Effect of nutritional conditions on extracellular protease production by the haloalkaliphilic archaeon Natrialba magadii

AIMS: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. METHODS AND RESULTS: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production. CONCLUSION: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.     

Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties

AIMS: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance. METHODS AND RESULTS: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l-1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4.7H2O, 0.1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60 degrees C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases. CONCLUSIONS: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.     

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     

Biocatalytic Knoevenagel reaction using alkaline protease from Bacillus licheniformis

Abstract

BLAP (alkaline protease from Bacillus licheniformis) was used as a biocatalyst in the Knoevenagel condensations of aromatic, hetero-aromatic and α;β-unsaturated aldehydes with less reactive acetylacetone or ethyl acetoacetate. The reactions were performed in organic solvent in the presence of water. The functionalized trisubstituted alkenes and α,β,γ,δ-unsaturated carbonyl compounds could be obtained in moderate to good yields with E/Z selectivities up to >99:1. This biocatalytic reaction provided an alternative pathway for Knoevenagel condensation, which also demonstrates a novel case of unnatural activity of existing enzymes.     

一株海洋细菌产碱性蛋白酶的研究

对1株产碱性蛋白酶的海洋细菌Pseudomonas sp.7-11发酵产生碱性蛋白酶的条件和酶的调控机制进行了初步研究。结果显示,7－11为好氧菌,具有嗜低温特性,且适宜在偏碱性的条件下生长并产酶,该菌产生碱性蛋白酶表现出初级动力学持征;酷蛋白培养基适合菌株产酶;K^ 对于菌株产酶有关键作用;复合氨基酸对产酶有抑制作用;初步显示,7－11产生蛋白酶受诱导及阻遏两种方向的调控。     

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.     

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     

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.     

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.     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

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。     

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.     

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。     

Modelling and optimization of fermentation factors for enhancement of alkaline protease production by isolated Bacillus circulans using feed-forward neural network and genetic algorithm

Aim: Modelling and optimization of fermentation factors and evaluation for enhanced alkaline protease production by Bacillus circulans. Methods and Results: A hybrid system of feed‐forward neural network (FFNN) and genetic algorithm (GA) was used to optimize the fermentation conditions to enhance the alkaline protease production by B. circulans. Different microbial metabolism regulating fermentation factors (incubation temperature, medium pH, inoculum level, medium volume, carbon and nitrogen sources) were used to construct a ‘6‐13‐1’ topology of the FFNN for identifying the nonlinear relationship between fermentation factors and enzyme yield. FFNN predicted values were further optimized for alkaline protease production using GA. The overall mean absolute predictive error and the mean square errors were observed to be 0·0048, 27·9, 0·001128 and 22·45 U ml^?1 for training and testing, respectively. The goodness of the neural network prediction (coefficient of R²) was found to be 0·9993. Conclusions: Four different optimum fermentation conditions revealed maximum enzyme production out of 500 simulated data. Concentration‐dependent carbon and nitrogen sources, showed major impact on bacterial metabolism mediated alkaline protease production. Improved enzyme yield could be achieved by this microbial strain in wide nutrient concentration range and each selected factor concentration depends on rest of the factors concentration. The usage of FFNN–GA hybrid methodology has resulted in a significant improvement (>2·5‐fold) in the alkaline protease yield. Significance and Impact of the Study: The present study helps to optimize enzyme production and its regulation pattern by combinatorial influence of different fermentation factors. Further, the information obtained in this study signifies its importance during scale‐up studies.     

Production of a protease inhibitor from edible mushroom Agaricus bisporus and its statistical optimization by response surface methodology

The production of a protease inhibitor from Agaricus bisporus through solid-state fermentation was studied. The purpose was to produce protease inhibitor from natural, cheap, and readily available carbon and nitrogen sources. Solid-state fermentation enhanced the mycelia growth and also gave a higher yield of the product. Further, fungal growth and other production parameters were statistically optimized. The specificity of the inhibitor was tested and was effective against trypsin. Screening of significant factors (wheat bran, cyanobacterial biomass, initial pH, temperature, incubation period, and moisture content and inoculum size) was performed using Plackett–Burman design. Central composite design was used to determine the optimized values of the significant variables which were found to be temperature (27.5°C), incubation time (156?hr), cyanobacterial biomass (1?g), and moisture content (50%) and gave a statistical yield of 980 PIU/g which was 25.6% higher than experimental yield (780 PIU/g). The inhibitor was purified by ammonium sulfate precipitation and diethylaminoethyl (DEAE) cellulose chromatography (yield 43.89% and 0.21%, respectively) and subjected to reversed-phase HPLC to validate its identity. Since protease inhibitors act against proteases, finding ample therapeutic roles; the isolated protease inhibitor from A. bisporus can also be a probable medicinal agent after its further characterization.     

Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1

AIMS: The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. METHODS AND RESULTS: Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. CONCLUSION: A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.     

Cloning and over-expression of an alkaline protease from Bacillus licheniformis

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

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.     

Screening and mutagenesis of a novel Bacillus pumilus strain producing alkaline protease for dehairing

AIMS: To characterize and optimize a novel Bacillus pumilus strain isolated from biological waste which produces protease with excellent dehairing effect. This newly isolated strain could be utilized in the industrial leather dehairing process. METHODS AND RESULTS: Bacterial strains secreting proteases were screened from biological wastes. Positive clones were further characterized by analysing their efficacy in dehairing and effects on collagen integrity. Among 171 colonies tested, a strain BA06, identified as B. pumilus, was picked owing to its efficient dehairing capabilities with minimal impact on collagen. By combined mutagenesis using UV, N-methyl-N'-nitro-N-nitrosdguanidine and Co(60)-gamma-rays, this strain was further improved with regard to its alkaline protease production. The alkaline protease activity of the mutant strain SCU11was greatly improved up to 6000 U ml(-1), in comparison with its parent strain BA06 of 1200 U ml(-1). CONCLUSIONS: By using screening and mutagenesis methods, we have successfully created a B. pumilus strain that can produce high levels of alkaline proteases that are able to efficiently remove hair from skin with minimal damage on the collagen. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain could be used in commercial alkaline protease production for leather dehairing.