Cloning and sequencing of the deacetylase gene from Vibrio alginolyticus H-8

A gene encoding deacetylase DA1 that is specific for N, N'-diacetylchitobiose was cloned using the shot-gun method with pUC118 and sequenced. The open reading frame encoded a protein of 427 amino acids including the signal peptide. The molecular mass of the mature enzyme estimated from the amino acid sequence data was 44.7 kDa, which is approximately similar to that, estimated by SDS-PAGE (48.0 kDa), of the purified enzyme reported previously. The N-terminal amino acid sequence deduced from the cloned deacetylase gene showed partial sequence homology with the Nod B protein from Rhizobium sp. (37% identity) and chitin deacetylase from Mucor rouxii (28%). It contained a domain, which showed homology with a chitin-binding domain of chitinase A from Bacillus circulans (39%).     

CLONING, EXPRESSION AND CHARACTERIZATION OF NOVEL LECTIN FROM ORYZA SATIVA

A lectin gene homolog of Oryza sativa was successfully cloned and expressed in Escherichia coli. The deduced amino acid sequence of the protein product showed a significant similarity with known chitin‐binding lectins. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 8 M urea in Tris/HCl buffer, pH 7.0 and renaturation by 10‐fold dilution in the same buffer. The recombinant lectin with His‐tag was simply purified to homogeneity by the process of affinity chromatography and was obtained with a yield of 6–8 mg/L culture. The recombinant lectin was a homo‐dimer composed of 22 kDa. The hemagglutination activity of the recombinant lectin was optimal at pH 4.0–7.0 and it was very sensitive to inhibition by N‐acetylneuraminic acid and thyroglobulin.     

Purification and characterization of N-Acetylglucosamine 6-phosphate deacetylase from Thermus caldophilus

N-Acetylglucosamine 6-phosphate deacetylase [EC 3.5.1.25] was purified and biochemically characterized from an extreme thermophile, Thermus caldophilus GK24. The optimum temperature and pH of the enzyme were 80 degrees C and 7.5, respectively. The enzyme is a tetramer composed of identical 45 kDa subunits. The N-terminal amino acid sequence of the purified enzyme was determined to be MSVDLKTLHRRHVLTP. It hydrolyzed GlcNAc-6-P, but not GlcNAc-1-P or chitin oligosaccharides. The deacetylase activity was completely inhibited by the addition of 1 mM Cu2+, but moderately activated by that of 1 mM Mn2+ and Co2+. Within 2 h of reaction, 2 mM GlcNAc-6-P was completely hydrolyzed to GlcN-6-P and acetate by the action of the deacetylase.     

Cloning, sequencing and expression of an alpha-L-arabinofuranosidase from Aspergillus sojae

The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae. It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75%), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.     

Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y

We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH₂-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene. Using the S. cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P. pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned. The open reading frame of the P. pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids. The molecular mass of the protein is calculated to be 59·44 kDa without sugar chains. The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites. The NH₂-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prc1p and S. cerevisiae Prc1p. Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity. Over-expression of the PRC1 gene under regulation of the P. pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY. The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987.     

草莓轻型黄边病毒外壳蛋白抗原表位预测、克隆、表达及其免疫原性分析

应用IEDB、DNAStar、DNAMAN和Snap Gene等生物信息学工具对草莓轻型黄边病毒外壳蛋白的氨基酸残基序列进行分析。选择一段抗原性较强的肽段(位于外壳蛋白27~38氨基酸残基处),依据大肠杆菌(Escherichia coli)的密码子偏好性化学合成了该肽段的DNA编码序列(AE)。AE片段克隆至E.coli表达载体pET32a(+)的Eco RⅠ和XhoⅠ位点,获得重组质粒pET32a(+)-AE。含AE片段的开放阅读框长561 bp,编码了一个187氨基酸残基的重组融合蛋白,理论分子质量为20.29kD。重组质粒pET32a(+)-AE分别在E.coli BL21(DE3)和E.coli Rosetta中进行了诱导表达和条件优化。重组融合蛋白在E.coli Rosetta中的最佳表达条件为1.5 mmol/L异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)、35℃诱导表达2 h,产率为13.22 mg/L;在E.coli BL21(DE3)中的最佳表达条件是为0.5 mmol/L IPTG、30℃诱导表达2 h,产率为9.55 mg/L。重组融合蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离、质谱鉴定表明,其含有所预测的草莓轻型黄边病毒外壳蛋白抗原表位肽段AE。AE重组融合蛋白经Ni~(2+)亲和色谱纯化、EK酶切、分子筛除去融合蛋白标签后,免疫产蛋鸡。从免疫鸡所产鸡蛋中提取鸡卵黄免疫球蛋白(immunoglobulin of yolk,Ig Y),Dot-Blot检测抗体结合活性。结果表明,所提取的IgY可特异性识别AE肽段和SMYEV外壳蛋白。这一结果表明所预测的AE肽段具有较好的免疫原性。     

Cloning and expression of the N-acetylmuramidase gene from Streptomyces rutgersensis H-46

The N-acetylmuramidase SR1 gene from Streptomyces rutgersensis H-46 was cloned in Escherichia coli JM109 and expressed in E. coli BL21(DE3)pLysS. An open reading frame included the leader peptide region encoding a polypeptide of 65 amino acid residues and the mature SR1 enzyme region encoding a polypeptide of 209 amino acid residues. The overall G + C content of the mature enzyme gene was 67.6%, with 98.1% of G or C in the third position of the codons. The calculated molecular weight of the mature enzyme was 23,057 Da. The amino acid sequence of the mature enzyme showed a significant level of identity with bacteriolytic enzymes from Streptomyces globisporus (50.9% identity), Chalaropsis species (40.2% identity) and Saccharopolyspora erythraea (31.0% identity). The mature enzyme gene cloned into plasmid pET26b carrying a signal peptide, peIB, was expressed in E. coli BL21(DE3)pLysS. The signal peptide region was cleaved during the production of the enzyme. Specific activity of the enzyme purified from the transformant was almost identical to that of the native enzyme. Furthermore, the SR1 enzyme gene cloned with the leader peptide gene into plasmid pET28a was also expressed in E. coli. In this case, a proform-like protein was partially processed; 35 amino acid residues were cleaved but 30 amino acid residues remained. This proform like protein has approximately one-nineteenth the activity of the native enzyme. These results indicated that the native SR1 enzyme was produced in the following manner in the cells of S. rutgersensis H-46. The SR1 enzyme gene was translated to a pre-proform protein followed by the deletion of a signal peptide. Finally, the proform-like protein was processed by deletion of the remaining leader peptide.     

重组N-乙酰鸟氨酸脱乙酰基酶包涵体蛋白的柱上复性

N-乙酰鸟氨酸脱乙酰基酶(NAO)具有广泛的底物选择性,可用于多种活性氨基酸的酶法拆分,具有广阔的工业应用前景。文中采用多种洗涤剂对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行洗涤去杂,并用DEAE Sepharose Fast Flow层析柱和三缓冲体系作为复性系统对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行复性实验。结果表明,4mol/L尿素与0.5%Triton X-100联合洗涤可以大量去除杂质;三缓冲体系能有效地实现重组N-乙酰鸟氨酸脱乙酰基酶包涵体的柱上复性。在流速为0.5 mL/min,尿素梯度为21 mL,尿素终浓度为1.8mol/L,蛋白质上样量为0.99 mg的实验条件下,蛋白回收率和复性酶比活分别达到52%和10.27 U/mg。     

Complete cDNA sequence of chitin deacetylase from Gongronella butleri and its phylogenetic analysis revealed clusters corresponding to taxonomic classification of fungi

A cDNA library containing a chitin deacetylase (CDA) gene from a zygomycete Gongronella butleri was constructed and the complete gene was sequenced. The complete gene contains an open reading frame of 1290 nucleotides which encodes a sequence of 430 amino acid residues. The gene sequence consists of nucleotides encoding a polysaccharide deacetylase domain located in the middle, covering 34% of the entire sequence. This domain shares high sequence similarities with CDAs of fungi and other deacetylases including the nodb-like protein from Sinorhizobium meliloti and Bacillus subtilis, xylanase D from Cellulomonas fimi, and acetylxylan esterase A from Streptomyces lividans. The deduced amino acid sequence shows homology with CDAs from Mucor rouxii (48% identity) and Phycomyces blakesleeanus (30% identity). Phylogenetic analysis reveals that CDAs are clustered in accordance with the taxonomic classification of the fungi.     

噬夏孢欧文氏菌(Erwinia uredovora)类胡萝卜素合成相关基因crtE的克隆及其在大肠杆菌中的表达

噬夏孢欧文氏菌基因crtE编码GGPP合成酶。通过PCR扩增获得crtE基因,克隆进表达载体,构建表达质粒pET-15bcrtE。重组质粒转化E.coli BL21(DE3),构建工程菌;重组GGPP合成酶在大肠杆菌中实现了高效表达,表达量占菌体总蛋白的42%。重组蛋白以包含体形式存在,包含体经洗涤、尿素溶解、复性并经镍离子亲和层析树脂纯化,得到了电泳纯的重组噬夏孢欧文氏菌GGPP合成酶,带有His-tag的该蛋白分子量为34kDa,pI值为6.3。     

利用his-tag纯化和复性基因工程产物肠激酶

构建了融合表达肠激酶轻链（EKLC）的基因工程大肠杆菌，将该菌于37℃在含30mg／L卡那霉素的LB培养基中培养，当细胞密度（OD600）达到0．5～0．6时加入最终浓度为0．4mmol／L的IPTG并降温至26℃进行诱导表达，4h后目的蛋白质的表达量可达菌体总蛋白质的40％以上，但所表达的重组产物大多以包涵体形式存在于菌体中。利用产物N端携带6个组氨酸标签与金属螯合层析介质中镍离子的亲和性，应用镍离子金属螯和层析对样品包涵体进行了纯化和复性研究。实验结果表明，在变性条件下纯化的样品在柱上不经洗脱，直接用复性液洗涤6h，可以使EKLC在得到提纯的同时实现复性，得到了电泳纯度为96％的具有生物活性的EKLC，最高活性为712U。     

产甲壳素脱乙酰酶赖氨酸芽孢杆菌的筛选及产酶初探

为了采用生物法降解天然甲壳素,本研究以甲壳素为唯一碳源,从自然发酵的虾皮中筛选产甲壳素脱乙酰酶的菌株,通过显色平板初筛和产酶活性复筛,利用形态学特征和16S rRNA序列分析对菌株进行鉴定。结果表明,筛选获得甲壳素脱乙酰酶产生菌X4经过形态学特征和16S rRNA序列分析确定为赖氨酸芽孢杆菌（Lysinibacillus sp.）。对该菌株进行发酵培养,其产生的甲壳素脱乙酰酶酶活为8.210 U/mL,对甲壳素的脱乙酰度为8.642%,研究结果对甲壳素的绿色生物利用具有较大的意义。     

Molecular cloning and high-level expression in Escherichia coli of fungal alpha-galactosidase from Absidia corymbifera IFO 8084

A cDNA encoding the alpha-galactosidase of Absidia corymbifera IFO 8084 was cloned and sequenced. The cloned DNA has a single open-reading frame consisting of 2190 base pairs, and the deduced amino acid sequence revealed that the mature enzyme consisted of 730 amino acid residues with a molecular mass of 82,712 Da. The native structure of the alpha-galactosidase of A. corymbifera IFO 8084 was determined to be a tetramer. Comparison with amino acid sequences of other alpha-galactosidase showed high homology with sequences of members of family 36. An expression vector, pET32Trx/galalpha, was constructed by introducing the cDNA coding region into a thioredoxin fusion system, pET32-Ek/LIC. The resulting transformant, pET32Trx/galalpha, overproduced the active enzyme as a thioredoxin fused form in the host Escherichia coli. By using His-binding metal affinity chromatography, recombinant alpha-galactosidase was purified to homogeneity in a single step. The purified recombinant fusion alpha-galactosidase showed properties very similar to the native alpha-galactosidase from A. corymbifera IFO 8084.     

密苏里游动放线菌葡萄糖异构酶基因xylA的克隆及其表达条件的优化

为克隆、表达密苏里游动放线菌葡萄糖异构酶(GI)基因xylA,并对其诱导表达条件进行初步优化。采用Slowdown PCR方法克隆得到密苏里游动放线菌(Actinoplanes missouriensis)CICIM B0118(A)的葡萄糖异构酶基因xylA,构建pET-28a(+)-xylA 表达载体,并转化至E. coli BL21 (DE3),经异丙基-β -D- 硫代半乳糖苷(IPTG)诱导表达,并对其表达产物进行SDS-PAGE 电泳。结果表明,融合蛋白分子质量约为45kD。在诱导时间9h、0.6mmol/L IPTG、30℃和OD600nm 值为0.8 的最适培养条件下,酶比活力最高达到62.42U/mL。     

Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris

A cDNA fragment encoding a mature neutral endoglucanase with 366 amino acids was cloned from Volvariella volvacea WX32. Online analysis of amino acid sequence homology showed that the endoglucanase, designated as NCel5A, belongs to glycoside hydrolase family 5. The recombinant plasmid, pPIC9K-ncel5A, was transformed into Pichia pastoris GS115 by electroporation. Screening of multiple copies of the gene ncel5A in transformants was performed on YPD plates containing geneticin G418. One transformant expressing the highest recombinant NCel5A (rNCel5A) activity, numbered as GSNCel4-3, was chosen for optimizing expression conditions. In optimized conditions, the expressed rNCel5A activity was up to 4612 U/ml. SDS-PAGE and enzyme activity assays demonstrated that the rNCel5A, a glycosylated protein with an M.W. of about 42 kDa, was extracellularly expressed in P. pastoris. The rNCel5A showed the highest activity at pH 7.5 and 55°C and was stable at a broad pH range of 6.0-9.0 and at a temperature of 55°C or below.     

1 株高产几丁质脱乙酰酶红球菌的基因组测序及其应用潜力分析

为高效利用虾蟹壳资源,推动壳聚糖的几丁质脱乙酰酶法生产,采用Illumina测序技术,对1 株高产几丁质脱乙酰酶的红球菌菌株11-3进行基因组测序,并进行系统的生物信息学分析,主要包括基因本体论（Gene Ontology,GO）、直系同源群集（Cluster of Orthologous Group,COG）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG）功能注释,碳水化合物活性酶注释以及几丁质降解相关酶基因的生物信息学分析。研究发现,红球菌11-3的基因组为6 089 866 bp,共5 904 个编码基因,GC含量为70.514%。经碳水化合物活性酶注释共识别了165 个基因,包括59 个碳水化合物酯酶基因,42 个糖基转移酶基因,36 个糖苷水解酶基因和28 个辅助氧化还原酶基因,其中,鉴定出1 个几丁质脱乙酰酶基因（gene4907）,4 个几丁质酶（EC 3.2.1.14）基因（gene1286、gene1287、gene3810、gene4754）和2 个壳聚糖酶（EC 3.2.1.132）基因（gene4921、gene5362）。gene4907与已报道的几丁质脱乙酰酶基因的序列一致性为26.60%～32.43%,为一种新的几丁质脱乙酰酶。因此,红球菌11-3菌株在几丁质资源的开发领域具有极大潜力。     

丝胶抗冻肽在大肠杆菌中的重组表达及其抗冻活性初探

为了高效制备抗冻肽,研究一种丝胶抗冻肽目的基因SerD在大肠杆菌BL21菌株中的重组表达,并分析表达产物的抗冻活性。首先合成SerD基因片段,经KpnI和XhoI双酶切后定向插入质粒载体Pet32a中,构建表达质粒Pet32a-SerD,然后电转化入大肠杆菌BL21(DE3),在IPTG诱导下进行目的基因表达,利用镍琼脂糖亲和层析对目的重组蛋白进行纯化,并对其进行抗冻活性分析。结果表明,最佳表达条件为重组菌在20℃诱导16 h;通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis,SDS-PAGE)和蛋白质免疫印迹试验(Western-Blot)鉴定重组蛋白表达成功且His-SerD融合蛋白表达的分子量在25~35 kDa之间;胞内表达His-SerD融合蛋白的大肠杆菌BL21-SerD复苏后的生长活性明显高于PBL空载菌;添加His-SerD融合蛋白可明显降低溶液中冰晶颗粒大小,具有较好的重结晶抑制效果。本文通过基因工程方法在大肠杆菌中成功构建丝胶肽抗冻肽的重组表达系统,并最终获得具有抗冷冻胁迫保护作用的His-SerD融合蛋白。     

Molecular cloning and characterization of thermostable beta-lactam acylase with broad substrate specificity from Bacillus badius

The gene (pac) encoding beta-lactam acylase from Bacillus badius was cloned and expressed in Escherichia coli. The pac gene was identified by polymerase chain reaction (PCR) using degenerated primers, on the basis of conserved amino acid residues. By using single specific primer PCR (SSP-PCR) and direct genome sequencing, a complete pac gene with its promoter region was obtained. The ORF consisted of 2415 bp and the deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a signal sequence, an alpha-subunit, a spacer peptide and a beta-subunit. The pac gene was expressed with its own promoter in different E. coli host strains and a maximum recombinant PAC (1820 U l(-1)) was obtained in E. coli DH5alpha. The recombinant PAC was purified by Ni-NTA chromatography and the purified PAC had two subunits with apparent molecular masses of 25 and 62 kDa. This enzyme exhibited a high thermostability with a maximum activity at 50 degrees C. This enzyme showed stability over a wide pH range (pH 6.0-8.5) with a maximum activity at pH 7.0 and activity on a wide beta-lactam substrate range. The K(m) values obtained for the hydrolysis of penicillin G and a chromogenic substrate, 6-nitro-3-phenylacetylamidobenzoic acid, from B. badius PAC were 39 and 41 microM, respectively. The PAC activity was competitively inhibited by PAA (K(i), 108 microM) and noncompetitively by 6-APA (K(i), 17 mM). The constitutive production of B. badius PAC in E. coli and its easier purification together with the advantageous properties, such as thermostability, pH stability and broad substrate specificity, make this as a novel enzyme suitable for beta-lactam industry.