Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm

When the capsules of a granulosis virus are fed together with the polyhedra of a nuclear-polyhedrosis virus to larvae of the armyworm, Pseudaletia unipuncta, the former enhances the infectivity of the latter virus, a synergistic interaction. The enhancement of infectivity depends upon the concentration of the polyhedra and the capsules. The factor responsible for the synergistic activity in the capsule can be dissolved in alkaline solution, separated from the virus particles by centrifugation, and further purified by Sephadex G-200 gel filtration with 4 m urea. The fraction obtained from Sephadex filtration and containing the synergistic factor can be separated into two components by disc-electrophoresis with 8 m urea. Both components possess synergistic activity. The ID₅₀ of the synergistic factor corresponds to 0.0015 OD₂₈₀. Its optimum pH is 8.5. Synergism is most evident when the factor is fed to larvae together with the polyhedra or is fed 24 hr prior to the ingestion of the polyhedra. The factor appears to be a simple or a conjugated protein of the capsule.     

Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar

After solubilization of polyhedra of Autographa californica, Lymantria dispar, and Mamestra brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0–30.0 kdalton), characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides.     

Alkaline protease from a non-toxigenic mangrove isolate of Vibrio sp. V26 with potential application in animal cell culture

Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae, V. mimicus, V. albensis and uncultured clones of Vibrio. Phenotypically also it resembled both V. cholerae and V. mimicus. Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administering APV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios.     

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

Effect of alkaline protease-producing Exiguobacterium sp. YS1 inoculation on the solubilization and bacterial community of waste activated sludge

A new approach to the solubilization of waste activated sludge (WAS) using an alkaline protease-producing bacterial isolate, Exiguobacterium sp. YS1, was investigated under controlled mild alkaline conditions at pH 10. Compared with the noninoculated experiment, the inoculated experiment in an anaerobic bioreactor increased soluble chemical oxygen demand concentration and alkaline protease activity by more than 40%, indicating a synergistic effect could be achieved when both bacterial inoculation and alkaline treatment were combined. Indeed, this combination led to 56.6% COD solubilization after 5 days of reaction time. However, the inoculant was not effective in the aerobic bioreactor. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA fragments revealed that the inoculated Exiguobacterium sp. YS1 became the predominant population in the bacterial community during the anaerobic solubilization processes. These results suggest that bioaugmentation of the organism might be useful for enhancing the solubilization of WAS at mild alkaline pH.     

Acylamines enhance the infection of a baculovirus of the armyworm, Pseudaletia unipuncta (Noctuidae,Lepidoptera)

The acylamines (cationic detergents) enhance the infection of a nuclear polyhedrosis virus (NPV) in larvae of the armyworm, Pseudaletia unipuncta. Those with 6 carbons (hexylamine) or 12 carbons (dodecylamine) in the acyl group are more effective in enhancing the NPV than those with 4, 8, or 10 carbons. Hexylamine and dodecylamine increase the rate of infection beyond the 50% level (ID₅₀) by 10 to 100 times when based on the concentration of the virus polyhedra (inclusion bodies).     

Effect of alkaline protease on the antigenic nature of wiseana nuclear polyhedrosis virus polyhedron protein

Polyhedron protein from Wiseana spp. nuclear polyhedrosis virus was found to be degraded by an alkali protease when polyhedra are dissolved in alkali. The protease activity did not occur at high pH (0.1 M NaOH) and was inactivated by heating polyhedra to 70°C for 3 h. The products from the protease degradation of Wiseana spp. nuclear polyhedrosis virus polyhedron protein retain the antigenicity of undegraded polyhedron protein as measured by the direct solid-phase radioimmunoassay and immunoadsorption. Degradation products below 27,000 daltons could not be detected by the sandwich radioimmunoassay, indicating that they are probably monovalent.     

Effect of dew from cotton and soybean foliage on activity of Heliothis nuclear polyhedrosis virus

The ionic composition of dew collected from foliage of cotton and soybean plants as well as its potential for inactivation of Heliothis NPV were compared. Cotton dew had a mean pH of 8.8 and was more alkaline than soybean dew (pH 7.8). Cotton dew had a higher concentration of ions that did soybean dew but there was no qualitative difference in the cation content of dew from the two hosts. In bioassay tests, no loss of activity occurred when polyhedra were held in dew of either plant species. If the dew in which polyhedra were suspended was air-dried and resuspended daily in deionized water, polyhedra in soybean dew remained active but in cotton dew retained little activity after 7 days. Also, electron microscopical examination of polyhedra pelleted from these cotton dew preparations showed much dissolution after 7 days. Although there was dissolution of polyhedra in cotton dew when dried, an examination of polyhedra on the upper surface of either plant species in the field showed little degradation after 7 days.     

Lime and Sulphide-Free Dehairing of Animal Skin Using Collagenase-Free Alkaline Protease from Vibrio metschnikovii NG155

The objective of this work was to isolate a microorganism producing alkaline protease that can be used as an ecofriendly alternative to chemicals in dehairing process of leather manufacture. Alkaline protease producing bacterium Vibrio metschnikovii NG155 was isolated from soil samples of leather industry. The protease was highly effective in dehairing of goat skin, completely eliminating the use of lime and sulfide. Histological studies of the skin after dehairing showed that the enzyme did not damage the collagen layer and brought good fiber opening. Absence of collagenase activity was confirmed by reacting pure collagen with the enzyme and analyzing it on SDS PAGE, which showed no degradation of collagen. The enzyme was stable in a wide range of pH (7–11) and temperature (10–50 °C), which makes it suitable for industrial application.     

Structural and kinetic analysis of pyrrolidine-based inhibitors of the drug-resistant Ile84Val mutant of HIV-1 protease

Human immunodeficiency virus (HIV) protease is a well-established drug target in HIV chemotherapy. However, continuously increasing resistance towards approved drugs inevitably requires the development of new inhibitors preferably showing no susceptibility against resistant HIV protease strains. Recently, symmetric pyrrolidine-3,4-bis-N-benzyl-sulfonamides have been developed as a new class of HIV-1 protease inhibitors. The most promising candidate exhibited a K_i of 74 nM towards a wild-type protease. Herein, we report the influence of the active-site mutations Ile50Val and Ile84Val on these inhibitors by structural and kinetic analysis. Although the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the important Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases, we were able to elucidate the structural basis of this phenomenon.     

海南近海水域产碱性蛋白酶菌株的分离筛选及发酵条件优化

【背景】微生物蛋白酶在工业生物技术上具有广阔的应用前景。在微生物蛋白酶中,碱性蛋白酶占全球酶总产量的50%以上,获取产碱性蛋白酶的新微生物资源意义重要。【目的】在海南近海贝类养殖基地海泥中筛选获得高产碱性蛋白酶的菌株,对其生长特性进行探究并优化菌株产酶条件,获得新的蛋白酶生产资源。【方法】以酪素培养基为筛选培养基,采用形态学结合系统发育分析鉴定菌株,通过响应面实验优化菌株的产酶条件。【结果】筛选获得一株高产碱性蛋白酶的菌株F3,鉴定为粘质沙雷氏菌（Serratia marcescens）。菌株在最优产酶条件下发酵酶活达到（339.36±4.30） U/mL。【结论】筛选获得的菌株粘质沙雷氏菌F3有较好的产碱性蛋白酶的能力。     

The removal of lipid from the surface of wool to promote the subsequent enzymatic process with modified protease for wool shrink resistance

Abstract

When treated with modified protease, wool shows shrink resistance without significant damage to the fiber. It was considered that if wool fiber was pre-treated to make it more hydrophilic, the subsequent treatment with modified protease would be more efficient. After wool was pre-treated with cetyltrimethylammonium bromide (CTAB) under alkaline conditions, the fiber became very hydrophilic due to the removal of surface lipid. After CTAB treatment, it was found that residual CTAB on the fiber significantly decreased enzyme activity. Therefore prior to enzyme treatment, CTAB was washed off the fiber with anionic surfactant under acidic conditions. It was found that the activity of modified protease towards wool improved if wool had been pre-treated with CTAB then washed with anionic surfactant. It was concluded that pre-treatment of wool with CTAB under alkaline conditions followed by washing with anionic surfactant improves the wettability of wool and therefore promotes more efficient treatment with modified protease, achieving improved levels of shrink resistance with no effect on strength of the fiber or coloration properties.     

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

Human α₂-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M’s unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible “bait region.” As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the “tabula rasa” bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.     

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.     

Optimization of novel and greener approach for the coproduction of uricase and alkaline protease in Bacillus licheniformis by Box–Behnken model

This study explores a novel concept of coproduction of uricase and alkaline protease by Bacillus licheniformis using single substrate in single step. Seven local bacterial strains were screened for uricase production, amongst which B. licheniformis is found to produce highest uricase along with alkaline protease. Optimization of various factors influencing maximum enzyme coproduction by B. licheniformis is performed. Maximum enzyme productivity of 0.386?U/mL uricase and 0.507?U/mL alkaline protease is obtained at 8?hr of incubation period, 1% (v/v) inoculum, and at 0.2% (w/v) uric acid when the organism is cultivated at 25°C, 180?rpm, in a media containing xylose as a carbon source, urea as a nitrogen source, and initial pH of 9.5. The statistical experimental design method of Box–Behnken was further applied to obtain optimal concentration of significant parameters such as pH (9.5), uric acid concentration (0.1%), and urea concentration (0.05%). The maximum uricase and alkaline protease production by B. licheniformis using Box–Behnken design was 0.616 and 0.582?U/mL, respectively, with 1.6- and 1.13-fold increase as compared to one factor at a time optimized media. This study will be useful to develop an economic, commercially viable, and scalable process for simultaneous production of uricase and protease enzymes.     

A dual protease approach for expression and affinity purification of recombinant proteins

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His₆-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His₆-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His₆ tag is removed by His₆-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His₆-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification.     

Isolation,partial purification,biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis,Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37?°C for 48?h with a constant agitation of 120?rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1?M ice cold CaCl₂ and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60?s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.     

Alkaline protease associated with virus particles of a nuclear polyhedrosis virus: assay, purification, and properties

Proteolytic activity was detected within polyhedra of the nuclear polyhedrosis virus of Spodoptera littoralis. The enzyme activity was detected by its ability to degrade the major structural polypeptide of polyhedra (polyhedrin). A quantitative assessment of activity was made by a radioassay technique using ³H-labeled polyhedrin as the substrate. Of the structural components of polyhedra, virus particles showed the greatest specific proteolytic activity. Preparations of purified nucleocapsids were inactive. The virus particle enzyme displayed a temperature optimum for proteolysis of 30 to 40°C and a pH optimum of 9.6. Its activity was inhibited by H²⁺ and Cu²⁺, but not by 2-mercaptoethanol. The enzyme was purified from detergent-treated virus particles by affinity column chromatography, using polyhedrin linked to cyanogen bromide-activated Sepharose. Three major envelope polypeptides (L107, L85, and L71) bound to the column at 4°C, but after incubation at 31°C, polypeptide L71 alone was eluted. The fractions containing this protein exhibited a specific enzyme activity more than 80-fold greater than that present in polyhedra. The possible significance of the alkaline protease, and other proteins with affinity for polyhedrin, is discussed.