来源于青霉XMZ-9两个低温脂肪酶的基因克隆、原核表达与性质测定

低温脂肪酶在低温条件下仍具有较高活性,在食品添加剂、洗涤添加剂及有机合成等产业具有非常独特的应用前景。从低温菌株中分离低温脂肪酶基因是开发新的低温脂肪酶的有效手段。首先利用油脂同化平板与三丁酸甘油酯-维多利亚蓝平板从冰川土样中筛选分离获得一株具有较高脂肪酶活性的真菌,18S rDNA鉴定其属于青霉属,命名为Penicillium sp.XMZ-9。根据真菌脂肪酶多序列比对获得的保守区,设计简并引物,利用降落PCR与染色体步移的方法从Penicillium sp.XMZ-9中克隆到2个完整的脂肪酶基因,分别记为LipA与LipB。LipA全长1 014 bp,无内含子,编码337个氨基酸。而LipB全长1 232 bp,cDNA长1 122 bp,含有2个内含子,编码373个氨基酸。将两基因的cDNA序列克隆到pET30a(+)载体上,转化大肠杆菌Escherichiacoli BL21(DE3)。经低温诱导表达后,LipA大部分表达为包涵体,包涵体经复性后具有脂肪酶活性,并表现出低温适应性;LipB则大部分表达为可溶性蛋白,Ni-亲和层析柱纯化后,其亦具有低温脂肪酶活性。青霉菌株XMZ-9的获得与低温脂肪酶的克隆表达研究,为研究低温菌株与低温酶的适冷机制提供了宝贵的资源,也为进一步开发利用低温脂肪酶奠定了基础。     

Molecular characterisation of the gene encoding an esterase from Bacillus licheniformis sharing significant similarities with lipases

An esterase gene from the moderate thermophilic strain Bacillus licheniformis LCB40 was cloned and expressed in Escherichia coli. Comparison of the amino acid sequence of the esterase with those of known lipases and esterases showed the presence of the well-conserved Gly-X-Ser-X-Gly pentapeptide, with an alanine replacing the first glycine. This substitution has never been reported for an esterase but it is present in the lipases from Bacillus subtilis, Bacillus pumilus and Galactomyces candidum. The amino acid sequence showed similarities with lipases and with mammalian lecithin-cholesterol acyltranferases and no similarities with esterases. The enzyme activity of a crude extract from a recombinant Escherichia coli strain showed hydrolysis of p-nitrophenyl caprylate (pNPC8) as for esterases, but not of p-nitrophenyl palmitate (pNPC16) or olive oil such as for lipases. Thus, the enzyme displays the original property of associating the activity of an esterase with a primary sequence showing high similarity with lipases.     

Molecular cloning of promoter-containing fragments from Bacillus stearothermophilus and their expression in Escherichia coli and Bacillus subtilis

Abstract Bacillus stearothermophilus DNA fragments containing a promoter were isolated in Escherichia coli using a shuttle promoter-probe vector. The molecular sizes of the isolated fragments ranged from 0.78 to 10 kb. The 0.78 and 1.1 kb fragments were selected and examined in some detail for promoter activity in both E. coli and Bacillus subtilis by analysis of expression of erythromycin-resistance (Em^r) and β-galactosidase. The results showed that the two fragments exhibit a high promoter activity in both bacteria. In vitro promoter activity of the 1.1 kb fragment was also shown by RNA syntheses catalyzed by RNA polymerases prepared from E. coli, B. subtilis and B. stearothermophilus .     

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Nitrile hydratase from a thermophilic Bacillus smithii

A novel thermophilic Bacillus smithii strain SC-J05-1, isolated from a hot spring, had the ability of hydrating nitrile to form amide. The nitrile hydratase was purified to homogeneity from the microbial cells of SC-J05-1 and was characterized. The enzyme was a 130-kDa protein composed of two different subunits (25.3 kDa and 26.8 kDa) and contained cobalt ions. This enzyme had the optimal temperature of 40°C and was stable up to 50°C. The optimal pH was in the alkaline region higher than pH 10. Received 02 September 1997/ Accepted in revised form 06 February 1998     

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.     

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     

淀粉液化芽孢杆菌b-1,3-1,4-葡聚糖酶基因的克隆及表达的优化

为了比较不同的表达系统对β-1,3-1,4-葡聚糖酶基因(bgl)的效果,本研究将高产β-1,3-1,4-葡聚糖酶的淀粉液化芽孢杆菌Bacillus amylolique faciens BS5582的bgl基因(GenBank Accession No.EU623974)克隆到3种不同的质粒载体中,即构建pEGX-4T-1-bgl、pET20b(+)-bgl和pET28a(+)-bgl重组质粒。比较了pEGX-4T-1-bgl在不同Escherichiacoli宿主中表达效果,以及pET20b(+)-bgl和pET28a(+)-bgl在E.coliBL21(DE3)中的表达效果。结果表明,E.coliBL21(DE3)-pET28a(+)-bgl能够表达最高的重组β-1,3-1,4-葡聚糖酶酶活,其总酶活可达(322.0±8.8)U/mL,是出发菌在最适摇瓶发酵条件下产酶活的40.1%。对该重组菌的产酶条件进行了分析,结合IPTG和乳糖协同的诱导作用,在基础产酶培养基中产最高总酶活为(1883.3±45.8)U/mL,表明其具有良好的工业应用价值。     

Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR)

AIMS: The aim of the work was to develop efficient method to identify polyhydroxyalkanoate (PHA)-producing species of Bacillus from numerous soil isolates of bacteria. Identification of the isolates and characterization of the PHA produced by strains positive on the polymerase chain reaction (PCR) was envisaged. METHODS AND RESULTS: Different bacteria isolated from soil were screened by PCR using two sets of primers designed for Bacillus megaterium. Amongst 23 isolates examined, the DNA of 12 isolates reacted positively with the primers giving amplicons identical in size to that obtained from B. megaterium. The isolates which were identified as strains of B. sphaericus, B. circulans, B. brevis and B. licheniformis, produced 11- 41% of PHA in biomass, in sucrose-containing medium, over a growth period of 24-72 h. The nature of the PHA thus produced was analyzed by Fourier transform infrared spectroscopy, gas chromatography and by nuclear magnetic resonance (NMR) and found to contain polyhydroxy butyrate and polyhydroxyvalerate. CONCLUSIONS: The results indicate that most of our isolates from different species contained the B. megaterium type of PHA synthase. Bacillus licheniformis appeared to belong to another group as it did not react with both sets of primers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the universality of the B. megaterium type of PHA synthase in soil isolates of Bacillus. Some variations were also found.     

在枯草杆菌原生质体中用鸟枪法进行基因克隆

用ＢａｍＨ１和ＣＩＰ处理ｐＣＢ２０质粒,Ｓａｕ３Ａｌ部分降解枯草杆菌抗阿霉素突变株染色体ＤＮＡ,Ｔ４连接酶连接载体和ＤＮＡ片断,构成表达文库。用这个文库分别转化枯草杆菌ＢＤ２２４菌株感受态细胞和原生质体。     

Enhancement of nematicidal potential through cloning and expression of chitinase gene from Bacillus subtilis subsp. Subtilis BTN7A strain

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.     

枯草芽孢杆菌群β-甘露聚糖酶基因克隆及同源性分析

本文对33株枯草芽孢杆菌群菌株进行β-甘露聚糖酶活性筛选,其中的32株具有β-甘露聚糖酶活性,只有1株无β-甘露聚糖酶活性.通过基因克隆测序的方法获得33株枯草芽孢杆菌群菌株β-甘露聚糖酶基因编码区全序列,对酶基因进行同源性分析并构建系统发育树;在β-甘露聚糖酶基因系统发育树中,33株枯草芽孢杆菌群菌株聚为3个分支,分别是枯草芽孢杆菌分支、地衣芽孢杆菌分支和解淀粉芽孢杆菌分支;枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌β-甘露聚糖酶基因种内同源性大于91%,而种间同源性为60%69%.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

耐热芽孢杆菌E2菌株纤维素酶基因克隆的研究

用质粒ｐＢＲ３２２作载体,大肠杆菌Ｅ．ｃｏｌｉ ＤＨ５αＦ’作受体,克隆到耐热孢杆菌Ｅ２菌株的羧甲基纤维素酶基因。重组质粒ＰＢＧ３从产生ＣＭＣａｓｅ的转化子中分离得到。克隆到的ＣＭＣａｓｅ基因位于４．０ｋｂＨｉｎｄⅠⅠⅠ片段上。功能状态ＣＭＣａｓｅ基因被亚克隆到２．４ｋｂＤＮＡ片段上。     

Isolation and characterization of Bacillus sp. BP-6 LipA,a ubiquitous lipase among mesophilic Bacillus species

AIMS: The aim of this study was to perform the isolation, cloning and characterization of a lipase from Bacillus sp. BP-6 bearing the features of a biotechnologically important group of enzymes. METHODS AND RESULTS: Strain Bacillus sp. BP-6, showing activity on tributyrin plates, was used for isolation of lipase-coding gene lipA by means of inverse and direct PCR. The complete 633 nucleotide ORF isolated was cloned in Escherichia coli for further characterization. The amino acid sequence of the cloned protein was 98% identical to B. subtilis and B. megaterium lipases, the enzyme also showing similar molecular and biochemical features. CONCLUSIONS: The gene coding for Bacillus sp. BP-6 LipA was found in all mesophilic Bacillus species assayed, indicating its ubiquity in the genus. The cloned enzyme displayed the same properties as those of homologous lipases. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall profile of Bacillus sp. BP-6 LipA was found to be that of a ubiquitous and highly conserved subfamily I.4 bacterial lipase. Previously described lipases within this family have shown to be well suited for biotechnological applications, suggesting that the cloned enzyme could be used accordingly.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

枯草芽孢杆菌寡聚—1，6—葡萄糖苷酶基因的克隆及其在大肠杆菌中的表达

本研究用鸟枪法构建了枯草芽孢杆菌（Bacillus subtilis)HB002的基因组文库,经平板法筛选得到了六株能水解合成底物对－硝基苯－α-D－葡萄糖吡喃糖苷的阳性克隆,经鉴定均含克隆了寡聚－1,6－葡萄糖苷酶基因的重组质粒（命名为pHBM001-pHBM006)。选择pHBM003,对其插入片段测序分析,此片段内有一编码561个氨基酸的开放阅读框,该 蛋白质的计算分子量为65.985kD。HB002的寡聚－1,6－葡萄糖苷酶的氨基酸序列与Bacillus sp.和凝结芽孢杆菌（Bacillus coagulans)的寡聚－1,6－葡萄糖苷酶的氨基酸序列一致性分别为81％、67％,相似性分别为89％、79％。从pHBM003中扩增出寡聚－1,6－葡萄糖苷酶基因,克隆到pBV220上,转化大肠杆菌（Escherichia coli)DH5α,得到三个能水解对－硝基苯－α－D－葡萄糖吡喃糖苷的阳性克隆HBM003－1～HBM003－3,将此三个菌株热诱导表达,SDS－PAGE电泳可检测到特异表达的蛋白质,其中HBM003－1、HBM003－2表达的蛋白约66kD,为完整的寡聚－1,6－葡萄糖苷酶,而HBM003－3表达的蛋白质偏小;表达的蛋白质均有寡聚－1,6－葡萄糖苷酶活性。