Synthesis of vinyl arabinose ester catalyzed by protease from Streptomyces sp.

The transesterification of 0.5 M divinyladipate with 0.25 M arabinose in dimethylformamide for 7 days was catalyzed by Streptomyces sp. alkaline protease to give 5-O-vinyladipoyl-d-arabinofuranose at ca. 50% yield. Only enzymatic transesterification of primary hydroxyl group of arabinofuranose proceeded without esterification of arabinopyranose.     

Selective enzymatic preparation of vinyl sugar esters using DMSO as a denaturing co-solvent

The protease-catalyzed transesterifications between hexoses and divinyladipate were examined. In dimethylformamide hexoses such as d-glucose, d-mannose, d-galactose and -methyl d-galactoside were esterified with divinyladipate by alkaline protease from Streptomyces sp. to give corresponding 6-O-vinyl adipoyl sugars. When the denaturing cosolvent, DMSO, was added to the solvent, galactose was selectively esterified at only the C-2 position.     

Preparation of vinyl thymidine ester catalyzed by protease and its chemical polymerization

Transesterification reaction of 0.25 M thymidine with 1 M divinyladipate in dimethylformamide (DMF) was catalyzed by an alkaline protease (5 mg ml^–1) from Streptomyces sp. (20 units mg^–1 min) at 30 °C for 7 days to give 5-O-vinyladipoyl thymidine (yield 77%) without formation of any by-products. Poly(vinyl alcohol) containing thymidine branches could be obtained by its free-radical polymerization.     

Regio- and stereo-selective synthesis of vinyl glucose ester catalyzed by an alkaline protease of Bacillus subtilis

The transesterification of -d-glucose with divinylsuccinate, divinyladipate and divinylsebacate in pyridine at 55 °C for 3 days was catalyzed by an alkaline protease from Bacillus subtilis to give corresponding 6-O-vinyl glucose esters at 30%, 53% and 35% yield, respectively. The stereo-selectivity of the alkaline protease toward the -anomer was affected by the acyl donor chain length. 6-O-Vinylsuccinyl-d-glucose was mixture of - and -anomers (/=44/56), the other two products were the pure -d-glucose derivatives.     

Transesterification of divinyladipate with glucose at various temperatures by an alkaline protease of Streptomyces sp.

The transesterification of 1 M divinyladipate with 0.25 M glucose in dimethylformamide (DMF) catalyzed by 5 mg ml^–1 alkaline protease (24 units mg^–1 min^–1) from Streptomyces sp. gave 6-O-vinyladipoyl d-glucose as the main product with yields are between 60 and 90%. The optimum temperature for the reaction was about 50 °C.     

Feasibility study for decomposition of gelatin layers on X-ray films by thermostable alkaline protease from alkaliphilic Bacillus sp.

Enzymatic decomposition of gelatin layers on X-ray films and repeated utilization of enzyme for potential industrialization were investigated using thermostable alkaline protease from the alkaliphilic Bacillus sp. B21-2. The decomposition of gelatin layers at 50 °C with the mutant enzyme (Ala187 was replaced by Pro) was higher than those of the wild-type and other mutant enzymes. In the repeated experiment for every 60 min (20 U ml^–1, 50 °C), the mutant enzyme could be satisfactorily used five times while three times for the wild-type enzyme.     

Bleach-stable, alkaline protease from Bacillus sp.

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.     

Michael addition of imidazole with acrylates catalyzed by alkaline protease from Bacillus subtilis in organic media

A new activity of alkaline protease from Bacillus subtilis for Michael addition reactions of imidazole, 4-nitro-1H-imidazole and 2-methyl-4-nitro-1H-imidazole with acrylates and acrylic acid was investigated. The reactions were carried out in pyridine at 50 degrees C for 72 h. Five N-substituted imidazole derivatives were obtained using acrylate esters, but not acrylic acid, in yields from 62% to 76%.     

包衣微丸型碱性蛋白酶的制备

开发了一种包衣微丸型碱性蛋白酶的制备工艺,结果表明:30 L发酵中罐发酵50 h比酶活可达4.26×104 U/mL,发酵液经絮凝处理、板框压滤、膜浓缩后可制成酶活达300 000 U/mL的酶浓缩液.经流化后制得含酶颗粒,再包裹薄层后,得包衣微丸型碱性蛋白酶.对制备的包衣微丸型碱性蛋白酶的稳定性、去污效果等指标进行了评估.制备的包衣微丸型碱性蛋白酶产品在严苛条件下的稳定性与国外产品( Savinase 8.0T、PuraFast 2000HS)相当,产品暴露在37℃、75％湿度下8周后仍可保持74％的酶活力,产品的去污效果优于国外产品.制备的包衣微丸型碱性蛋白酶颗粒大小均匀,流动性和分散性好,对外界高温、高湿等不良环境具有很强的抵抗能力,适于工业化生产.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

低温碱性蛋白酶QDAPr的生物信息学分析及同源模建

对一株来自黄海的假单胞杆菌QD80所产的碱性蛋白酶QDAPr进行了生物信息学分析,并用生物信息学软件对其空间结构进行了模拟,同时对模建结果进行了结构质量的分析和检测。最后对模建的结果进行了初步的实验验证。结果表明：该碱性蛋白酶的等电点为8．52,外源性氨基酸为32．9％,二级结构中α螺旋占5．88％,β折叠片占14．6％,与铜绿假单胞菌的碱性蛋白酶的同源性为76％。三维结构检测表明此模型的结构符合立体化学和生物学功能。同源性分析表明该模建模型含有HEXXHXUGUXH的基元（motif）,通过BrAc修饰实验初步验证了活性中心的功能。     

短小芽孢杆菌2080碱性蛋白酶的纯化与性质

短小芽孢杆菌（Bacillus pumilus）2080碱性蛋白酶的发酵液经超滤、硫酸铵沉淀、CM Sepharose Fast Flow和DEAE Sepharose Fast Flow离子交换层析得到了纯化的组分。SDS-PAGE电泳分析显示其分子量约为61kDa。酶学性质研究表明,该纯化酶的最适pH为10．5,最适温度为50℃。     

Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S-1

AIMS: The present study was conducted by screening soil bacteria in an attempt to isolate a bacterium that produced extracellular alkaline protease, and for purification and characterization of the protease. METHODS AND RESULTS: Soil bacteria were screened by growth on casein as the sole carbon source. Characterization of a strain isolated from soil of Abashiri, Japan indicated a taxonomic affiliation to Stenotrophomonas maltophilia, and was named S-1 strain. The purified S-1 protease, designed S. maltophilia Protease-1 (SmP-1), exhibited an optimal pH of 12.0, optimal reaction temperature of 50 degrees C and a molecular mass of approximately 40 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cleavage sites of the oxidized-insulin B chain by SmP-1 were identified as Leu6-Cys7, Cys7-Gly8, Tyr16-Leu17 and Leu17-Val18. The N-terminal amino acid sequence of the purified alkaline protease was determined as NH2-SASAPMVSGVAALVLE. CONCLUSION: A novel extracellular alkaline serine protease was isolated from S. maltophilia strain S-1. The optimal pH of the proteolytic activity was pH 12.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The extremely high optimal pH and heat stability of the alkaline serine protease SmP-1 might make it widely applicable to food and other industries.     

地衣芽孢杆菌碱性蛋白酶的研究 Ⅰ.碱性蛋白酶高产菌的筛选及产酶条件初探

从成都佳丰食品厂等处采集的样品中平板分离初筛到124株碱性蛋白酶产生菌,进一步复筛出一株高产,且稳定的碱性蛋白酶产生菌株B．L．JF-ld,初步鉴定为地衣穿孢杆菌（Bacilluslicheniformis）。该菌的最适产酶条件为：培养基（％）为麦芽糖7．5,酵母膏3,NaCl0．5,K2HPO4·3H2O0．53,NaHPO4·2H2O0．03,Na2CO30．056,MnSO4l×10－4mol／L,pH8．7,通气量为（1：0．5）～（1：1）（v／v）,37℃发酵40h,酶活力单位高达7180U／ml。     

Kinetic resolution of esters of amino acids in t-butanol containing 5% water catalyzed by a stable industrial alkaline protease

We developed a procedure for the resolution of esters of amino acids in 95% t-butanol, followed by saponification of the unreacted esters to afford both enantiomers with high yield and optical purity. The hydrolysis, catalyzed by alkaline protease, was conducted in a mixture of t-butanol (95%) and water (5%) at 25°C, with a pH controlled at pH 8.5 by the addition of NaOH (2 M). The hydrolyzed L -amino acid, which was insoluble under these conditions, precipitated during the course of hydrolysis. After separation of the precipitate, the pH of the filtrate was adjusted to 11.5 to saponify the unreacted ester. The D -antipode precipitated at pH 6.2–6.5. Both optically pure antipodes were obtained with high enantiomeric excesses and yields by simple filtration. © 1994 Wiley-Liss, Inc.     

Synthesis and activity of isoxazoline vinyl ester pseudopeptides as proteasome inhibitors

The ubiquitin–proteasome pathway (UPP) influences essential cellular functions including cell growth, differentiation, apoptosis, signal transduction, antigen processing and inflammatory responses. The main proteolytic component of the UPP is the 26S proteasome, which is responsible for the turnover of many cellular proteins and represents an attractive target for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Natural and synthetic proteasome inhibitors having different chemical structures and potency have been discovered. We report herein the synthesis, proteasome inhibition and modelling studies of novel C‐terminal isoxazoline vinyl ester pseudopeptides. Some new compounds that contain a C‐terminal extended conjugation inhibit β1 and especially β5 proteasomal catalytic subunits with IC₅₀ values ranging from 10 to 100 µm . These results will permit further optimization based on these structural moieties to develop more active and selective molecules. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Thermostability of an alkaline protease, AprP, is enhanced by replacements of Ser307 and Ser331 at the cleavage sites

The thermostability of an alkaline protease, AprP from Pseudomonas sp. KFCC 10818, was improved by replacing Ser307 and Ser331 at the autoproteolytic cleavage sites with various amino acids. Six mutant enzymes were purified and characterized. Two of these had half-lives four and three times longer than the wild-type protease at 55 °C in the presence of 1 mM CaCl₂. Three mutant enzymes had half-lives twice as long as the wild-type under the same condition.     

Production of a thermophilic,extracellular alkaline protease by Bacillus stearothermophilus AP-4

A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mm CaCl₂. The enzyme was completely inactivated by PMSF, EDTA and -mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India     

Immobilization of alkaline protease from Conidiobolus macrosporus for reuse and improved thermal stability

Alkaline protease from Conidiobolus macrosporus was immobilized on polyamide using glutaraldehyde as a bifunctional agent. The immobilized enzyme was optimally active at a higher temperature of 50°C than the free enzyme (40°C ) and showed a ten-fold increased thermostability at 60°C compared to that of the free enzyme. The efficiency of immobilization was 58% under the optimal conditions of pH and temperature. There was a 14-fold decrease in the K _m of immobilized enzyme compared to the free enzyme. The immobilized enzyme was fully active even after twenty-two cycles of repeated use. It retained 80% activity at 50°C in presence of 8 M urea exhibiting its stability to the denaturant and was compatible with several commercial detergents.