灰色链霉菌中乙酰木聚糖酯酶基因的克隆、表达及酶学性质研究

根据Gen Bank数据库中已报道的灰色链霉菌(Streptomyces griseus)全基因组序列,分析得到假定的乙酰木聚糖酯酶基因序列并设计引物;利用分子克隆的方法得到该菌株基因组中乙酰木聚糖酯酶基因,并构建原核表达载体p ET28a-Sgraxe,经IPTG诱导表达重组Sgr Axe,Ni-NTA亲和层析法纯化该蛋白。结果显示,克隆得到乙酰木聚糖酯酶基因axe,其序列全长1 008 bp,编码336个氨基酸。SDS-PAGE检测带有p ET28a-Sgraxe转化菌株诱导表达产物相对分子量约为37 k D,与理论值相符。纯化的重组Sgr Axe酶学性质表明,该酶最适反应温度为50℃,最适p H8.0,热稳定性较强,p H作用范围广;金属离子对酶均表现为抑制作用,尤其是Zn2+严重抑制酶活力;重组酶特征的分析揭示了其在工业中潜在的应用价值。     

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (K_m) and V_max for α-naphthyl acetate were 1.54 mM and 360 μmol min⁻¹ mg of protein⁻¹, respectively.     

白色链霉菌乙酰木聚糖酯酶基因的克隆及生物信息学分析

乙酰木聚糖酯酶可以水解乙酰化木聚糖中的O-乙酰取代基团,消除该基团对木聚糖酶水解的空间阻碍作用,增强木聚糖酶对木聚糖的亲和力和降解能力。以白色链霉菌基因组为模板,利用简并PCR和TAIL-PCR扩增获得长约741 bp阅读框片段,编码247个氨基酸。生物信息学分析表明,该多肽片段具有AXE1家族蛋白保守区域;与已知的乙酰木聚糖酯酶蛋白C端区相比,相似性较高,二级和三级结构空间排布特点极为相似;初步判定该多肽片段为白色链霉菌乙酰木聚糖酯酶的C端区域。     

Purification and Characterization of a Lovastatin Esterase from Clonostachys compactiuscula

An esterase from the fungus Clonostachys compactiuscula selectively hydrolyzes lovastatin, a clinically useful antihypercholesterolemic agent. Lovastatin or lovastatin-related compounds were required to induce the activity of the lovastatin 8(prm1)-((alpha)-methylbutyryloxy) esterase. The 46-kDa esterase was purified from mycelial extracts by centrifugation and a single anion-exchange chromatographic separation. Maximal lovastatin esterase activity was found at pH 9.0 to 9.6 and at 25 to 30(deg)C. The addition of 5 to 20% methanol resulted in greater lovastatin hydrolysis, while the addition of other solvents (ethanol, isopropanol, butanol, ethyl acetate, isopropyl acetate, or tetrahydrofuran) decreased hydrolysis. Lovastatin was selectively hydrolyzed even in the presence of an excess of simvastatin, another antihypercholesterolemic agent that is structurally very similar to lovastatin. This lovastatin 8(prm1)-((alpha)-methylbutyryloxy) esterase can be used to prepare a core intermediate for the generation of novel antihypercholesterolemic agents or to purify simvastatin prepared by C methylation of the 2(S)-methylbutyryloxy side chain of lovastatin.     

Thermostable Flavin Reductase That Couples with Dibenzothiophene Monooxygenase,from Thermophilic Bacillus sp. DSM411: Purification,Characterization, and Gene Cloning

Flavin reductase is essential for the oxygenases involved in microbial dibenzothiophene (DBT) desulfurization. An enzyme of the thermophilic strain, Bacillus sp. DSM411, was selected to couple with DBT monooxygenase (DszC) from Rhodococcus erythropolis D-1. The flavin reductase was purified to homogeneity from Bacillus sp. DSM411, and the native enzyme was a monomer of M_r 16 kDa. Although the best substrates were flavin mononucleotide and NADH, the enzyme also used other flavin compounds and acted slightly on nitroaromatic compounds and NADPH. The purified enzyme coupled with DszC and had a ferric reductase activity. Among the flavin reductases so far characterized, the present enzyme is the most thermophilic and thermostable. The gene coded for a protein of 155 amino acids with a calculated mass of 17,325 Da. The enzyme was overproduced in Escherichia coli, and the specific activity in the crude extracts was about 440-fold higher than that of the wild-type strain, Bacillus sp. DSM411.     

Purification,Characterization, and Molecular Cloning of Tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.     

Purification, Characterization and Cloning of Phospholipase D from Peanut Seeds

We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K _m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl₂ and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.     

Cloning,Expression, Purification,and Characterization of a Novel Esterase from <Emphasis Type=Italic>Lactobacillus plantarum</Emphasis>

Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His₆-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40°C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.     

Overexpression, Purification, and Biochemical Characterization of the Esterase Est0796 from Lactobacillus plantarum WCFS1

Lactobacillus plantarum is a lactic acid bacterium that is encountered in an extensive range of foods such as fermented dairy products, meat, vegetables, and bakery products. Given the little molecular information available on the lipolytic activity of L. plantarum, the aim of this study was to clone, purify, and biochemically characterize the esterase coded by gene lp_0796 (Est0796). The esterase was cloned in pET28a and purified in two steps, using solid ammonium sulfate and His tag affinity chromatography. The molecular mass of the purified Est0796 was 28.7 kDa (by SDS-PAGE) and 26.6 kDa (by gel filtration chromatography), pointing to a monomeric structure. Est0796 showed maximum activity at pH 8.0 and 35 °C and toward shorter acyl chain lengths (C₂–C₄). The activity was resistant to organic solvents and cations, suggesting that this esterase may play a role in the fermentation of food products.     

Gene Cloning, Purification, and Characterization of a Phosphodiesterase from Delftia acidovorans

A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65°C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a K_m of 2.9 ± 0.1 mM and a k_cat of 879 ± 73 min⁻¹. The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E. coli. The IS1071 transposon insertion sequence was found downstream of pdeA.     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.     

Screening, Nucleotide Sequence, and Biochemical Characterization of an Esterase from Pseudomonas fluorescens with High Activity towards Lactones

A genomic library of Pseudomonas fluorescens DSM 50106 in a λRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43°C. The showed highest purified enzyme activities towards lactones. The activity increased from γ-butyrolactone (18.1 U/mg) to -caprolactone (21.8 U/mg) to δ-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.     

Production and Characterization of a Recombinant Cold-Active Acetyl Xylan Esterase from Psychrophilic Paenibacillus sp. R4 Strain

Applied Biochemistry and Microbiology - Acetyl xylan esterases (AXEs) hydrolyze the specific ester linkages between acetic acid and xylose units, leading to the deacetylation and depolymerization...     

重组人可溶性B淋巴细胞刺激因子细菌表达、纯化及其对B淋巴细胞的增殖作用

采用PCR法从人胎盘cDNA文库中扩增出人B淋巴细胞刺激因子（hBLyS）的cDNA;再用巢式PCR法（Nest-PCR）扩增出hBLyS的膜外可溶性功能部分hBLyS,152aa）的DNA片段,经过纯化及测序鉴定后,双酶切法定向插入原核表达载体pET-30a（＋）;然后转化λDE3溶原宿主菌并诱导其高效表达（分析得知靶蛋白约占总蛋白质的46％）出带有His-tag的重组融合蛋白。采用本室自行研制的能与His-tag特异性结合的固定化Ni^2 亲和层析胶（Ni^2-IDA）纯化重组融合蛋白,经SDS－PAGE鉴定为单一区带。生物学功能检测得知,在anti-hIgM的协同作用下,hsBLyS具有明显地刺激人B淋巴细胞的增殖作用。     

L-谷氨酸氧化酶的克隆表达、纯化及酶学性质研究

为提高微生物产L-谷氨酸氧化酶水平,将Streptomyces sp.X-119-6的L-谷氨酸氧化酶基因(LGOX)与表达载体pET28a连接,导入E.coli BL21(DE3),实现LGOX的高效表达.采用HisTrapTM FF亲和层析柱对重组L-谷氨酸氧化酶进行纯化,并对纯酶的酶学性质进行了研究.结果表明:在IPTG终浓度为0.4 mmol/L下,30℃诱导6h,可以获得比酶活为1.1 U/mg的粗酶液;重组酶的最适反应温度和pH分别为37℃和5.O;km值为2.12 mmol/L,Vmax为1.06μmol/min·mg,对L-谷氨酸具有专一性;具有良好的应用前景.     

Purification and Characterization of a Novel Cholesterol Esterase from Pseudomonas aeruginosa,with Its Application to Cleaning Lipid-stained Contact Lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53°C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25°C, and retained its activity up to 53°C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate>oleate>stearate>palmitate>caprylate>myristate> laurate, caprate>caproate>butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

Purification, Characterization, and Antifungal Activity of Chitinase from Streptomyces venezuelae P10

Streptomyces venezuelae P₁₀ could produce extracellular chitinase in a medium containing 0.6% colloidal chitin that was fermented for 96 hours at 30°C. The enzyme was purified to apparent homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography and DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 66 kDa. The chitinase was characterized, and antifungal activity was observed against phytopathogens. Also, the first 15 N-terminal amino-acid residues of the chitinase were determined. The chitin hydrolysed products were N-acetylglucosamine and N, N’-diacetylchitobiose.     

Cloning, purification, and characterization of a thermostable alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari

The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V _max and K _m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu²⁺ and Hg²⁺. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.     

Purification,Characterization, and Molecular Cloning of a Thermostable Superoxide Dismutase from Thermoascus aurantiacus

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS–PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.