Enzymes have attracted attention from researchers all over the world because of the wide range of physiological, analytical and industrial applications. Among these enzymes, proteases execute a large variety of functions and have important biotechnological applications. Proteases represent one of the three largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes . For instance, Fish protein hydrolyzate, Soy protein hydrolyzate, and zein have been produced with high nutritive value and therapeutic effects by the application of microbial protease in food [8, 17, 21].
Protease are widespread in nature, microbes serve as a preferred source of these enzymes. The proteases from microorganisms were widely studied because of their broad biochemical diversity, feasibility of mass culture and ease of genetic manipulation . Bacillus produces a wide variety of extra-cellular enzymes, including proteases. Several Bacillus species involved in protease production are e.g. B. cereus, B. sterothermophilus, B. mojavensis, B. megaterium and B. Subtilis [1, 2, 9, 20, 23, 24].
The octopus valgaris (common octopus) is the most studied commercial octopus of all species. The major consumer counties of octopus are Japan, South Korea, followed by Argentina and China. During the process of octopus digestion, the endogenous digestive enzymes, which are secreted to the lumen of the alimentary canal, originate from the oesophageal, gastric, phloric caeca and intestinal mucosa and from the pancreas . The presence of endogenous digestive enzymes in aquatic organisms has been reported in many studies [4, 5]. Although many protease-producing bacterial have been studied, the discovery of new ones is still significant for both commerce and research. This is especially true for protease-producing bacterial from extreme environments such as the deep sea and Antarctica, because of their novel characteristics.
In this study, intestinal protease producing strains were isolated from octopus itself. Morphological and molecular biological identifications were carried out. The protease produced by the intestinal bacteria was purified and different parameters were detected to obtain the protease properties
Materials and Methods
Octopus vulgaris purchased from seafood market in Qingdao city was bred for 3 days in laboratory before experiment, the culture temperature was under 10℃, and water has to be changed twice a day.
The octopus vulgaris were dissected. The intestinal tracts were separated from the octopus body and rinsed five times with sterile sea water to remove intestinal debris. The tracts were shredded and grinded with sterile water to obtain bacterial suspension.
The bacterial suspension prepared above was diluted and applied on screening medium . The mediums were sealed and put into the thermostat incubator under 25℃ for 3 days. The well growing colony which has the largest proteolytic transparent circle on the casein medium was selected.
Measurement of protease activity
Method used for protease activity measurement was Azocasein method . 1% Azocasein was dissolved in 0.02 mol/l pH 7.0 Phosphate Buffered Saline (PBS) as the substrate, 50 μl of crude enzyme was mixed with azocasein buffer thoroughly, the mixture was incubated at water bath oscillator at a speed of 140 rpm, under 37℃ for 1 hour. The reaction was terminated by adding 300 μl 10% (w/v) trichloroacetic acid (TCA) to the mixture. The mixture was allowed to stand at room temperature for 15 min, and then was centrifuged at 10,000 rpm for 5 min, 100 μl supernatant was mixed with 100 μl of 1 mol/l NaOH. After vortexing, the absorbance (A) was analyzed under 450 nm wavelength to measure enzyme activity.
Identification of strains
Physiological and biochemical identification
The classification and identification were performed based on the morphological, physiological and biochemical characteristics of the bacteria that isolated from octopus gut, referring to “Bergey’s Manual Bacterial Identification” and “System Identification”.
Genomic DNA extraction and 16S rRNA analysis
The identification was conducted by 16S rRNA analysis. Genomic DNA from strain obtained from the tract of octopus was prepared using a Genome Extraction Kit (Bioteke, China). The primers for the PCR reaction were universal bacteria primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’- GGTTACCTTGTTACGACTT- 3’). The amplification was conducted by subjecting the samples to an initial denaturation step of 4 min at 98℃ and then 30 cycles of 35 seconds of denaturation at 95℃, annealing at 55℃ for 1.5 min, and 1.5 min at 72℃ for extension. The final step consisted of 10 min at 72℃ and storage at 4℃. The amplified 16S rRNA was cloned into strain E.coli 110 and sequenced by Sangon Biotech Company (China)
Preparation of crude enzyme solution
Strain QDV-3 was incubated for 3.5 days with initial medium pH 8.0 and culture temperature 30℃. The fermentation broth was centrifuged at a speed of 10,000 rpm for 10 min to remove cells and other insoluble materials, and the supernatant was referred to as crude enzyme solution, which has to be placed in 4℃ before using.
Purification of protease
The organism was grown for 3.5 days as described previously. The cells were separated by centrifugation, and the supernatant was fractionated by precipitation with an ammonium sulfate solution of 80% of saturation. The ammonium sulfate was gradually added into the enzyme solution with slowly stirring until it dissolved completely. All the subsequent steps were carried out at 4℃. The protease was resuspended in 50 mM phosphate buffer, pH 7.0, and dialyzed against the buffer for three days.
The sample after ammonium sulfate precipitation was loaded into the cellulose CM-52 cation exchange column (1.2 cm × 40 cm). Firstly, the enzyme was eluted with a 0.02 mol/l phosphate buffer (pH 7.0), and then was fractionated with a linear gradient of 0 to 2 mol/l sodium chloride in the same buffer at a flow rate of 60 ml/h. A fraction collector was used to collect the purified protease, and the enzyme activity from each receiving tube was measured.
For further purification, the activated resultant protease received from Cellulose CM-52 cation exchange chromatography was loaded onto a DEAE-Sephadex A50 anion exchange column (1.6 cm × 40 cm) which had been equilibrated with a 0.02 mol/l Tris-HCl buffer (pH 8.5), then the unadsorbed materials were washed from the column with the same buffer. The rest was eluted with a linear gradient of 0 to 2 mol/l sodium chloride in the same buffer at a flow rate of 60 ml/h. The protease fractions were collected and scanned for their A280, and assayed for protease activity.
Determination of molecular weight
The molecular weight of purified protease was determined by sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) with casein as standard protein. A 3.75% stacking gel and a 12% separation gel (containing 0.05% of casein substrate) with a thickness of 0.75 mm were prepared. The purified protein samples collected from DEAE-Sephadex A50 anion exchange chromatography were run on a SDS-PAGE. After completion of the electrophoresis, the gel with protein standard bands was cut down for conventional staining and destaining.
Effects of temperature on proteases and thermal stability
The proteases were placed at different temperatures from 10 to 70℃ for one hour, and the activity of proteases was measured. For thermal stability, the purified proteases were put under the condition in which the temperature ranged from 10 to 80℃ respectively for 60 min. The reaction was stopped in ice water and the residual protease activity was measured under standard assay condition mentioned before.
Effects of pH on proteases
The pH effect of the protease was studied from 7.0 to 13.0, and the buffers with different pH were mixed with the purified protease at a ratio of 1:1. The mixture was stored at 4℃ for one hour, and the method mentioned before was utilized as the measurement for enzyme activity value.
Effects of metal ions and surfactants on proteases
The protease was pre-incubated for 30 min with different metal ions including Ca2+, Mg2+, Ba2+, Mn2+, Cu2+, and Zn2+, at an optimized concentration of 2.0 mM and the remaining activity was determined after the incubation period. Phenyl methyl sulfonyl fluoride (PMSF) and ethylene diamine tetra acetic acid (EDTA) were also tested against the enzyme under optimum reaction conditions. Aliquots of the protease were pre-incubated with the different protease inhibitors at 5.0 and 2.5 mM for 30 min at 37℃ and the residual activity of the enzyme was assayed.
Results and Discussion
Screen of protease producing bacteria from octopus vulgaris gut
The ability of enzyme production was demonstrated by the diameter of transparent circle in screening medium produced by protease producing bacteria. In this experiment, there were total 6 strains were detected to produce transparent cycles on casein medium, and the protease activity of each strain was detected. As the results shown in Table 1, the D/d value of QDV-3 and QDE-5 were higher than that of the others. The activity produced by stain QDV-3 was observed to be the highest, followed by QDE-5 and QDE-7. Therefore, QDV-3 was selected for further experiments.
Table 1.Screening results of protease-producing strains from tract of Octopus vulgaris
Identification of strains
The bacterial morphology was observed after 24 hours by microscope QDV-3 strain was rod-shaped, rounded at both ends, with spores. The Colony was white, round, convex, smooth and with neat edges (results not show). The QDV-3 strain was identified as Gram-positive bacteria.
Further characterization was confirmed with its 16S rRNA gene sequence, where the extracted genomic DNA of the strain QDV-3 was tested with 1% and 1.5% agarose gel electrophoresis. An approximately 1500 bp of the gene sequence showed 99.2% similarity with Bacillus flexus (DQ376024). The NJ-based phylogenetic tree showed that the isolated QDV-3 stood alone in unidentified strains of Bacillus sp. clade (Fig. 1). Hence, strain QDV-3 of the present study was identified as Bacillus sp. QDV-3 and the sequence was submitted to GenBank under the accession number JQ836666.
Fig. 1.Phylogenetic tree of strain QDV-3 on 16S rRNA gene sequences. Genomic DNA was prepared using a Genome Extraction Kit (Bioteke, China). The primers for the PCR reaction were universal bacteria primer 27F and 1492R. The amplification was conducted by subjecting the samples to an initial denaturation step of 4 min at 98℃ and then 30 cycles of 35 seconds of denaturation at 95℃, annealing at 55℃ for 1.5 min, and 1.5 min at 72℃ for extension. The final step consisted of 10 min at 72℃ and storage at 4℃. The amplified 16S rRNA was cloned into strain E.coli 110 and sequenced by Sangon Biotech Company (China).
Purification of protease and molecular weight determination
An extracellular alkaline protease was purified from the 3.5 days culture filtrate of Bacillus sp. QDV-3. A three-step purification process was followed for the purification of the protease. The crude enzyme was precipitated with 80% ammonium sulphate with a recovery of 79.4% of activity that amounted to nearly a purification fold of 1.1. The precipitated enzyme was loaded onto a Cellulose CM-52 cation exchange column. The chromatogram showed two activity peaks named FI and FII were detected (Fig. 2), which gave 36.5% recovery of the enzyme activity with nearly 2.3 fold purification. In the final step, the active fraction FI collected from the gel filtration were loaded to the DEAE-Sephadex A50 anion exchange column and eluted for the active fractions. Such active fractions were then concentrated and analyzed, and then four active fractions were obtained as shown in Fig. 3, which were named as FI-1, FI-2, FI-3, and FI-4. The enzyme was purified up to 2.5 fold with a final recovery of 12.5% whose specific activity was found to be 9075.5 U/mg proteins
Fig. 2.Ion-exchange chromatogram on Cellulose CM-52 of protease. The sample was loaded into the cellulose CM-52 cation exchange column (1.2 cm × 40 cm). Firstly, the enzyme was eluted with a 0.02 mol/l phosphate buffer (pH 7.0), and was fractionated with a linear gradient of 0 to 2 mol/l NaCl in the same buffer at a flow rate of 60 ml/h. A fraction collector was used to collect the purified protease. Protease activity was measured by Azocasein method. 1% Azocasein was dissolved in 0.02 mol/l pH 7.0 PBS , 50 μl of crude enzyme was mixed with azocasein buffer and was incubated at a speed of 140 rpm, under 37℃ for 1 hour. After adding 300 μl 10% (w/v) TCA, it stood at room temperature for 15 min, and centrifuged at 10,000 rpm for 5 min, 100 μl supernatant was mixed with 100 μl of 1 mol/l NaOH., The absorbance was analyzed under 450 nm wavelength to measure enzyme activity.
Fig. 3.Ion-exchange chromatogram on DEAE-Sephadex A 50 of protease SDS-PAGE electrophoresis strain QDV-3. The activated resultant protease received from Cellulose CM-52 cation exchange chromatography was loaded onto a DEAE-Sephadex A50 anion exchange column (1.6 cm × 40 cm) which had been equilibrated with a 0.02 mol/l Tris-HCl buffer (pH 8.5), then the unadsorbed materials were washed from the column with the same buffer. The rest was eluted with a linear gradient of 0 to 2 mol/l sodium chloride in the same buffer at a flow rate of 60 ml/h. The protease fractions were collected and scanned for their A280, and assayed for protease activity.
The purified protease FI was shown to have three clear bands by SDS-PAGE electrophoresis with apparent molecular masses which were estimated to be approximatedly 96.6 kDa, 75.8 kDa, and 61.6 kDa (Fig. 4). The protease FI was further purified through DEAE-Sephadex A50 anion exchange column, and FI-2 was obtained as one of the activity fractions with a single clear band in SDS-PAGE. The molecular weight of FI-2 was determined to be 61.6 kDa, and it was named as QDV-E. In general, the molecular weights of previously found protease are rarely more than 60 kDa [6, 13, 24]. However, molecular mass of protease QDV-E is about 61.6 kDa, indicating that the protease is novel protease. The overall results of the purification procedures are summarized in Table 2.
Fig. 4.SDS-PAGE of FI ＆ FI-2 fraction by strain QDV-3. The molecular weight of purified protease was determined by SDS-PAGE with casein as standard protein. A 3.75% stacking gel and a 12% separation gel with a thickness of 0.75 mm were prepared. The purified protein samples collected from DEAE-Sephadex A50 anion exchange chromatography were run on a SDS-PAGE. After completion of the electrophoresis, the gel with protein standard bands was cut down for conventional staining and destaining.
Table 2.Summary of purification of protease
Effect of pH on proteases
The QDV-E protease was active in the pH range of 7.0-13.0, with optimum activity at pH 9.0 (Fig. 5), suggesting that it belongs to alkaline protease. At the range of pH 9.0-9.5, the enzyme showed more than 96% activity, whereas at pH 7.0 and 13.0, the protease activities were decreased by nearly 53.3% and 64.2%, respectively. Currently, most of the alkaline proteases’ optimum pH range from 8.5 to 10.0, only a few is more than 11.0, even up to 12.0. Shikha et al. have reported a strain of Bacillus pantotheneticus could produce protease with an optimum pH at 8.5 . Kumar et al. have isolated Bacillus sp. NCDC180 from soil which could secrete two kinds of proteases named as AP1 and AP2. The optimum pH values for both proteases were 11.0 and 12.0, respectively . The protease of B. licheniformis NH1 has also been reported to have similar properties . The alkaline proteases which have high commercial value are those that have pH optima in the range of 9.0-12.0.
Fig. 5.Effects of different pH values on protease activity of QDV-E by strain QDV-3. The pH effect of the protease was performed from 7.0 to 13.0, and the buffers were mixed with the purified protease at a ratio of 1:1. The mixture was stored at 4℃ for one hour, and azocasein method used for protease activity.
Effect of temperature on proteases
The effect of temperature on QDV-E was shown in Fig. 6. The optimum temperature for protease QDV-E to have the highest activity was 40℃, which indicated the proteases belong to mesophilic enzyme. Generally, microbial proteases have a broader optimal temperature range from 30 to 75℃ depend on different genera of bacteria. Uttam et al have isolated a protease producing short Bacillus subtilis from water sample, the optimal temperature of the protease was 37℃ . Another researcher Jung also has reported a Pseudomonas sp. KFCC 10818 strain which could secrete protease whose optimum temperature was 70℃ .
Fig. 6.Effects of different temperatures on protease activity of QDV-E by strain QDV-3. The proteases were placed at different temperatures from 10 to 70℃ for one hour, and the activity of proteases was measured by azocasein method.
The thermal stability of proteases
To examine the thermal stability of the QDV-E protease, the enzyme solution was allowed to stand for 60 min at various temperatures, and the residual activity was measured. The QDV-E protease showed good stability at 50℃ (Fig. 7) with 82% retained activity. However, as the temperature was increased to 60℃, the retained activity was dropped to 40%, and was rapidly inactivated at higher temperature. The protease activity was completely inactivated at 70℃. The temperature of best thermal stability was same with the protease isolated from Exiguobacterium sp. SKPB5 . However it was found to be higher than that of earlier reported protease from Azospirillum sp.  and Shewanella strain Ac10 .
Fig. 7.The thermal stability on protease activity of QDV-E by strain QDV-3. The purified proteases were put under the condition in which the temperature ranged from 10 to 80℃ respectively for 60 min. The reaction was stopped in ice water and the residual protease activity was measured by azocasein method
Protease is desirable to be stable in the presence of commercial detergents. The suitability of any protease in detergent formulation is dependent on its stability and compatibility with the detergent components. Besides, the enzyme should be alkaline and thermostable in nature. However, the stability and compatibility of the enzyme with the components alone should not be considered as the only pre-requisite for its inclusion in detergent formulation. Therefore, more experiments about active state of protease at room temperature are necessary to be taken place in the future.
Table 3.Effects of inhibitions and metal ions on QDV-E
Effects of metal ions and inhibitors on proteases
The effects of various metal ions, at a concentration of 2.0 mM, on the activity of QDV-E were analyzed (Table 4). It was observed that activity of the enzyme was enhanced by nearly 15% and 90% in the presence of Mg2+ and Mn2+ ions. On the other hand, the heavy metals like Ba2+, Zn2+, and Cu2+ had inactivated the enzyme. Among the different protease inhibitors, EDTA did not completely inhibit the enzyme. In contrast, the protease was strongly inhibited by the PMSF, thus confirming the finding that the protease QDV-E of Bacillus sp. QDV-3 was that of serine protease.
The effect of metal ions and various compounds on protease isolated from Azospirillum sp. and Bacillus sp. APR-4 have been reported. Azospirillum sp. was stabilized by Mg2+ , and that from Bacillus sp. APR-4 was stabilized by Ca2+ . In this study, a stimulatory effect on the activity was shown by Mg2+ and Mn2+ ions. Between two of them, Mn2+ showed the maximum increase in activity. Because EDTA had no much effect on the activity, the enzyme could not be classified as a metalloprotease. This also suggests that metal ions were not the essential requirement for the activity, but at their addition, the enzyme showed a stimulatory effect. An 89% increase in activity was observed when the enzyme was incubated in presence of Mn2+.
Traditionally, microbial proteases have been applied in many areas in the food industry. Alkaline protease is used in the production of hydrolyzed protein, which has high nutritional values on the blood regulation and infant milk formula. After that, treatment food products, fortified juices, and soft drinks have been developed with the alkaline proteases. For instance, Fish protein hydrolyzate, Soy protein hydrolyzate, and zein have been produced with high nutritive value and therapeutic effects by the application of alkaline microbial protease in food.
Currently, the use of microorganisms or proteolytic enzymes for deproteinization of marine produce wastes is a modern trend in conversion of waste into useful biomass. It is a simple and inexpensive alternative to chemical methods employed in the preparation of various products such as: peptide and chitin. Selection of organism for the commercial production of protease relies not only on its ability to produce the enzyme with desired characteristics, but also on the cost-effective methods of production of the enzyme. The results in the present study indicated that the protease production pattern varied with the type of marine-residues. This could be attributed to the marine products, which play a dual role; that is, supply of nutrients to the microbial culture and anchorage for the growing cells.