芽胞杆菌BSD-8肌氨酸氧化酶的纯化与性质

对一株从土壤中分离到的芽胞杆菌Bacillus sp.BSD-8菌株所产生的热稳定性较高的肌氨酸氧化酶进行纯化,并对该酶的特性进行了研究。通过硫酸铵分级沉淀、DEAE-纤维素离子交换柱、Toyopearl疏水层析柱和Sephadex G-75分子筛层析,使酶提纯25倍,比活力达到5.3U/mg。研究了纯化后的酶的生化特性,确定了该酶的主要特性:该酶为黄素蛋白,与黄素以非共价键的方式结合,由单一亚基组成,其亚基分子量为51kDa。酶的最适反应温度及pH分别为60℃与8.5。该酶在60℃及pH8.0~10.0条件下稳定。以Lineveaver-Burk作图法求得该酶米氏常数Km值为3.1mmol/L。Ag+、Hg2+、SDS及Tween80对该酶有强抑制作用,而Tween20和Triton X-100对酶活性无影响。该肌氨酸氧化酶在耐热性质上比以前所报道的肌氨酸氧化酶有很大的提高,在酶法肌酐测定应用中有明显的优势。     

Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli

Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.     

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino- terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. H(6)SCChi-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were 50°C and pH 8.0, respectively.     

Purification and characterization of a novel thermostable lipase from Bacillus sp

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.     

A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization

A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60–65 ^∘C and retained 100% activity after incubation at 60 ^∘C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ^∘C and 23 min incubation at 80 ^∘C. Catalytic function of lipase was activated by Mg⁺⁺ (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, βME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m and V _max of 0.5 mM and 0.139 μM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.     

High level expression of a thermostable Bacillus xylose (glucose) isomerase in Escherichia coli

Summary A thermophilic Bacillus sp. producing xylose (glucose) isomerase has been isolated. Its xy/A gene when cloned in Escherichia coli and expressed gave 37.5 and 12.8 units/ mg protein respectively for xylose and glucose isomerase activities at 85°C. A single heat treatment of the crude extract purified the enzyme further yielding the highest ever recorded activities of 150 and 49.02 units /mg protein.     

Cofactors in sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD⁺, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.     

Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0

A Bacillus spp. strain SPS-0, isolated from a hot spring in Portugal, produced an extracellular xylanase upon growth on wheat bran arabinoxylan. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange, gel filtration, and affinity chromatography. The optimum temperature and pH for activity was 75 degrees C and 6.0. Xylanase was stable up to 70 degrees C for 4 h at pH 6.0 in the presence of xylane. Xylanase was completely inhibited by the Hg(2+) ions. beta-Mercaptoethanol, dithiothreitol, and Mn(2+) stimulated the xylanase activity. The products of birchwood xylan hydrolysis were xylose, xylobiose, xylotriose, and xylotetraose. Kinetic experiments at 60 degrees C and pH 6.0 gave V(max) and K(m)values of 2420 nkat/mg and 0.7 mg/ml.     

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.     

Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11

An alkalophilic, environmental micro-organism, Bacillus sp. BG-11, has been isolated and characterized. It produced 76 U ml-1 of chitinase in liquid batch fermentation after 72 h of incubation at 50 degrees C using chitin-enriched medium. The molecular weight of purified chitinase was estimated to be 41 kDa by SDS-PAGE. The pH and temperature optima of chitinase immobilized on chitosan and calcium alginate were 8.5 and 50 degrees C, respectively, which were same as that of free enzyme. The pH and thermostability of immobilized chitinase were enhanced significantly. The chitinase immobilized on chitosan was stable between pH 5.0 and 10.0, and the half-life of chitosan-immobilized enzyme at 70, 80 and 90 degrees C was 90, 70 and 60 min, respectively. The end-products formed during the enzyme-substrate reaction were identified by 13C-NMR, and N-acetyl-D-glucosamine was found to be the major end-product. GlcNAc (GlcNAc)2 and (GlcNAc)3 inhibited the chitinase activity by 32, 25 and 18%, respectively, at a concentration of 10 mmol l-1. The shelf-life of chitinase (retained 100% activity) at 4 degrees C was 8 weeks in the presence of either sodium azide (100 microgram ml-1), sodium metabisulphite (0.1% w/v) or KCl (15% w/v). The enzyme was resistant to the action of proteases and allosamidin.     

Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes

The gene kerA (1,047 bp) encoding the main keratinase from Bacillus licheniformis was cloned into two conventional vectors, pET30α and pET32α, and expressed in Escherichia coli. From SDS-PAGE analysis, the recombinant keratinases were 45 and 55 kDa. They had different optimal pH values (7.5 and 8.5) but the same optimum temperature of 50 °C. The recombinant keratinase produced in E. coli pET30α-kerA was more stable than that produced in E. coli pET32α-kerA, and retained approx. 70 % of its total enzyme activity after 30 min at 70 °C.     

A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies

An extracellular, thermostable, alkaline lipase was partially purified from a thermophilic Bacillus strain J 33. It was optimally active at pH 8.0 at 60°C, retaining 50% activity at 70°C for 30 min. It had native molecular mass of 45 kDa. The lipase was stable in 90% (v/v) hexane or benzene mixtures in water. It converted 66% oleic acid at 0.25 M with 0.4 M methanol in hexane to methyl oleate at 60°C in 16 h. Activity was stimulated by Mg2 (10 mM) but inhibited by EDTA (10 mM) and PMSF (10 mM). It was stable in Triton X-100, Tween 20 and Tween 80 (0.1% v/v). © Rapid Science Ltd. 1998     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Cloning of genes encoding heterotetrameric sarcosine oxidase from Arthrobacter sp.

Summary An isolate of Arthrobacter sp. produced the sarcosine oxidase which was purified to homogeneity. SDS-PAGE indicated that the enzyme was composed of four dissimilar subunits with molecular weights of 106, 43, 24, and 15 kDa. The genes encoding the four subunits of sarcosine oxidase were isolated and expressed in E. coli.     

微杆菌属ZZJ4-1菌株的耐热尿酸氧化酶基因的克隆及重组酶性质

为了研究微杆菌Microbacterium sp.ZZJ4-1菌株的耐热尿酸氧化酶(Uox)的性质,克隆其基因(uox),得到1个894 bp的开放阅读框。该基因与多数已报道的uox无明显同源性,仅与球形节杆菌Arthrobacterglobiformis的uox有72%的同源性。将基因插入质粒pET-15b构成pET-15b-uox表达载体,转化至Escherichiacoli BL21(DE3)中诱导表达。对重组Uox的主要理化性质研究表明:该酶由大小约为35 kDa的亚基组成;其最佳反应温度和pH分别为30℃和7.5;在65℃以下和pH 8.5～11.0范围内稳定;以尿酸为底物的Km值为0.22 mmol/L;Ag+、Zn2+、Cu2+和SDS均能完全抑制酶活,Tween 20、Tween 80和Triton X-100对酶活有一定的促进作用。该重组酶的耐热性是目前报道的重组Uox中最好的,这一特性有利于其在诊断治疗中的开发应用。     

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.     

Expression,purification and partial characterization of a xanthine oxidase (XOD) in Arthrobacter sp.

A xanthine oxidase (XOD) was expressed, purified and partially characterized from Arthrobacter sp. with a negative immune protocol. To determine the optimal inducer for XOD, xanthine, hypoxanthine and uric acid were added into the medium of cultivation. The results revealed that with the inducement of about 14 mM xanthine, the highest XOD activity could be detected. To separate XOD from Arthrobacter sp., the cells were first cultured without any inducement; then the total proteins of the collected cells were extracted and immunized to rabbits for the polyclonal antibodies. These antibodies were then coupled with sepharose CL 6 B, and the medium was further employed to deplete most of the cells’ back ground proteins. Began with ～20 mg crude protein from disrupted cells was subjected to the antibody medium, and ～1.45 mg protein was detected in unbinding fractions with ～92.0% of activity. The extracted xanthine oxidase was ～85% pure with native-PAGE analysis, and ～90% pure with SDS-PAGE analysis, the yield of protein was ～7.4%. The specific activity of the enzyme was 36.0 U/mg. The native enzyme should be a dimer (～280 kDa) of a protein composed with two different peptides with the mass of approximately 55.5 and 85.5 kDa, respectively. The optimal pH and temperature of this enzyme were determined at about pH 7 and 50 °C. Furthermore, EDTA revealed almost no influences on the activity.     

Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The alpha subunit is composed of two domains, contains NAD(+), and binds folinic acid. The beta subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10A apart. The gamma subunit is in contact with two domains of alpha subunit and has possibly a folate-binding structure. The delta subunit contains a single atom of zinc and has a Cys(3)His zinc finger structure. Based on the structures determined and on the previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.     

Cloning and expression in Escherichia coli of chromosomal mercury resistance genes from a Bacillus sp.

A 7.9-kilobase (kb) chromosomal fragment was cloned from a mercury-resistant Bacillus sp. In Escherichia coli, in the presence of a second plasmid carrying functional transport genes, resistance to HgCl2 and to phenylmercury acetate (PMA) was expressed. Shortening the cloned fragment to 3.8 kb abolished resistance to PMA but not to HgCl2. In Bacillus subtilis, the 3.8-kb fragment produced mercuric reductase constitutively but did not produce resistance to HgCl2 or to PMA.     

Purification and characterization of thermostable xylose(glucose) isomerase from Bacillus thermoantarcticus

Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K _m of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85°C. Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for its activity and thermal stability. Mn²⁺, Co²⁺ or Mg²⁺ were of comparable efficiency for xylose isomerase reaction, while Mg²⁺ was necessary for glucose isomerase reaction. Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240. Received 18 March 2001/ Accepted in revised form 03 July 2001