Influence of cultivation conditions on the production of a thermostable extracellular lipase from Amycolatopsis mediterranei DSM 43304

Among several lipase-producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture conditions and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 ± 0.103 IU/ml of culture medium was obtained in 96 h at 28°C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. Under optimal culture conditions, the lipase activity was enhanced 12-fold with a twofold increase in lipase specific activity. The lipase showed maximum activity at 60°C and pH 8.0. The enzyme was stable between pH 5.0 and 9.0 and temperatures up to 60°C. Lipase activity was significantly enhanced by Fe³⁺ and strongly inhibited by Hg²⁺. Li⁺, Mg²⁺ and PMSF significantly reduced lipase activity, whereas other metal ions and effectors had no significant effect at 0.01 M concentration. A. mediterranei DSM 43304 lipase exhibited remarkable stability in the presence of a wide range of organic solvents at 25% (v/v) concentration for 24 h. These features render this novel lipase attractive for potential biotechnological applications in organic synthesis reactions.     

Short-chain flavour ester synthesis by immobilized lipase in organic media

Summary Mucor miehei lipase has been adsorbed on Celite and covalently bound to nylon. The obtained derivatives have been studied regarding their ability for synthetize several flavouring esters in biphasic aqueous/organic media. The influence of the immobilization procedure on the synthetic activity of the derivatives was considered. Solvent hydrophobicity and water content in the biphasic system influenced both enzyme stability and equilibrium displacements. In this way, solvents with log P>3.5 and less than 1% water were optimal. It was important to consider pH effects on enzyme microenvironment when using acidic substrates. Optimum temperature and reuse of the catalyst were also checked.     

地中海拟无枝菌酸菌(Amycolatopsis mediteranei)U-32硝酸还原酶的定位、纯化及性质

在力复霉素ＳＶ研究中,发现硝酸盐对抗生素合成呈现多效性作用,不仅大幅度提高产量,还对产生菌——地中海拟无枝菌酸菌生理产生多方面的影响,从而提出整体性调节的结论．这一多效性作用是由硝酸盐所引起的,为此对硝酸还原酶进行了研究．首先,通过原生质体渗透裂解发现地中海拟无枝菌酸菌Ｕ－３２的硝酸还原酶是一个胞质酶．该酶极不稳定,缓冲液中加入硝酸钾、甘油等保护剂能极大地提高其稳定性．通过硫酸鱼精蛋白沉淀,硫酸铵分级分离,Ｐｈｅｎｙｌ－ＳｅｐｈａｒｏｓｅＣＬ４Ｂ、Ｂｉｏ－ＧｅｌＡ１．５ｍＤＥＡＥ－Ｓｅｐｈａｃｅｌ和ＳｅｐｈａｄｅｘＧ－７５柱层析等多步纯化得到了电泳纯的硝酸还原酶．该酶为一７９ｋＤ的单亚基酶,每分子酶含有约２．２９原子的钼,但并不含有非血红素铁、酸不稳定硫、ＦＭＮ及ＦＡＤ,其等电点为６．２,反应最适ｐＨ为７．２,最适温度为４０℃．对硝酸根的Ｋｍ值为１３．３μｍｏｌ／Ｌ．同时分析了该酶的吸收光谱．     

地中海拟无枝酸杆菌甲基丙二酰CoA转羧基酶的纯化及酶学特性

经硫酸铵沉淀,ＤＥＡＥ－纤维素吸附,磷酸纤维素吸附和Ｓｅｐｈａｒｏｓｅ４Ｂ分子筛层析四步从地中海拟无枝酸杆菌纯化得到电泳纯ＭＣＴ酶,酶比活力为３．２１Ｕ／ｍｇ,纯化倍数１７８,酶活回收１４．９％。酶反应的最适ｐＨ和温度分别为７．０和３５℃。纯化ＭＣＴ酶对底物丙酰ＣｏＡ和草酰乙酸的米氏常数分别为０．０２７ｍｍｏｌ／Ｌ０．０３５０９ｍｍｏｌ／Ｌ．经ＳｅｐｈａｄｅｘＧ－１５０测定酶分子量为２０００００,ＳＤＳ－取丙烯酰胺电泳凝胶显示一条分子量６８０００的亚基蛋白带,说明该酶由三个等大小亚基组成．薄层等电聚焦测定酶等电点为ｐＩ６．０．二价金属离子Ｃｏ￣（２＋）和Ｆｅ￣（３＋）促进酶活力．采用原生质体裂解的方法发现ＭＣＴ酶是可能分布于胞浆和细胞膜上．     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095.

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

Purification and properties of DAHP synthase from Nocardia mediterranei

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the first enzyme of the shikimate pathway was isolated from Nocardia mediterranei. It has a molecular weight of approx. 135,000, and four identical subunits, each with a molecular weight of 35,000. The Km values for phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E-4-P) were 0.4 and 0.25 mM, respectively, and kinetic study showed that LTrp inhibited DAHP synthase activity, but was not competitive with respect to PEP or E-4-P. The enzyme activity was inhibited by excess of E-4-P added in the incubation system. D-ribose 5-phosphate (R-5-P), D-glucose 6-phosphate (G-6-P) or D-sedoheptulose 7-phosphate (Su-7-P) etc. inhibited DAHP synthase in cell-free extract, but on partially purified enzyme no inhibitory effect was detected. The indirect inhibition of R-5-P and other sugar phosphates was considered to be due to the formation of E-4-P catalyzed by the related enzymes present in cell-free extract.     

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Novel minor lipase from Rhizopus chinensis during solid-state fermentation: Biochemical characterization and its esterification potential for ester synthesis

Rhizopus chinensis produces two lipases that catalyze ester synthesis when cultured under solid-state fermentation. The Lip2 was purified to homogeneity by ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. It has an apparent molecular weight of 33 kDa estimated from SDS–PAGE and 32 kDa calculated from analytical gel permeation, with synthetic activity and purification fold of 96.8 U/mg and 138.3, respectively. Maximum hydrolytic activity was obtained at pH 8.0–8.5 and 40 °C using pNPP as substrate. Slight activation of the enzyme was observed when Mn²⁺ is present. The enzyme was most active on p-nitrophenyl laurate (C12). The purified lipase exhibited maximum synthetic activity at pH memory of 6.0 and 30 ^oC. Most of ethyl esters synthesized by lyophilized enzyme achieved good yields (>90%), and caprylic acid served as the best acyl donor. The enzyme presented a particular affinity for ethanol, n-propanol and n-hexanol, with conversion of 92%, 93% and 92%, respectively, after 20 h incubation.     

Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch cultures

The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis mediterranei in batch and fed-batch culture was investigated. Fed-batch culture using glucose as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with constant feeding rate, in bioreactor level, proved to be an alternative production system with a significant increase in both volumetric and specific antibiotic production. The maximal concentrations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained in fed-batch culture in bioreactor level under non-oxygen limitation. On the other hand, the rate of rifamycins production was increased from 6.58 to 12.13 mg/l x h for rifamycin B and from 9.47 to 31.83 mg/l x h for rifamycin SV on the bioprocess transfer and improvement from the conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.     

Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis

A novel and simple method was developed for the preparation of magnetic Fe₃O₄ nanoparticles by chemical co-precipitation method and subsequent coating with 3-aminopropyltrimethoxysilane (APTMS) through silanization process. Magnetic Fe₃O₄-chitosan particles were prepared by the suspension cross-linking and covalent technique to be used in the application of magnetic carrier technology. The synthesized immobilization supports were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Using glutaraldehyde as the coupling agent, the lipase from R. oryzae was successfully immobilized onto the functionalized magnetic Fe₃O₄-chitosan beads. The results showed that 86.60% of R. oryzae lipase was bound on the synthesized immobilization support. This immobilized lipase was successfully used for the esterification of phenolic acid which resulted in esterification of phenolic acid in isooctane solvent reaction system for 8 consecutive cycles (totally 384 h), 72.6% of its initial activity was retained, indicating a high stability in pharmaceutical and industrial applications.     

Purification and properties of an acetyl specific carboxylesterase from Nocardia mediterranei

Summary An acetyl specific carboxylesterase has been purified from Nocardia mediterranei. The purified enzyme is homogeneous as shown by SDS polyacrylamide gel electrophoresis. The esterase has a molecular weight of 68,000 and is composed of two identical subunits. The enzyme exhibits optimal activity at pH 7.5 and at 35°C and is stable below 40°C. The enzyme activity is inhibited by several sulfhydryl reagents. The esterase hydrolyzes preferentially acetyl esters. Propionyl esters are cleaved very slowly whereas butyryl esters are no substrates at all. In addition, the esterase shows a pronounced regiospecificity. On the other hand the enantiospecificity is rather low as demonstrated by the hydrolysis of prochiral and racemic substrates.     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei.

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Production of rifamycin B and SV by free and immobilized cells of Amycolatopsis mediterranei

The effect of growth conditions (incubation time, inoculum size, initial pH value) and some nutrient concentrations on the growth and rifamycin B and SV production by free and immobilized cells of Amycolatopsis mediterranei CBS 42 575 was studied. In alginate beads, the immobilized cells behaved like the free cells, but a pronounced difference was observed in antibiotic production and cell growth. The rifamycin production by the immobilized cells was higher than that obtained by the free cells. The immobilized cells were also reused repeatedly for six batch cultivations with a fresh medium charged into flasks at the beginning of each batch. It was found that the immobilized cells were stable, and the rifamycin yield was almost constant during the first three batches and then decreased.     

pH-imprinted lipase catalyzed synthesis of dextran fatty acid ester

The application of enzymatic catalysis for the synthesis of polysaccharide-based surfactants was investigated. The polysaccharide dextran, a neutral bacterial polysaccharide consisting of -1,6 linked glucose units, was chemically modified by the attachment of hydrophobic groups through a transesterification reaction with a vinyl decanoate. A screening of commercially available lipases and protease for the synthesis of amphiphilic polysaccharides in DMSO suggested that lipase AY from Candida rugosa modified dextran T-40 with vinyl decanoate at the highest conversion. A pH-adjustment in a phosphate buffer at pH 7.5 prior to use is crucial to make this enzyme active in DMSO. The effect of enzyme concentration and mole ratio of fatty ester to dextran T-40 on the conversion and the rate of reaction were studied. Finally, investigation of the kinetics and regioselectivity of lipase AY-catalyzed modification offer a possibility to regulate the position and the extent of hydrophobic group attached to dextran. These two properties are fundamental for controlling the physico-chemical properties of the final polymeric surfactants.     

Purification and some properties of Pseudomonas fluorescens lipase

Lipase (triacylglycerol lipase, EC 3.1.1.3) has been purified from Pseudomonas fluorescens wild strain by chromatography on DEAE-cellulose and octyl-Sepharose CL-4B. The yield was 21% and the specific activity of the purified enzyme 4780 U/mg protein. It showed a Mr of about 45 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is active over a wide pH range and at 50-55 degrees C.     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.