Research Article

Trace Elements Optimization for Production of Fibrinolytic Protein from Wild and Mutant Streptococcal Strains

Ghulam Akbar1*, Muhammad Anjum Zia1, Amer Jamil1 and Faiz Ahmad Joyia2

1Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan; 2Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan.

Received | October 14, 2022; Accepted | November 22, 2022; Published | December 26, 2022

*Correspondence | Ghulam Akbar, Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan; Email: [email protected]

Citation | Akbar, G., Zia, M.A., Jamil, A., and Joyia, F.A., 2022. Trace elements optimization for production of fibrinolytic protein from wild and mutant Streptococcal strains. Journal of Innovative Sciences, 8(2): 301-310.

DOI | https://dx.doi.org/10.17582/journal.jis/2022/8.2.301.310

Keywords | Fibrinolytic, Streptokinase, Trace elements, Optimization, Enzyme production

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction

It was investigated that SK produced by various Streptococcal strains activates the plasminogen by different ways because of its different structural domains make up (α, β and γ) having various functional activity. It was also suggested that SK protein is made up of two structural domains analyzed by calorimetric scanning analysis. One domain has NH2-terminal including a minimum plasminogen affinity and a protein chain of 60-414 amino acids is responsible for the synthesis of plasminogen complex. The other is CO2-terminal domain which plays important role in recognition and activation of substrate: Plasminogen complex (Rafipour et al., 2020) and specifically the section of Asp 41 to His 48 interlink to plasminogen (Kim et al., 2000). For the activation of plasminogen the important region is the γ-domain coiled region of the SK with the proper conformational structure where SK links with plg by lysine residue (Wu et al., 2001; Sharma et al., 2020).

The SK directly has no proteolytic effect however, its combination to form complex with plasminogen with 1:1 ratio breaks down the Arg 561-Val 562 of circulating plasminogen to transform them into plasmin and this activated plasmin degrades the fibrin (Figure 1) (de Souza et al., 2013). As it was already described that SK is a non-specific in its action however, its not only activate plasminogen but also have ability to induce hyper-plasminemia, which is condition of exhaustion of circulating fibrinogen and coagulation factors V and VIII (Figure 2) with the relative enhancement of degradation products of plasminogen (Lin et al., 2020). SK dose of 1500 000 IU despite the systemic lytic condition have an equal chance of hemorrhagic complications like other fibrinolytic drugs which exhibit higher degree interaction for fibrin (Aslanabadi et al., 2018). The removal of SK from the circulatory system is biphasic. First is very fast phase in which SK inactivation occurs by particular antibodies that is about of four minutes. In 2nd phase when once SK-Plg complex is synthesized then after half hour of its average life SK is degraded and removed from the blood circulation (Erdoğan et al., 2006).

 

The two basic problems of SK therapy are bleeding, which depends on dose and administration duration and immunogenicity of SK, due to its bacterial source, which may cause allergic reactions. On the basis of carbohydrate composition of bacterial antigens found on their cell wall lance grouping have been arranged which includes coagulase-negative and catalase negative bacteria. This system made by Rebecca Lancefield was established for different species of Streptococcaeae in which Streptococcus and Lactococcus genera included. However, now it is exceeded due to identification of much number of Streptococci. Kozińska and Sitkiewicz (2020) used the both classical and molecular analysis techniques for the detection and characterization of group A S. pyogenes bacterial strain. The most of group A, C and G species are beta hemolytic which cause complete hemolysis of red blood cells. 

 

Thrombolytic enzyme activity depends upon the capacity of enzyme to convert plasminogen into plasmin and this capability of plasmin hydrolysis for specific duration is directly linked to the concentration of streptokinase. There are many substrates that are cleaved by the plasmin have a composition of fibrin clot, casein and esters that are good for finding the enzymatic activity (Arshad et al., 2018). SK is the major fibrinolytic drug which is commonly used for myocardial infarction treatment in several countries and about 0.5 patients have been successfully cured with SK treatment per year (Couto et al., 2004). The mortality and the morbidity rates occurred from cardiovascular diseases are very high throughout the World and especially in underdeveloped and undeveloped countries are more than 80%. However, acute myocardial infarction (AMI), ischemic heart disease, arrhythmias and stroke are primary reasons of mortality (Go et al., 2013). The blockage of arteries and veins may cause the indication of blood clots with serious conditions which can leads to death. In some clinical studies streptokinase and tPA were compared but there was no clear preference was observed by any one of them. SK is more efficient and cost-effective as compared to the tissue plasminogen activators (tPA).

The use of streptokinase directly into the coronary artery was uncommon until 1979. However, direct administration of streptokinase into coronary vessels causes serious harmful side effects such as brain hemorrhage and digestive tract bleeding. These side effects generally appear in the older heart patients treated with high doses of streptokinase (Aslanabadi et al., 2018). There is a balance between blood clot formation and prevention and this process which equilibrates both these conditions is called hemostasis. Outside the body blood forms blood clot while inside the body fibrinolytic agents are present which prevent blood clot formation. Imbalance in hemostasis causes either bleeding or thrombus formation. There are many fibrin degradation enzymes like urokinase, streptokinase (SK), plasminogen activators (tPA) are the main medicines used for the treatment of cardiovascular diseases (Nedaeinia et al., 2019). Tissue plasminogen activators are expensive but more specific for plasminogen which made it popular in advanced countries like British, Germany, America and Japan. While streptokinase is nonspecific and less costly due to its microbial origin so it’s widely used in undeveloped and under developed countries such as continent African and Asian continents. The most of the people in these countries are poor and can’t afford expensive medicine. So patients of heart diseases in these low income areas preferred the use of streptokinase. This is the main reason of saving lives of million to trillions of cardiovascular disorders patients (Yan et al., 2020).

In the blood circulatory system, the formation of a thick and sticky clump which blocks the flow of blood through vessels this process is known as thrombosis. After injury of a blood vessel, platelets, fibrin and red blood cells play a key role in the formation of a blood clot to stop bleeding. A piece of the blood clot broken from thrombus start to travel in the blood vessels is named as embolus (Furie and Furie, 2008). Blood clot formation may occur in the veins or in arteries. Insoluble Fibrin is formed from soluble fibrinogen by the action of thrombin protein. This fibrinogen is polypeptide made up of two subunits which are connected by disulphide linkages consisting of molecular weight, 340 kDa. Each subunit contains three large peptide chains (Aα, Bβ, and γ) (Vasilyeva et al., 2020).

The fibrin protein in blood matrix clots the blood in blood vessels is known as thrombosis. Thrombolytic medications such as streptokinase, urokinase and plasminogen activator (tPA) act with plasminogen to form plasmin which acts on this clot and breaks down it (Table 1). This procedure is called thrombolysis. In mammalian cells, the protein responsible for thrombolysis is plasmin, which is a serine protease analog to trypsin (Emberson et al., 2014).

 

Plasmin is functional form modified from inactive zymogen (plasminogen) and this transformation of

 

Table 1: Properties of thrombolytic enzymes.

Enzyme	Mol. mass ( kDa)	Substrate specificity	Source of origin	Half-life (Min)	Cure for diseases	References
Streptokinase	47	Plasmin	Streptococcus species	20	Myocardial infarction, pulmonary embolism,	Tillet et al., 1933
Nattokinase	27.7	Plasmin	Bacillus subtilis	240	Remedy for heart diseases	Fujita et al., 1993
Alteplase	59	Plasmin	Human melanoma cell line	5	Ischemic stroke, pulmonary emboli. myocardial infarction	McCartney et al., 2019
Urokinase	31	Plasmin	Human urine	15	Pulmonary embolism, myocardial infarction	Degryse, 2011
Reteplase	39	Plasmin	Escherichia coli	15	Myocardial infarction, ischemic stroke.	Noble et al., 1996
Anistreplase	131	Plasmin	Streptococcus + plasma product	70	Myocardial infarction and pulmonary embolism	Hannaford et al., 1995
Tenecteplase	70	Plasmin	Mammalian cell line	15	Myocardial infarction, pulmonary emboli.	Melandri et al., 2009
Lanoteplase	39	Plasmin	Recombinant	30	Investigated for use/treatment in myocardial infarction.	Hazzard et al., 1999
Amediplase	346 Da	Plasmin	Animal source	16	Myocardial infarction, thrombosis.	Guimarães et al., 2001
Saruplase	46	Plasmin	Kidney and liver	9	Arterial thrombosis	Grignani et al., 1994
Subtilisin DJ-4	29	Plasmin	B. licheniformis	30	Myocardial, cardiovascular infarction	Kim and Choi 2000
Subtilisin QK-2	28	Plasmin	Bacillus subtilis	30	Prevent and control thrombosis diseases.	Ko et al., 2004
Subtilisin DFE	28	Plasmin	Bacillus amyloliquefacien	10.5	Deep venous thrombus and Atherosclerosis	Peng et al., 2004

 

plasminogen into plasmin the Arg 561-Val 562 bond is the site of proteolytic cut by several plasminogen activators and this activated plasmin degrades fibrin molecules as shown in Figure 3. The most of the group C beta hemolytic Streptococcal species produce streptokinase protein which is not found in human body only appears after medical treatment. There is no direct effect of fibrinolytic agents such as, tPA, uPA and SK, however their clinical effect is performed by the transformation of the plasminogen into plasmin (Law et al., 2012).

2. Materials and Methods

The wild Streptococcus mutans was subjected to physical mutation by UV-radiations and Ethidium bromide with chemical random mutations. The optimization trace elements in the nutrient agar medium of all these strains were performed and obtained optimum levels of these elements on wild and mutants’ bacterial strains.

2.1 Trace element K2HPO4

The different concentrations of K2HPO4 (Spectrum Chem. Mfg. Corp.) (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0%) were added in the fermentation medium containing 250mL Erlenmeyer flasks. These flasks were incubated for 24h and after that centrifugation were performed. The supernant and pellet were subjected for enzyme activity (Arshad et al., 2018).

2.2 Trace element CH3COONa. 3H2O

The various concentrations of hydrated sodium acetate (CH3COONa. 3H2O) (Elsen Golden Laboratories, assay 99%) (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0%) were added in the fermentation medium (nutrient agar medium) containing Erlenmeyer flasks. These flasks were incubated for 24h and then centrifugation was done. The supernant and pellet were subjected for enzyme activity.

2.3 Trace element KH2PO4

The various quantities of KH2PO4 (Elsen Golden Laboratories, assay 99%) (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0 %) were added in the fermentation medium (nutrient agar medium) containing 250mL Erlenmeyer flasks. These flasks were incubated for 24h and then centrifugation was done. The supernant and pellet were subjected for enzyme activity.

2.4 Trace element NaHCO3

The NaHCO3 (Pure ChemsTM M.W: 84) (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0 %) were added in the fermentation medium (nutrient agar) containing 250mL Erlenmeyer flasks. These flasks were incubated for 24h and then centrifugation was done. The supernant and pellet were subjected for enzyme activity.

2.5 Trace element FeSO4.7H2O

The FeSO4.7H2O (Sigma Aldrich ACS reagent, ≥99.0%) (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0 %) were added in the fermentation medium containing 250mL Erlenmeyer flasks. These flasks were incubated for 24h and then centrifugation was done. The supernant and pellet were subjected for enzyme activity.

2.6 Trace element CaCO3

The CaCO3 (Minday Materials, assay 99%) (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0 %) were added in the fermentation medium (nutrient agar) containing 250mL Erlenmeyer flasks. These flasks were incubated for 24h and then centrifugation was done. The supernant and pellet were subjected for enzyme activity.

2.7 Statistical analysis

The data was statistically observed in one way ANOVA by using Graph Pad InStat software.

3. Results and Discussion

The bacterial strain Streptococcus mutans was collected from EBL stock University of Agriculture Faisalabad. This strain was re-cultured on nutrient agar media again and again to get a healthy and pure strain culture. The optimization of trace elements was performed after the mutagenesis of wild strain.

3.1 Optimization of trace elements in the fermentation medium

3.1.1 Optimization of K2HPO4

In a fermentation medium with the addition of dipotassium hydrogen phosphate (K2HPO4) it was shown that the amount of enzymes produced was observed in an elevated level. In this research work various concentrations of K2HPO4 from 0.1 to 1.0% have been added in the fermentation media of parental and mutant Streptococcus mutans shown in Figure 4 and Table 2. The largest amount of SK was found from UV-EtBr-mutant with enzyme activity 675.01 UmL-1 by adding 0.3 % K2HPO4.

 

Table 2: ANOVA for influence of K2HPO4 on SK yield from wild and mutagenic Streptococcus mutans.

S.O.V	DF	Sum of square	Mean square	F-value	p-value
Source	2	1162355	581178	181.98	0.0000**
Levels	1	69685	69685	21.82	0.0000**
Source×Level 2	22574	11287	3.53	0.0336*
Erro	84	268270	3194
Total	89

 

*, Significant (P<0.05); **, Highly significant (P<0.01).

 

3.1.2 Optimization of CH3COONa. 3H2O

The CH3COONa.3H2O had a significant effect in the production of SK in optimized fermentation media. The amount of CH3COONa.3H2O from 0.01 to 0.1 was applied to optimize its concentration. The optimum production of SK 676.0 U/ml was observed by using 0.01% of CH3COONa.3H2O in the UV-EtBr Streptococcus mutans fermentation medium (Figure 5 and Table 3).

 

 

Table 3: ANOVA for influence of CH3COONa.3H2O on SK yield from wild and mutagenic Streptococcus mutans.

S.O.V	DF	Sum of square	Mean square	F value	p-value
Source	2	568320	284160	42.09	0.0000**
Level	1	148759	148759	22.03	0.0000**
Source ×level	2	93513	46756	6.93	0.0016**
Error	84	567098	6751
Total	89

 

*, Significant (P<0.05); **, Highly significant (P<0.01).

 

3.1.3 Optimization of KH2PO4

Throughout the continual improvement of enzymes from bacterial strains in the fermentation process, salt concentrations even play a crucial role. In various trials, a spectrum of KH2PO4 concentrations was introduced around 0.01 to 0.1 in the fermentation medium of S. mutans (Figure 6 and Table 4). The 0.06% quantity of such a salt as given for the maximum SK enzyme output with an activity of 680.0 UmL-1.

 

Table 4: SK yield from wild and mutagenic Streptococcus mutans on diverse KH2PO4 amount.

S.O.V	DF	Sum of square	Mean square	F value	p-value
Source	2	1301099	650549	592.23	0.0000**
Level	1	25056	25056	22.81	0.0000**
Source× level	2	17811	8906	8.11	0.0006**
Error	84	92271	1098
Total	89

 

*, Significant (P<0.05); **, Highly significant (P<0.01).

 

3.1.4 Optimization of NaHCO3

The Influence of NaHCO3 was detected for the utmost production of SK from wild and mutant bacteria in the fermented medium. The ten various concentrations were applied from 0.01 to 0.1 percent (Figure 7 and Table 5). An amount of 0.04% NaHCO3 in the fermentation medium showed the extreme yield of SK with activity of 675.0 UmL-1.

 

Table 5: ANOVA for influence of NaHCO3 on SK yield from wild and mutagenic Streptococcus mutans.

S.O.V	DF	Sum of square	Mean square	F-value	p-value
Source	2	1006447	503224	166.89	0.0000**
Level	1	73845	73845	24.49	0.0000**
Source× level	2	19929	9965	3.30	0.0415*
Error	84	253279	3015
Total	89

 

*, Significant (P<0.05); **, Highly significant (P<0.01).

 

3.1.5 Optimization of FeSO4.7H2O

The effect of FeSO4.7H2O on enhanced SK output in the fermentation process was also identified. In the fermented medium, the different concentrations of this salt were analyzed from 0.01 to 0.1 percent shown in Figure 8 and Table 6. A concentration of 0.01% added in the fermentation medium expressed the highest quantity of enzyme with activity of 663.02UmL-1.

 

3.1.6 Optimization of CaCO3

The impact of calcium carbonate in the fermented product on SK yield through bacterial strains has been reported. The calcium carbonate concentrations range has been examined between 0.01 and 0.1% and the highest production with SK activity of 652.01UmL-1 was found with a level of 0.02% CaCO3 (Figure 9 and Table 7).

 

Table 6: ANOVA for influence of FeSO4.7H2O on SK yield from wild and mutagenic Streptococcus mutans.

S.O.V	DF	Sum of square	Mean square	F-value	p value
Source	2	891739	445869	97.54	0.0000**
Level	1	96694	96694	21.15	0.0000**
Source× level	2	28724	14362	3.14	0.0483*
Error	84	383988	4571
Total	89

 

*, Significant (P<0.05); **, Highly significant (P<0.01).

 

 

Table 7: ANOVA for influence of CaCO3 on SK yield from wild and mutagenic Streptococcus mutans.

S.O.V	DF	Sum of square	Mean square	F value	p-value
Source	2	835333	417667	150.35	0.0000**
Level	1	75864	75864	27.31	0.0000**
Source× level	2	23453	11727	4.22	0.0179*
Error	84	233349	2778
Total	89

 

*, Significant (P<0.05); **, Highly significant (P<0.01).

 

El-Mongy and Taha (2012) and Faran et al. (2015) who found maximum SK production (331.71UmL-1) and (467. 73 UmL-1) from mutated Streptococcus equisimilis by adding 0.25% and 0.05% K2HPO4 in the fermentation medium.

Faran et al. (2015) obtained maximum SK production (365.33U/mL) by using 0.15% CH3COONa.3H2O in the fermented medium of mutated S. equisimilis and Madhuri et al. (2011) obtained highest amount of SK production by using 0.1% CH3COONa.3H2O in the medium. Abdelghani et al. (2005) and Faran et al. (2015) are significantly associated who observed optimum SK output at 0.25% and 0.15% KH2PO4 concentrations and analyzed the decrease in SK output by raising the concentrations.

Patel et al. (2011), who got the highest output of SK from bacterial strain by adding 0.2% NaHCO3 in the fermentation medium, Madhuri et al. (2011) who observed maximum production streptokinase by using 0.15% NaHCO3 in the media and also correlates with the findings of Faran et al. (2015) who obtained maximum SK quantity with activity (457.01UmL-1 by using 0.15% NaHCO3 in the optimized fermentation medium. Yazdani and Mukherjee (2002) who achieved optimum SK output in the fermentation process by adding 0.06% FeSO4.7H2O concentration and also related to the results of Madhuri et al. (2011) and Faran et al. (2015) that accomplished optimum SK activity upon the addition of 0.04% FeSO4.7H2O in the fermentation media.

Baewald et al. (1975) and Faran et al. (2015) that found better SK yield from the fermented medium by the addition of 0.004% CaCO3 and also narrowly interlinked with the findings of Elmongy and Taha (2012) who attained maximum SK yield upon addition of 0.005% of CaCO3 in the fermentation medium.

Conclusions and Recommendations

The addition of trace elements like KH2PO4, K2HPO4, NaHCO3, CaCO3, CH3COONa.3H2O and FeSO4. 7H2O in the fermentation medium expressed the highly significant results. Among all these trace elements, K2HPO4 has shown the highest enzymatic activity with UV-Et mutant 680.0 UmL-1 and with CaCO3 UV-Et mutant has been found to give the relatively lowest enzyme activity 652.01UmL-1. While UV-mutant has shown maximum enzyme activity 275 UmL-1 with the addition of K2HPO4 and this mutant shown minimum 57 UmL-1 with the addition of FeSO4.7H2O. The parental strain expressed maximum enzyme activity with 108UmL-1 FeSO4 minimum enzymatic activity 5UmL-1 with the addition of CH3COONa.3H2O. However, the trace elements play key role for the production of specific metabolites from microbial source.

Acknowledgement

I am thankful to Enzyme Biotechnology Laboratory for the facilitation of this research study.

Novelty Statement

As for as the novelty is concerned, first time I have developed this strain from locally isolated strain. In enzyme biotechnology use of novel strain for enzyme production and enhancement in enzyme activity is its novelty as well. Hence, developing a novel strain and achieving higher levels of enzyme production than the previous ones is the novelty of my work.

Author’s Contribution

Ghulam Akbar performed all the research work, Muhammad Anjum Zia Supervised research work, Amer Jamil provided research plan and technical assistance, Faiz Ahmad Joyia helped research work to perform some part in his lab and also provided guide lines in paper writing and reviewed this article before submission.

Conflict of interest

The authors have declared no conflict of interest.

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