|Title of Invention||
A PROCESS FOR THE INDUCTION OF A NOVEL ALKALINE PROTEASE IN A NOVEL STRAIN OF BACILLUS SPECIES
|Abstract||The present invention relates to an alkaline protease from Bacillus species MTCC 3606. The invention also relates to a process for the preparation of the said alkaline protease. The alkaline protease according to the invention is suitable for use in detergent compositions for cleaning and washing purposes.|
|Full Text|| Field of invention
The present invention relates to a novel alkaline protease from Bacillus species and a process for the preparation thereof.
The invention relates ro the field on an oxidaticn stable detergent alkaline protease from a bacteria and methods for producing the enzyme. The alkaline protease according to the invention is suitable for its use in detergent compositions for cleaning and washing purposes. Further the present investigation is directed towards a detergent composition comprising of this oxidation stable detergent protease.
The overall cost of enzyme production and downstream processing is the major obstacle in the successful application of any technology in the enzyme industry. Researchers use several methods to increase the yields of alkaline proteases with respect to their industrial requirements. Recent approaches for increasing protease yield include screening for hyper-producing strains, cloning and over-expression, fed-batch, chemostdt fermentations, and by optimization of the fermentation medium through statistical approach such as response surface methodology. Some other methods, such as, cell immobilization, aqueous two-phase systems composing of PEG, solid state fermentation and biphasic growth systems have been used for improving protease production from different microorganisms. The alkaline proteases
of the prior art have been obtained in particular by cultivation of Bacillus species bacteria
such as Bacillus alcalophilus, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, which produce alkaline proteases and secrete them into the culture medium.
Attempts have been made in the prior art to obtain high yields of alkaline proteases using combinations of methods such as controlled batch and fed-batch fermentations using simultaneous controls of glucose, ammonium ion concentration, oxygen tension, pH and salt availability (Hubner et al. 1993; vanPutten et al. 1996) and chemostat cultures (Frankena et al. 1986) have been successfully used for improving protease production for long time incubations from different microorganisms. In order to scale up the protease production from microorganisms at industrial level, biochemical and process engineers use several strategies to obtain high yields of protease in a fermentor Generally proteases produced from
microorganisms are constitutive or partially inducible in nature and under most culture conditions, the Bacillus sp. produces extracellular proteases during post-exponential and stationary phases. Extracellular protease production in microorganisms is also strongly influenced by med'a components, viz.. variation in C/N ratio, presence of some easily metabolizable sugars, such as glucose and metal ions. The protease synthesis is also effected by rapidly metabolizable nitrogen sources such as amino acids in the medium. Besides these, several other physical factors, such as aeration, inoculum density, pH. temperature and incubation also affect the amount of protease produced. In recent years, the statistical approach methods using different statistical softwares during process optimization studies with the aim of obtaining high yields of alkaline protease in the fermentation medium have been used (deConinck et ai. 2000). The application of properly designed approaches with multi-factor models allow process engineers and biochemical engineers to design scale-up strategies for increasing enzyme production.
Proteciytic enzymes are ubiquitous in occurrence, being found in ail living organisms ami are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Though there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available such as subtilisin Carlsberg, subtilisin BPN' and Savinase with their major application as detergent enzyme. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations viz., Durazym, Maxapem and Purafect have been launched using techniques of site-directed mutagenesis and/or random mutagenesis.
Many attempts have already been made in the prior art to obtain new alkaline proteases having desired properties. For instance, a series of natural and artificially (genetically) altered alkaline and highly alkaline proteases is already known. However, there remains a need for new, alkaline proteases having beneficial properties, particularly with respect to their washing behavior. Lot many commercial alkaline proteases are available in market, however only few of them exhibit stability in presence of bleaching components and chemicals used in detergents, hi the past
few years, the stability of proteases in bleaching agents has been improved by use of genetic engineering and protein engineering techniques (e.g. random mutagenesis, site directed mutagenesis, and directed evolution) by replacement of amino acid residues and various microbial strains have been genetically engineered to express high levels of extracellular proteolytic enzymes in the fermentation medium. Site directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. Replacement of an oxidation sensitive methionine at position 222 by oxidation resistant amino acids, such as, alanine, serine or glutamine in the linear sequence of B. amyloliquefaciens subtilisin BPN' resulted in prevention of subtilisin inactivation by oxygen bleaches (Estell et al. 1985). vonderOsten et al. (1993) performed protein engineering of two alkaline Bacillus proteases, Savinase and Esperase (commercially produced by Novo Nordisk A/S) to improve the storage stabilities of enzymes, lower the isoelectric point of the enzymes to approach the pH of the liquid detergent formulation so as to improve their wash performance in laundry detergents, and to improve the thermal stabilities of the enzymes. Aehle et ai. (1993), based on a modeled 3-D structure have designed a model of a highly alkaline subtilisin-like protease (opticlean) from B. alcalophilus using a computer-aided protein design process of 'modeling by homology' starting with the structure of subtilisin Carlsberg 1CSE.BRK from Brookhaven protein databank with an aim to increase the wash performance of enzyme. The random mutagenesis approach on mutant M222A gene was used (Yang et al. 2000b), to develop a thermally stable and oxidation-resistant mutant of subtilisin E. The heat stability of this new mutant was 5-fold greater than that of the wild-type enzyme. In addition to reports in literature, several international patents have also been filed on various strategies used by different researches for obtaining more oxidation stable mutants of subtilisin. Bech et al. (1993) reported a novel process for stabilizing detergent enzymes for improved stability towards oxidative agents. The subtilisin-309 was modified by replacing the methionine residue at position 222 with cysteine having a HS-group, and this cysteine residue v/as further modified by replacing H-atom of the HS-group of the formula R ner et al. (1996) generated several mutations in amino acid sequence of matured subtilisin BPN' to construct several subtilisin mutants for their uses in enzymatic detergent and cleaning compositions.
Alkaline proteases are valuable industrial products with advantageous applications, in particular in the detergent industry, since they remove protein-containing contaminants. In order to be effective, these proteases must not only have proteolytic activity under washing conditions (pH value, temperature), but they must also be compatible with other detergent constituents, e.g. other enzymes, surfactants, builders, bleaching agents, bleaching agent activators and other additives and adjuvants. In particular, the proteases must possess sufficient stability with respect to these detergent constituents and sufficient washing effectiveness in their presence. Enzymes have long been of interest to the detergent industry for their ability to aid in the removal of proteinaceous stains as well as to deliver unique benefits that cannot otherwise be obtained with conventional detergent technologies. Applications of detergent proteases have grown substantially and the largest application is in the household laundry detergent formulations. Increased reliance of detergent manufacturers on enzyme technology is because of consumer-recognizable cleaning benefits, addition of completely new performance benefits, fabric restoration, and increased performance/cost ratio because of the availability of more efficient enzymes and the industry trend toward reduced pricing. Current market trends and consumer needs are influencing the development of enzymes for detergent applications, with the emphasis on enzymes that have improved performance/cost ratios, increased activity, and improved compatibility to other detergent ingredients. In addition, enzyme suppliers and detergent manufacturers are actively pursuing the development of new enzyme activities that address the consumer-expressed need for improved cleaning, fabric care, and antimicrobial benefits. However, apart from their use in laundry detergents they are also popular in formulation of household dishwashing detergents, industrial and institutional cleaning detergents
Brief disclosure of the invention
The object of this invention is to provide oxidation stable-alkaline protease from a wild type strain of Bacillus sp, which is stable in strong oxidizing agents and bleaches an 3 process for preparing the same or use in detergents. Said proteaio exhibits stability in the temperature range up to 60°C.
Another object of this invention is to provide a process for protease production from strain of Bacillus sp. The protease production by Bacillus sp. is inducible in presence of casamino acids and is repressible by glucose and ammonium, ammonium ions being more effective repressers. The time of addition of inducer had greater influence on protease induction, as cells under de-repressed state reacted best to induction as compared to cells growing in active phase. An increase in protease production could be obtained by separating biomass production phase and enzyme production phase, i.e. by de-repression and subsequent induction. The de-repression and induction under fed-batch operations regulate the protease synthesis in Bacillus sp. This would be of interest for applied and industrial microbiologists, and process engineers for designing scale-up strategies for process optimization.
Another object of this invention is to provide a suitable statistical method to for protease production from strain of Bacillus sp. The protease production in Bacillus sp. can be improved by controlling various factors simultaneously viz., casamino acids, agitation and time and this interaction could only be well understood by suitable selection of response surface method.
Another object of this invention is to propose a process for the preparation of an alkaline protease that can be purified in a single-step using an anion exchanger column.
Yet another object of this invention is to propose an alkaline protease and a process for preparation of alkaline protease which is compatible with several surfactants, oxidants, bleaches, and other ingredients of detergent compositions.
To achieve the afore-said objectives, the present invention provides an oxidation stable alkaline protease derived from Bacillus species having an N-terminal amino acid sequence of A-Q-T-V-P-H-G-I-P-L-I-K-A-D-K (SEQ ID NO. 1)
Said protease is characterized by the following properties:
(a) an apparent molecular weight of 30kDa on SDS-PAGE after silver staining;
(b) an N-terminal amino acid sequence of A-Q-T-V-P-H-G-I-P-L-I-K-A-D-K
(SEQIDNO. 1); (c ) having an alkaline pH stability at a pH range between 7-11.5;
(d) having a stability in the temperature range up to 60 °C
(e) having a thermostability at a up to 65 °C in the presence of stabilizers and
(f) the enzyme substrate constant varies between 0.0250 - 0.0360 mg/ml and a
maximum enzyme velocity between 70- 125 (ig/ml/min at temperature ranging
between 45- 60°C.
(g) The energy of activation (Ea) was more between 45-55°C (9747 cal/mol)
compared to Ea between 50-60°C (4162 cal/mol).
Said protease exhibited a pH optima of 10.5 . The stabilizers are selected from calcium, polyethylene glycol, starch, sucrose, mannitol, sucrose and glycerol. The activity of said protease is increased in presence of 2-mercaptoethanol, dithiothereitol and glutathione up to 2-fold, while the proteolytic activity was completely inhibited in presence of iodoacetic acid and phenyimethylsulfonylflouride. Said protease is a thiol-dependent serine protease.
The enzyme-substrate constant (Km) 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml and 0.0250 mg/ml at 45, 50, 55 and 60°C, respectively. The maximum enzyme velocity (Vmax) between 74.07, 99.01, 116.28 and 120.48 ng/ml/min at temperature at 45, 50, 55 and 60°C, respectively. The energy of activation (£a) was more between 45-55°C (9747 cal/mol) compared to Ea between 50-60°C (4162 cal/mol).
The present invention further comprises a process for preparing an oxidation stable alkaline protease derived from Bacillus species having an N-terminal amino acid sequence of A-Q-T-V-P-H-G-I-P-L-I-K-A-D-K (SEQ ID NO. 1) comprising (a) cultivating a Bacillus sps. strain in a minimal medium containing organic nitrogen sources and (b) isolating the protease;
After step (b), the said protease is concentrated and thereafter purified. Said protease can be produced in either batch or fed-batch methods. Said organic nitrogen source is casein, casamino acids, yeast extract, peptone, or skim milk, preferably casein or
casamino acids. The isolation of the alkaline protease is carried out by cold centrifugation. The concentration is by ammonium sulphate precipitation.
The purification step is by anion exchange chromatography.
The protease is used for removal of stained cotton fabric and results analyzed by change in reflectance. The best stain removal is obtained at 60°C and 45°C of old blood slains.
The present invention further relates to a detergent composition comprising up to 3 % by weight of alkaline protease according to the present invention Said detergent composition further comprises
up to 4- 6 % by weight of a surfactant or surfactant mixture.
up to 5 % by weight of a bleaching agent or bleaching agent mixture.
up to 50-60 % by weight of a builder or builder mixture
up to 10-15 % by weight of tripolyphosphate or additional constituents such
as adjuvants to make up to 100 % by weight.
Said surfactant is Tween-80, Triton X-100. Said bleaching agent is sodium perborate. Said builder is sodium sulfate.
Description of drawings
The present invention will now be explained in more detail with references to the
Figure 1: shows the partial 534 bp sequence of the soil microorganism, Bacillus sp.
Figure 2: shows the neighbour-joining tree of the Bacillus sp. with other bacteria.
Figure 3: shows the elution profile of protease from Bacillus sp. on Q-sepharose anion exchange column pre-equilibrated with glycine-NaOH buffer of pH 10.5 (50 mM).
Figure 4: shows the SDS-PAGE of purified rmtease from Bacillus sp. showing single band of 30 kDa. Lane 1: stand. arkers of known molecular weights (from Sigma); Lane 2: purifk saline protease after ion-exchange chromatography; Lane 3: ammonium sulphate precipitated protease; Lane 4: crude protease
Figure 5: shows the optimum pH and pH stability of protease from Bacillus sp.
Figure 6: shows the optimum temperature of protease from Bacillus sp. Figure 7: shows the temperature stability of protease from Bacillus sp.
Detailed disclosure of the invention
The alkaline protease according to the invention has been obtained in high amounts using combination of batch and a fed-batch method, the organism being a soil Bacillus, purified in a single step on an anion exchange column, said enzyme being stable towards commercial bleaches, surfactants and detergents, and is suitable for its use in detergent compositions for cleaning and washing purposes.
The alkaline protease according to the invention is produced from soil Bacillus, which is identified as close to Bacillus mojavensis on the basis of partial 16S rRNA alignment with GenBank database. The 534 bp 16S rRNA sequence of the organism has been deposited in the Genbank under accession number AF44Q779. The culture has been deposited in the Microbial Type Culture Collection (MTCC) center of Institute of Microbial Technology, Chandigarh, India under accession no. MTCC 3606.
The protease production from Bacillus sp. was inducible in presence of organic nitrogen and was maximum in casein and casamino acids and was repressed by glucose and ammonium ions. A fed-batch strategy was adopted to enhance protease synthesis by Bacillus sp. using intermittent de-repression and induction during the growth of the organism. In the first biomass production phase, the growth of the bacterium was achieved in non-inducible conditions for 6 h in presence of glucose and ammonium ions. Feeding of casamino acids at this stage resulted in a 2.8-fold increase in protease yield after 36 h compared to protease yield in batch culture. This protease production was further enhanced to 4-fold in the second fed-batch operation on the onset of second stationary phase by a glucose feed (5 mg/ml) at 33 h followed by a .asamino acids feed (5 mg/ml) at . 3 h. The overall fermentation process was highly sensitive to agitation and the protease production was drastically inhibited at low agitation conditions and only negligible amounts of protease was produced in static culture of Bacillus sp.
The alkaline protease production in Bacillus sp. was improved up to 4.2-fold in a 14-1 bioreactor during validation of predicted statistical model. The maximum enzyme yield was obtained within 10-12 h compared 18 h in shake flask cultures. The statistical optimization of protease production was carried out in two steps using Response surface methodology. The first step suggested that the protease production was subjected to catabolite repression by glucose. The response surface curves also suggested that maximum levels of casamino acids and midlevel of glucose as best C/N combination for optimal enzyme production (Table 1). Hence, after increasing casamino acid concentration in second step, an optimum level of 11 mg/ml casamino acids supported maximum protease yield (Table 2). High agitation rates concomitant with low inoculum density supported maximum protease production. Protease production was drastically reduced at low agitation rates. The present study provides useful information about the regulation of protease synthesis through manipulation of various physieochemicai factors.
An N-terminal sequence analysis of said protease revealed that it was up to 91% similar to most of the other popular subtilisin-type alkaline proteases. The said alkaline protease was also compatible with several surfactants, oxidants, bleaches, and other ingredients of detergent compositions, retains 90% activity in presence of 1 mM EDTA, good washing effectiveness of protein-based stains.
The alkaline protease according to the invention may be produced by cultivating the Bacillus sp. in a minimal medium containing any organic nitrogen source such as casein, casamino acids, yeast extract, peptone, or skim milk, preferably casein and casamino acids. The protease according to the invention can both be produced either in batch or by following the fed-batch methods. The isolation of the alkaline protease from the culture supernatant can be carried out by cold centrifugation, the protease is concentrated by ammonium sulphate precipitation, purified by anion exchange jhromatography. The protease elutes out in unbound fractions from the column pre-equilibrated with high pH buffer, may be 9, 10, 10.5, preferably buffer of pH 10.5 with 17-fold purification (Figure 3; Table 4) and showed a single band of approximately 30 kDa on SDS-PAGE after silver staining (Figure 4). The N-terminal
sequence analysis (A-Q-T-V-P-H-G-I-P-L-I-K-A-D-K) of the purified protease from Bacillus sp. shows 91% homology to the most popular alkaline protease, Subtilisin Carlsberg (Table 5).
The protease according to invention was characterized with reference to its properties related to its use as detergent enzyme. The enzyme exhibited pH optima of 10.5 and was also stable between a wide pH range of 7-11.5 for more than 48 h at room temperature (Figure 6). The temperature optima of protease was found to be 60°C (Figure 7). The protease retained 86% of its activity at 60°C after 1 h and exhibits half-life of 2.5 h, 15 mm and 7 mm at 60, 65 and 70°C, respectively (Figure 8). The thermostability of protease at high temperature up to 65°C according to the invention can be enhanced in presence of stabilizers such as calcium, polyethylene glycol, starch, sucrose, mannitol, sucrose and glycerol (Table 6). The kinetic parameters (Km and Vmax) of the enzyme were determined by a statistical approach using a central composite circumscribed design using casein concentration and temperature as two variables (Table 7). The protease exhibited Km of 0.0357 nig/ml, 0.0270 mg/ml, 0.0259 mg/ml and 0.0250 mg/ml at 45,-50, 55 and 60°C, respectively, whereas Vmia values at these temperatures were 74.07, 99.01, 116.28 and 120.48 ug/ml/min, respectively (Table 7). The energy of activation (Ea) was more between 45-55°C (9747 cal/mol) compared to Ea between 50-60°C (4162 cal/mol). The activity of purified enzyme was increased in presence of 2-mercaptoethanol, dithiothereitol and glutothione up to 2-fold, while the proteolytic activity was completely inhibited in presence of iodoacetic acid and phenylmethylsulfonylflouride suggesting it to be a thiol-dependent serine protease. Several specific protease inhibitors such as bestatin, chymostatin and N-bromosuccinimide inhibited the enzyme up to more than 90%. The enzyme activity was increased in presence of TPCK and TLCK (Table 10). The protease was also stable in presence of various metal ions and also retained 90% activity in 1 mM EDTA. Besides pH and temperature stability's, a good detergent enzyme should also be stable in the presence of various commercially available bleaching agents, detergents and surfactants. The stability of protease in presence of various commercial detergents, bleaches and surfactants were tested and it was found to be stable in most of these commercial preparations. The protease from Bacillus sp. was also stable towards several commercially available laboratory bleaches and
surfactants such as fyO:, sodium perborate, tweens, Triton X-100, sodium choleate, etc (Table 11). The protease also showed its compatibility towards all commercial detergents and bleaches used in laundries, such as Surf, Nirma, Ariel, Rin, Wheel, Fena, Revel, Ala and Robin Liquid bleach (Table 11). Lot many commercial alkaline proteases are available in market, however only few of them exhibit stability in presence of bleaching components and chemicals used in detergents (Table 12). hi the prior art, the bleach- or oxidation stability in the protein or enzyme has been obtained by techniques of site-directed mutagenesis and protein engineering (Table 12) by replacing 'methionme' with 'cysteine' and making it richer in free sulfhydryl (-SH) groups, so that the protein become resistant to attack by oxidizing chemicals. The present inventions pertains to a wild-type alkaline protease, which already has these desired properties of being resistant to oxidizing and bleaching agents. This is one protein, which has sufficient rich number of -SH groups. All those properties thatare engineered in a protein by site-directed mutagenesis are already occurring in this enzyme. In addition, the protease also hydrolyzed several native proteinacious substrates such as gelatin, eiastin, albumin, haemoglobin and skim milk to a significant extent with maximum specificity towards casein and skim milk (Table 13).
The wash performance analysis of enzyme revealed that it could effectively remove a
variety of stains such as blood, grass and beetle (Table 14). The soiling containing
blood, beetle and grass stain are removed equally well at 60°C. No significant
impairment of the washing effectiveness of the proteases according to the invention
by the other constituents contained in the detergent formulation can be detected in
washing tests. For these applications, the invention provides a novel alkaline protease
having beneficial properties, by means of which the protein-containing soiling,
especially hospital stains can advantageously be removed. Thus, in brief it can be
concluded that this protease could have potential use as detergent additive. In general,
all currently used detergent compatible enzymes are alkaline and thermostable in
nature with a high pH optimum because the pH of laundry detergents is generally in
the range of 9-12 and varying thermostat at laundry temperatures (50-70°C).
The present enzyme could be promising fbt application in detergents for washing at ambient temperatures (45-50°C), as well as temperatures up to 60°C as it is active over a broad temperature range.
The detergent proteases work best by hydrolyzing large insoluble protein fragments in the bulk wash liquor. These fragments are initially removed from the fabric surface either by components of the detergent matrix, or by water alone. Depending upon the size of the resulting fragments, they are either solubilized into oulk solution or they deposit themselves back to the fabric. Hence the best detergent protease provides improved protein hydrolysis, resulting in better stain removal and antideposition benefits.
Using different protein stains, such as blood, beetle and grass, in the present invention, on the cotton fabric was evaluated. Treatment with enzyme and detergent in combination gave the best stain removal of all the treatments, though protease treatment alone was also at par the combine treatment of 'detergent+enzyme' in all the stains, while treatment of stained cloth with either enzyme or detergent alone, exhibited some and slight stain removal, respectively. This shows that although protein stain may be fragmented by protease alone, the sizes of fragments are too large to be solubilized in the absence of detergent. Similarly the role of detergent is to solubilize large protein fragments, but they must be small fragments to become completely removed with the wash water, or redeposition occurs. The best removal was observed when both protease and detergent work together and to remove hydrolysis fragments as they are formed. The results suggest that detergent proteases serve a multifunctional role-they hydrolyze proteins absorbed to fabric, making the protein more easily removed by the detergent and they inhibit protein redeposition to the clean fabric.
In addition to the detergent enzyme already mentioned, the detergents and cleaning agents of the invention may also contain the detergent constituents which are conventional in the prior art, such as surfactants, bleaching agents or builders, and also additional conventional adjuvants for detergent formulations in conventional quantities.
Such detergent formulations may be lormulated in conventional manner. The proteases according to the invention may additionally be mixed with the other
constituents of the detergent formulation in a known manner, for instance in the lyophilized form or in the form of granules or tablets.
The protease according to the invention from Bacillus sp. is distinguished by advantageous properties. At alkaline pH values, it has a high stability. The pH optimum of the proteases according to the invention lies in a range of pH 7-11.5, which is advantageous for use in detergent compositions. Furthermore, the protease according to the invention have a temperature optimum in the range of about 50-60°C. It has good stability, even in washing solutions. Due to its beneficial activity at temperatures of up to 60°C, the Bacillus protease according to the invention is particularly suitable for use in detergent compositions which are intended to be used at temperatures up to ambient (45-60°C) and 60°C. Such detergent and cleaning agent compositions, which contain a protease according to the invention demonstrate beneficial washing effectiveness with respect to protein stains which are to be removed.
Materials and methods
The activity of the enzyme according to the invention was determined by following procedure: The enzyme Was allowed to react with standard casein solution under controlled conditions. The enzyme activity was calculated by the extent of casein hydrolysis by spectrophotometrically measuring the amount of liberated products from casein. 1 ml enzyme solution was mixed with 1 ml of 1% casein solution at pH 10.5 and incubated for exactly 10 minutes at temperature of 60°C. 4 ml of 5% TCA was added to the reaction mixture to stop the reaction. Thereafter the contents were centrifuged at 5000 rpm for 5 minutes and the 1 ml supernatant was mixed with 5 ml of 0.4 N sodium carbonate solution, followed by addition of 0.5 ml of 1:1 diluted Folin's Ciocalteu's reagent. Tubes are incubated in dark and after 30 minutes the absorbance of each sample was determined at 660 nm. 1 unit of protease is defined as amount of enzyme required to release one microgram of products from the casein equivalent to tyrosine per minute per ml of the reaction mixture. The following examples will further illustrate the present invention.
EXAMPLE 1 Fed-batch operations
These operations were carried out in two phases: (a) biomass production phase, and (b) protease induction phase. Cells were first grown in a minimal medium mentioned above (except casamino acids) for 6 h so that the cells reached a stationary phase (O.D.550nn,=3.0) and the final concentrations of glucose reached to less than 3 mg/ml from initial concentration of 5 mg/ml, and NH4+ ion concentration declined to 0.0093 mmol/ml from initial 0.093 mmol/ml. This low concentration of these two components reached after 6 h of growth was not inhibitory to protease synthesis. Glucose concentration used was standardized in presence of 5 mg/ml of NHiCl (=0.093 mmol ml"1 NItt4) to obtain maximum specific growth rate of the organism. At this moment the cells were fed with 5 mg/ml casamino acids. In the second feeding operation, the cells were first fed with 5 mg/ml of glucose at 33 h followed by feeding with 5 mg/ml casamino acids at 36 h at the onset of second stationary phase and course of protease synthesis was monitored periodically.
Statistical approach using response surface methodology for protease production Five chemical and physical variables, namely, casamino acids, glucose, inoculum age, incubation time and agitation were observed, which mainly controlled the protease production by Bacillus sp. under batch fermentation were taken for optimizing protease production using face-centered central composite design by a two step experimental design for improving total protease production from Bacillus sp. The statistical software package 'Design-Expert® 6.0', Stat-Ease, Inc., Minneapolis, USA-was used to analyze the experimental design.
hi the first step, Face-centered central composite design (FCCCD-1), five independent variables were used to approach the interaction among different factors. For a 25 FCCCD with five factors, including 6 center points, a set of 32 experiments was carried out. All the variables were taken at a central coded value considered as zero. The minimum and maximum ranges of variables investigated and the full experimental plan with respect to their values are listed in Table 1 along with the predicted and observed response. Upon completion of experiments, the average maximum protease yield was taken as the dependent variable or response (Y). A
second order polynomial equation was then fitted to the data by multiple regression procedure. This resulted in an empirical model that related the response measured to the independent variables to the experiment. For a five-factor system, the model equation was: Y=
where, Y, predicted response; Po, intercept; Pi, p2. Pa, P4, PS, linear coefficients; pu,
P22, p33, P44, Pss, squared coefficients; pl2, pi3, PH, Pis, 023, P24, P25, P34, Pss,
P4S, interaction coefficients.
ANOVA (analysis of variance) was then performed on results of Table 1 to reach the optimal points (Table 3).
In the second design (FCCCD-2), only two variables, namely, casamino acids and incubation time were taken, and rest all the factors were fixed on the basis of the data obtained from the first set of experimental design. In this case, a set of 13 experiments, including 5 center points was carried out. Both the variables were taken at a central coded value considered as zero. The minimum and maximum ranges of both variables and the full experimental plan with respect to their values are listed in Table 2 along with the predicted and observed response. After completion of experiment, the average maximum protease production was taken as independent variable or response (Y). Regression analysis was then performed on the data obtained, which gave the following equation for the 2-factor interaction. Y= po+piA+p2B+puA2+p22B2+pi2AB where, Y, predicted response; Po, intercept; Pi, p2, linear coefficients; Pi i, p22, squared
coefficients; Pi2, interaction coefficient.
ANOVA (analysis of variance) was then performed of Table 2 to reach the optimal points (Table 3).
The validation of statistical approach on protease production from Bacillus sp. was carried out in a 14-1 bioreactor (Ch AG, Switzerland) with a working volume of 10-1. The bioreactor was equipped with two rushton type turbines and baffles. The optimized medium (pH 7.0) was sterilized in situ at 121°C for 30 min. Glucose was sterilized separately and was mixed aseptically with other components of
the medium in the bioreactor. The medium was inoculated with 2% of inoculum (A550nm=0.250) and fermentation was carried out at 50°C for 20 h at uncontrolled pH. The impeller speed was initially adjusted to 400 rpm and compressed sterile air was sparged into the medium @ 4-wm. The dissolved oxygen was not allowed to go below fixed set point of 20%. The samples were withdrawn periodically at an interval of 2 h and analyzed for protease production, residual glucose and biomass estimation. Other fermentation parameters, such as temperature, pH, dissolved oxygen and air flow were continuously monitored using microprocessor-controlled probes.
Purification of the protease
The 500-ml of crude enzyme preparation was subjected to ammonium sulfate saturation (0-85%) by slow continuous stirring in a cold room. The saturated solution was left overnight at 4°C, cenrrifuged and the precipitate was then dissolved in minimum amount of glycine-NaOH buffer (pH 10.5, 50 rnM) and dialyzed against the same buffer for 24 h with 6-8 changes. The anion-exchanger Q-sepharose column (Pharmacia Biotech. Upssala, Sweden; 1.5 cm x 12 cm) with a void volume of 8 cm3 column was pre-equilibrated with glycine-NaOH buffer (pH 10.5, 50 mM) and 2 ml of dialyzed protein (9.4 mg protein) was loaded and the flow rate was adjusted to 30 ml/h. the enzyme was eluted out from the column by using a continuous NaCl gradient (0-0.5 M) and 96 fractions of 2-ml each were collected. The protein was measured at 280 nm on a Shimadzu spectrophotometer (model UV 160 A). The peak of protease activity was in the unbound fractions. These unbound fractions were pooled, concentrated, and then used as source of protease for further characterization of enzyme. Finally the column was again equilibrated and stored in the same buffer before its reuse.
Polyacrylamide gel electrophoresis
The mirified protein sample was subjected Sodium dodecyl sulfate-polyarrvlamide
gel electrophoresis (SDS-PAGE) on Bio-Rad system using 12% acrylamide gel. The
protein samples were denatured by heating them at 100°C with sample buffer for 5
min before loading them onto the gel. The electrophoresis was carried out at 20 mA
before tracking dye reached the bottom of the gel. The relative molecular mass of the protein was calculated using standard protein markers (Sigma, St. Louis, MO) run simultaneously. The protein bands were visualized by silver staining of the gel.
EXAMPLE 5 Enzyme characterization
The temperature and pH kinetics of the Bacillus sp. protease were determined by assaying the protease activity at different temperatures (30-80°C) and pH's (7-12), respectively. The enzyme was assayed in the pH range of 7-12 using buffers of different pH range (phosphate buffer, 7-8; Tris-HCl buffer, 7.5-9; borate-NaOH buffer, 8-9; carbonate-bicarbonate buffer, 9-11; and glycine-NaOH buffer, 8.5-12). pH stability was determined by incubating the enzyme with equal amount of buffers of different pHs for 48 h at room temperature and thereafter the relative activity was determined under standard assay conditions at pH 10.5 and temperature of 60°C. The temperature stability was determined by incubating enzyme sample at various temperatures in range on 50-70°C for 2 h and the relative enzyme activity was estimated at regular intervals under standard assay conditions at pH 10.5 and temperature of 60°C.
Effect of stabilizers on temperature stability
Various stabilizers and additives (polyethylene glycol 6000, glycerol, sucrose,
mannitol, sorbitol and sucrose) were mixed with enzyme at concentration of 1%
(w/v), whereas, calcium ions were mixed with enzyme in the concentrations of 1 mM,
5 mM and 10 mM, and the enzyme sample was then incubated at different
temperatures of 60 and 65 °C for 6 h and the relative enzyme activity was estimated at
regular intervals under standard assay conditions at pH 10.5 and temperature of 60°C.
Determination of kinetic constants
Statistical approach using a central composite circumscribed design (CCCD) was
adopted for determining kinetic constants for alkaline protease activity, where two
independent variables (casein concentration and reaction temperature) were varied
simultaneously relative to the chosen center point (1 mg/ml casein and 60°C), there being 4 replicates at the center points and a single run for each of the other combinations, i.e., a set of 12 experiments were carried out. The minimum and maximum ranges of both the variables were investigated and the full experimental plan with respect to their values are listed in Table 7 along with the predicted and observed response. The statistical software package 'Design-Expert® 6.0', Stat-Ease, Inc., Minneapolis, USA was used to analyze the experimental design. Upon completion of experiments, the average maximum protease yield was taken as the dependent variable or response (Y). A second order quadratic equation was then fitted to the data by multiple regression procedure. This resulted in an empirical model that related the response measured to the independent variables in the experiment. For a two-factor system, the model equation was: Y=p0+Pi A+p2B+pi iA2+p22B2+pi2AB where, Y, predicted response; Po, intercept; Pi, PI, linear coefficients; Pi i, $22, squared
coefficients; $\2, interaction coefficients.
ANOVA (analysis of variance) was then peformed on above equation to reach the optimal points (Table 8).
Effect of inhibitors and metal ions
The effect of various inhibitors of serine, cysteine and trypsin-type proteases [Phenyl methyl sulphonylfluoride (PMSF), p-chloromecrucic benzoate (pCMB), etc.] and chelators of divalent cations [Ethylene diamine tetra acetate (EDTA)] (All from Sigma, St. Louis, MO) were determined by incubating them with protease for 30 min at room temperature and the relative activity was determined by standard assay methods. All the inhibitors were used at 1-mM final concentration unless otherwise stated. The effect of metal ions (1 mM) on protease activity was determined by incubating the enzyme with different compounds at room temperature for 60 min and thereafter the relative activities were determined under standard assay conditions at pH 10.5 and temperature of 60°C.
Detergents and bleach compatibility
The alkaline protease was tested for detergents, surfactants and bleach-stability by incubating enzyme samples with different ionic and non-ionic surfactants (Tween-20, -40, -60, -80, -85, Triton X-100, sodium choleate, sodium deoxycholeate. sodium perborate, sodium dodecyl sulphate), bleaches (hydrogen peroxide, sodium hypochlorite, robin liquid bleach, ala bleach), and commercial detergents (Arial, Fena, Henko, Nirma, Revel, Rin, Surf, Tide, Wheel) for 60 min and relative proteolytic activity was assayed under standard assay conditions at pH 10.5 and temperature of 60°C. Similar tests for comparison of bleach, surfactant and detergent-compatibility were also carried out on two other commercially available alkaline proteases: Savinase from Novozymes, Denmark and other alkaline protease from Advance Biochemicals, Thane, India.
EXAMPLE 10 Wash performance analysis
Wash performance analysis of protease on small square pieces (7cm x 7cm) of a new cotton fabric was performed. The washing effectiveness of the enzyme on cotton test fabric was determined by measuring the reflectance of the washed test fabric. The concentrated sap from grass was obtained by macerating fresh grass in a mortar and pestle. Samples of human blood, grass sap and beetle were used to stain the fabric. The stained cloth pieces were subjected to wash treatments at 25, 45 and 60°C with tap water, buffer (pH 10.5), detergent (1%), enzyme (100 U/ml), and a combination of enzyme and detergent for 30 min. The enzyme-containing solution acted on the stained test fabric in a beaker, kept at a particular temperature in incubator shaker at 100 rpm. After the washing process, the test fabric was rinsed twice with tap water and then ironed. The washing effectiveness achieved by the enzyme is indicated by the higher reflectance of the test fabric washed with the enzyme-containing solution. The wash performance was analyzed on reflectance meter (Model no. UEC-1018, Universal Engineering Corporation, India).
1. A process for the induction of a novel alkaline protease in a novel strain of Bacillus
species MTCC 3606, wherein the steps comprising:
[a] culturing Bacillus species MTCC 3606 in an nutrient medium at a temperature of 45 to 55 degree C for a period of 15 to 25 hours under constant aeration characterized in that the organic nitrogen source is selected from casein, casamino acids, yeast extract, peptone or skim milk;
[b] harvesting the supernatant from the culture medium of step [a] to obtain a crude enzyme extract;
[c] subjecting the crude enzyme extract of step [b] to ammonium sulphate precipitation, wherein the concentration of ammonium sulphate is 1 to 85% under continuous stirring at a temperature of 2 to 8 degree C for 12 to 24 hours;
[d] purifying the precipitate as obtained in step [c] by known methods to obtain the desired alkaline protease.
2. A process as claimed in claim 1, wherein the nitrogenous source is preferably casein or casamino acids.
3. A process as claimed in claim 1, wherein the optimum concentration of casamino acids in the medium is 11mg/ml to induce maximum protease yield.
4. A novel alkaline protease from Bacillus species MTCC 3606 represented by SEQ ID No. 1 having an N-terminal amino acid sequence of A-Q-T-V-P-H-G-l-P-L-l-K-A-D-K as prepared by the process as claimed in claim 1.
5. A protease as claimed in claim 1, wherein its pH optima is in the range of 7.0 to 11.5.
6. A protease as claimed in claim 1, wherein its temperature optima is in the range of 25 to 60 degree C.
7. A protease as claimed in claim 1, wherein its molecular weight is 30 KDa.
8. A protease as claimed in claim 1, wherein the protease is a thiol dependent serine protease.
9. A protease as claimed in claim 1, wherein the activity of the said protease increased up to 2-folds in the presence of 2-mercaptoethanol and dithiothereitol and was completely inhibited in the presence of iodoacetic acid.
10. A protease as claimed in claim 1, useful for the preparation of cleaning compositions.
11. A process for the induction of a novel alkaline protease in a novel strain of Bacillus species MTCC 3606substantially as herein described with reference to the foregoing examples.
|Indian Patent Application Number||417/DEL/2002|
|PG Journal Number||50/2008|
|Date of Filing||28-Mar-2002|
|Name of Patentee||COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH|
|Applicant Address||RAFI MARG, NEW DELHI-110 001,INDIA.|
|PCT International Classification Number||C11D 3/386|
|PCT International Application Number||N/A|
|PCT International Filing date|