a molecular mass of about 44.8 kDa, as determined by SDS-PAGE analysis. The lipase hydrolyses short and long-chain triacylglycerols and naphthol derivates at comparable rates. A specific activity of 1787 U mg⁻¹ and 475 U mg⁻¹ was measured with triolein and tributyrin as substrates, respectively, at pH 8.0 and 30°C in the absence of colipase. The lipase showed a K _{m, app} of 3.22 mM and k _{cat, app}/K _{m, app} of 0.303 × 10³ mM⁻¹ s⁻¹ using triolein as substrate. Natural detergents, such as sodium deoxycholate, act as potent inhibitors of the lipase. This inhibition can be reversed by adding fresh oil emulsion. Result with tetrahydrolipstatin, an irreversible inhibitor, suggests that the lipase is a serine enzyme. Peptide sequences of the lipase were determined and compared with the full-length sequence of lipase which was obtained by the rapid amplification of cDNA ends method. The full cDNA of the pvl was 1,186 bp, with a deduced protein of 362 amino acids that includes a consensus sequence (GXSXG) of the lipase superfamily of α/β-hydrolase. The gene exhibits features of conserved catalytic residues and high homology with various mammalian and insect lipase genes. A potential lid sequence is suggested for pvl.     

Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader <Emphasis Type="Italic">Bacillus</Emphasis> sp. HH-01

An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4–11 and up to 30°C, and its optimum pH and temperature were 8–9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl₂, in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996     

Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1

The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced. Nucleotide sequence analysis and alignments of amino acid sequences suggest that Lip Ais a member of bacterial lipase family I.1 and that LipB is a lipase activator of LipA. The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB. In the hydrolysis of p-nitrophenyl esters and triacylglycerols, using the reactivated LipA, the optimum chain lengths for the acyl moiety on the substrate were C14 for ester hydrolysis and C10 to C12 for triacylglycerol hydrolysis.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.     

A novel psychrophilic lipase from Pseudomonas fluorescens with unique property in chiral resolution and biodiesel production via transesterification

A lipase-producing bacterium strain B68 screened from soil samples of China was identified as Pseudomonas fluorescens. With GenomeWalker, the open reading frame of lipase gene lipB68, encoding 476 amino acids, was cloned and expressed in Escherichia coli BL21 (DE3). By affinity chromatography, the recombinant LipB68 protein was purified to the purity of 95%. As a member of lipase subfamily I.3, LipB68 has a unique optimum temperature of 20 °C, which was the lowest in this subfamily. In chiral resolution, LipB68 effectively catalyzed the transesterification of both α-phenylethanol and α-phenylpropanol at 20 °C, achieving E values greater than 100 and 60 after 120 h, respectively. Among all the known catalysts in biodiesel production, LipB68 produced biodiesel with a yield of 92% after 12 h, at the lowest temperature of 20 °C, and is the first one of the I.3 lipase subfamily reported to be capable of catalyzing the transesterification reaction of biodiesel production. Since lipase-mediated biodiesel production is normally carried out at 35–50 °C, the availability of a highly active lipase with a low optimal temperature can provide substantial savings in energy consumption. Thus, this novel psychrophilic lipase (LipB68) may represent a highly competitive energy-saving biocatalyst.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

Purification and characterization of a lipase from Pseudomonas aeruginosa KKA-5 and its role in castor oil hydrolysis

An extracellular lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) from Pseudomonas aeruginosa KKA-5 hydrolyzed castor oil by 90%. Purification of this castor oil-hydrolyzing lipase included ammonium sulfate precipitation and successive hydroxylapatite column chromatography. The enzyme was purified 518-fold. It was homogeneous electrophoretically and its molecular weight was estimated to be 30 kDa. The enzyme was stable up to 45°C and retained its activity in the alkaline pH range. Lipase was highly stable in the presence of aqueous organic solvents like methanol and ethanol. It was weakly inhibited in the presence of acetone. The anionic surfactant, sodium dodecyl sulfate, was inhibitory while the cationic surfactants, Triton X-100 and Tween-80 appreciably enhanced activity. Lipase was stabilized significantly by Ca²⁺. Inactivation of the enzyme by EDTA was overcome by sequential CaCl₂ treatment. This finding suggests the existence of a calcium-binding site in Pseudomonas aeruginosa KKA-5 lipase. Received 22 January 1998/ Accepted in revised form 27 April 1998     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10⁻² U mg⁻¹, respectively. The Michaelis–Menten constant (K _m values) and the maximal reaction velocities (V _max values) of CelEdx16 were 47.1 μM and 9.6 × 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 × 10⁻⁵ μmole min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.     

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases from Thermosyntropha lipolytica

Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH^25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH^25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C_18:1) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca²⁺ and one Mn²⁺ ion per monomeric enzyme unit. The addition of 1 mM MnCl₂ to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Biotechnological application of Pseudomonas aeruginosa lipase: efficient kinetic resolution of amines and alcohols

 Pseudomonas aeruginosa secretes an extracellular lipase (EC 3.1.1.3), which has been isolated from culture media of either industrial fermentation of wild-type P. aeruginosa PAC1R or an overexpressing P. aeruginosa strain carrying a plasmid with the cloned lipase gene. Both culture supernatants contained enzymatically active lipase protein, as demonstrated by determination of hydrolytic activity using p-nitrophenylpalmitate and 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester as substrates and analysis by sodium dodacyl sulphate/polyacrylamide electrophoresis and Western blotting. Immobilization by entrapment into chemically inert hydrophobic silica gels was tested with crude enzyme preparations. A matrix consisting of tetramethoxysilane and propyltrimethoxysilane at a molar ratio of 1 : 5 yielded the highest enzyme activity as determined by esterification of lauric acid with 1-octanol in isooctane. The biotechnological potential of P. aeruginosa lipase to catalyse the kinetic resolution of chiral compounds was tested by enantioselective acylation of two different model compounds, racemic 1-phenylethanol and 2-pentylamine. Both compounds were acylated with high efficiency giving enantiomeric excess rates of more than 99% for the alcohol and 96% for the amine with an average conversion rate of 50%. These results demonstrated that P. aeruginosa lipase is an extremely useful enzyme for application in synthetic organic chemistry. Received: 5 February 1996/Received revision: 1 April 1996/Accepted: 15 April 1996     

Identification and characterization of a novel nitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pf-5

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min⁻¹ mg protein⁻¹ for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K _m = 17.9 mM and k _cat/K _m = 0.5 mM⁻¹ s⁻¹ for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.     

Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu²⁺, Co²⁺, Ni²⁺, and Zn²⁺, and this inhibition was reversed by EDTA.M Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996     

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.     

High-level expression of the thermoalkalophilic lipase from Bacillus thermocatenulatus in Escherichia coli

An efficient expression system for the previously only weakly expressed thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was developed for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYTEXP1 (pT1) expression vector downstream of the temperature-inducible λ promoter P_L. Three different expression vectors were constructed: (i) pT1-BTL2 containing the mature lipase gene, (ii) pT1-preBTL2 containing the prelipase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fused to the signal peptide of the OmpA protein, the major outer membrane protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expression levels of 7000–9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 660 000 soluble lipase U/g cells was produced, whereas, with E. coli DH5α and BL321, production levels of 30 000 U/g cells were achieved. However, most of the lipase remained insoluble but active after cell breakage because of the unprocessed OmpA signal peptide. A simple cholate extraction followed by proteinase K cleavage and ultrafiltration allowed the isolation of 1.15 × 10⁶ units of 90% pure mature lipase/wet cells. Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997