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81 Review Article Vitreoscilla Sp. Hemoglobin (Vhb) Expression In Qaralleh et al., 2019 J. basic appl. Res biomed 5(2): 81-88 BAAR JournalJ of Basic and Applied Research in Biomedicine ISSN 2413-7014 Review Article Vitreoscilla sp. hemoglobin (VHb) Expression in Heterologous Hosts: Some Aspects of Their Biotechnological Application Haitham Qarallehb, Muhamad O. Al-limouna, Khaled M. Khleifata, Khaled Y Alsharafaa, Amjad A. Al c Tarawneh aBiology Department, Mutah University, Mutah, Karak, 61710, Jordan; bDepartment of Medical Laboratory Sciences, Mutah University, Mutah, Karak, 61710, Jordan; cPrince Faisal Center for Dead Sea, Environmental and Energy Research, Mutah University, Karak, Jordan *Corresponding author: [email protected] Received: 3-7-2019 Abstract: It is worth mentioning that the high output of different physiological Revised: 3-9-2019 responses under the expression of vgb, may have a considerable effect on the Published: 15-9-2019 enzyme productivity, dairy industry, heavy metal uptake, biodegradation of different organic pollutants and other applications. The expression of bacterial Keywords: Vitreoscilla, haemoglobin is useful in lessening the load of perceived toxic conditions such Haemoglobin, as high oxygen levels. This in turn probably has the same impact on some Biodegradation peripheral toxic materials. This, hemoglobin biotechnology can be extended to enhance production of pollutants degrading enzymes or production of some valuable manufacturing materials on the case-by-case bases. It is likely that the mechanism of bacterial hemoglobin (VHb) effects is intermediated via an oxygen trapping action. This may drive the enrichment of ATP production, which is mostly required for higher productivity of needed substances for that activity. Cite this article as Qaralleh, H., Al-limoun, M.O., Khleifat, K.M, Alsharafa, K.Y., Al Tarawneh, A.A. (2019). Vitreoscilla sp. hemoglobin (VHb) Expression in Heterologous Hosts: Some Aspects of Their Biotechnological Application. Journal of basic and applied Research in Biomedicine, 5(2 ): 81-88 This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Introduction space (Khosla et al., 1990), an ideal location for its Vitreoscilla sp. are strictly aerobic Gram-negative proposed function. Vgb was also successfully bacteria that live in oxygen-limited environments cloned and expressed in members of the such as deep pools and cow dung (Pringsheim, Pseudomonadaceae (Liu et al., 1995; Dikshit et al., 1951). Vitreoscilla hemoglobin (VtHb) was 1990). In some cases, the presence of vgb enhanced identified as hemoglobin by amino acid sequencing the growth and metabolism of benzoic acid by (Wakabayashi et al., 1986). Further studies showed members of this group (Liu et al., 1995; Kallio and that VtHb is composed of two identical subunits Bailey, 1996). Degradation of nonpolar aromatic each containing a protoheme IX molecule; each compounds usually requires the addition of oxygen subunit has a molecular weight of 16,392 Daltons. at one or more steps (Suen et al., 1996). The In Vitreoscilla or E. coli transformed with vgb, presence of vgb/VtHb may provide oxygen directly VtHb levels were found to be increased when the to those oxygenases or stimulate benzoic acid cells were grown at low oxygen concentrations metabolism by enhancing growth. (Boerman and Webster, 1982; Dikshit et al., 1990). Further studies demonstrated that VtHb-containing The presence of hemoglobin protein in prokaryotic E. coli cells grow more quickly and to a higher organisms was first recognized in the gram- density compared with non-VtHb containing cells negative bacterium Vitreoscilla (Wakabayashi et (Khosla et al., 1990; Khosravi et al., 1990; Joshi al., 1986). This Vitreoscilla hemoglobin is the most and Dikshit, 1994). It is thought that VtHb binds widely studied bacterial hemoglobin, including its oxygen (particularly at low oxygen concentrations) potential use in biotechnological applications and feeds it to the terminal oxidases of the (Khleifat and Abboud, 2003). Enhanced respiratory chain (Wakabayashi et al., 1986). This biosynthesis of VHb is mediated at the theory was proposed based on the evidence transcriptional level by an oxygen-sensitive mentioned above, for example, the fact that promoter which is turned on under hypoxic Vitreoscilla cells grown under oxygen limited conditions in the native and recombinant host of E. conditions exhibit an approximately 50 fold coli (Ramandeep et al., 2001; Joshi and Dikshit, increase in hem content (Boerman and Webster, 1994; Kallio et al., 1994).The expression of VHb 1982). Additionally, nearly half of the VtHb in vgb- hemoglobin may result in the enhancement of cell bearing E. coli is located within the periplasmic density, oxidative metabolism, bioremediation and 81 Qaralleh et al., J. basic appl. Res. Biomed. 5(1): 81-88 engineered product formation especially under The general effect of VHb is based on the fact that oxygen-limited conditions. A particular application a bacterial cells engineered with the gene (vgb) of VHb expression involved the increased encoding Vitreoscilla hemoglobin (VHb) typically production of α-amylase (Khleifat and Abboud, produce more protein than vgb-lacking cells 2003), the enhanced degradation of toxic wastes (Khosla et al., 1990; Khleifat and Abboud, 2003) such as 2,4- DNT and benzoic acid degradation by Pseudomonads (Nasr et al., 2001). It is believed Tentative purification of VHb that the putative function of VHb is to trap oxygen The purification data of Vitreoscilla hemoglobin and feeds it to a membrane terminal oxidase from a culture of E. coli JM109 [pUC8:16] grown (Hwang et al., 2001). Presence of Vitreoscilla in terrific broth are shown in Table 1. The purified hemoglobin gene (vgb) on the plasmids minimized VtHb was examined by SDS-polyacrylamide gels the negative impact conferred by such plasmid stained with Coomassie blue R-250 (Fig. 1). Two (Khosla and Bailey, 1989; Dikshit et al., 1992). bands of similar intensity were seen. Their Initial works on the VHb expressing gene, vgb and molecular weights are 18,500 and 16,000 as promoter activity in E. coli confirmed that the determined by a standard curve of the same expression of the vgb gene is regulated by oxygen proteins (data not shown). Solutions of the purified (Dikshit et al., 1989). It has been, also, suggested VtHb were dark red, and the CO-difference that besides its effective oxygen deliverity potential spectrum obtained for the partially purified protein and transport function, VHb has generated was used to calculate the VtHb concentration (data fundamentally for the detoxification purpose not shown). The ratio of A410/A280 after Sephadex (Minning et al., 1999). However, it has been G-100 was almost 1.5 which indicated that the use hypothesized that VHb has the ability to trap of Sephadex G-l00 as a single step after ammonium oxygen molecules from neighboring domain and sulfate fractionation might be enough for partially feeds it to the cellular activities in heterologous purified protein. The additional Sephadex G-75 hosts due to its exceptional kinetic parameters for step produced a preparation with a A410/A 280 ratio oxygen binding and freeing (Kd = 72 μM) of almost 2.0, indicating a highly purified VHb (Webster, 1988; Frey et al., 2000; Kallio et al., preparation (data not shown), although this 1994; Tsai et al., 1995; Akbas, 1997; Patel et al., conclusion was clouded by the presence of two 2000; Abboud et al., 2010; Khleifat et al., 2019a). bands in these fractions. Table 1. Purification Table Of Vitreoscilla Hemoglobin (Khleifat, 2010) Processes Total A 410 Total A280 Ratio Yeild Purification A410 : A280 ( % ) ( Fold ) Crude Extract 1800 40450 0.044 100 1 45% Ammonium Sulphate Supernatant 1599 24980 .0.064 89 1.54 70% Amonium Sulphate Precipitate 1200 9540 0.126 67 2.86 Sephadex G-100 769.5 524.39 1.47 42 33.4 Sephadex G-75 350.1 175.4 1.996 19 45.4 Figure 1. Purified VtHb by SDS-polyacrylamide gels stained with Coomassie blue R-250 82 Qaralleh et al., J. basic appl. Res. Biomed. 5(1): 81-88 Does highly purified Vitreoscilla hemoglobin molecular oxygen (De Modena et al., 1993). VtHb would in vitro affect partially purified DN'T has also been linked to enhanced degradation of dioxygenase benzoate by Xanthomonas (Liu et al., 1996) and Activity was tested at different concentrations of 2,4-DNT by Burkholderia (Patel et al, 2000) and VtHb. The assays were run at both 100% and 25% E. coli bearing the DNT dioxygenase gene (Fish et air saturation (250 and 62.5 M oxygen, al., submitted). All three processes require respectively). VtHb inhibited DNT dioxygenase molecular oxygen. In the latter case, which was the activity in a dose dependent manner, almost precursor to the work reported here, the stimulation abolishing it at 6 M (350 g/ml) VtHb; the results occurred during growth or when whole cells or are similar at both levels of oxygen. DNT whole cell extracts were tested and was probably dioxygenase was completely inhibited (at both due simply to an increase in DNT dioxygenase 100% and 25% air saturation) by 25, 100 and 150 synthesis due to a VtHb produced increase in ATP g/ml horse hemoglobin and 25, 100 and 150 levels in vivo (Tsai et al., 1995). Because the direct g/ml horse myoglobin. In another independent interaction of VtHb with DNT dioxygenase experiment, DNT dioxygenase was shown to be as resulted in the inhibition of DNT conversion to active in the presence of boiled VtHb compared MNC (Fig. 2), this interpretation, rather than a with that VtHb absence, while the same amount of direct stimulation of DNT dioxygenase by VtHb, is active VtHb completely inhibited the enzyme. The supported. This could be a unique tool causes Kd ‘s for both VHb and dioxygenase can be induction of the degradation ability of high described by Kd and Kd’, respectively, below.
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