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Article

Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production

by
Zixuan He
1,2,†,
Yang Sun
3,†,
Min Chu
1,
Jing Zhu
1,
Yu Zhang
4,
Qiyong Tang
1,
Ghenijan Osman
1,
Ling Jiang
3,* and
Zhidong Zhang
1,2,3,*
1
Xinjiang Key Laboratory of Special Environmental Microbiology, Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China
2
College of Life Sciences and Technology, Xinjiang University, Urumqi 830046, China
3
College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China
4
Plant Protection Station of Xinjiang Uygur Autonomous Region, Urumqi 830023, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2023, 9(5), 410; https://doi.org/10.3390/fermentation9050410
Submission received: 15 March 2023 / Revised: 16 April 2023 / Accepted: 20 April 2023 / Published: 26 April 2023
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
Fibrinolytic enzymes are a kind of proteolytic enzymes that can hydrolyze fibrin and dissolve blood clots. They could be used as a therapeutic agent for treating thrombosis. It is important for the treatment of cardiovascular disease to find and develop new thrombolytic drugs. In order to explore new fibrinolytic enzymes, a strain named 214L-11 with protease and fibrinolytic enzyme activity, which was isolated from the Flaming Mountain of Xinjiang Province, was screened using the skimmed milk plate, the blood powder agarose plate and the fibrin plate methods. Phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), which indicated that it represented a potential novel species in the Streptomyces genus. The fibrinolytic enzyme produced by 214L-11 displayed thrombolytic and anticoagulant activities, and it could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure. The fermentation medium optimized through response surface methodology was 15 g/L soluble starch, g/L KNO3 0.58, 0.43 g/L peptone, 0.01 g/L FeSO4·7H2O, 0.5 g/L MgSO4·7H2O, 0.2 g/L Mn2+, 0.5 g/L NaCl and 1 L distilled water, pH 8, and the maximum amount of fibrinolytic enzyme produced by strain 214L-11 in the optimal fermentation medium was 1255.3 FU/mL. Overall, the fibrinolytic enzyme-producing strain was screened from the Flaming Mountain of Xinjiang for the first time, which provided a basis for further research and the development of new efficient and safe hemolytic drugs.

1. Introduction

Fibrinolytic enzymes are a kind of protein hydrolase that break down fibrin and dissolve blood clots and are an active ingredient in the treatment of thrombotic diseases [1]. Thrombosis is strongly associated with most cardiovascular diseases such as myocardial infarction, ischemic heart diseases, deep vein thrombosis and venous thromboembolism. According to the World Health Organization (WHO), 17.9 million people died from cardiovascular diseases (CVDs) in 2016, which represents 31% of world deaths [2]. With changes in people’s living environment and habits, the age at which people experience CVDs is becoming lower. Thrombolytic agents such as tissue-type plasminogen activator (t-PA), a urokinase-type plasminogen activator (u-PA), and streptokinase (bacterial plasminogen activator) have been developed and widely used for the treatment of CVDs. In addition, nattokinase is also commonly used for primary prevention of thrombotic events [3,4]. However, there are a lot of imperfections, such as a short half-life, low specificity to fibrin, gastrointestinal bleeding and other shortcomings [5,6]. Hence, over the past few decades, exploring novel thrombotic drugs and fibrinolytic enzymes has become a hotspot for researchers.
A promising application of microbial-derived thrombolytic and fibrinolytic enzymes is the prevention and treatment of vascular occlusion, due to their advantageous cost–benefit ratio and large-scale production. A lot of strains with fibrinolytic enzyme activity have been obtained, such as Bacillus [7], Lactobacillus [8], Aspergillus versicolor [9] and Streptomyces [10], etc. In recent years, microbes from abnormal environments have been one of the most important sources for the screening of fibrinolytic enzyme-producing strains. In particular, the actinomycetes were the most important sources of fibrinolytic enzyme-producing strains. A novel, safe and non-toxic fibrinolytic enzyme produced by marine Streptomyces sp. P3 isolated from marine soil was obtained, and enzyme characterization analyses suggested that it was a new option for the development of thrombolytic drugs [11]. The fibrinolytic enzyme of Streptomyces sp. SD5 from high-temperature hot springs could effectively degrade fibrin in a short time, showing strong thrombolytic ability [12]. Dhamodharan et al. [13] characterized and purified the fibrinolytic enzyme produced by Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifer, and it is similar to that of the Streptomyces radiopugnans that first isolated from the Gobi radiation zone and named by our group [14]. Unquestionably, the abnormal environment is a living environment that presents exciting opportunities to future microbiology and biotechnology research.
Response surface methodology is used to determine the optimal process parameters through the analysis of response surfaces and contours, and a polynomial model is developed to fit the functional relationship between the factors and the response values in a certain region [15]. Among them, the Plackett–Burman design method is mainly based on two levels in order to quickly and efficiently screen out the main influencing factors from multiple factors and utilizes the least number of trials, so it is often used to evaluate the main effect of factors.
Therefore, in the current study, the aim is to obtain a safe, high-specificity, and effective fibrinolytic enzyme from extreme environment strains. In this study, the molecular identification of the isolated strain 214L-11 was carried out, the taxonomic status of the strain was initially clarified, the preliminary purification of the fibrinolytic enzyme from its fermentation broth was performed and studies on its thrombolytic and anticoagulant activities were carried out. Finally, the optimum conditions for enzyme production in fermentation were analyzed using response surface analysis, the feasibility of the fermentation conditions were optimized using response surface analysis and the relationship between enzyme production and the process of mycelial growth and glucose consumption was demonstrated through fermentation kinetics experiments. Without a doubt, the abnormal environment is a living environment with great potential for future microbiology and biotechnology study.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

All strains, named 214L-11, G163 and G85, were isolated from the Flaming Mountain of Xinjiang, as described in a previous study [16]. Briefly, in July 2021, samples were collected from the Flaming Mountain, Turpan, Xinjiang Uyghur Autonomous Region. As the Flaming Mountain is surrounded by the famous scenic spots of Turpan, sample sites in an unfrequented area were chosen to avoid human influence. Five sites in a 3 km radius around the Flaming Mountain were chosen. Three samples (1 kg soil for each sample) were collected randomly within a 5 × 5 area at each site, and each sample was collected at a depth of 10–15 cm from the surface. The soil sample (2 kg) was mixed well at each site after removing impurities, obtained using the quartering method [17] and stored at 4 °C within 24 h. Subsequently, actinomycetes were isolated and purified from the soil samples, and strains 214L-11, G163 and G85 were isolated from samples collected in the area. Strains 214L-11, G163 and G85 were conserved in the Microbiological Culture Collection Center of Xinjiang (MCCCX). Among them, Streptomyces sp. 214L-11 has been deposited in the China Center for Type Culture Collection (CCTCC) under collection number CCTCC M 20221543. Strains were inoculated on GAUZE’s agar plates (soluble starch 20 g, KNO3 1 g, FeSO4·7H2O 0.01 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, H2O 1 L, agar 15 g, pH 7.4) at 37 °C for 3–5 days.

2.2. Morphological and Molecular Identification of Strains

The cell morphology of strains was observed under a light microscope using the morphological observation method [18]. 16S rRNA gene sequencing and the determination of a reasonably sized sequence was performed [19]. DNA was extracted using the TIANamp Bacteria DNA Kit (Tiangen Biochemical, Beijing, China), following the manufacturer’s instructions. DNA was eluted in 100 µL of elution buffer. The 16S rRNA gene was PCR-amplified using the universal primers 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R 5′-GGTTACCTTGTTACGACTT-3′. Amplification reactions were performed in a final volume of 30 µL. The amplification reaction conditions were as follows: predenaturation at 94 °C for 5 min, then 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 45 s, and, finally, extension at 72 °C for 20 min. The PCR-amplified products were sent to Sangon Biotech (Sangon Biotech, Shanghai, China).
The 16S rRNA gene sequences obtained were assembled using the SeqMan program (DNAStar software) and compared with the corresponding sequences of validly described species in the EZbioCloud 16S database (https://www.ezbiocloud.net) (accessed on 14 March 2023) and NCBI (https://www.blast.ncbi.nlm.nih.gov) (accessed on 14 March 2023). To determine the phylogenetic relationships of strain 214L-11, multiple alignments of their sequences with related type strains were performed using the Clustal X software package [20]. Phylogenetic and molecular evolutionary analyses were performed using the neighbor-joining model with the software package MEGA version 7.0 [21].

2.3. Preparation of the Crude Enzyme Solution and Assay of Enzyme Activity

The strains 214L-11, G163 and G85 were inoculated into 50 mL of GAUZE’s liquid medium in a 250 mL conical flask and incubated in a shaker for 5 days at 37 °C and 150 rpm. After fermentation, the crude enzyme solution was prepared at 4 °C through filtration of the culture supernatant using sterile filter paper and a 0.22 μm sterile filter.
To screen the best strain from strains 214L-11, G163 and G85 for the subsequent research, an assay of enzyme activity using the agar diffusion assay [22] was performed. Protease activity was determined through the skimmed milk plate method (skimmed milk agar: 20 g/L skimmed milk powder and 20 g/L agar). Blood powder agarose agar plates (to prepare the plates, fresh pig blood from a market was clumped, pound, dried, ground and sieved, then 1.0 g of the powder was added to 100 mL agarose solution (1.2%) heated in advance and bathed in water at 60 °C) were used to initially screen for fibrinolytic enzyme. Fibrinolytic enzyme was determined through the blood fibrin agarose plate method with slight modifications. Fibrin plates were prepared by mixing 6.67 mg/mL of fibrinogen in 3 mL saline, 3 mL of thrombin (20 U/mL) and 10 mL of agarose (1% (w/v). The mixture was poured into Petri dishes and kept at room temperature for 1 h for clot formation. Then, 50 µL enzyme samples were carefully placed into a well (5 mm diameter) made on the plates, and plates were incubated at 37 °C for 18 h. The size of the hydrolysis circle area produced on the plate was observed, and, finally, the hydrolysis circle area was substituted into the standard curve of nattokinase to calculate the enzyme activity.

2.4. Determination of Fibrinolytic and Protease Activities

The protease activity in the crude enzyme solution produced by strain 214L-11 was determined with reference to the Folin method for the determination of protease activity provided in the national standard SBT10317-1999 [23]. Fibrinolytic enzyme activity was estimated according to the method of Astrup and Mullertz [24] with slight modifications. A calibrated standard curve was established using nattokinase as standard. Then, 50 µL enzyme samples were carefully placed into a well (5 mm diameter) made in the gel. For standard curve calibration, nattokinase (5000 FU) was diluted with normal saline in the range 200–1000 (200, 400, 600, 800 and 1000) FU/mL.

2.5. In Vitro Anticoagulant and Thrombolytic Activity

Li’s method was used with slight modifications [4]. First, 200 µL of saline and 200 µL of crude enzyme solution produced by strain 214L-11 were mixed with 1 mL of fresh rabbit blood in a sterile tube and incubated at 37 °C. The tube was tilted every 5 s to observe whether the blood moved along the tube and stopped at the time of coagulation, which was the external coagulation time.
Fresh rabbit blood was added to ampoule tubes with a 6 mm inner diameter to a height of 1 cm, and kept upright until it clotted naturally. Then, 3 mL of sterile crude enzyme solution was added to the tubes and they were incubated at 37 °C, observed at 2 h intervals, shaken and tilted. At 8 h, the liquid in the tube was fully mobile without visible clots. Inactivated crude enzyme solution was used as negative control and saline as blank control.

2.6. Thrombolysis and Casein Degradation through SDS-PAGE

The proteins in the fermentation broth of strain 214L-11 were denatured and precipitated using the ethanol precipitation method [24], and the volume ratio of crude enzyme solution to anhydrous ethanol precipitate was 1:4. The fermentation broth was allowed to stand for 6 h at 4 °C and centrifuged at 8000 rpm for 20 min, then the supernatant was discarded and the protein precipitate was dried. Finally, the protein precipitate was dissolved in 1 mL sterile water. Fresh rabbit blood (0.5 mL) was taken and allowed to clot, and then 1 mL of the crude enzyme solution was added and treated in a constant-temperature water bath at 37 °C for 4 h until the clot dissolved, then it was stored at 4 °C for SDS-PAGE analysis. Then, 1 mL of crude enzyme solution and 0.5 mL casein solution (0.5%) prepared using 0.02 M pH 7.2 phosphate buffer were mixed in a tube and treated for 4 h at 37 °C in a constant-temperature water bath, then stored at 4 °C for SDS-PAGE analysis. Inactivated crude enzyme solution was used as negative control.
Under denaturing and reducing conditions, the molecular weight of the purified enzyme was determined through SDS-PAGE, using a 12% gel (Solarbio Science & Technology, Beijing, China) as described by the instruction manual. Gel was stained with CBB R-250 (3 h), decolorized with a decolorizing solution (ultrapure water: 50%, 95% glacial acetic acid: 45%, ethanol: 5%) for 3 h and then decolorized overnight with 7% (v/v) glacial acetic acid for gel imaging.

2.7. Single-Factor Condition Optimization of Fibrinolytic Enzyme Production

A single-factor test was used to investigate the effects of carbon and nitrogen source, pH (4.0–10.0), temperature (25–45 °C), carbon/nitrogen ratio (5:1, 10:1, 15:1, 20:1, 25:1), metal ions (Mn2+, Fe2+, Fe3+, K+, Cu2+), liquid volume (10%, 20%, 30%, 40%, 50%) and inoculum size (2%, 4%, 6%, 8%, 10%, 12%) on the fermentation yield with GAUZE’s synthetic medium No.1 as the basal medium. The medium was rationed according to 20 g/L carbon source and 1 g/L nitrogen source. Six carbon sources, glucose, cottonseed sugar, sucrose, soluble starch, dextrin and corn starch, and six nitrogen sources, peptone, yeast powder, potassium nitrate, beef paste, ammonium sulfate and ammonium chloride, were examined, along with carbon/nitrogen ratio, metal ions and pH, using the initial medium as a control. Liquid volume, inoculum size and temperature were investigated, using the original fermentation conditions (Inoculum size 2%, 250 mL conical flask with 100 mL medium, temperature 37 °C, pH 7.4) as the control.

2.8. Experimental Design

Based on the above single-factor test, the seven components (soluble starch, KNO3, peptone, inoculum size, liquid volume, metal ions and initial pH) of the fermentation medium were analyzed using Design expert 10.0 software. The experiments were conducted with fibrinolytic enzyme activity as the response value, and the two levels were taken as nearly ±25% of the original level, respectively, as shown in Table 1. Based on the results of the Plackett–Burman experiment, a 3-factor, 3-level interaction was performed for potassium nitrate, peptone and metal ions with fibrinolytic enzyme activity as the response value. Each group was tested 3 times in parallel, and the factor levels are shown in Table 2.

2.9. Response Surface Model Validation

The optimum fermentation conditions for the fibrinolytic enzyme production of the strain selected using the response surface method were tested to verify the effectiveness of the model. Strain 214L-11 was grown at 35 °C in a rotary shaker at 150 rpm in 30 mL fermentation medium (15 g/L soluble starch, 0.58 g/L KNO3, 0.43 g/L peptone, 0.01 g/L FeSO4· 7H2O, 0.5 g/L MgSO4· 7H2O, 0.2 g/L Mn2+, 0.5 g/L NaCl, 1 L distilled water, pH 8) for 120 h. The production of fibrinolytic enzyme was determined at different fermentation time intervals (0, 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112 and 120 h) in triplicate.

2.10. Determination of Fermentation Process Curve

The optimal medium formula was obtained using the response surface methodology, and the fermentation process curve of the strain was determined to further verify the optimization result. Fibrinolytic activity in the fermentation broth was determined using the fibrin plate method [25]. The mycelium dry weight of strain 214L-11 was determined, and its growth curve was drawn under the condition of 250 mL conical flask with 50 mL medium and an inoculum size of 2% [26].
Chen et al.’s method was used with slight modifications [27]. Briefly, the residual sugar content in the fermentation broth was determined quantitatively, and the standard glucose solution of 20 mg/100 mL was prepared. A certain amount of glucose standard solution (0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL) was added to 2 mL distilled water, and 1 mL phenol solution (5%) and 5 mL concentrated sulfuric acid were added, respectively. They were shaken well and left to stand for 30 min, after which 2 mL distilled water was taken as blank control, absorbance at 490 nm was determined and the standard curve was drawn. Then, the glucose content in the fermentation broth of strain 214L-11 was determined using the same method.
The fibrinolytic activity, dry weight of mycelium and residual sugar content in the fermentation broth of strain 214L-11 were measured every 8 h.

2.11. Statistical Analysis

Experiments were carried out at least twice, with a minimum of three biological replicates. Error bars represent S.D. from three independent experiments and Microsoft Excel 2010 was used to perform weighted regression, Student’s t-tests, and one-way ANOVA with Tukey’s posttest. Response surface methodology data were collated and statistically analyzed using Design Expert 10.0.

3. Results

3.1. Screening of Fibrinolytic Enzyme-Producing Actinomycetes

Strains 214L-11, G163 and G85 belong to the actinomycetes that were isolated from the Flaming Mountain, and they displayed transparent rings with protease activity on the skimmed milk plate (Figure 1A). The primary and secondary screening of fibrinolytic enzyme-producing strains was performed on blood powder agarose plates and blood fibrin agarose plates, respectively. Transparent rings were observed when strains 214L-11, G163 and G85 were grown on blood powder agar plates (Figure 1B). The transparent rings produced by strain 214L-11 on the fibrin agar plates was better than those of G163 and G85 (Figure 1C). In a word, strain 214L-11 was found to have significant fibrinolytic enzyme activity in this study, and not only had strong protease activity, but could also hydrolyze blood clots and blood fibrin, exhibiting better fibrinolytic enzyme activity. Therefore, we chose strain 214L-11 for the subsequent research.

3.2. Morphological and Phylogenetic Analysis of the Strain

Strain 214L-11 grew on GAUZE’s medium with abundant aerial mycelium and white colonies (3 days), and dry, filamentous blue-green colonies were formed after 5 days. Spore filaments were formed through the differentiation of aerial mycelium in chains or spirals (Figure 2). Phylogenetic analysis showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), and 98.81% similarity with Streptomyces pseudogriseolus NRRL B-3288T and Streptomyces werraensis NBRC 13404T, 98.66% similarity with Streptomyces viridiviolaceus NBRC 13359T and 98.58% similarity with Streptomyces fimbriatus NBRC 15411T. Although it has a high degree of similarity to Streptomyces fumanus NBRC 13042T, phylogenetic analysis (Figure S1) shows that it forms a different branch from the most homologous strain of Streptomyces fumanus NBRC 13042T, and taxonomic status cannot be determined at this time. The 16S rRNA gene sequence of the strain is registered as OP615214.

3.3. In Vitro Fibrinolytic Effect on Anticoagulant Activity and Blood Clots

The anticoagulant activity of the enzyme was measured by incubating the enzyme with fresh blood. In the blank control sample, blood was coagulated in 15 min (Figure 3A), but no coagulation was observed in the sample with enzyme until 24 h. The blood sample with the enzyme appeared as a free-flowing solution without coagulation (Figure 3B). The result indicates that the enzyme can serve as an anticoagulant.
The blood clots were dissolved by the enzyme, and the dissolution substantially increased with time. The blood clots were almost completely dissolved after 8 h. However, in the samples of inactivated crude enzyme solution and saline, only a few erythrocytes ruptured due to prolonged immersion, and the thrombus remained unchanged (Figure 3C). The thrombolytic effect of the enzyme in vitro is good, with the potential for development of thrombolytic drugs.

3.4. Fibrinolytic and Casein Degradation Activity through SDS-PAGE Analysis

The fibrinolytic and protein activities of the crude enzyme solution were determined according to item 2.6 (Figure 4A). The fibrinolytic activity of the crude enzyme solution of strain 214L-11 was 939.9 FU/mL, while the protease activity was 137.5 U/mL, indicating that the enzyme had good fibrinolytic activity and protease activity. The effects of enzyme on blood clots and caseinase were analyzed using SDS-PAGE. The enzyme significantly degraded a single protein of about 85 KDa in the blood clots (Figure 4B), and no significant effects on human immunoglobulin G (IgG) (15–19 KDa), human serum albumin (HSA) (68 KDa) or other proteins that are important in the blood and no clear protein band were observed after the crude enzyme solution was concentrated 20-fold. The result indicated that the fibrinolytic enzyme is more specific as it degraded one specific protein in the blood clots, caused fibrin degradation and dissolved blood clots. The enzymatic digestion of casein showed that the enzyme has good protease activity and it can efficiently degrade the major protein molecules in casein to fewer than 10 KDa molecular peptide breaks, resulting in no visible bands in SDS-PAGE.

3.5. Optimization of Fermentation Conditions

Microorganisms obtain energy from carbon and nitrogen sources, and the ideal carbon and nitrogen sources required by various bacteria vary. As shown in Figure 5A, there were significant differences in the effects of six carbon sources on fibrinolytic activity, and the highest fibrinolytic activity was found when soluble starch was used as the carbon source. Soluble starch is cheap and easy to obtain, so it could be used as a fermentation material to save on costs. From Figure 5B, the fibrinolytic activity of strain 214L-11 was the highest when peptone was used as the carbon source, while the addition of NH4Cl, (NH4)2SO4 and yeast powder was not conducive to the production of fibrinolytic activity. When peptone and KNO3 were used as nitrogen sources, no significant differences were found in their relative enzyme activities. Therefore, soluble starch was chosen as the fermentation medium’s carbon source, while peptone and potassium nitrate were chosen as the fermentation medium’s combined nitrogen source. Based on the above studies, this study also investigated the effects of different carbon/nitrogen ratios on the fibrinolytic enzyme activity of strain 214L-11. As shown in Figure 5C, the highest fibrinolytic enzyme activity of strain 214L-11 was observed at a carbon/nitrogen ratio of 15:1. Therefore, a carbon/nitrogen ratio of 15:1 was selected for the preparation of the fermentation medium.
Subsequently, the temperature (25–45 °C), inoculum size (2–12%), liquid volume (10–50%), metal ions and pH (6–10) were optimized using GAUZE’s medium as the basic medium. As shown in Figure 6A, the enzyme activity increased with increasing temperature (25–35 °C), with the best enzyme production at 35 °C, which was significantly higher than the enzyme production in fermentations at temperatures other than 40 °C. There was no significant difference in enzyme production between the fermentation temperature of 35 °C and 40 °C, a temperature range close to the human physiological environment. Subsequently, as the temperature increased, the enzyme activity decreased. As shown in Figure 6B, there was no significant difference in enzyme production when the inoculum size was 2–6%, but the best enzyme production was achieved when the inoculum size was 2% (Figure 6B). However, when the inoculum size exceeded 6%, the enzyme activity of the fibrinolytic enzyme started to decrease. It was initially concluded that the strain was aerobic and when the inoculum size was too large, it would cause insufficient dissolved oxygen and affect the synthesis of the product. From Figure 6C, it can be seen that there was no significant difference in enzyme production when the liquid volume was 20% or 30%, but the best enzyme production was achieved when the liquid volume was 20%. The decrease in enzyme activity with increasing liquid volume from 30% is further evidence of the aerobic nature of strain 214L-11. Metal ions are necessary cofactors for many catalytic reactions, and as shown in Figure 6D, the best enzyme production was achieved when Mn2+ was added to the medium. Elevated enzyme activity was also observed for the characterization of fibrinolytic enzymes derived from E. coli when a final concentration of 1 mm Mn2+ was added [28]. A pH range of 6.0–10.0 was chosen for the single-factor optimization experiments. The effect of different pH on the fibrinolytic enzyme activity of strain 214L-11 was tested. As shown in Figure 6E, there was no significant difference in enzyme production when the pH was 8.0 versus 9.0, but the best enzyme production was observed at pH 8.0. The relative enzyme activity of the fibrinolytic enzymes produced by this strain, 214L-11, was significantly higher under alkaline conditions than under acidic conditions, and the fibrinolytic enzymes produced by this strain were likely alkaline proteases.
The 11 factors (N = 12, 7 actual and 4 virtual factors) in the fermentation process were chosen to be analyzed in the Plackett–Burman experiment with fibrinolytic enzyme activity (Y FU/mL) as the response value. The design table and the results of the test are shown in Table S1, and the effects of each factor are shown in Table 3. The effects of KNO3, peptone and metal ions on fibrinolytic enzyme activity were 25.40%, 19.76% and 15.13%, respectively, and the effects of KNO3 and peptone on fibrinolytic enzyme activity were positively correlated in that fibrinolytic enzyme activity increased with their contents; metal ions were negatively correlated. Therefore, these three main factors were selected for the next experiment (Table 3).
Subsequently, KNO3, peptone and metal ions were analyzed through the Box–Behnken test design with 3 center points (15 tests). The design table and results of the test with fibrinolytic activity as the response value are shown in Table S2, and the ANOVA is shown in Table S3. The ANOVA results showed that the order of factors affecting fibrinolytic enzymes was peptone > KNO3 > metal ions. The p < 0.05 and the coefficient of determination R2 = 0.9524 were significant; meanwhile, the misfit term p = 0.8112 > 0.05 and R2Adj = 0.8667 indicated that the model fit was good and could reflect the change in response values, which could be used for the analysis and prediction of the fermentation process. The final equation in terms of coded factors is as follows:
Y = 1178.5 + 54.8 × A + 66.71 × B−7.79 × C − 33.27 × A× B + 31.32 × A × C + 56.15 × B × C − 69.53 × A2 − 165.25 × B2 − 108.8 × C2
The interaction between the factors is shown in Figure 7. The optimal levels of KNO3 and peptone were 0.58 g/L and 0.43 g/L, respectively, at a metal ion level of zero. Additionally, the optimal level of metal ions was 0.20 g/L at peptone and KNO3 levels of zero, respectively. The fibrinolytic activity was inversely proportional to metal ions within a certain range. Additionally, the interaction showed that the highest fibrinolytic enzyme activity was 1249.5 Fu/mL at 0.58 g/L of KNO3, 0.43 g/L of peptone and 0.2 g/L of metal ions.

3.6. Validation of the Models

To further confirm the influence of optimized medium on the production of the fibrinolytic enzyme, strain 214L-11 was subjected to fermentation for the production of fibrinolytic enzyme in the fermentation medium. It is apparent from the process curve of fermentation that 0~40 h is the lag phase of the strain, 40~88 h is the logarithmic phase and thereafter is the stationary phase (Figure 8). Meanwhile, from the relationship between the growth of the bacterium and the production of the fibrinolytic enzyme, in the main growth phase of the bacterium (logarithmic phase), glucose was consumed a lot, the volume of the bacterium increased rapidly and the fibrinolytic enzyme activity also increased; the highest fibrinolytic activity (1249.5 FU/mL) was reached at about 96 h, and the enzyme activity decreased with the increase in time after 96 h.
The enzyme yield of the fermentation supernatant of strain 214L-11 before optimization was 939.9 FU/mL, and the three factors with greater influence, potassium nitrate, peptone and metal ions, were finally obtained using the Plackett–Burman method. The optimized fibrinolytic enzyme yield was 1249.5 FU/mL, which was improved by about 32.9%. The optimized medium showed that the optimal nitrogen source was 0.58 g/L for potassium nitrate, 0.43 g/L for peptone and 0.2 g/L for metal ions. Fermentation kinetics experiments showed that the fermentation yield of the fibrinolytic enzyme using the optimal conditions was 1192.1 FU/mL, 1239.6 FU/mL and 1255.3 FU/mL, respectively, with a good fit. During the fermentation process, soluble starch was continuously decomposed and utilized by the strain, and the final residual sugar amount was 3.23 g/L at 120 h. The mycelial weight and fibrinolytic enzyme activity of the strain increased rapidly after 40 h into the logarithmic growth phase, which was similar to those of Streptomyces sp. MY0504 [29].

4. Discussion

It necessary to search for abundant microbial resources from unusual habitats. Recent studies revealed that abundant and diverse functional enzymes and natural metabolites were produced by microbes isolated from extreme habitats (marine environments, hot springs, deserts) [30,31,32], providing an important source for industrial enzyme development and novel drug discovery. It is hard to survive in hyper-arid areas, which were once considered to be “exclusion zones”. In recent years, it has been reported that there is an abundance of microbial diversity and functional enzymes in arid and hyper-arid environments [33,34,35].
The Turpan Flaming Mountain of Xinjiang has a unique geographical location and basin topography. The region is hot and dry, with a substantial temperature gap between day and night and serious soil desertification. The highest temperature in summer is as high as 47.6 °C, the highest surface temperature is as high as 89 °C and the MAR/MAE is only 0.005 [36], which make it the hottest and most hyper-arid place in China. The Flaming Mountain area contains abundant protease-producing bacteria [16]. Our previous research found that the enzyme activity of XJT9503 isolated from the Turpan Flaming Mountain reached 13,240 U/mL after pilot fermentation and the enzyme had optimal activity at 65 °C and pH 7.2 [37]. However, the protease activity was not as high as 13,240 U/mL when the strain was first discovered. Through strain mutagenesis and continuous fermentation optimization, the enzyme activity production increased significantly, which promoted a series of industrial applications of the strain. Although the protease and fibrinolytic enzyme production of strain 214L-11 were not very high, the study of strain XJT9503 provided a theoretical basis for our subsequent studies to improve the enzyme activity.
Streptomyces is the largest genus in actinomycetes, most of which have a variety of hydrolytic enzymatic activities (proteases, amylases and cellulases) [38,39,40], and most antibiotics are also produced by Streptomyces. It is also the main source of novel secondary metabolites, including polyketides, peptides, terpenoids, alkaloids and saccharides [41,42,43,44]. There are currently 706 species and subspecies of the Streptomyces genus (www.bacterio.net/genus/streptomyces, accessed on 14 March 2023), which have been further explored with the development of macrogenomics, culturomics and other technologies. A number of actinomycetes were screened from the Flaming Mountain of Xinjiang Province [16,36], and among them, strain 214L-11 was screened and found to have significant protease and fibrinolytic activity. Phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), which indicated that it represented a potential novel species in the Streptomyces genus. Kim et al. [45] found that 98.65% 16S rRNA gene sequence similarity can be used as the threshold for differentiating two species. If it is higher than 98.65%, it may belong to the same species or a different species, which should be determined by using other methods such as average nucleotide identity (ANI) and DNA–DNA hybridization. In this study, we conclude that it represented a potential novel species in the Streptomyces genus. However, whole-genome sequencing, physiological and biochemical investigation, DNA–DNA hybridization and ANI are required for further analysis and validation.
Fibrin clots can be degraded into fibrin degradation products in the blood by fibrinolytic enzymes, which are considered to have significant promise and value in the treatment of thrombolytic diseases. Related studies have shown that the majority of the thrombolytic drugs have low specificity towards fibrin, streptokinase (SPK) is not site-specific, lysing thrombus anywhere in the body, and tissue-type plasminogen activator (t-PA) has a short half-life, needs continuous infusion to achieve its greatest efficacy and also causes a marginal increase in stroke rate. In recent years, screening for fibrinolytic enzymes has also been further increased. Li et al. [4] and Liu et al. [46] found significant fibrinolytic activity for Agrocybe aegerita and Cordyceps militaris. However, they also found that HSA and IgG were partially degraded by their fibrinolytic enzymes, which may cause some mild effects on humans. Currently, multiple strains of Streptomyces have been reported to produce fibrinolytic enzymes. The enzyme produced by SD5 [12] has a strong in vitro thrombolytic effect; it was compared with a thrombolytic drug and was found to be faster than 500 IU of urokinase and 350 IU of streptokinase. The enzyme had optimal activity at 55 °C and pH 8.0. Tang et al. [47] found that the strain of Streptomyces albus HS1 has good in vitro thrombolytic activity; the fibrinolytic activity of the fermentation liquid of HS1 reached 207.92 IU/mL. Silva et al. [48] found that Streptomyces sp. DPUA 1576 had fibrinolytic activity of 304 mm2/1.12 g, and the enzyme produced by Streptomyces sp. DPUA 1576 exhibited optimal fibrinolytic activity at 28 °C and pH 8.0. They are not adapted to the human physiological environment. The fibrinolytic enzyme activity of Streptomyces sp. MY0504 [29] was 129.1 U/mg, and the enzyme had optimal activity at 37 °C and pH 8.0. This is close to the physiological environment of humans, but the in vitro thrombolytic and anticoagulant functions of MY0504 have not been studied. Therefore, one of the research directions is focused on newer fibrinolytic drugs that are safer and have improved effectiveness.
The fibrinolytic activity of the crude enzyme solution of strain 214L-11 was 939.9 FU/mL, while the protease activity was 137.5 U/mL. Moreover, the fibrinolytic enzymes produced by 214L-11 displayed thrombolytic and anticoagulant activities, and the fibrinolytic enzymes of 214L-11 analyzed through SDS-PAGE could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure to boost blood flow. Meanwhile, the enzymatic characterization showed that the fibrinolytic enzyme exhibits strong activity at 35–40 °C and pH 7.0–9.0, which might mean it is a metalloproteinase enzyme and retains good fibrinolytic activity in human physiological conditions. Studies on fibrinolytic enzymes have shown that the application of fibrinolytic enzyme activity varies for different strains and that the enzyme activity units are positioned differently (IU, U) [2,12,49]. Li et al. [50] found that the ratio of nattokinase to urokinase was 1:1.36. For comparison, in this study, the fibrinolytic activity of 214L-11 was significantly higher than most of the reported fibrinolytic enzyme activities of Streptomyces [13,47]. This provides a scientific basis for further strengthening of the intellectual property protection of fibrinolytic enzymes and increasing their development and exploitation.
The enzyme yield of the fermentation supernatant of strain 214L-11 before optimization was 939.9 FU/mL, and the three factors with greater influence, KNO3, peptone and metal ions, were finally obtained using the Plackett–Burman method. The optimized fibrinolytic enzyme yield was 1249.5 FU/mL, which was improved by about 32.9%. The optimized medium showed that the optimal nitrogen source was 0.58 g/L for KNO3, 0.43 g/L for peptone and 0.2 g/L for metal ions. Fermentation kinetics experiments showed that the fermentation yield of the fibrinolytic enzyme using the optimal conditions was 1192.1 FU/mL, 1239.6 FU/mL and 1255.3 FU/mL, respectively, with a good fit. During the fermentation process, soluble starch was continuously decomposed and utilized by the strain, and the final residual sugar amount was 3.23 g/L at 120 h. The mycelial weight and fibrinolytic enzyme activity of the strain increased rapidly after 40 h into the logarithmic growth phase, which was similar to that of Streptomyces sp. MY0504 [29]. The optimization of fibrinolytic enzyme production was already investigated by other authors. By optimizing the fermentation conditions of Streptomyces HS1, Tang et al. [47] found the optimal fermentation conditions for this strain and increased the enzyme activity in the supernatant of fermentation broth 1.09-fold. Verma et al. [10] applied response surface methodology to Streptomyces rubiginosus VITPSS1 to optimize enzyme production and found that the strain had the highest fermentation enzyme activity at glycerol 2.5 g/L, soybean meal 12 g/L, pH 9.2 and cultivated temperature 37 °C, which resulted in a twofold increase in fibrinolytic enzyme activity.

5. Conclusions

A strain with fibrinolytic enzyme activity was isolated for the first time from the Flaming Mountain of Xinjiang, and phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.8%), which indicated that it represented a potential novel species in the Streptomyces genus. Moreover, the fibrinolytic enzymes produced by 214L-11 displayed thrombolytic and anticoagulant activities, and could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure. In addition, the medium optimized through response surface methodology showed that the optimal nitrogen source was 0.58 g/L for KNO3, 0.43 g/L for peptone and 0.2 g/L for metal ions. Fermentation kinetics experiments demonstrated that the amount of fibrinolytic enzyme produced by fermentation using the optimal conditions was 1192.1 FU/mL, 1239.6 FU/mL and 1255.3 FU/mL, respectively. Based on these studies, the fibrinolytic enzyme produced by strain 214L-11 has great potential to be developed as a natural agent for the prevention and treatment of thrombolytic diseases.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9050410/s1, Figure S1: Phylogenetic analysis; Table S1: Design and results of Plackett–Burman experiment; Table S2: Design and results of Box–Behnken experiment; Table S3: Analysis of variance.

Author Contributions

Conceptualization, Z.Z. and L.J.; methodology, Z.H. and Y.S.; software, G.O.; validation, M.C., Y.Z. and Q.T.; formal analysis, J.Z. and L.J.; investigation, Z.Z. and L.J.; data curation, Z.H.; writing—original draft, Z.H. and Y.S.; writing—review and editing, Z.Z. and L.J.; project administration, M.C.; funding acquisition, Z.Z., L.J. and Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2021YFC2102700), the National Natural Science Foundation of China (32060004, U2106228), the “Outstanding Youth Fund” of Xinjiang Natural Science Foundation (2022D01E19), the Xinjiang Academy of Agricultural Sciences Science and technology innovation key cultivation project (xjkcpy-2021002, xjkcpy-2022004), the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTC2205), the Third Xinjiang Scientific Expedition Program (2022xjkk1200) and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (22KJB550008).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Screening of fibrinolytic enzyme-producing strains. (A) Skimmed milk plate; (B) dried blood agarose plate; (C) fibrin agarose plate.
Figure 1. Screening of fibrinolytic enzyme-producing strains. (A) Skimmed milk plate; (B) dried blood agarose plate; (C) fibrin agarose plate.
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Figure 2. Morphology of the strain. (A) Spore morphology under a microscope; (B) colonies’ morphology after 3 days of culture; (C) colonies after 5 days of culture.
Figure 2. Morphology of the strain. (A) Spore morphology under a microscope; (B) colonies’ morphology after 3 days of culture; (C) colonies after 5 days of culture.
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Figure 3. Anticoagulant activity and thrombolytic activity of strain 214L-11 in vitro. (A) Blood samples with saline; (B) blood samples with crude enzyme. (C) The first one is just crude enzyme and the second to fourth are saline, inactivated crude enzyme and crude enzyme after 12 h of thrombotic degradation.
Figure 3. Anticoagulant activity and thrombolytic activity of strain 214L-11 in vitro. (A) Blood samples with saline; (B) blood samples with crude enzyme. (C) The first one is just crude enzyme and the second to fourth are saline, inactivated crude enzyme and crude enzyme after 12 h of thrombotic degradation.
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Figure 4. Analysis of fibrinolytic and casein degradation activity of fibrinolytic enzyme. (A) Fibrinolytic enzyme activity and protease activity in the crude enzyme of strain 214L-11. (B) Analysis of SDS-PAGE. A is maker, B is protein in fermentation broth obtained through ethanol precipitation, C is thrombus + inactivation crude enzyme solution, D is thrombus + crude enzyme, E is casein + inactivation crude enzyme solution and F is casein + crude enzyme.
Figure 4. Analysis of fibrinolytic and casein degradation activity of fibrinolytic enzyme. (A) Fibrinolytic enzyme activity and protease activity in the crude enzyme of strain 214L-11. (B) Analysis of SDS-PAGE. A is maker, B is protein in fermentation broth obtained through ethanol precipitation, C is thrombus + inactivation crude enzyme solution, D is thrombus + crude enzyme, E is casein + inactivation crude enzyme solution and F is casein + crude enzyme.
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Figure 5. Optimization of carbon and nitrogen sources. (A) Effects of different carbon sources on fibrinolytic enzyme. (B) Effects of different combined nitrogen on fibrinolytic enzyme. (C) Effects of different C/N on fibrinolytic enzyme. The differences between groups are expressed by “a”, “b”, “c”, “d”, “e”, those lower case letters in the figure. p < 0.05.
Figure 5. Optimization of carbon and nitrogen sources. (A) Effects of different carbon sources on fibrinolytic enzyme. (B) Effects of different combined nitrogen on fibrinolytic enzyme. (C) Effects of different C/N on fibrinolytic enzyme. The differences between groups are expressed by “a”, “b”, “c”, “d”, “e”, those lower case letters in the figure. p < 0.05.
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Figure 6. Optimization of temperature, inoculum size, liquid volume, metal ion and pH. (A) Effects of different temperatures on fibrinolytic enzyme. (B) Effects of different inoculum sizes on fibrinolytic enzyme;.(C) Effects of different liquid volumes on fibrinolytic enzyme. (D) Effects of different metal ions on fibrinolytic enzyme. (E) Effects of different pH on fibrinolytic enzyme. The differences between groups are expressed by “a”, “b”, “c”, “d”, “e”, those lower case letters in the figure. p < 0.05.
Figure 6. Optimization of temperature, inoculum size, liquid volume, metal ion and pH. (A) Effects of different temperatures on fibrinolytic enzyme. (B) Effects of different inoculum sizes on fibrinolytic enzyme;.(C) Effects of different liquid volumes on fibrinolytic enzyme. (D) Effects of different metal ions on fibrinolytic enzyme. (E) Effects of different pH on fibrinolytic enzyme. The differences between groups are expressed by “a”, “b”, “c”, “d”, “e”, those lower case letters in the figure. p < 0.05.
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Figure 7. Activity of interaction factors in response surface methodology with strain 214L-11.
Figure 7. Activity of interaction factors in response surface methodology with strain 214L-11.
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Figure 8. Changing curves of sugar residue, fibrinolytic enzyme and dry cell weight during fermentation production of strain 214L-11.
Figure 8. Changing curves of sugar residue, fibrinolytic enzyme and dry cell weight during fermentation production of strain 214L-11.
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Table
Table 1. Plackett–Burman experimental design.
Table 1. Plackett–Burman experimental design.
FactorsCodesLevel
−1+1
Soluble starch (g/L)A11.2518.75
KNO3 (g/L)B0.3750.625
Peptone (g/L)C0.3750.625
Inoculum size (%)D1.52.5
Liquid volume (%)E1525
Mn2+ (g/L)F0.150.25
Initial pHG7.58.5
Virtual factorH~L--
Table
Table 2. Response surface design.
Table 2. Response surface design.
FactorsCodesLevel
−101
KNO3 (g/L)A0.450.50.55
Peptone (g/L)B0.450.50.55
Mn2+ (g/L)C0.180.20.22
Table
Table 3. Effect of factors in the Plackett–Burman design for strain 214L-11.
Table 3. Effect of factors in the Plackett–Burman design for strain 214L-11.
TermStdized EffectSum of SquaresContribution (%)Importance
A: Soluble starch40.924370.852.64
B: KNO3115.5842005.9725.40Important
C: Peptone101.6232679.2919.76Important
D: Inoculum size−4.387.974.820 × 10−3
E: Liquid volume−15.42990.450.60
F: Metal ions−88.5825023.5115.13Important
G: Initial pH87.0221304.3012.88
H: (Virtual factor)−14.0015236.149.21
I: (Virtual factor)−21.021695.041.03
J: (Virtual factor)54.589860.195.96
K: (Virtual factor)60.9812184.547.37
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He, Z.; Sun, Y.; Chu, M.; Zhu, J.; Zhang, Y.; Tang, Q.; Osman, G.; Jiang, L.; Zhang, Z. Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production. Fermentation 2023, 9, 410. https://doi.org/10.3390/fermentation9050410

AMA Style

He Z, Sun Y, Chu M, Zhu J, Zhang Y, Tang Q, Osman G, Jiang L, Zhang Z. Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production. Fermentation. 2023; 9(5):410. https://doi.org/10.3390/fermentation9050410

Chicago/Turabian Style

He, Zixuan, Yang Sun, Min Chu, Jing Zhu, Yu Zhang, Qiyong Tang, Ghenijan Osman, Ling Jiang, and Zhidong Zhang. 2023. "Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production" Fermentation 9, no. 5: 410. https://doi.org/10.3390/fermentation9050410

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