Impact of microbial proteases on biotechnological industries

Abstract

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.

Keywords:

• Microbial proteases
• sources
• protease engineering
• purification strategies
• biological characteristics
• industrial applications

Introduction

Enzymes are biological macromolecules that catalyze the chemical reactions. Dr Anselme Payen, the French chemist who first discovered the enzyme diastase in 1833. A few decades later, Louis Pasteur concluded that a vital force converts the sugar into alcohol during fermentation and termed it as ‘ferments’. At last, Dr Wilhelm Kühne first coined the term enzyme in 1877. Nowadays, the demands of enzymes as biocatalysts in various sectors are increasing rapidly, due to several advantages such as (i) efficiency (work at lower concentration), (ii) selectivity (positional, chiral, and functional), (iii) mild reaction condition (lower pH and temperature), (iv) modification (can be modified to enhance activity and stability), and environment friendly nature. The industries that consume the major amount of enzymes include food processing industries, beverage, textile, leather, bio-fuel, detergent, and paper industries. According to Jemli, Ayadi-Zouari, Hlima, and Bejar (2016), the hydrolases are the most used industrial enzymes till date. Among these enzymes (amylase, cellulase, phytase, lipase, chitinase), protease receive special attention due to their potential application in various industries, including biotechnological and medical sectors (Gupta, Beeg, Khan, & Chauhan, 2002; van der Maarel, van der Veen, Uitdehaag, Leemhuis, & Dijkhuizen, 2002; Rajagopalan & Krishnan, 2008; Husain, 2010; Padmapriya & Williams, 2012; Souza et al., 2015). A diverse group of plants, animals, and microbiota are reported to be good source of enzymes. Though, people are interested in enzymes obtain from bacteria and fungi due to lower production cost, extracellular nature, availability, and stability (Gupta, Gigras, Mahapatra, Goswami, & Chauhan, 2003; Banerjee, Mukherjee, Bhattacharya, & Ray, 2016). Subba Rao, Sathish, Ravichandra, and Prakasham (2009) have stated that the approximate value of industrial enzymes was $220 billion in 2009. Industrial enzymes produced in bulk, generally require little downstream processing, and hence are relatively crude preparations. The commercial use of these enzymes does not require purification but enzymes used in pharmaceutical and clinical sectors require high purity.

Proteolytic enzymes are ubiquitous in occurrence, present in all living organisms and essential for cell growth, metabolism, cell signaling, differentiation and other physiological purpose. Enzymes derived from micro-organisms may be located intracellular, periplasmic, or extracellular (Kohlmann, Nielsen, Steenson, & Ladisch, 1991; Ray, Ghosh, & Ringø, 2012). Proteases are considered to be the most important classes of industrial enzymes and account for nearly 60% of the total enzyme sale globally (Escobar & Barnett, 1993; Paul et al., 2016). In this direction, Beg and Gupta (2003) have reported that microbial proteases have highest commercial value over enzymes obtained from animals and plants sources. A wide group of bacteria and fungi produce extracellular proteases (Raut, Sen, Kabir, Satpathy, & Raut, 2012; Sharma et al., 2015; Souza et al., 2015), however most of the protease producing candidates belongs to Bacillus species such as B. subtilis ATCC 14416 (Bhunia, Basak, & Dey, 2012), B. sphaericus (Surendran, Vennison, Ravikumar, & Syed Ali, 2011), B. licheniformis (Banerjee, Ray, Askarian, & Ringo, 2013), B. subtilis RSKK96 (Pant et al., 2015), B. cohnii APT5 (Tekin et al., 2012), B. stearothermophilus Ap-4 (Qudar, Shireen, Iqbal, & Tuzun, 2009), B. firmus (wishard et al., 2014), etc. In general, extracellular proteases have advantages over intracellular one in term of cost and availability. The production rate of bacterial enzymes depends on several culture parameters like temperature, pH, vitamins, moisture, inoculums size, carbon, and nitrogen sources (Roy, Banerjee, Dan, & Ray, 2013) and might be enhanced through optimization. In this present review, we have summarized the sources of bacterial and fungal proteases, their purification strategies, pH stability, temperature optima, and their application in several industries.

Sources of microbial proteases

Microbes live in such an environment where the nutrient available is in macromolecular form (protein, lipid, and carbohydrate), and thus need to convert it into smaller ones. The breakdown of such macromolecules is completed by the action of different types of enzymes (Conlon & Bird, 2015). Proteases are the most important class of enzymes that convert complex protein into smaller peptides or amino acids. Proteases produced by microbiota differ greatly in structure and function, and can be divided into several classes such as, serine protease, cysteine protease, acid protease, and metallo protease (Salevesen & Nagase, 1983; Kalisz, 2006; Bhunia et al., 2012). Serine protease are characterized by the presence of a serine residue at active site and are reported to be most common form of bacterial protease (Salevesen & Nagase, 1983; Hershey et al., 2016). In a recent investigation, Zhang et al. (2015) have demonstrated the diversity of serine protease producing bacteria in the sediment of the eutrophied Jiaozhou Bay, China. Results of their study reported that the dominant bacterial species belongs to the genus Pseudoalteromonas, Psychrobacter Photobacterium, Vibrio, Halobacillus, Bacillus, Microbulbifer, and Shewanella. Till date, a wide range of bacterial and fungal species have been identified as an active producer of serine proteases (Table 1). Generally, serine proteases are alkaline in nature and optimally active at pH 7.0–11.0 (Khan, Ahmad, Zafar, Nasir, & Qadir, 2011; Padmapriya, Thamaraichelvan, Nandita, & Raj, 2012; Mothe & Sultanpuram, 2016). Whereas, cysteine proteases contains cysteine residue at their active site and are mostly produced by fungal species like Aspergillus oryzazae and Sporotrichum pulverulentum (Kalisz, 2006; Souza et al., 2015). On the other hand, metallo proteases are another class of enzymes that requires a metal ion (usually zinc, cobalt, nickel and manganese) as cofactor. Several types of micro-organisms (B. subtilis, B. cereus, B. megaterium, Streptomyces griseus, Eupenicillium javanicum, Microsporum canis) have been identified as good producer of metallo enzymes (Brouta et al., 2001; Saxena & Singh, 2011; Neto, Motta, & Cabral, 2013). In this present review, we have focused most of our priority to serine proteases, as it has high demand in industries.

Table 1. Serine proteases producing microbial isolates.

Microorganisms	Isolation sources	References
Vibrio etschnikovii	NM	Jellouli et al. (2009)
Bacillus sp. CEMB10370	Industrial soil sample	Khan et al. (2011)
Aspergillus tamari	NM	Sharma and De (2011)
Pseudomonas sp.	Antarctic water sample	Martinez-Rosales and Castro-Sowinski (2011)
Alternaria solani	Agricultural field	Chandrasekaran and Sathiyabama (2014)
Bacillus alcalophilus LW8	Hypersaline Lake water	Rathod and Pathak (2014)
Bacillus licheniformis NMS	Soil from hot water spring	Mathew and Gunathilika (2015)
Marinomonas arctica PT-1	Marine environment	Yoo and Park (2016)

Note: NM – not mentioned.

Proteases production/secretion by bacterial isolates is a common phenomenon and has been investigated extensively (Table 2). There are several sources of bacterial proteases; however, Bacillus strains have commercial recognition in most of the cases (Gupta et al., 2002; Pant et al., 2015; Tavea, Fossi, Fabrice, Ngoune, & Ndjouenkeu, 2016). Due to high demand, the isolation and characterization of protease producing candidates is an important research area and several projects are going on in that particular direction. In a detail investigation, Padmapriya et al. (2012) have characterized an alkaline protease produced by the bacterium Bacillus sp. isolated from Perna viridis. Similarly, Chu, Lee, and Shu-Tsu (1992) and (Boyer & Byng, 1996) have also reported the production of alkaline protease from bacteria like Bacillus amyloliquefaciens, Bacillus proreolyticus, Bacillus subtilis. Not only bacteria, a wide range of fungi, such as Aspergillus flavus, Fusarium graminarum, Penicillium griseofulvin, Aspergillus niger, Chrysosporium keratinophilum, Scedosporium apiosermum and Aspergillus melleu were also considered to be good sources of serine proteases (Ellaiah, Srinivasulu, & Adinarayana, 2002; Sharma & De, 2011; Novelli, Barros, & Fleuri, 2016). In a study conducted by Kalpana Devi, Rasheedha Banu, Gnanaprabhal, Pradeep, and Palaniswamy (2008) has characterized the protease producing fungi, A. niger isolated from soil sample. Similarly, Charles et al. (2008) also have reported the proteolytic fungus Aspergillus nidulans HA-10 isolated from poultry farm. On the other hand, Anand (2016) has isolated and characterized several strains of A. flavus from soil and optimized their protease producing capacity through solid state fermentation. Whereas, Penicillium griseofulvum and Scedosporium apiospermum were also recognized to be good producer of proteases (Dixit & Verma, 1993; Larcher et al., 1996; Jisha et al., 2013; Souza et al., 2015). We are surrounded by a microbial world and very little information has been documented about their diversity and community, even most of the bacterial isolates (culturable and un-culturable) have not identified till date. The water, soil and gastrointestinal tract (GI) of animal are considered to be the richest sources of several bacterial and fungal candidates (Banerjee et al., 2016). Choudhary and Jain (2012) have investigated the diversity of proteolytic fungus at different habitats and reported that Aspergillus sp. and Fusarium sp. were the most predominant genera. Researchers have reported the protease producing candidates in the GI tract of several animals, which play a crucial role in host nutrition and defense (Ray et al., 2012; Banerjee et al., 2013 Banerjee et al., 2016). Fungal sources of proteases also have high industrial demand due to stability, broad diversity, and substrate specificity. Compared to bacterial cells, separation of mycelium is easy and thus it is cost-effective. The available important sources of fungal proteases were given in Table 3.

Table 2. Important sources of bacterial proteases.

Bacterial strains	References
Alteromonas sp.	Tsujibo, Miyamoto, Okamoto, Orikoshi, and Inamori (2000)
Thermomonospora fusca YX	Kim and Lei (2005)
Chromohalobacter sp.	Vidyasagar, Prakash, Jayalakshmi, and Sreeramulu (2007)
Vibrio alginolyticus	Rui et al. (2009)
Paenibacillus tezpurensis	Rai, Roy, and Mukherjee (2010)
B. subtilis, B. licheniformis, Vibrio sp.,	Ray et al. (2012)
Brevibacterium linens	Shabbiri et al. (2012)
Vibrio parahaemolyticus	Miyoshi (2013)
Tepidimonas fonticalsi	Chen et al. (2013)
Virgibacillus, Halobacillus, Oceanobacillus	Taprig, Akaracharanya, Sitdhipol, Visessanguan, and Tanasupawat (2013)
Lactobacillus helveticus	Griffiths and Tellez (2013)
Pseudomonas aeruginosa	Ariole and Ilega (2013)
Lactobacillus acidophilus	Gandhi and Shah (2014)
Lactobacillus delbrueckii	Gandhi and Shah (2014)
Streptococcus thermophilus	Gandhi and Shah (2014)
Microbacterium sp.	Lu et al. (2014)
Pseudomonas stutzeri	Dian Siti, Ihsanawati, and Hertadi (2015)
Engyodontium album	Souza et al. (2015)
Bacillus firmus	Geng et al. (2016)

Table 3. Proteases production by fungal isolates.

Fungal isolates	References
Fusarium poae	Marzan, Manchur, Hossain, and Anwar (2001)
Curvularia lunata	Tripathi, Kukreja, Singh, and Arora (2009)
Aspergillus fumigates	Oyeleke, Egwim, and Auta (2010)
Piromyces sp.	Paul, Kamra, and Sastry (2010)
Aspergillus flavus	Negi, Gupta, and Banerjee (2011)
Paecilo myceslilacinus	Khalil, Allam, and Barakat (2012)
Entomophthora coronate	Jisha et al. (2013)
Aspergillus oryzae	Leng and Xu (2013)
Cladosporium herbarum	Naji, Abdullah, AlZaqri, and Alghalibi (2014)
Aspergillus sojae	De Castro and Sato (2014)
Fusarium salani	Al-Askar, Abdulkhair, and Rashad (2014)
Aspergillus niger	de Castro et al. (2015)
Penicillium chrysogenum	Sethi and Gupta (2015)

Proteases engineering

Proteases engineering is an important step that lead to the development of novel tool for therapeutic and biotechnological applications. In general, the activity and stability of any enzymes including proteases depend on the amino acid composition and their respective position. Protein engineering modifies the protease structure in such a way which enhances the activity, specificity and stability of proteases (Pogson et al., 2009). Engineering of proteases for diverse industrial purposes has been extensively investigated (Table 4). Currently, two different methods are used for making engineered proteases: directed evolution and random design (Böttcher & Bornscheuer, 2010). Thermal stability of protease is very important, which can been increased using different engineering strategies such as disulfide bond formation, substrate cite amino acid modification, modification of metal binding site, and comparing sequence homology with other enzymes produced by mesophiles and thermophiles (Li, Yi, Marek, & Iverson, 2013). Similarly, pH stability (surface charge engineering) and activity in organic solvent environment (disulfide bond engineering) of proteases have also been reported (Takagi, Hirai, Maeda, Matsuzawa, & Nakamori, 2000; Li et al., 2013; Vojcic et al., 2015). Cummings, Murata, Koepsel, and Russell (2014) have used dual block polymer-based protein engineering to enhance the pH and thermal stability of protease. The need of biocatalyst varies greatly (in term of stability and activity at different temperature and pH) from one industry to another, and protein engineering is the way to make it possible.

Table 4. Engineered proteases for industrial use.

Microbial candidates	Proteases nature	Modifications	Benefits	References
Bacillus amyloliquefaciens	Alkaline protease	Replacement of aspartic acid with serine at position 99	Alter the pH optima	Thomas, Russell, and Fersht (1985)
Bacillus stearothermophilus	Neutral protease	Replacement of asparagines with leucine at position 241	Increase thermal stability	Eijsink, vander Zee, vanden Burg, Vriend, and Venema (1991)
Bacillus subtilis	Neutral protease	Replacement of sysine with Serine at positions 249 and 290	Increase thermal stability	Eijsink, Vriend, vanden Burg, vander Zee, and Venema (1992)
Lysobacter enzymogenes	Serine protease	Random mutation in substrate binding site	Increase activity	Graham et al. (1993)
B. subtilis	Subtilisin	Alteration of disulfide bond	Increase stability in organic solvent	Takagi et al. (2000)
Bacillus subtili	Subtilisin	Modification at metal binding site	Increase stability	Strausberg, Ruan, Fisher, Alexander, and Bryan (2005)
P. aeruginosa PST-01	PST-01 protease	Substitution of tyrosine to phenylalanine at position 114	Increase activity	Ogino, Tsuchiyama, Yasuda, and Doukyu (2010)
NM	Subtilase	Alteration of multiple amino acid in its less stable counterparts	Increase thermal stability and activity	Xu, Shao et al. (2015)

Note: NM – not mentioned.

Purification strategies of microbial protease

Biotechnological industries produce different types of microbial enzymes in bulk amount and do not require high purity. However, enzymes used in pharmaceutical, medical and research purpose require high purity. Several types of classical methods have been employed for enzyme purification from microbial sources. However, the general method of enzyme purification was given in Figure 1. Enzyme purification is not a single step procedure, it require combination of several technologies and methods. Purification details of proteases from several microbial sources are given in Table 5. Few important techniques that are generally used for microbial enzyme purification are described below.

Figure 1. Diagram demonstrates the general procedure of microbial protease and amylase purification.

Table 5. Purification strategies of microbial proteases.

Microbial sources	Method of purification	Purification fold	Recovery (%)	References
Lactobacillus plantarum A6	50–70% (NH4)2SO4, DEAE-Cellulose	19.5	35.6	Giraud, Gosselin, Marin, Parada, and Raimbault (1993)
Penicillium expansum	Acetone, Sephadex G-100, DEAE-Sephadex A-50	96.23	48	Umar Dahot (1994)
B. subtilis	80%(NH4)2SO4, DEAE-Sepharose, Sephacryl S-200	37	18	Yang, Shih, Tzeng, and Wang (2000)
Chaetomium thermophilum	90% (NH4)2SO4, DEAE-Sepharose, Phenyl-Sepharose (PRO33)	19.3	4	An et al. (2007)
A. niger	74% (NH4)2SO4, DEAE-Cellulose	34.42	32	Kalpana Devi et al. (2008)
Chromohalobacter sp.	Ethanol, Phenylsepharose 6B column, Gel permeation G-100	180	22	Vidyasagar, Prakash, Mahajan, Shouche, and Sreeramulu (2009)
A. flavus	70% (NH4)2SO4, DEAE-Cellulose	5.8	3.2	Muthulakshmi et al. (2011)
Bacillus cereus	60% (NH4)2SO4, SephadexG- 200, SephadexG-100	–	–	Senthilraj and Saravanakumar (2011)
Bacillus megaterium	60% (NH4)2SO4, DEAE-Cellulose, Sephadex G-200	7.72	11	Asker, Mahmoud, El Shebwy, and Abd el Aziz (2013)
B. licheniformis B18	70% (NH4)2SO4, DEAE-Cellulose, SephadexG-100	26.33	10.6	Lakshmi and Prasad (2015)
Corynebacterium alkanolyticum	60% (NH4)2SO4, Sephadex G-50, DEAE cellulose	26.03	15.21	Banerjee et al. (2016)

Gel filtration chromatography

It is very common method of enzyme purification. Several types of gels or beads have been used for column packing such as Sephadex (G-25, G-50, G-75, G-100, G-200), Sepharose, Sephacryl, etc. The selection of packing material depends on molecular weight of the enzyme. Such as, lower molecular weight enzymes (10–80 kDa) require Sephadex G-25 to G-75. Whereas, enzymes having higher mass require G-100 to G-300. Packing of column is very important for proper purification. Bubble in the column hampers the flow rate and thus before packing, gel should be properly de-gassed. Readymade columns are also available from different company such as, Sigma Pvt. Ltd, Merck Pvt. Ltd, HiMedia Pvt. Ltd, Bio-RAD Pvt. Ltd, etc.

Affinity chromatography

It is a very popular method for enzyme and protein purification. Several types of packing material are used depending on the nature of molecules. The most common matrices for bacterial neutral enzymes are Sephadex-4-phenylbutylamine. The others available matrices are Casein agarose, N-benzoyloxycarbonyl, lectin, etc. The major drawbacks of the affinity chromatography are high purification cost and labile nature of some ligands.

Ion-exchange chromatography

In ion-exchange chromatography, separation or purification of enzymes depends on ionic nature (positive or negative). Generally two types of beads are used, DEAE-sephades/cellulose (anion) and CM-Sephadex/cellulose (Cation). Mostly microbial enzymes are positively charged and require anion exchanger. Elution of bounded molecules is done by increasing salt concentration (usually NaCl and CaCl₂) or pH gradient.

Aqueous two-phase system chromatography

Microbial alkaline protease can be purified using a combination of two mixtures such as, polyethylene glycol (PEG) and dextran or PEG and different types of salts. This method has several advantages: provides mild condition for biological macromolecule, higher concentration, less time taking, and cost-effective.

High-pressure and low-pressure liquid chromatography

High-pressure liquid chromatography (HPLC) and low-pressure liquid chromatography (LPLC) are sophisticated technique for protein purification. HPLC differ from LPLC in term of pressure. Both of these techniques are useful for molecular grade purification of protein. Depending on the purpose, HPLC might be divided into three categories: analytical, semi-preparative, and preparative.

Fast protein liquid chromatography

It is one of the most popular chromatography instrument, usually used to purify the proteins and enzymes. Like liquid chromatography, it also contains a liquid mobile phase or buffer (phosphate buffer and citrate buffer) and a stationary phase which contains resin. The flow rate of buffer is controlled by a positive-displacement pump. Selection of column is important in Fast protein liquid chromatography (FPLC) and may be varied (size exclusion, hydrophobicity, ion exchange, reverse phase, etc.) according to the nature of separating molecules.

Temperature and pH optima of microbial protease

The production rate of microbial enzymes are highly dependent on various culture parameters like temperature, pH, moisture content, inoculums size, duration, agitation speed, carbons, and nitrogen sources. The optimization of these factors are important in response to product enhancement, cost-effectiveness and time. Among several parameters, temperature and pH are considered to be most crucial for maximum enzyme activity. Proteases produced by bacteria and fungi vary greatly in structure, substrate specificity, molecular weight, and physical characteristics. The optimum temperature for proteases produced by different Bacillus species has been reported by several researchers such as, 50 °C for Bacillus licheniformis (Sareen & Mishra, 2008), 45 °C for Bacillus thermoruber (Manachini, Fortina, & Parini, 1998), 70 °C for marine Bacillus sp. (Padmapriya et al., 2012) and 70 °C for B. subtilis 50a (Azlina & Norazila, 2013). Oseni (2011) has isolated four fungal isolates (A. flavus, A. niger, Aspergillus fumigatus, and Penicillium italicum) and investigated their temperature optima, which lied between 30 and 60 °C.

Like temperature, the activity of microbial proteases also depend on pH of the reaction media such as pH 9.0 for B. subtilis 50a and B. licheniformis 50a (Oseni, 2011), pH 12.0 for Bacillus clausii (Joo, Kumar, Park, Paik, & Chang, 2003) and pH 7.0 for Pseudomonas fluorescens (Padmapriya et al., 2012). Oseni (2011) also has stated that proteases produced by A. niger and P. italicum have acidic pH optima. While, proteases obtained from Aspergillus versicolor have exhibited optimum activity at pH 9.0 (Choudhary & Jain, 2012). The sources of protease and their temperature and pH optima were presented in Table 6.

Table 6. Temperature and pH optima of proteases produced by the selected micro-organisms.

Microorganisms	Temperature optima (°C)	pH optima	Molecular weight (kDa)	References
Aspergillus terreus (IJIRA 6.2)	37	8.5	37	Chakrabarti, Matsumura, and Ranu (2000)
Penicillium chrysogenum (Pg222)	45	6	35	Benito et al. (2002)
Stenotrophomonas maltophilia S-1	50	12	40	Miyaji et al. (2005)
Xenorhabdus nematophila	30	8.5	39	Mohamed (2007)
Aspergilius niger	45	8.5	38	Kalpana Devi et al. (2008)
Aspergillus nidulans HA-10	35	8	42	Charles et al. (2008)
Chromohalobacter sp. TVSP101	75	8	66	Vidyasagar et al. (2009)
Pseudomonas thermaerum GW1	60	8	43	Gaur, Agrahari, and Wadhwa (2010)
Myceliophthora sp.	40–45	9	28.2	Zanphorlin et al. (2011)
Salimicrobium halophilum	80	10	30	Li and Yu (2012)
Bacillus sp.	70	7	37	Padmapriya et al. (2012)
B. megaterium	50	7.5	28 & 25	Asker et al. (2013)
A. oryzae BCRC 30118	60	3	41	Yin, Chou, and Jiang (2013)

Application of protease in industrial sectors

Microbial enzymes, especially proteases have several important roles in industrial sectors such as paper mill, leather processing, liquid detergent preparation, food processing, and medical sector. Among different classes, alkaline and heat stable serine proteases are considered to be most important. The contribution of proteases in different industrial sectors is highlighted below.

Leather processing industries

Leather industries consume a significant amount of protease for raw leather processing, which consists of several steps like soaking, liming, dehairing, deliming, bating, degreasing, and pickling. Alkaline proteases produced by micro-organism are very useful for removing hair and this is quite safer than the traditional method that used hazardous chemicals like, sodium sulfide, which creates environmental pollution (Ellaiah et al., 2002; Sawant & Nagendran, 2014; Souza et al., 2015). Alkaline proteases hydrolyze non-collagenous constituent of skin and also remove non-fibrillar proteins such as globulin and albumin that are needed for leather processing (Jridi et al., 2014). Jisha et al. (2013) have reported the use of some industrially important proteases producing microbes such as Streptomyces sp., A. flavus, Bacillus amyloliquefaciens and Bacillus subtilis. Ellaiah et al. (2002) have stated that tanning of raw leather should be done in high alkaline condition (pH 12.0) and for that purpose alkaline proteases produced by micro-organism is a better choice. However, genetically modified strains have higher priority in industries and are used randomly for getting better results (Khan, 2013; Adrioo & Demain, 2014). For example, addition of pBX96 plasmid in Bacillus pumilus c172–14 (Ng, 2001), site directed mutagenesis in Bacillus amyloliquefaciens (Estell, Graycar, & Wells, 1985; Xu, Dai et al. 2015) and modification of enzyme producing gene in B. licheniformis (Fleming, Tangney, Jorgensen, Diderichsen, & Priest, 1995; Zhang, Duan, & Wu, 2016) gives better performance in leather industries.

Waste management

Microbial sources of keratinases are very useful in waste management due to its broad substrate specificity. In general, keratinases convert the feather or other waste materials (such as scale, hair, wool, etc.) into karatin, which has high demand in poultry and aquaculture industries as feed ingredient (Deivasigamani & Alagappan, 2008; Motyan, Toth, & Tozser, 2013). In a review, Gopinath et al. (2015) have explored the microbial sources of keratinases and their function in different industrial sectors. Karatin is an insoluble protein; however, the conversion of insoluble karatin to soluble from is possible through submerged fermentation using keratinases produced microbes (Suntornsuk & Suntornsuk, 2003). Due to special characteristics, keratinases have been widely used as nematicidal agents, biofuel production, and fertilizer production from poultry waste (Verma et al., 2017). Dalev (1994) has reported an alkaline keratinase from B. subtilis which might be useful in processing of feather in poultry industries. In a similar study, Verma, Singh, Anwar, Ansari, and Agrawal (2014) have also reported the feather degrading alkaline keratinase from Thermoactinomyces sp. RS1, which can be used in feed preparation in poultry farm. Other keratin degrading protease were also reported for various function, such for peptide production (Motyan et al., 2013), recycling waste (Verma et al., 2016), food processing industries (Vanitha, Rajan, & Murugesan, 2014) and waste water treatment (Jisha et al., 2013).

Food industries

In recent year, microbial proteases have been randomly used in food industries like meat tenderization and cheese preparation (Shi, Coyne, & Weiner, 1997; Adrioo & Demain, 2014). Milk protein coagulation is the first step in cheese preparation, which is accomplished with the help of protease enzyme. Cheese making industries uses microbial source of proteases; however, it has two drawbacks: low yield and bitterness upon long storage. Among several classes of proteases, aspartic proteases are considered to be best for cheese industries (Claverie-MartÌn & Vega-Hernàndez, 2007; Feijoo-Siota et al., 2014). Microbes produce several types of proteases which are non-specific and thus cause bitterness in storage food. Nissen (1986) has reported that Alcalase (an alkaline protease) remove bitterness from food stuff and make it sweeter. Whereas, protease produced by B. amyloliquefaciens is useful for making methionine-rich protein hydrolysate from chickpea protein (George, Sivasankar, Jayaraman, & Vijayalakshmi, 1997; Wang et al., 2016). Proteases obtained from microbial sources are used for the fortification of fruit juices or soft drinks and in preparation of protein rich therapeutic diets also (Adamson & Reynolds, 1996; Amore & Faraco, 2015). Wheat flour is the major component of baking process which contains an insoluble protein, gluten that determines the bakery dough’s properties. Protease produced by Aspergillus oryzae hydrolyzes the gluten and increases the product quality in terms of color, softness, and texture (Sawant & Nagendran, 2014; Souza et al., 2015).

Silver recovery

Silver is a valuable metal and major cost component in the film, used in photography, MRI, CT scan, and X ray. X-ray film content approximately 1.5 to 2% silver in its gelatin layer. Recovery of silver from used X-ray film is quite difficult and laborious. There are several chemical methods for silver recovery such as burning of film and oxidation of metallic silver, but these are not environment friendly. Alkaline proteases are used for this particular purpose, which actively degrade the gelatin layer of the film and release the bounded silver (Kumaran, Mahalakshmipriya, & Rajan, 2013). Shankar, More, and Seeta Laxman (2010) have isolated and characterized an alkaline protease from Conidiobolus coronatus and reported its silver recovering efficiency. They also have stated that silver hydrolysis by this protease was completed at pH 10.0 within 6 min. While, Singh, Vohra, and Sahoo (1999) have investigated the silver recovering efficiency of protease obtained from Bacillus sphaericus, which was able to hydrolyze the silver at pH 11.0–12.0 within 8 min. Bacillus coagulans and Bacillus sp. 21-2 were also reported to be important bacterial strains in silver recovering industries (Fuziwara & Yamamoto, 1987; Gajju, Bhalla, & Agarwal, 1996; Jisha et al., 2013; Vanitha et al., 2014). Similarly, Anju, Kondari, and Sarada (2014) also have reported the efficiency of silver recovery from X-ray and MRI plate by protease producing Bacillus sp. isolated from soil sample. Due to availability, eco-friendly nature, and low cost microbial sources of proteases have great demand in silver recovery industries.

Detergent industries

Different types of enzymes like protease, amylase, lipase, etc. are used in detergent industries. Among these, proteases account for approximately 60.0% of total enzyme sales in detergent industries (Ibrahim et al., 2016). In 1913, Otto Ro¨hm discovered that pancreas extract was useful to degrade protein and can be used for washing clothes. Later on, in 1956 bacterial enzyme containing detergent was introduced in market under the trade name Bio-40. The domestic detergent contains different types of toxic chemicals such as polycarboxylates, chlorine, phosphonates, etc. which are not good for environment and can cause skin diseases. The performance of enzyme in detergent depends on various factors such as, washing temperature, type of stain, water hardness, and washing procedure (Hasan, Ali Shah, Javed, & Hameed, 2010; Li, Yang, Yang, Zhu, & Wang, 2012). The ideal protease that can be used in detergent industries should have two characteristics; stable at alkaline pH and compatible with detergent (Niyonzima & More, 2015). Proteases obtained from Bacillus sp. are very useful in manufacturing laundry detergent such as Era-plus (Procter & Gamble), Dynamo and Tide (Colgate Palmotive). In recent year, protease produced from two potential bacterial strains, B. clausii KSM-k16 and Bacillus sp. KSM-KP43 have been incorporated in laundry industries (Hasan et al., 2010; Bialkowska et al., 2016). Demand of detergent-compatible protease is high and thus, the screening of protease producing bacteria is a continuous process. From environmental perspective, use of microbial proteases in manufacturing detergent is good for health and nature.

Chemical industries

Proteases play a major role in synthetic chemical industry in form of biocatalyst. However, a major drawback of this application is reduction of enzyme activity under anhydrous condition. Several researchers have reported the catalysis of peptide synthesis in organic solvent by alkaline proteases (Motyan et al., 2013; Anbu, 2016). Patil, Rethwisch, and Dordick (1991) reported a novel alkaline protease (proleather) produced by Bacillus sp. which is very useful in case of sucrose polymer synthesis under anhydrous condition and transesterification of D-glucose into pyridine. On the other hand, alkaline proteases from C. coronatus has potential role in replacing subtilisin from various reaction mixture (Sutar, Srinivasan, & Vartak, 1991; Bhunia et al., 2012; Bialkowska et al., 2016).

Medical purpose

The applications of protease in medical sectors are accepted worldwide. Proteases from microbial sources were detected to be effective in treatment of various diseases like tumor, inflammation, cancer, etc. A. oryzae is an important fungal isolate which produces a novel protease that is useful in treatment of certain enzyme deficiency syndromes (Palanivel, Ashokkumar, & Banagurunathan, 2013; Gurung, Ray, Bose, & Rai, 2013). In last few years, the concept of protease action has been changed dramatically. Apart from the catalytic activity, proteases are considered to be important agent in several biochemical pathways and physiological functions such as, cell signaling, apoptosis, cell migration, cell adhesion, and metastasis. Researchers have reported that inactivation of such proteases using microbial protease inhibitor might be an alternative way in developing anti cancer drug (Pervaiz, Hirpara, & Clement, 1998; Rakashanda & Amin, 2013; Petra & Monika, 2016). From toxicity point of view, use of enzyme in cancer treatment has advantage over chemotherapeutic agents. Sawant and Nagendran (2014) have reported that Clostridial collagenase or subtilisin has been randomly used in preparation of several antibiotics, whereas, aspargine removing protease, asparginase, produced by E. coli is effective in treatment of lymphocytic leukemia (Kishore, Nishita, & Manonmani, 2015). Blood clotting in vascular pipeline block blood supply and it is a major problem that causes cerebral and cardiac attack. Researchers have reported that fibrinolytic activity exhibited by several bacterial and fungal species are important in cardiovascular treatment (Table 7). The molecules which dissolve these clots are some kind of proteases called thrombolytic agent. In last few years, several thrombolytic agents from microbial sources like streptokinase (SK) and staphylokinase (SAK) were developed to overcome this critical situation (Srilakshmi, Madhavi, Lavanya, & Ammani, 2014; Mane & Tale, 2015). Protease also plays a critical role as an anti-inflammatory agent. The bacterium Serratia sp. produced a special protease called serratiopepetidase, which is able to reduce inflammation rapidly. In a review, Patyar et al. (2010) have stated that genetically engineered bacteria are very much effective in different types of cancer therapy, including targeted cell signaling inhibition by bacterial protease such as streptokinase production from Streptococcoi through recombinant DNA technology.

Table 7. Fibrinolytic activity exhibited by micro-organisms.

Microbial isolates	Isolation sources	References
B. amyloliquefaciens DC-4	Douchi, a soybean food	Peng, Huang, Zhang, and Zhang (2003)
B. licheniformis kJ-31	Traditional Jeot-gal food	Hwang, Choi, Kim, Park, and Cha (2007)
B. subtilis natto B-12,	Natto, a fermented food	Wang et al. (2009)
Aspergillus sp.	Soil sample from Eastern Ghat	Palanivel et al. (2013)
Bionectria sp.	Las Yungas rainforest, Peru	Rovati, Delgado, De Figueroa, and Fariña (2013)
Bacillus pumilus 2.g	Gembus, a fermented food	Afifah et al. (2014)
Penicillium citrinum S13, Fusarium sp. FH3	Hibiscus plant Leaves	Ahmad, Noor, and Ariffin (2014)
Mucor subtillissimus UCP 1262	Soil of Caatinga-PE, Brasil	Nascimento et al. (2015)
Aspergillus japonicum	Soil sample, Bangalore, India	Yadav and Siddalingeshwara (2015)
B. subtilis	Fermented food sample	Obeid, Alawad, and Ibrahim (2015)

Other uses

In recent year, proteases from biological source have been used randomly for research purpose also, such as alkaline protease produced by C. coronatus is the substitute of trypsin in animal cell culture (Sawant & Nagendran, 2014).

Future scope

Proteases are unique classes of enzymes that perform different role in industries, as well as maintain physiological condition. Till date, researchers have investigated and reported the production of protease by several micro-organisms. In present day, a wide variety of molecular biological and biotechnological methods have been introduced for enhancing the microbial enzyme production through genetic modifications and protein engineering. Cloning, addition of plasmid, site-directed mutagenesis and deletion of repressor gene are modern techniques that are used to manipulate the gene and control their expression. Along with gene editing, protein engineering is also very important and has huge impact in improving proteases properties. The goal of the protein engineering is to change the structure of the enzyme to maximize the industrial production. A huge number of micro-organisms have been reported as protease producer, but few have been accepted as commercial application. The combination of DNA recombinant technology, protein engineering, and gene cloning can overcome this situation. Nowadays more than 50% of industrially important enzymes are produced by genetically modified strains or engineered protein. Thus, targeted modification of such enzyme producing gene and alteration of amino acid position might be effective for better production.

Conclusion

Due to huge industrialization, demand of enzymes increase rapidly. In this present review, we have summarized the importance of protease in different sectors. Production of enzyme from different group such as animal, plant, and microbes were already established, but people are interested in microbial sources of enzymes due to several reasons such as, higher production through fermentation, availability, diversity, stability, and lower cost. Bacteria and fungi were reported to be good sources of different types of enzymes like protease, amylase, lipase, cellulase, pectinase, chitinase, phytase, β-galactosidase, etc. In our review, we have included only protease due to greater importance. Microbes produce different types of proteases but alkaline serine protease has much more importance. Among various bacterial strains, Bacillus species were reported to be most important in enzyme production. Similarly, the fungal isolates belongs to the genus Aspergillus were industrially valuable. Day by day, the use of enzyme in industries will increase, and thus to fulfill the demand, screening of new enzyme producing microbial isolate is important.

Disclosure statement

No potential conflict of interest was reported by the authors.