Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene.

Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a beta-mannanase (mannan endo-1,4-beta-mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several beta-mannanases in glycoside hydrolase family 5. The beta-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The beta-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae alpha-factor signal sequence. The beta-mannanase was produced in a 14-L fermenter with an expression level of 900 mg.L-1. The expression level is strongly affected by the induction temperature. A two-step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure beta-mannanase. However, due to post-translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulis beta-mannanase was close to that of the wild-type enzyme. Also pH and temperature optima were the same as for the native protein. In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel beta-mannanase. This is the first time a mollusc beta-mannanase has been characterized at the DNA level.     

棒曲霉Asp-195v产蛋白酶的酶学性质研究

对液体发酵的棒曲霉Asp-195v菌株所产蛋白酶的活力进行了研究,并通过分离纯化获得了电泳纯的酶蛋白。研究结果表明,该蛋白酶的最适反应温度为40℃,在30-50℃温度范围内相对活力可保持在70%以上;最适pH为7,pH稳定范围在4-8;Mn2+对该蛋白酶活力有明显的激活作用,K+、Ag+、Cu2+、Fe2+、Mg2+、Zn2+、Ca2+、Al3+和Fe3+离子则有明显的抑制作用,尤其是Hg2+和Pb2+对酶活的抑制作用更加强烈;其他试剂如葡萄糖、EDTA对酶活的抑制作用不明显,而蔗糖、SDS和Tween-20对酶活的抑制明显;以酪氨酸为底物采用双倒数作图法测得Vmax为30.40mmol/min,Km为97.53mmol/L。该酶的表观分子量为30.1kDa。     

Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp.

The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.     

枯草芽孢杆菌中性β—甘露聚糖酶的产生及性质

由土壤中分离出一株产中性β甘露聚糖酶的枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）,编号ＢＭ９６０２。该菌在液体培养条件下,产生中性β甘露聚糖酶。多糖能作为碳源,而单糖不能作为碳源;有机氮源优于无机氮源。产酶最适培养基组成：魔芋粉４％,牛肉蛋白胨和酵母膏各１％。产酶最适培养条件：培养基起始ｐＨ８５,３５℃,振荡培养３６ｈ。以槐豆胶为底物,培养滤液中性β甘露聚糖酶活力为９６ＩＵ／ｍＬ。酶在ｐＨ５０～１００和５０℃下稳定;作用最适条件为ｐＨ６０和５０℃;水解魔芋粉和槐豆胶均产生寡聚糖。     

A gene encoding a novel multidomain beta-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.     

芽孢杆菌M50产生β—甘露聚糖酶的条件研究

从土壤中分离到９株产生β－甘露聚糖酶的芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐ．）。Ｂａｃｉｌｌｕｓ ｓｐ．Ｍ５０２５０ｍＬ三角瓶摇瓶培养试验,以４％的魔芋粉为碳源,１．０％（ＮＨ４）２ＳＯ４为氮源,０．３５％Ｎａ２ＣＯ３,３０～３４℃培养６０ｈ产酶达到高峰。酶活力为１８０～２２０ｕ／ｍＬ。１００Ｌ罐发酵,在３０～３２℃,１：０．７５ｖｖｍ通气量,２００ｒ／ｍｉｎ条件下,发酵液酶活力高达３３０ｕ／ｍＬ。     

Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum"

A lambda recombinant phage expressing beta-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of Mr 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6 and 80 degrees C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the "C. saccharolyticum" genome by homologous recombination.     

枯草芽孢杆菌β-甘露聚糖酶在毕赤酵母中的分泌表达

目的:研制高效分泌表达枯草芽孢杆菌β-甘露聚糖酶的毕赤酵母基因工程菌株。方法与结果:将优化设计的枯草芽孢杆菌MA139β-甘露聚糖酶基因用EcoRⅠ/XbaⅠ双酶切,克隆到诱导型表达载体pPICzαA中α因子信号肽编码序列的下游,转化大肠杆菌筛选重组质粒,转化毕赤酵母X-33感受态细胞,经Zeocin筛选,获得重组表达菌株X-33/mann。将重组菌株在10L全自动发酵罐中进行高密度发酵培养,甲醇诱导72h发酵活力达到2100U/mL。重组甘露聚糖酶的最适催化温度为40℃,最适催化pH值为6.0。结论:枯草芽孢杆菌β-甘露聚糖酶在毕赤酵母中获得了高效分泌表达,具有开发作为饲料添加剂的潜能。     

Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.     

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.     

耐碱性甘露聚糖酶基因的克隆及其在毕赤酵母中的表达

通过功能平板从土壤中筛选得到含甘露聚糖酶基因的耐碱菌株。构建其基因组文库,从中筛选到甘露聚糖酶基因TM1并测序分析,用BLAST分析表明,TM1的氨基酸序列与其他在GenBank发表的甘露聚糖酶的氨基酸序列的同源性均低于60%,故确定其为一个新的甘露聚糖酶基因(GenBank登录号为AY623903)。将此基因去除信号肽后的编码序列克隆到表达载体pHBM905C上,得到重组质粒pHBM1201。经SalⅠ酶切后分别转化毕赤酵母(Pichiapastoris)KM71、GS115、SMD1168,得到分泌表达的重组毕赤酵母。挑选相对表达量最高的重组毕赤酵母SMD1168-3在摇瓶中诱导产酶,对该酶的粗酶进行酶学性质分析表明,其最适反应温度为55℃,最适PH值为7.5,以魔芋粉为底物所测得的最高酶活为41.8U,半衰期为1h,在80℃保温5min其酶活由最初酶活的77%下降到11%,温度下降到55℃后活性可恢复到最初酶活的60%以上。     

Expression, purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris

The DEX gene encoding an extracellular dextranase from Lipomyces starkeyi was cloned into vector pPIC9k-His6 and was expressed in Pichia pastoris GS115 strain under the control of AOX1 promoter. After 107 h of the 5L-scaled fermentation, wet cells weight of the recombinant P. pastoris Mut(+) strain reached to 588.6g/L, and the concentration of dextranase and enzyme activity in the supernatant were 0.46 g/L and 83900 U/L, respectively. The activity of dextranase was improved 17.56-fold by cation-exchange chromatography only with a final yield of 71.61% and the specific activity of the purified enzyme was 181.96 U/mg. The purified dextranase, analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. Then the factors affecting the dextranase activity were evaluated. The optimal temperature and pH was 30 degrees C and pH 4.5, respectively. Metal ions Al(3+), Cu(2+), Fe(3+), and SDS could completely inhibit the enzyme activity, whereas Mg(2+) enhanced 145% of the enzyme activity. These characters are much different from what was previously reported for the L. starkeyi dextranase that was either expressed in S. cerevisiae or purified from natural L. starkeyi.     

Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme

An endoglucanase from Bacillus akibai I-1 was successfully overexpressed in Bacillus subtilis 168 and the expression level of the recombinant enzyme was greatly enhanced by using the sucrose-inducible sacB promoter. The endoglucanase activity in the culture supernatant of recombinant B. subtilis by using itself promoter (HpaII) in plasmid pMA5 was 3U/ml. Interestingly, with the addition of sacB promoter at downstream from the HpaII promoter or the replacement of HpaII promoter by the sacB promoter, the endoglucanase activities reached 62 and 60U/ml, respectively, under the optimal culture conditions. These results demonstrated that the sacB promoter might be more efficient for the expression of the endoglucanase than the HpaII promoter. More interestingly, the purified native enzyme had broad pH stability, good thermostability and resistibility to various metal ions and chelating agents examined, while the recombinant enzyme had improved resistibility to SDS, which was stable in 0.2% (w/v) laundry detergent and thus showed great potential in detergents industry.     

Purification and characterization of keratinase from recombinant Pichia and Bacillus strains

The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 degrees C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K(m) and V(max) of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.     

Characterization of a thermostable Bacillus stearothermophilus alpha-amylase

Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.     

重组毕赤酵母表达棘孢木霉几丁质酶Tachi1的酶学性质研究及表达条件优化

【目的】对转棘孢木霉几丁质酶基因tachi1的毕赤酵母工程菌GS-tachi1-K进行诱导表达,研究重组几丁质酶Tachi1的酶学性质,优化表达条件。【方法】对GS-tachi1-K进行甲醇诱导培养,纯化目的蛋白Tachi1进行几丁质酶酶学性质的研究;通过单因素和正交试验对GS-tachi1-K菌株产几丁质酶Tachi1表达条件进行优化。【结果】GS-tachi1-K表达的几丁质酶Tachi1表观分子量约为44 kDa,酶反应最适的温度和pH分别为50℃和5.5,具有较宽的温度、pH适用范围;50℃以下保持较高的酶活力,在碱性条件下稳定性较差;受Ag+、Hg2+、Cu2+、Fe2+和高浓度的SDS及β-巯基乙醇强烈抑制。该菌株的最佳表达条件为:pH为6.5,甲醇诱导浓度为0.5%,起始细胞浓度为OD600=2,甲醇诱导时间为180 h;几丁质酶Tachi1活力可达17.93 U/mL,蛋白表达量为6.19 g/L。【结论】成功实现了棘孢木霉新几丁质酶基因tachi1的毕赤酵母高效分泌表达,工程菌GS-tachi1-K具有高表达量和表达产物酶活性高两个特点,明确了几丁质酶Tachi1的酶学性质和最佳诱导表达条件,为该几丁质酶及其基因的深入研究和开发利用奠定了基础。     

巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化

青霉素酰化酶（ＰＧＡ）在医药工业起着重要的作用,它能够水解青霉素Ｇ产生６－氨基青霉烷酸（６－ＡＰＡ）和苯乙酸,６－ＡＰＡ是半合成青霉素的关键中间体．该酶广泛存在于各种微生物中如真菌和细菌中．国际上对Ｅ．ｃｏｌｉ、Ａｒｔｈｒｏｂａｃｔｅｒｖｉｓｃｏｓｕ...     

重组超耐热酸性α-淀粉酶的分离纯化及其性质研究

基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3 、Fe2 、Cu2 抑制酶的活性,Ca2 对酶活无影响。EDTA和DTT对酶的活性无影响。     

Purification and properties of Bacillus subtilis SA-22 endo-1, 4-beta-D-mannanase.

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.     

Isolation of a highly active preparation of beta-D-galactosidase

Methods for isolation and purification of beta-galactosidase from Bacillus subtilis, st. IBP-101 are described. The bacterial cells were disrupted by different procedures such as freezing and thawing with subsequent autolysis at 37 degrees C, disrupting in a French press DKM-3 or in ultrasonic disintegrators UZDN-1 (USSR) and Soniprep-150. It is shown that the specific activity and yield of the enzyme depends to a great extent on the disrupting procedure used. The best results were obtained in case of sonication. The preparation was purified by precipitation with ammonium sulphate (25-75% saturated) and chromatography on DEAE-cellulose and DEAE-Sephadex. The purified enzyme had a specific activity of 3155 units per mg protein. The molecular weight of the homogeneous according to gel polyacrylamide electrophoresis preparation was 215,000, as estimated by gel filtration, and 105,000, as estimated by SDS gel electrophoresis. The enzyme retains the activity in the presence of Na+, Mn2+ or Mg2+ ions or the thiolic reagents, dithiothreitol or 2-mercaptoethanol. The pH optimum of the enzyme activity is 6.3 and it is stable in water solutions at pH from 6 to 9 and can be lyophilized. The given preparation of beta-galactosidase has a high affinity for synthetic substrates such as o- and p-nitrophenyl-beta-D-galactopyranosides and 4-methylumbelliferyl-beta-D-galactopyranoside.