Impact of microbial proteases on biotechnological industries

The demand of enzymes in industrial sectors is increasing rapidly due to their economical and ecological advantages. Micro-organisms produce different types of extracellular enzymes for maintaining their own metabolism, defense, and normal physiological condition. Among several enzymes, proteases have gained special attention in industrial sectors. Several sources of extracellular enzymes are reported by various researchers, but enzymes obtain from microbial sources have high demand in industries due to lower cost, high production rate, availability, stability, and diversity. Among micro-organism, bacteria and fungi are reported to be good sources of different types of proteases such as alkaline protease, cysteine protease, aspartate protease, and metallo protease. In this review, we have summarized the available information about the sources of bacterial and fungal proteases, their purification strategies and their temperature and pH optima. Due to huge competition, companies are trying to reduce their manufacturing cost and that’s why microbial sources of enzymes are important. However, genetically engineered strains or engineered proteases have much more importance over natural isolates/protease in industries due to higher production rate and other advantages. Here we have also summarized the important applications of protease in different industries such as, paper mill, starch degrading sector, food processing factories, and detergent making companies.     

Search for novel proteolytic enzymes aimed at textile and agro-industrial applications: An overview of current and novel approaches

The types and sources of proteolytic enzymes, enzyme assays, strategies for fermentation yield improvement, and novel proteases and their applications in industrial sectors are widely covered in this review. We give a special focus on alkaline proteases for the textile and detergent industries, as well as for the degradation of keratin-rich wastes.     

Immunochemical characterization of commercial protease products

Total protease activity at pH 7 and 10.3 of 23 commercial grade enzymes was determined. The type and amount of enzymatic activity varied widely among the products. The wide variation in pH 7.0/pH 10.3 proteolytic activity ratios among products indicated that the products studied contained differing levels of alkaline and neutral proteases. Antisera were prepared against the purified enzyme in detergent grade Enzyme AP, neutral protease from B. megaterium, detergent grade ALK Enzyme, and Thermolysin. The commercial (unpurified) products were classified as neutral subtilopeptidase A and subtilopeptidase B from three Bacillus species using these antisera. It was concluded that standard immunochemical techniques provide rapid and sensitive methods for the preliminary identification of sources and types of proteases present in commercial enzyme products.     

Preparation of Immobilized Protease Inhibitor (S-SI) and Application to Enzyme Purification

Microbial alkaline protease inhibitor, S-SI, was immobilized by covalent binding with Sepharose (agarose spheres) which was previously activated by cyanogen bromide. S-SI-Sepharose, thus obtained, contained 7.2 mg of S-SI in 1 ml of settled volume, and its subtilisin-combining capacity was 16.6 mg per ml. Stability of S-SI did not be lowered by immobilization. Affinity of immobilized S-SI for various proteases was examined, and it was revealed that α-chymotrypsin, as well as microbial alkaline proteases, had affinity for immobilized S-SI. To determine the most effective condition for dissociation of coupled subtilisin BPN’, effects of pH, ionic strength, protein denaturants, and sodium dodecyl sulfate (SDS) were examined. Dissociated subtilisin BPN’ with high specific activity was obtained when SDS was used as dissociating agent and was removed with Dowex 2-X10 column from dissociated enzyme solution. S-SI-Sepharose was applied to purifications of B. subtilis S04 alkaline protease and α-chymotrypsin, and purified enzymes with high specific activity were obtained.     

An overview on fermentation,downstream processing and properties of microbial alkaline proteases

Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8-12 and at temperatures between 50 and 70 degrees C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80-90 degrees C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8-11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures.     

A serine alkaline protease from the fungusConidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.     

A new approach for alteration of protease functions: pro-sequence engineering

Several proteases, including the bacterial serine protease subtilisins, require the assistance of the N-terminal pro-sequence of precursors to produce active, mature enzymes. Upon completion of folding, the pro-sequence is autocatalytically degraded because it is not necessary for the activity or stability of folded, mature cognates of the original enzymes. Therefore, the pro-sequence functions as an intramolecular chaperone that guides correct folding of the protease domain. Interestingly, Shinde et al. proposed a new theory of "protein memory" in which an identical polypeptide can fold into an altered conformation with different secondary structure, stability and specificities through a mutated pro-sequence [Shinde et al. (1997) Nature 389:520–522]. We also showed that the autoprocessing efficiency was improved by modifications in the pro-sequence of mutant subtilisins with altered substrate specificity. Further, the pro-sequence from a subtilisin homologue was found to chaperone the intramolecular folding of denatured subtilisin. These results indicate that engineering of the pro-sequence, i.e., site-directed and/or random mutagenesis, chimeras and gene shuffling between members of the family, would be a useful method for improving the functions of autoprocessing proteases. Conventional protein engineering techniques have thus far employed mutagenesis in the protease domain to modify the enzymatic properties. This new approach, which we term "pro-sequence engineering", is not only an important tool for studying the mechanism of protein folding, but also a promising technology for creating unique proteases with various beneficial properties.     

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.     

Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detegents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, α-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed. Received: January 22, 1998 / Accepted: February 16, 1998     

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.     

Optimization of Protease Extraction from Horse Mango (Mangifera foetida Lour) Kernels by a Response Surface Methodology

Protease is one of the most important industrial enzymes with a multitude of applications in both food and non-food sectors. Although most commercial proteases are microbial proteases, the potential of non-conventional protease sources, especially plants, should not be overlooked. In this study, horse mango (Mangifera foetida Lour) fruit, known to produce latex with a blistering effect upon contact with human skin, was chosen as a source of protease, and the effect of the extraction process on its protease activity evaluated. The crude enzyme was extracted from the kernels and extraction was optimized by a response surface methodology (RSM) using a central composite rotatable design (CCRD). The variables studied were pH (x(1)), CaCl(2) (x(2)), Triton X-100 (x(3)), and 1,4-dithryeitol (x(4)). The results obtained indicate that the quadratic model is significant for all the variables tested. Based on the RSM model generated, optimal extraction conditions were obtained at pH 6.0, 8.16 mM CaCl(2), 5.0% Triton X-100, and 10.0 mM DTT, and the estimated response was 95.5% (w/w). Verification test results showed that the difference between the calculated and the experimental protease activity value was only 2%. Based on the t-value, the effects of the variables arranged in ascending order of strength were CaCl(2)< pH < DTT < Triton X-100.     

Microbial alkaline proteases: from a bioindustrial viewpoint

Alkaline proteases are of considerable interest in view of their activity and stability at alkaline pH. This review describes the proteases that can resist extreme alkaline environments produced by a wide range of alkalophilic microorganisms. Different isolation methods are discussed which enable the screening and selection of promising organisms for industrial production. Further, strain improvement using mutagenesis and/or recombinant DNA technology can be applied to augment the efficiency of the producer strain to a commercial status. The various nutritional and environmental parameters affecting the production of alkaline proteases are delineated. The purification and properties of these proteases is discussed, and the use of alkaline proteases in diverse industrial applications is highlighted.     

Genomic and exoproteomic analyses of cold‐ and alkaline‐adapted bacteria reveal an abundance of secreted subtilisin‐like proteases

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography–tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin‐like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20°C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity.     

Biochemical features of microbial keratinases and their production and applications

Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.     

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology

Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf‐enzyme products and their validation with 15 commercial microbial‐enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude‐leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast‐derived enzymes are stable in crude‐leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude‐extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker‐free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site‐specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf‐production platform offers a novel low‐cost approach by the elimination of fermentation, purification, concentration, formulation and cold‐chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.     

Peptide diazomethyl ketones are inhibitors of subtilisin-type serine proteases

Peptide diazomethyl ketones, well known as specific cysteine protease inhibitors are also potent inhibitors of the microbial serine proteases thermitase (EC 3.4.21.14) and subtilisin Carlsberg (EC 3.4.21.14). The affinity of the enzymes towards the synthetic inhibitors Z-Ala(n)-PheCHN2 (n = 0, 1, 2) depends on the chain length and is in the same range as for the corresponding chloromethyl ketones. Both kinds of inhibitors react irreversibly in a 1:1 ratio with the enzymes and covalently bind to the active site histidine of both subtilisin Carlsberg and thermitase despite the fact that thermitase contains an active-site cysteinyl residue. The mechanism of the inhibition reaction is discussed.     

Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.     

The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments.

It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus, whereas subtilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALP I enzymes by mutating the amino acid residues specific for subtilisin ALP I to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C-terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and, on thebasis of three-dimensional modelling, that they are involved in stabilizing the conformation of the C-terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis, stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes.     

Biotechnological applications and prospective market of microbial keratinases

Keratinases are well-recognized enzymes with the unique ability to attack highly cross-linked, recalcitrant structural proteins such as keratin. Their potential in environmental clean-up of huge amount of feather waste has been well established since long. Today, they have gained importance in various other biotechnological and pharmaceutical applications. However, commercial availability of keratinases is still limited. Hence, to attract entrepreneurs, investors and enzyme industries it is utmost important to explicitly present the market potential of keratinases through detailed account of its application sectors. Here, the application areas have been divided into three parts: the first one is dealing with the area of exclusive applications, the second emphasizes protease dominated sectors where keratinases would prove better substitutes, and the third deals with upcoming newer areas which still await practical documentation. An account of benefits of keratinase usage, existing market size, and available commercial sources and products has also been presented.