Cloning and sequencing of beta-mannosidase gene from Aspergillus aculeatus no. F-50

The manB gene, coding for a unique beta-mannosidase (MANB) of Aspergillus aculeatus, was cloned from genomic and cDNA libraries, and sequenced. The gene consists of 2,811 bp encoding a polypeptide of 937 amino acid residues with a molecular mass of 104,214 Da. The A. aculeatus MANB shared amino acid sequence identity with MANB of human (24%), goat (24%), bovine (24%), and Caenorhabditis elegans (22%). When the A. aculeatus MANB was compared with other related enzymes, a Glu residue corresponding to the active site identified by the Escherichia coli beta-galactosidase and the human beta-guclonidase was conserved. This is the first fungal gene that encodes MANB.     

A beta-mannanase from Bacillus subtilis B36: purification, properties, sequencing, gene cloning and expression in Escherichia coli

MANB36, a secrete endo-beta-1,4-D-mannanase produced by Bacillus subtilis B36, was purified to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 degrees C. The enzyme activity of MANB36 is remarkably thermostable at 60 degrees C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoethanol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted molecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.     

分支杆菌噬菌体D29 Lysin B的表达、纯化及酶学性质分析

克隆表达噬菌体D29 LysinB(LysB)并对其酶学性质进行研究。以噬菌体D29基因组为模板,用PCR方法扩增lysB基因,与表达载体pET22b连接,将重组质粒转化至Escherichiacoli BL21(DE3)中表达,镍柱亲和层析(Ni-NTA)纯化可溶性表达产物,并对重组蛋白的活性进行分析检测。结果表明:成功构建了pET22b-lysB表达载体,并从1L的LB培养物中获得了33.2mg高纯度重组蛋白(His-LysB);His-LysB具有分解脂肪的能力,属于脂肪酶;生物化学特性分析表明:丁酸对硝基苯(pNPB)为水解底物,His-LysB热稳定性不佳,30℃以下比较稳定,随着温度的升高,稳定性逐渐降低;该蛋白具有较高的pH值适应性,pH5.0~9.5范围内稳定性较高;在23℃和pH7.5时酶活力最高,其比酶活为1.3U/mg;金属离子Zn2+、Cu2+、Mg2+、Mn2+和苯甲基磺酰氟(PMSF)抑制剂对酶活具有强烈的抑制作用。本研究为开发新的治疗结核药物提供了一个新的选择。     

Gene cloning, expression, and characterization of a novel beta-mannanase from Bacillus circulans CGMCC 1416

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoded 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with endo-beta-1,4-D-mannanase from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by Ni2+ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa beta-mannanase having a specific activity of 481.55 U/mg. The optimal activity of the purified protein, MANB48, was at 58 degrees C and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.     

Gene Cloning,Expression, and Substrate Specificity of an Imidase from the Strain <Emphasis Type="Italic">Pseudomonas putida</Emphasis> YZ-26

A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni²⁺–NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。     

Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1

A novel lipase-producing thermophilic strain TW1, assigned to Geobacillus sp. TW1 based on 16S rRNA sequence, was isolated from a hot spring in China. Based on this strain, a lipase gene encoding 417 amino acids was cloned. Subsequently, the lipase gene was expressed in Escherichia coli and purified as a fusion protein with glutathione S-transferase. The results showed that the recombinant lipase had an activity optimum at 40 degrees C and pH at 7.0-8.0. It was active up to 90 degrees C at pH 7.5, and stable over a wide pH ranging from 6.0 to 9.0. The recombinant lipase was stable in 1 mM enzyme inhibitors (EDTA, 2-ME, SDS, PMSF or DTT), as well as in 0.1% detergents (Tween 20, Chaps or Triton X-100). Its catalytic function was enhanced in the presence of Ca(2+), Mg(2+), Zn(2+), Fe(2+) or Fe(3+), but inhibited by Cu(2+), Mn(2+), and Li(+). By comparison with the crude lipase, the recombinant lipase had similar properties and was characteristic of thermostable enzymes. Our study presented a rapid overexpression and purification of the lipase gene from thermophile, aimed at improving the enzyme yield for industrial applications.     

菠菜乙醇酸氧化酶基因的克隆及表达

采用RT-PCR技术从菠菜总RNA中分离扩增了乙醇酸氧化酶（GO）基因的cDNA序列,首先克隆到质粒pMD18T,进行了测序。然后将乙醇酸氧化酶的cDNA分别亚克隆至质粒pThioHisC、 pTIGTrx、pBV220和pET-2b(+),分别转化大肠杆菌DH5α和BL21（DE3）,并对重组乙醇酸氧化酶在大肠杆菌中的表达进行了研究。SDSPAGE和酶活分析表明,菠菜乙醇酸氧化酶在E.coli BL21 (DE3) （pTIGTrxGO）和E.coli BL21(DE3) （pET-22b(+)GO）里得到了高水平的表达,其中E.coli BL21(DE3) （pET-22b(+)GO）的乙醇酸氧化酶活性较高。     

Development of an efficient production method for beta-mannosidase by the creation of an overexpression system in Aspergillus aculeatus

AIM: To develop an overexpression system in Aspergillus aculeatus in order to establish an efficient overproduction method of beta-mannosidase (MANB). METHODS AND RESULTS: An overexpression plasmid for the manB gene, encoding A. aculeatus MANB, was constructed and introduced into A. aculeatus cells. The gene was overexpressed under an improved promoter containing 12 copies of Region III cis-elements of Aspergillus oryzae in the transformant, and it secreted 2.56 mg MANB ml(-1) in liquid culture, which obtained a 9.4-fold higher productivity than that achieved in an overexpression system in A. oryzae. Most of the secreted protein in the cultured medium of the transformed A. aculeatus was the overproduced enzyme. CONCLUSIONS: Aspergillus aculeatus with the introduced overexpression plasmid produced 2.56 mg MANB ml(-1) in cultured medium. The improved promoter with A. oryzae Region III functioned in A. aculeatus; thus the strain is an expectant host for recombinant protein productions. SIGNIFICANCE AND IMPACT OF THE STUDY: The overexpression system with the improved promoter in A. aculeatus brought the highest productivity of MANB reported to date. The expression system would be a strong bioindustrial tool for protein production.     

Beta-mannosidase (EC 3.2.1.25) activity during and following germination of tomato (Lycopersicon esculentum Mill.) seeds. Purification,cloning and characterization

Beta-mannosidase, a high-salt-soluble enzyme, increases in activity in seeds of tomato prior to the completion of germination. This increase occurs in both the lateral and micropylar endosperm and becomes more evident during post-germinative seedling growth. The beta-mannosidase activity profile is similar to that of endo beta-mannanase although it is the first to increase in the lateral endosperm. Tomato seed beta-mannosidase was purified to homogeneity and its cDNA (LeMside1) obtained by 3'-RACE PCR using oligonucleotide sequences based on four peptide sequences obtained from the purified enzyme. The derived amino acid sequence of the tomato beta-mannosidase shows the enzyme is a member of the Glycosyl Hydrolases Family 1 (GHF1) but has a very low sequence identity with that of beta-mannosidases from non-plant sources; no other plant sequence for the enzyme is known. There appears to be only one gene encoding beta-mannosidase in tomato, the sequence of which has been determined (LeMSide2). Its expression occurs first in the micropylar endosperm, and then declines after germination. This is followed by an increase in its expression in the lateral endosperm, which precedes that of the gene for endo beta-mannanase. Expression of the beta-mannosidase gene increases appreciably in the growing seedling embryo. With this report, the cloning of all three of the enzymes involved in galactomannan mobilization (endo beta-mannanase, alpha-galactosidase and beta-mannosidase) in tomato seeds has now been achieved.     

信号肽对短杆菌胆固醇氧化酶基因在大肠杆菌中表达的影响

从Brevibacterium sp．DGCDC-82染色体DNA中扩增出含信号肽序列的胆固醇氧化酶结构基因,插入大肠杆菌表达载体pET28a（＋）中,构建重组质粒pET28a—COD（s＋）。以pET28a-COD（s＋）为底物,扩增出不含信号肽的胆固醇氧化酶结构基因,构建成重组质粒pET28a-COD（s-）。两种重组载体转化大肠杆菌BL21（DE3）通过IPTG诱导均获得活性表达,SDS-PAGE分析,目的产物表达量都占到了细胞总蛋白的50％以上,Brevibacterium sp．DCR2DC-82胆固醇氧化酶的信号肽对重组酶的空间构象和表达量并没有太大的影响。     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.     

小鼠Nanog基因的克隆及其在大肠杆菌中的表达

按照nanog基因编码序列设计合成引物,利用RT-PCB从小鼠的囊胚期胚胎中扩增得到该 基因,并将该基因克隆到pET-28b(+)载体上,获得pET-28b(+)-nanog原核表达重组质粒,限制 性酶分析和DNA序列测定均证实该克隆插入片段为nanog基因编码序列。重组质粒转化大肠杆 菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得了高效表达,western杂交证实该 蛋白具有6-His抗原活性,从而证实目的蛋白为Nanog蛋白。     

地衣芽孢杆菌普鲁兰酶编码基因的鉴定

对地衣芽孢杆菌基因组序列分析显示。其中标注为amyX的基因可能编码普鲁兰酶。以PCR方法,从地衣芽孢杆菌染色体DNA中扩增出amyX基因蛋白编码区,插入大肠杆菌表达载体pET28aT7启动予下游。含重组质粒的大肠杆菌BL21（DE3）在IPTG诱导下表达出有活性的普鲁兰酶。酶学性质初步分析表明,重组普鲁兰酶最适反应温度为40℃,最适pH值为6．0。     

胆固醇氧化酶基因的克隆及在E.coli中的表达

根据NCBI中报道的BrevibacteriumsterolicumATCC21387胆固醇氧化酶基因序列,采用PCR方法以Brevibacteriumsp.DGCDC-82的基因组为模板,扩增得到了编码胆固醇氧化酶的基因,该基因与来源于BrevibacteriumsterolicumATCC21387的胆固醇氧化酶基因(choB)同源性为98%。将得到的基因定向克隆到pET28a载体中,转化至含有编码argU和proL基因的大肠杆菌BL21-CodonPlus(DE3)-RP中表达。经过IPTG诱导后,经SDS-PAGE检测在约55kD处有一蛋白表达条带,目的蛋白表达量约占总蛋白的16%,经测定酶活为340U/L。     

棉花GhDHAR2基因克隆、功能序列分析及原核表达

通过RT-PCR方法从棉花纤维组织中克隆得到脱氢抗坏血酸还原酶基因GhDHAR2的cDNA,该基因开放阅读框为639 bp,编码212个氨基酸的蛋白质。同源性序列对比分析显示,GhDHAR2蛋白具有较高的保守性,具有典型的功能结构域,包括GST-N家族和GST-C-DHAR家族的功能结构域;进化树分析显示GhDHAR2和拟南芥AtDHAR2在进化关系上较近。将GhDHAR2基因连接到原核表达载体pET-28a中,将重组载体pET28a-GhDHAR2转入到表达菌株BL21(DE3)中,通过IPTG诱导表达出重组GhDHAR2蛋白,SDS-PAGE凝胶电泳分析显示重组蛋白大小约为28 kD,诱导表达的重组蛋白具有较高的DHAR活性。首次克隆了棉花GhDHAR2基因,通过结构域分析其可能的作用,并成功进行蛋白体外表达及酶活性分析。     

New effective sources of the Staphylococcus simulans lysostaphin

The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.     

不同来源的谷氨酸棒杆菌氨基脱氧分支酸合成酶的活性分析

分别以高产L-丝氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)SYPS-062与模式菌株谷氨酸棒杆菌(Corynebacterium glutamicum) ATCC 13032的基因组DNA为模板,运用PCR技术扩增出氨基脱氧分支酸合成酶(ADC synthase)的编码基因pabAB。实验结果表明：来源于SYPS-062和ATCC 13032的pabAB片段全长均为1863bp,编码620个氨基酸。两片段存在16个碱基的差异,引起了7个氨基酸的突变。将pabAB连接表达载体pET-28a(+),构建表达质粒pET-28a-pabAB,并转化E.coli BL21(DE3),在IPTG诱导下,E.coli BL21(DE3)(pET-28a-pabAB)高效表达分子量约为67kDa的可溶性蛋白。表达产物带有His-tag标记,选用Ni柱对表达产物进行纯化,纯化后酶活测定结果表明,来源于SYPS-062氨基脱氧分支酸合成酶的比酶活低于ATCC 13032达46.6%。     

Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3.

The gene encoding the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the D-amino-acid amidase. This enzyme, DaaA, is composed of 363 amino-acid residues (molecular mass 40 082 Da), and the deduced amino-acid sequence exhibits homology to alkaline D-peptidase from Bacillus cereus DF4-B (32% identity), DD-peptidase from Streptomyces R61 (29% identity), and other penicillin-recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active-site motifs identified in the penicillin-binding proteins and beta-lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell-free extracts of E. coli was 33.6 U. mg-1 with D-phenylalaninamide as substrate, which is about 350-fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel-filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward D-amino-acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the DD-peptidase and beta-lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3.