Production of hydantoinase fromPseudomonas fluorescens strain DSM 84

Summary Pseudomonas fluorescens strain DSM 84 was selected as a good hydantoinase (dihydropyrimidinase E.C. 3.5.2.2.) producer from a screening involving 60 collection strains. Optimization of the culture and growth conditions were performed in order to increase the enzyme production. A mineral medium supplemented with 10 g/l of yeast extract having an initial pH of 7.1±0.1 but containing no additional carbon source or inducer was devised. The strain DSM 84 was found to produce the maximal level of hydantoinase in the defined mineral medium within 15 h of incubation at 27°C. When using 5-isopropylhydantoin as substrate, N-carbamyl-valine was detected as the end product of the crude hydantoinase. Conditions leading to the isolation and conservation of a crude hydantoinase as well as its temperature and pH stability are described.     

Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747.

The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.     

Conditions used with a continuous cultivation system to screen for d-hydantoinase-producing microorganisms

The continuous cultivation technique has been used to screen for microorganisms producing d-hydantoinase, a biocatalyst involved in the production of optically active amino acids. Pseudomonas putida strain DSM 84 was used as a model hydantoinase producer to establish selective culture conditions through the addition of various pyrimidines, dihydropyrimidines, hydantoins and 5-monosubstituted hydantoins. Thymine induced more activity than all cyclic amides tested. Addition of thymine as a non-metabolised inducer at a concentration of 0.05 g l^–1 in a continuous culture of P. putida stimulated hydantoinase production up to 80 times the basal level. Using continuous culture conditions established with the model strain, a different strain of P. putida having hydantoinase activity was isolated from commercial mixed cultures of microorganisms. DNA fingerprinting revealed that this new isolate was distinct from strain DSM 84. When used as a probe, the d-hydantoinase gene of strain DSM 84 hybridized with the DNA of the new P. putida isolate.     

Production of L-tryptophan from D,L-5-indolylmethylhydantoin by resting cells of a mutant of Arthrobacter species (DSM 3747).

The reaction parameters and the stereospecificity of the enzymatic cleavage of D,L-5-indolylmethylhydantoin in producing L-tryptophan with resting cells of Arthrobacter sp. DSM 3747 were studied. When intact cells were tested, the optimal pH was between 8.5 and 9.0 and the optimal temperature was 50 degrees C. Both, L-N-carbamoylase and hydantoinase could be stabilized over 24 h at 30 and 40 degrees C by the addition of D,L-5-indolylmethylhydantoin. Furthermore, the hydantoinase was stable over 24 h at 50 degrees C by the addition of 0.5 mM Mn2+ ions. The treatment with sodium desoxycholate turned out to be successful in overcoming the poor availability of D,L-5-indolylmethylhydantoin for the cells. The optimal temperature with permeabilized cells decreased to 30 degrees C and therefore ensured a good enzyme stability. While the L-N-carbamoylase proved to be absolutely L-specific, the hydantoinase led to a mixture of enantiomers of N-carbamoyltryptophan. The produced D-N-carbamoyl-tryptophan caused an inhibition of the L-N-carbamoylase. The transformation yield from D,L-5-indolylmethylhydantoin always reached 100%.     

Thermostable hydantoinase from a hyperthermophilic archaeon, Methanococcus jannaschii

A hyperthermophilic hydantoinase from Methanococcus jannaschii with an optimum growth at 85°C was cloned and expressed in E. coli. The recombinant hydantoinase was purified by affinity and anion-exchange chromatography and determined to be homotetrameric protein by gel filtration chromatography. The best substrate for the hydantoinase was D,L-5-hydroxyhydantoin, which has the specific activity of 183.4 U/mg. The optimum pH and temperature for the hydantoinase activity was 8.0 and 80°C, respectively. The half-life of the hydantoinase was measured to be 100 min at 90°C in the buffer containing 500 mM KCl. Manganese ions were the most effective for the hydantoinase activity. Stereospecificity was determined to be L-specific for the 5-hydroxymethylhydantoin and 5-methylhydantoin by chiral TLC. The activity yields as well as the operational stabilities of the thermostable M. jannaschii hydantoinase could be significantly improved by immobilization method.     

Catalytic and structural function of zinc for the hydantoinase from Arthrobacter aurescens DSM 3745

Metal dependency of the hydantoin amidohydrolase (hydantoinase) from Arthrobacter aurescens DSM 3745 has been analyzed based on kinetic studies of metal/chelator-caused enzyme inactivation, denaturation and reactivation, accompanied by the identification of specific metal binding ligands. The enzyme can be inactivated by metal chelating agents and—apart from the loss of its activity—completely dissociates into its subunits. Enzyme activity can be restored from recollected monomers by the addition of cobalt, manganese or zinc-ions, whereas nickel and magnesia remain ineffective. Subjection of the hydantoinase to metal analysis reveals a content of 10 mol zinc per mol enzyme. Zinc plays an essential role not only for the catalytic activity but also for the stabilization of the active quarternary structure of the hydantoinase. Histidine-specific chemical modification of the enzyme causes a complete loss of the catalytic activity and reveals histidine residues as putative zinc binding ligands. Both, the metal/chelator-caused enzyme inactivation as well as the metal-caused enzyme reactivation, can be reduced in the presence of the substrate. Therefore, it is very likely that at least one metal-ion acts specifically near or at the active site of the enzyme.     

Optimization of the immobilization parameters and operational stability of immobilized hydantoinase and l-N-carbamoylase from Arthrobacter aurescens for the production of optically pure l-amino acids

2The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His(6)-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH(2))-immobilized hydantoinase and EAH-Sepharose-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements.In comparison to the free enzymes, temperature and pH-optima were increased by 10 degrees C and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60 degrees C (60 degrees C).In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor.     

Thermal stability of beta-xylanases produced by different Thermomyces lanuginosus strains

The thermostability of beta-xylanases produced by nine thermophilic Thermomyces lanuginosus strains in a coarse corn cob medium was assessed. The xylanase produced by T. lanuginosus strain SSBP retained 100% of its activity after 6 h at temperatures up to 65 degrees C. In comparison seven ATCC strains and the DSM 5826 strain of T. lanuginosus only retained 100% xylanase activity at temperatures up to 60 degrees C. Culture filtrates of T. lanuginosus strain SSBP grown on coarse corn cobs, oatspelts xylan, birchwood xylan, wheatbran, locust beangum, and sugar cane bagasse, retained 100% xylanase activity at temperatures up to 60 degrees C. The xylanase produced on corn cobs was the most thermostable and showed an increase of approximately 6% from 70 degrees C to 80 degrees C. The T(1/2) of all strains at 70 degrees C at pH 6.5 varied greatly from 63 min for strain ATCC 28083 to 340 min for strain SSBP. The xylanase of strain SSBP was much less thermostable at pH 5.0 and pH 12.0 with T(1/2) values of 11.5 min and 15 min, respectively at 70 degrees C. At 50 degrees C, the enzyme of T. lanuginosus strain SSBP produced on coarse corn cobs was stable within the pH range of 5.5-10.0. Furthermore, the enzyme retained total activity at 60 degrees C for over 14 days and at 65 degrees C for over 48 h. The xylanase of T. lanuginosus strain SSBP possesses thermo- and pH stability properties that may be attractive to industrial application.     

Thermostable d-hydantoinase from thermophilic Bacillus stearothermophilus SD-1: characteristics of purified enzyme

One thousand thermophiles isolated from soils were screened for hydantoinase and its thermostability. One thermophilic bacterium that showed the highest thermostability and activity of hydantoinase was identified to be Bacillus stearothermophilus SD-1 according to morphological and physiological characteristics. The hydantoinase of B. stearothermophilus SD-1 was purified to homogeneity via ammonium sulfate fractionation, anion-exchange chromatography, heat treatment, hydrophobic-interaction chromatography, and preparative gel electrophoresis. The relative molecular mass of the hydantoinase was determined to be 126 kDa by gel-filtration chromatography, and a value of 54 kDa was obtained as a molecular mass of the subunit on analytical sodiumdodecylsulfate/polyacrylamide gel electrophoresis. The hydantoinase was strictly d-specific and metal-dependent. The optimal pH and temperature were about 8.0 and 65°C respectively, and the half-life of the d-hydantoinase was estimated to be 30 min at 80°C, indicating the most thermostable enzyme so far.     

解糖热解纤维素菌F32降解未预处理秸秆及产纤维素酶特性分析

【目的】明确极端嗜热厌氧木质纤维素降解菌解糖热解纤维素菌F32代谢特征,并分析其产酶特性。【方法】使用细胞计数法绘制菌株的生长曲线,使用离子色谱及气相色谱进行产物和残糖量分析,以DNS法及对硝基苯酚法检测菌株胞外蛋白的酶活性。【结果】解糖热解纤维素菌F32在以葡萄糖、微晶纤维素和未经预处理小麦秸秆为碳源时生长状况优于解糖热解纤维素菌DSM 8903。在以葡萄糖为碳源进行培养时,与菌株DSM 8903相比,菌株F32具有产乳酸较多,而产氢气较少的特点。在以微晶纤维素和未经预处理小麦秸秆为碳源进行培养时,与菌株DSM 8903相比,菌株F32胞外蛋白具有较高的内切纤维素酶活性和木聚糖酶活性。【结论】解糖热解纤维素菌F32表现出较强的木质纤维素降解能力,其与DSM 8903的产物组成及胞外蛋白的酶活性具有明显差异。     

Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine

Using directed evolution, we have improved the hydantoinase process for production of L-methionine (L-met) in Escherichia coli. This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst. The selectivity of all known hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid. We used random mutagenesis, saturation mutagenesis, and screening to convert the D-selective hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme and increased its total activity fivefold. Whole E. coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h. The improved hydantoinase increased productivity fivefold for >90% conversion of the substrate. The accumulation of the unwanted intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway. Highly D-selective hydantoinase mutants were also discovered. Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.     

Arthrobacter K1108乙内酰脲酶反应条件和立体选择性研究

研究了Arthrobacter K1108乙内酰脲酶的反应条件,结果表明,K1108乙内酰脲酶的最适反应温度为55℃,最适pH为7.0,Co^2 和Fe^2 对该酶有激活作用,而Ca^2 有严重抑制作用。K1108乙内酰脲酶的底物专一性较强,其最适底物为5－苄基乙内酰脲,5－苯基乙内酰脲和5－吲哚甲基乙内酰脲均不能作为其有效底物。对K1108乙内酰脲酶立体反应机制研究结果表明,其乙内酰脲水解酶不具立体选择性,决定产物立体构型的酶是N－氨甲酰氨基酸水解酶。     

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.     

Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp.

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3'-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.     

Thermostable xylanase from Marasmius sp.: purification and characterization

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as 90 degrees C. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of 90 degrees C. When using xylan from birchwood as substrate, it exhibits Km and Vmax values of 2.6 +/- 0.6 mg/ml and 428 +/- 26 U/mg, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to 70 degrees C. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at 70 degrees C for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.     

Burkholderia piCkettii中D-乙内酰脲酶基因(dha)的克隆、测序及表达

对一株能转化D,L－对羟基苯乙内酰脲为D－对羟基苯甘氨酸的菌株MMR003进行了细菌分类学鉴定,该菌为皮氏伯克霍尔德氏菌（Burkholderia pickettii)。实验通过Southern杂交,部分文库构建和筛选,并经一系列亚克隆分析,获得一长度为1374bp的完整开放阅读框,编码458个氨基酸的D－乙内酰脲酶基因。用该基因序列构建的高表达质粒xXZPH2转化E.coliBL21(DE3),经IPTG诱导后,检测到D－乙内酰脲酶活性。该基因编码的氨基酸序列经Blast同源比较分析与放射形土壤杆菌NRRL B11291所产相应酶有85％的同源性。以D,L－对羟基苯乙内酰脲为底物测得的表达酶的活力为0.66u/mL,比相同条件下所测出发菌株MMR003的酶活提高了2倍。     

Cellobiohydrolase from Clostridium thermocellum, synthesized by a recombinant E. coli strain]

Clostridium thermocellum cellobiohydrolase was isolated in preparative amounts from the recombinant strain of E. coli K12 C600 carrying plasmid pCU 304 with a C. thermocellum chromosomal DNA insertion. The isolation procedure included chromatography on Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose CL-6B, rechromatography on Ultrogel and FPLC on Mono Q resulting in a 17.6% yield and 1530-fold purification. According to data from sodium dodecylsulfate polyacrylamide gel electrophoresis performed under nondenaturing conditions and analytical gel isoelectrofocusing, the enzyme preparation contains only one active protein band with Mr 56.2 +/- 1.0 kDa and pI 4.15. The enzyme does not reduce the viscosity of the CM-cellulose solution but forms reducing sugars from this soluble substrate. Cellobiose (93-97%) is the major component produced by the enzyme from crystalline and amorphous cellulose (specific activity 2.3 x 10(-3) and 2.8 x 10(-2) U/mg, respectively). The activity optimum of the enzyme is at pH 5.6, 60 degrees C. The half-inactivation time at 60 degrees C and 65 degrees C is 450 and 15.5 min, respectively. The action pattern of the enzyme on the low molecular fluorogenic cellooligosaccharides suggests that the enzyme pertains to typical cellobiohydrolases.     

Selective inactivation of the deoxyadenosine phosphorylating activity of pure human deoxycytidine kinase: stabilization of different forms of the enzyme by substrates and biological detergents

Deoxycytidine kinase, purified from human leukemic spleen to apparent homogeneity, is a multisubstrate enzyme that also phosphorylates purine deoxyribonucleosides [Bohman & Eriksson (1988) Biochemistry 27, 4258-4265]. In the present investigation we show that the stability and temperature dependence of dCyd kinase activity differed appreciably from the dAdo kinase activity of the same pure enzyme. Selective inactivation of dAdo activity was observed upon an incubation of the enzyme at both 4 and 37 degrees C. The half-life of dAdo activity at 4 degrees C increased from 36 to 84 h, when the protein concentration was increased by addition of bovine serum albumin. However, the half-life of dCyd activity increased from 72 h to more than 7 days under the same conditions. dCyd activity was stable for at least 6 h at 37 degrees C while the half-life of dAdo activity was 2 h. The presence of substrates like ATP, dTTP, or dAdo stabilized dAdo activity at both temperatures, and full maintenance of both activities at 37 degrees C was obtained by the addition of the zwitterionic detergent CHAPS. Furthermore, thermal inactivation of the dAdo activity occurred at a lower temperature (48 degrees C) as compared to the dCyd activity (54 degrees C). The presence of protease inhibitors had no effect on enzyme inactivation, nor was there a difference in the subunit structure of the selectively inactivated enzyme as compared to the fully active form, as revealed by size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium

Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C. The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).