Substrate specificity of SAPB and KERAB.
Enzymes have long been used as alternatives to chemicals to improve the efficiency and cost-effectiveness of a wide range of industrial systems and processes. They are currently used in basic and applied arenas of research as well as in a wide range of product design and manufacturing processes, such as those pertaining to the food, beverage, pharmaceutical, detergent, leather processing, and peptide synthesis industries (Gupta et al., 2002). Of particular interest to the aims of the present work, proteases have often been reported to constitute a resourceful class of enzymes with promising industrial applications. According to recent estimates, these enzymes account for nearly 65% of total worldwide enzyme sales (Anonyme, 2007; Rao et al., 1998). They are widely distributed in nature and play a vital role in life processes. They are particularly known for their capacity to hydrolyze peptide bonds in aqueous environments and to synthesize peptide bonds in non-aqueous biocatalysis.
Proteases have been employed in a wide array of applications for many years with satisfactory results. They constitute a large family of enzymes present in a wide range of living organisms, such as plants, animals and microorganisms. In biotechnologically oriented systems and processes, however, proteases from microbial origins have often been reported to have distinct advantages when compared to plant or animal proteases, particularly because they possess almost all the characteristics desired for biotechnological applications. Among these biocatalysts, high-alkaline proteases, which alone account for about 40% of the total worldwide enzyme sales (Kirk et al., 2002), proved particularly suitable for industrial use. This is mainly due to their high stability and activity under harsh conditions.
Nowadays, the use of alkaline protease-based detergents is preferred over the conventional synthetic ones. This is partly because of their better cleaning properties, higher performance efficiency at lower washing temperature, and safer dirt removal conditions (Gupta et al., 2002). Typically, a detergent protease needs to be active, stable, and compatible with the alkaline environment encountered under harsh washing conditions: pH 9 - 11, temperature of 20 - 60°C, as well as high concentrations of salt, bleach, and surfactant. Some of the alkaline proteases that are particularly preferred in contemporary detergent formulations include Savinase™ (Subtilisin 309), Subtilisin Novo (BPN′), Alcalase™ (Subtilisin Carlsberg; SC), Maxacal™ (Novozymes A/S, Denmark), BLAP Sb (Henkel, Germany) and Properase™ (Genecor Int. USA). They are often reported to be stable at conditions of elevated temperatures and pH. Most of them have, however, been criticized for their limited efficiency in the presence of liquid or solid laundry detergents wherein their stability decreases (Beg and Gupta, 2003; Maurer, 2004). Therefore, the search for and screening of alternative microorganisms that produce detergent-stable enzymes and preserve their high activity and stability at extreme conditions would be highly desired, particularly within the framework of the persistent aspirations that consumers, industrialists and, by extension, researchers, have towards improved laundry detergents with powerful, safe and healthy cleansing abilities.
Various alkaline proteases have been reported to constitute appropriate additives for a variety of detergent, laundry and cleansing supplies as well as other leather processing, dyeing, and finishing applications. Keratinases are a group of mostly extracellular serine-proteases that have often been reported for their excellent potency to degrade keratins, a group of fibrous, insoluble and abundant structural proteins that constitute the major components of structures growing from the skin of vertebrates, such as hair, wool, nails, hooves, horns and feather quills. In fact, due to their high degree of cross-linking to disulphide bonds, hydrogen bonds, and hydrophobic interactions, these proteins show high stability and resistance to proteolytic hydrolysis (Coulombe and Omary, 2002).
Large amounts of keratin containing wastes are discharged every year from poultry, leather and meat processing industries. Current estimates indicate that the global annual discharge of feather from the poultry processing industry alone reaches millions of tons (C.A.S.T., 1995; Freeman et al., 2009). This keratinous poultry waste is degraded very slowly in nature and is, therefore, considered hazardous to the environment. Seeing that keratinous waste represents a valuable source for proteins and amino acids, several steam pressure and chemical treatment processes have been developed to convert feathers into feather meal for animals (Hess and FitzGerald, 2007). These physico-chemical conversion methods have, nevertheless, been reported to involve costly treatments under harsh temperature and pressure conditions that result in the loss of essential amino acids (Onifade et al., 1998). Alternatively, feather biodegradation processes have been proposed as viable substitutes (Ignatova et al., 1999; Xie et al., 2010).
Keratinolytic microorganisms can be employed in the manufacture of nutritious, cost-effective, environmentally safe feather meal for poultry, as well as in the enhancement of drug delivery, hydrolysis of prions, construction of biodegradable films, and production of biofuels (Brandelli et al., 2010). Additionally, these keratinolytic enzymes have a variety of current and potential applications in a wide range of biotechnological processes that involve keratin hydrolysis, including the enzymatic dehairing and catalysis for leather and cosmetic industries, the breaking down of recalcitrant matter for the laundry and detergent industries, the slowing down of nitrogen release for fertilizer and pesticide industries, and the production of biohydrogen and rare amino acids for animal feed and foodstuff industries (Bertsch and Coello, 2005).
Several microorganisms that possess keratinolytic activity have been reported to accede to the biodegradation of keratin waste by secreting keratinolytic peptidases into the culture medium and to offer valuable tools for the development of efficient and cost-effective keratin waste bioconversion methods (Onifade et al., 1998). In this respect, various keratinases have been purified from different microorganisms, namely fungi, such as
Despite this large flow of data on keratinases, however, little information has so far been reported on the characterization and purification of keratinases from
The present chapter aim to provide an overview on the current quest for novel natural bacterial alkaline proteases with special emphasis on the purification and characterization of two enzymes, namely SAPB and KERAB, from isolated alkaline proteinase and keratinase producing microbial strains, whose promising properties and attributes are likely to open new pathways in current and future research and new possibilities for the improvement of current detergent formulations and leather processing industries. In fact, both SAPB and KERAB showed valuable operational characteristics that made them strong potential candidates for future application as additives in biotechnological applications and processes, particularly in detergent formulations and in dehairing during leather processing. They also showed relatively high stability in the presence of organic solvents, a feature which is highly desired in applications involving the biocatalysis of non-aqueous peptides. Accordingly, this chapter intends to report on the screening, identification, and phylogenetic analysis of the
2. Screening and identification of alkaline proteinase and keratinase producing microbes
The isolation and screening of micro-organisms from naturally occurring alkaline habitats and keratinacious biowaste is likely to help identify potential microbial strains capable of producing active and stable enzymes that can resist the aforementioned harsh substances and conditions present in detergent formulations and leather dehairing processes.
2.1. Screening of alkaline protease and keratinase producing strains
A recent work by the authors (Jaouadi et al., 2009; Badis et al., 2009) involved the screening of about 125 bacterial strains (Bacilli and Actinomyces), originating from a collection of bacterial strains at the CBS and other strains that were previously isolated from surface soil samples at the Mitidja plain, North of Algeria (Badis et al., 2010), for protease and keratinase activities. Based on the ratio of the diameter of the clear zone (onto skimmed milk or keratin-containing medium agar plates at pH 9.) and that of the colony, only 24 isolates, which exhibited the highest ratio (> 3 mm), were selected for further assays pertaining to protease or keratinase production in liquid media. The two bacterial strains that displayed the highest extracellular protease and keratinase activity were termed as strain CBS (from the CBS bacterial strain collection) and strain AB1 (from Algerian soil samples) and retained for all subsequent experimental assays.
2.2. Identification and molecular phylogeny of the microorganisms
The two newly isolated bacterial strains, CBS and AB1, were submitted to identification and typing by molecular and catabolic techniques. The data from the morphological, biochemical and physiological characterization tests, performed on the isolates in accordance with the methods described in the Bergey’s Manual of Systematic Bacteriology, showed that the CBS and AB1 strains appeared in a bacilli and filamentous form, respectively, that are aerobic, endospore-forming, Gram-positive, catalase+, oxydase+ and motile rod-shaped. The findings from API 50 CH gallery tests revealed that the CBS isolate metabolized l -arabinose, d-tagatose, ribose, and mannitol in addition to several other simple sugars. The AB1 strain, on the other hand, could use galactose, sucrose, maltose, cellobiose, fucose, raffinose, d-xylose, l-arabinose, and d-ribose, but not lactose, starch, l-rhamnose, erythritol, adonitol, and inositol. The results from API ZYM tests revealed that strain AB1 also exhibited alkaline phosphatase, esterase lipase (C8), leucine arylamidase and valine arylamidase activities, but no lipase (C14), trypsin, α-chymotrypsin,
A molecular approach was used to establish further support for the identification of the CBS and AB1 isolates. Two 16S rRNA gene fragments, namely 1,497 bp (Jaouadi et al., 2009) and 1541 bp (Jaouadi et al., 2010a), were amplified from the genomic DNA of the CBS and AB1 isolates, respectively, and then cloned and sequenced on both strands. The 16S rRNA gene sequences obtained were subjected to GenBank BLAST search analyses, which yielded strong homologies of up to 98 and 99% with those of several cultivated strains of
3. Production, purification and biochemical characterization of SAPB and KERAB enzymes
3.1. SAPB and KERAB production
Different carbon and nitrogen sources and trace elements were assayed to optimize the culture growth conditions for the production of the enzymes. In the medium containing (g/l): gelatin 10, yeast extract 5, CaCl2 1, K2HPO4 1, and KH2PO4 1, the addition of 0.1% (v/v) trace elements [composed of (g/l): ZnCl2, 0.4; FeSO4 7H2O, 2; H3BO3, 0.065; and MoNa2O4 2H2O, 0.135] at pH 10 was noted to bring about a significant enhancement of 1.32 folds in SAPB production, which reached 6,500 U/ml under the optimal conditions used (pH 10.6 and 65°C), after 24 h of incubation at 37°C and 250 rpm (Jaouadi et al., 2009). In medium containing trace salts with feather as carbon and nitrogen source (g/l): NaCl, 0.5; KH2PO4, 0.5; K2HPO4, 0.5; KCl, 0.1; MgSO4 7H2O, 1; and chicken feather meal, 10; at pH 9, KERAB production was observed to undergo a significant improvement, reaching a maximum of 9,500 U/ml under the optimal conditions used (pH 11.5 and 75°C) after 96 h of incubation at 30°C and 200 rpm (Jaouadi et al., 2010a). Under these particular conditions, the production of the SAPB and KERAB enzymes started after a 6- and 10-h lag phase, respectively. These productions were then noted to increase exponentially and concomitantly with the increase of cellular growth and to reach the maxima within 24 h of cultivation for SAPB (Fig. 2) and 96 h for KERAB (data not shown).
Compared to the production yields obtained in flask cultivations, the use of a 7-litre fermentor containing the optimized medium after 24-h cultivation at 37°C, an aeration of 1.5 vvm, and an agitation of 600 rpm was noted to improve SAPB production by about 4-folds. It is worth noting here that the cell densities obtained in both cases (Rotary flask and fermentor) were almost the same (about O.D. = 10.9). Based on this particular finding, it was possible to infer that the improvement of enzyme production was related not only to the cell’s growth but also to the stability of fermentation parameters (pH and pO2).
3.2. SAPB and KERAB purification and characterization
The purification protocols used for the purification of each enzyme were conducted at temperatures not exceeding 4°C. Five-hundred ml of 24 h and 96 h cultures of
Purification to homogeneity was achieved for SAPB by HPLC using Shodex Protein WK 802-5 column. The analysis indicated that enzyme achieved a degree of purity that was about 38-fold greater than that of the crude extract. Under the optimal assay conditions used, the purified enzyme preparation exhibited a yield of about 12% with a specific activity of 25,500 U/mg (Jaouadi et al., 2008). As far as KERAB was concerned, the insoluble material was then removed by centrifugation. The supernatant obtained was incubated for 1 h at 50°C and insoluble material was removed by centrifugation. The supernatant was loaded on a Sephacryl S-200 column equilibrated with buffer B. The elution of protease was performed with the same buffer. The fractions containing keratinase activity were then pooled and applied to a Q-Sepharose column equilibrated in buffer D. The column was rinsed with 500 ml of the same buffer and the adsorbed material was eluted with a linear NaCl gradient. At the final purification step, Keratinase activity was eluted between 0.15 and 0.3 M NaCl. The purity of the enzyme was estimated to be about 86-fold greater than that of the crude extract. The purified enzyme preparation contained about 24% of the total activity of the crude enzyme and had a specific activity of 67,000 U/mg (Jaouadi et al., 2010a). These preparations were homogeneous enzymes with high purity as they exhibited single protein bands on native PAGE and unique elution symmetrical peaks on gel filtration chromatography.
For determination of their molecular weight, enzyme preparations were treated with 1 mM PMSF prior to electrophoresis to inhibit possible autolysis during electrophoresis. Electrophoresis under denaturing conditions (SDS-PAGE) also revealed single bands with molecular masses estimated as 34 kDa for SAPB (Jaouadi et al., 2008) and 30 kDa for KERAB (Jaouadi et al., 2010a). The exact molecular masses obtained for the purified SAPB and KERAB were confirmed by MALDI-TOF mass spectrometry as being 34598.19 and 29850.17 Da, respectively. Zymogram activity staining also revealed two clear zones of proteolytic activity at 34 and 30 kDa for the SAPB and KERAB, respectively. These observations indicated that SAPB extracted from the newly isolated bacterium
The molecular mass of SAPB determined by SDS-PAGE (~ 34000 Da) and conducted by MALDI-TOF mass spectrometry (34598.19 Da) were not close to that calculated from the primary sequence of the mature polypeptide (27789 Da), which strongly suggested that the protein underwent noteworthy post-translational changes that were presumably pertaining to glycosylation. Similar differences between experimental and theoretical determinations were previously observed for several
3.3. Physico-chemical and kinetic properties of SAPB and KERAB
Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DIFP) were noted to strongly inhibit SAPB and KERAB, which indicated that both enzymes belonged to the serine proteases family. While the optimal pH and temperature values of 10.6 and 65°C were determined for SAPB using casein as a substrate, those obtained for KERAB were 11.5 and 75°C with keratin azure as substrate. The thermoactivity and thermostability of KERAB were also demonstrated to be enhanced in the presence of 5 mM Mg2+ against 2 mM Ca2+ for SAPB. One of the distinguishing properties of SAPB was its catalytic efficiency (
3.4. Substrate specificity of SAPB and KERAB
The activity of the purified SAPB and KERAB enzymes towards various natural and modified protein substrates is summarized in Table 1. Among the proteinaceous substrates tested, casein and keratin were most efficiently hydrolyzed by SAPB and KERAB, respectively. When SAPB and KERAB activities against casein and keratin were taken as 100%, the hydrolysis rates of gelatine and casein were 95 and 92%, respectively. Poor BSA hydrolysis rates were, however, noted in both cases. A similarly low hydrolysis level was also observed with gluten and egg albumin. Using modified proteins as substrates, the highest activities observed for SAPB and KERAB were with azocasein and keratin azure, respectively. Previous reports also showed that alkaline serine proteases from
The cleavage specificities of SAPB and KERAB toward various oligopeptidyl and ester substrates were also investigated. The findings revealed that SAPB exhibited both esterase and amidase activities on oligopeptides, with Tyr or Phe at position P1 (the amino acid residue at the N-terminal side of the scissile peptide bond). This included
In the same way, the purified KERAB was noted to exhibit a preference for aromatic and hydrophobic amino acid residues, such as Phe, Leu, Ala, and Val, at the carboxyl side of the splitting point in the P1 position. KERAB was, therefore, active against leucine peptide bonds. When Suc-(Ala)n-
|Keratin||10 g/l||65 ± 1.4||100 ± 3.0|
|Casein||20 g/l||100 ± 2.5||92 ± 2.0|
|Gelatine||20 g/l||95 ± 2.4||79 ± 1.4|
|BSA||20 g/l||52 ± 1.3||66 ± 1.4|
|Albumin (egg)||20 g/l||15 ± 0.7||26 ± 0.9|
|Gluten (wheat)||20 g/l||20 ± 0.8||11 ± 0.8|
|Keratin azure||10 g/l||63 ± 1.4||100 ± 3.0|
|Azo-casein||20 g/l||100 ± 2.5||94 ± 2.5|
|Collagen type Ic||1 mg/ml||0 ± 0.0||0 ± 0.0|
|Collagen type IIc||1 mg/ml||0 ± 0.0||0 ± 0.0|
|BAEE||4 mM||0 ± 0.0||100 ± 3.0|
|BTEE||3 mM||71 ± 1.4||10 ± 1.5|
|ATEE||3 mM||100 ± 2.5||20 ± 0.9|
|Suc-Tyr-Leu-Val-||2 mM||30 ± 1.1||100 ± 3.0|
|Suc-(Ala)2-Pro-Phe-||3 mM||100 ± 2.5||17 ± 0.9|
|Suc-(Ala)2-Pro-Leu-||3 mM||45 ± 1.3||13 ± 0.8|
|Suc-(Ala)2-Val-Ala-||3 mM||39 ± 1.2||50 ± 1.8|
|Suc-(Ala)2-Val-||3 mM||25 ± 0.9||56 ± 2.0|
|Suc-(Ala)3-||2 mM||10 ± 0.4||67 ± 2.2|
|Suc-(Ala)2-Phe-||2 mM||17 ± 0.5||89 ± 2.5|
|BAPNA||2 mM||0 ± 0.0||66 ± 1.3|
4. Molecular cloning of
sapBgene and engineering of more efficient SAPB mutant enzymes
5. Potential and prospects for SAPB and KERAB in detergent formulations
5.1. Effect of detergents on the activity and stability of SAPB and KERAB
With the aim of evaluating the performance of the purified proteases in real life-like detergents, SAPB and KERAB were pre-incubated at 40°C and in the presence of several commercially available laboratory non-ionic surfactants, denaturing agents or anionic surfactants, and bleach agents for 24 and 72 h, respectively. The residual activity was determined at pH 10.6 and 65°C (for SAPB) and pH 11.5 and 75°C (for KERAB). The findings revealed that the SAPB enzyme exhibited high stability at 10% of oxidizing agents (Tween 60 or Triton X-100) as well as against strong anionic surfactants, particularly sodium dodecyl sulphate(SDS) and linear alkylbenzene sulfonate (LAS) (Jaouadi et al., 2008). In fact, SAPB retained its activity upon treatment with 0.8% SDS and 0.5% LAS. In addition, 80 and 65% residual activity were obtained after incubation with 1.5% SDS and 1% LAS, respectively. The SAPB and KERAB enzymes were also highly stable against bleaching agents for they retained 110 and 115% of their initial activity after treatment with 15% hydrogen peroxide, respectively. This is an important behaviour of SAPB and KERAB because oxidant-, surfactant-, and bleach-stable wild-type enzymes are rarely reported.
By way of comparison, the alkaline protease from alkalophilic
|None||–||100 ± 2.5||100 ± 2.5||100 ± 2.5||100 ± 2.5|
|H2O2||15%||140 ± 3.7||155 ± 3.8||110 ± 2.6||115 ± 2.6|
|Sodium perborate||2% (w/v)||85 ± 2.2||110 ± 2.6||55 ± 2.0||85 ± 2.2|
|SDS||1.5%||110 ± 2.6||125 ± 3.0||80 ± 2.2||109 ± 2.6|
|LAS||1% (w/v)||79 ± 2.2||120 ± 3.2||65 ± 2.1||103 ± 2.5|
|Sulfobetaine||30 mM||105 ± 2.5||130 ± 3.3||90 ± 2.3||113 ± 2.6|
|Tween 40||5% (v/v)||111 ± 2.6||135 ± 3.4||101 ± 2.5||119 ± 2.8|
|Tween 60||10% (v/v)||120 ± 3.0||126 ± 3.3||105 ± 2.5||111 ± 2.6|
|Triton X-100||10% (v/v)||101 ± 2.5||132 ± 3.5||94 ± 2.3||112 ± 2.7|
|TAED||10% (w/v)||115 ± 2.8||128 ± 3.5||103 ± 2.5||117 ± 2.7|
|Na2·CMC||5% (w/v)||109 ± 2.6||137 ± 3.5||101 ± 2.5||112 ± 2.6|
|Zeolithe||1% (w/v)||99 ± 2.5||100 ± 2.5||94 ± 2.3||95 ± 2.3|
|STPP||1% (w/v)||88 ± 2.3||90 ± 2.3||80 ± 2.2||82 ± 2.2|
|Perfume||1% (v/v)||115 ± 2.6||116 ± 2.6||103 ± 2.5||104 ± 2.5|
|Na2·CO3||100 mM||50 ± 2.0||113 ± 2.4||42 ± 1.8||100 ± 2.5|
|Zwittergent 3-12||10 mM||107 ± 2.5||116 ± 2.6||100 ± 2.5||109 ± 2.5|
|CHAPS||15 mM||121 ± 3.0||133 ± 3.1||106 ± 2.5||115 ± 2.6|
|CTAB||25 mM||104 ± 2.5||107 ± 2.3||95 ± 2.4||100 ± 2.5|
|TTAB||25 mM||99 ± 2.4||105 ± 2.6||90 ± 2.3||98 ± 2.3|
5.2. Compatibility of SAPB and KERAB enzymes with various commercial laundry detergents
To check the compatibility and stability of the alkaline proteases towards detergents, the enzymes were pre-incubated in the presence of various commercial laundry detergents of different compositions for 1 h at 40°C. The laundry detergents were diluted in tap water to a final concentration of 7 mg/ml to simulate washing conditions. The endogenous proteases were inactivated by incubating the diluted detergents for 1 h at 65°C, prior to the addition of the SAPB and KERAB enzymes, or the SB 309 commercial enzyme, which was used for comparison (Table 3). The findings showed that SAPB and KERAB were relatively more stable and compatible with some commercial liquid detergents than the commercial enzyme. In fact, while SB 309 retained 100, 85, 70 and 90% of its initial activity in the presence of Axion, Dinol, Nadhif, and Lav+, SAPB retained about 100, 95, 94, and 85% and KERAB about 87, 90, 75, and 95% of their initial activities, respectively. The SAPB and SB 309 enzymes were, however, less stable in the presence of Axion, where they were totally active. Furthermore, SAPB and KERAB showed excellent stability and compatibility in the presence of some commercial solid detergents, namely OMO, New Det, and Skip, with SAPB retaining about 96, 82, and 69% of its initial activity, and KERAB about 88, 93, and 95%, respectively. SAPB and KERAB were, however, less stable in the presence of Ariel, retaining about 55 and 51% of their initial activities, respectively. Nevertheless, the compatibility and stability exhibited by SAPB and KERAB were much more significant than that of SB 309, which retained only 70, 68, and 84% of its initial activity in the presence of OMO, New Det, and Skip, respectively. Incubated in the same conditions in the presence of New Det, the NH1 protease was reported to retain 60% of its initial activity (Hadj-Ali et al., 2007) and, in the presence of Ariel, the VM10 (Venugopal and Saramma, 2006) and SSR1 (Singh et al., 2001) proteases were reported to retain only 42 and 37% of their initial activities, respectively. Overall, the results obtained clearly indicated the superior performance of SAPB and KERAB enzymes in detergents compared to currently commercialized or previously described proteases. A minor discordance was, however, reported as present with regards this performance, which was presumably correlated to the nature and concentration of the laundry detergent compounds used.
|None||100 ± 2.5||100 ± 2.5||100 ± 2.5||100 ± 2.5||100 ± 2.5||100 ± 2.5|
|Dinol||95 ± 2.4||90 ± 2.2||85 ± 2.2||81 ± 1.4||80 ± 2.2||77 ± 2.0|
|Lav+||85 ± 2.2||95 ± 2.4||90 ± 2.2||75 ± 2.0||81 ± 2.2||75 ± 2.0|
|Nadhif||94 ± 2.4||75 ± 2.0||70 ± 2.0||77 ± 2.0||62 ± 1.8||60 ± 1.7|
|Axion||100 ± 2.5||87 ± 2.1||100 ± 2.5||91 ± 2.3||66 ± 1.9||85 ± 2.2|
|New Det||99 ± 2.5||94 ± 2.4||68 ± 2.0||82 ± 2.2||93 ± 2.4||58 ± 1.7|
|Skip||75 ± 2.0||100 ± 2.5||84 ± 2.2||69 ± 2.0||95 ± 2.4||72 ± 2.2|
|Ariel||65 ± 1.8||60 ± 1.7||61 ± 1.7||55 ± 1.6||51 ± 1.5||50 ± 1.5|
|OMO||100 ± 2.5||95 ± 2.4||70 ± 2.2||96 ± 2.4||88 ± 2.1||61 ± 1.7|
5.3. Wash performance analysis of SAPB
In order to evaluate the performance of SAPB in terms of ability to remove harsh stains, namely those caused by chocolate or human blood, several pieces of stained cotton cloth were incubated at different conditions (Fig. 3). The findings from these assays revealed that the blood and chocolate stain removal levels achieved with the use of SAPB alone were more effective than the ones obtained with detergent (Det) alone. In fact, SAPB facilitated the release of proteinacious materials in a much easier way than the commercialized SB 309 protease (Jaouadi et al., 2009). Furthermore, the combination of SAPB and the Det detergent resulted in complete stain removal (Fig. 3). In fact, a similar study has previously reported on the usefulness of alkaline proteases from
5.4. Storage stability of the spray-dried and lyophilized SAPB
|t = 2 months||t = 12 months|
|Residual activity (%)|
|SAPB + Xylitol||88||70|
|SAPB + Det||64||50|
|SAPB + Det + Xylitol||78||70|
|SAPB + Xylitol||80||68|
|SAPB + Det||61||50|
|SAPB + Det + Xylitol||75||65|
The findings indicated that spray-dried SAPB, from fermentor culture, lost about 3% of its original activity; lyophilized SAPB lost about 10% (Jaouadi et al., 2009). Several of the additives used during the spray drying and lyophilizing processes were noted to improve SAPB stability (Table 4). However, the best results were actually obtained with 1% of xylitol, maltodextrin, and PEG 8000, which preserved about 100, 99 and 97% of its proteolytic activity, respectively. The stability of the spray-dried and lyophilized SAPB during subsequent storage in the presence of 1% xylitol showed that, after incubation at room temperature for 12 months, the enzymes lost only about 20 and 25% of their original activity, respectively, against 35% for the control without additives. The non-treated enzyme was rapidly inactivated, losing about 50% of its initial activity after 2 months of incubation. Moreover, compared to the treated enzyme and in the absence of additives, 1% xylitol clearly enhanced SAPB stability during storage within the Det solid detergent (Jaouadi et al., 2009). In fact, after being incubated for 12 months at room temperature in the presence and absence of xylitol, the spray-dried enzyme retained 68 and 55% of its initial activity, respectively. The level of stability enhancement achieved for the lyophilized SAPB by xylitol was, on the other hand, less pronounced, since the enzyme retained only 65% of its initial activity.
6. Potential and prospects for SAPB and KERAB in the leather processing industry
6.1. Keratin-degradation profile of
Bacillus pumilusCBS and Streptomycessp. AB1
Keratinacious substrates, such as keratin and keratin azure, were previously reported to be significantly hydrolyzed by SAPB (Jaouadi et al., 2008) and KERAB (Jaouadi et al., 2010a). It was also demonstrated that the
The feather-meal degradation rate achieved by
An increase simultaneous to keratin degradation was noted in protein levels and sulfhdryl groups (Jaouadi et al., 2009). Higher levels of keratin degradation resulted in high sulfhydryl group formation. The results obtained, therefore, suggested that
The use of enzymatic and/or microbiological methods for the hydrolysis of feathers is an attractive alternative to the currently used methods of feather meal preparation which involve high temperature and pressure treatments that result in the loss of essential amino acids (Hess and FitzGerald, 2007). The ability of the
6.2. Dehairing utility of SAPB
The incubation of the SAPB protease with skin from goat (Fig. 5), bovine (Jaouadi et al., 2009), and sheep (Jaouadi et al., 2009) for dehairing showed that after 24 h-incubation at 37°C, hair was removed very easily for all skins, compared to the corresponding controls, with no observable damage on the collagen. Therefore, the dehaired skins obtained exhibited clean hair pore and clear grain structure (data not shown). Again, these results confirmed that SAPB alone could accomplish the whole dehairing process.
The dehairing operation in leather processing is generally carried out under a relatively high pH value of about 8 - 10 (Dayanandan et al., 2003). This criterion was also satisfied by SAPB. In fact, approximately similar results were reached with the
7. Effect of organic solvents on the activity and stability of SAPB and KERAB
In addition to the key areas of application discussed for proteases above, the latter constitute a highly resourceful class of enzymes for various industrial sectors. They are, for instance, necessary in the biocatalysis of various peptide coupling reactions, which are of an extremely pharmaceutical and nutritional interest, namely those involved in the synthesis of several drug precursors such as the enkephalin (Kimura et al., 1990) and aspartame precursors (Nakanishi et al., 1990). However, the ultimate application of proteases in the synthesis of peptides has often been curtailed by the poor levels of specificity and instability in the presence of organic solvents so far reported in the literature. Accordingly, various water-miscible organic solvents and alcohols at final concentrations of 50% were assayed for their effect on SAPB and KERAB activity at pH 10 and 60°C. Buthanol, acetonitrile, and ethyl acetate had significant inhibitory effects on the activity of both enzymes (Table 5). By contrast, dimethylformamide (DMF), DMSO, and hexane were noted to enhance the activity and stability of both enzymes while isopropanol and ethanol enhanced those of SAPB and KERAB, respectively (Table 5). Hence, good stability rates of 115, 97, 90 and 85% were exhibited by SAPB in the presence of DMF, hexane, isopropanol and DMSO, respectively. Equally good stability rates of 150, 125, 115 and 105% were displayed by KERAB in the presence DMSO, DMF, ethanol, and haxane, respectively. Acetonitrile, however, exerted a considerably negative effect on enzyme stability. Compared to SAPB, NH1 (Hadj-Ali et al., 2007) seemed less efficient for it exhibited only 181.5 and 94.5% of its initial activity and stability in the presence of 25% DMSO, respectively. The exception was observed with the organic solvent-tolerant protease BG1 (Ghorbel-Frikha et al., 2005; Ghorbel et al., 2003), which showed a half-life of 50 days of its activity in the presence of 25% DMSO. While the only report available to date on organic solvent protease from
|None||100 ± 2.5||100 ± 2.5||100 ± 2.5||100 ± 2.5|
|Methanol||100 ± 2.1||80 ± 2.1||85 ± 2.2||75 ± 2.0|
|Ethanol||75 ± 2.0||132 ± 3.2||55 ± 2.6||115 ± 2.6|
|Buthanol||50 ± 1.4||79 ± 2.0||38 ± 1.4||63 ± 1.5|
|Isopropanol||115 ± 2.6||25 ± 0.5||90 ± 2.3||15 ± 0.8|
|Acetonitrile||25 ± 1.0||20 ± 1.0||10 ± 0.8||0 ± 0.1|
|Ethyl acetate||85 ± 2.2||66 ± 1.5||72 ± 2.0||58 ± 1.5|
|DMF||200 ± 5.0||155 ± 3.7||115 ± 3.0||125 ± 3.0|
|DMSO||150 ± 3.7||195 ± 4.9||85 ± 2.2||150 ± 3.7|
|Hexane||170 ± 4.0||160 ± 3.8||97 ± 2.5||105 ± 2.5|
A combination of high esterase and low amidase activities is necessary for several synthetic applications of proteases, including peptides coupling (Plettner et al., 1999). In addition to demonstrating its organic tolerance, the findings presented above show that both SAPB and KERAB exhibited powerful esterase activities on BTEE and on BAEE. Furthermore, no amidase activity was detected for SAPB and KERAB on BAEE with P1= Arg and ATEE with P1 = Tyr, respectively. These findings, in addition of the observed activity and stability in certain organic solvents strongly suggested that SAPB and KERAB are potential strong candidates for use in peptide synthesis reactions in low water systems.
This chapter described the valuable advantages inherent in proteases and the promising opportunities they offer for the enhancement of a variety of industrial and consumer product applications. This was illustrated by an overview on the purification and characterization of two extracellular extremozyme serine alkaline proteinases, namely SAPB and KERAB, which were isolated from
This work was funded by the Tunisian Ministry of Higher Education and Scientific Research (contract program CBS-LEMP, grant no. RL02CBS01) and the Algerian Ministry of Higher Education and Scientific Research (CNEPRU project grant no. JO100420070004). The authors wish to express their sincere gratitude to Pr. Anouar Smaoui, from the English department at the Sfax Faculty of Science for carefully structuring, proofreading, and polishing the language and format of the present book chapter.