Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов

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СКАЧАТЬ E. coli BW25113 (pTAHEF) 5.14 Lee et al. (2009)

Ferulic acid is well known as a potential substrate for bioconversion to vanillin, which is available abundantly in the natural sources. Ferulic acid is converted to vanillin by the catalytic activity of feruloyl‐CoA synthetase (fcs) and enoyl‐CoA hydratase/aldolase (ech). Yoon et al. (2005a) generated a recombinant E. coli expressing with fcs gene of Amycolatopsis sp. HR104 and ech gene of Delftia acidovorans under the control of P_BAD promoter that produced the highest yield of vanillin (580 mg/l) under the optimized conditions in LB medium, which is 3.6‐folds higher than the yield obtained before media optimization. Meantime, another E. coli genetically engineered to express fcs gene of Amycolatopsis sp. HR104 and ech gene of D. acidovorans under the control of IPTG‐inducible trc promoter in complex medium (2YT) supplemented with ferulic acid (0.2% w/v) has produced highest amount of vanillin (1.1 g/l) (Yoon et al., 2005b). Improvement of the vanillin production has been investigated further by Yoon et al., 2007 by constructing recombinant E. coli carrying plasmid pTAHEF cloned with both fcs and ech genes of Amycolatopsis sp. and tested with ferulic acid (2.0 g/l) that produced a maximum of 1.0 g/l vanillin in 48 hours. In due course, bacterial growth and vanillin yield decreased due to vanillin toxicity. In order to avoid vanillin toxicity, generation of NTG mutant and removal of excess vanillin from the culture medium using polystyrene resin XAD‐2 has been followed. The vanillin production by NTG mutants has increased three times higher when compared with its wild‐type strain. Following the use of polystyrene resin (XAD‐2) with NTG mutagenized E. coli, the production of vanillin has attained a maximum yield (2.9 g/l), which is twofolds higher than that obtained without using the resin (Yoon et al., 2007). It has been presumed that shortage of CoA for catalyzing conversion of ferulic acid to feruloyl‐CoA due to its utilization as acetyl‐CoA, could be a reason the scoring less yield of vanillin biotechnologically. To circumvent this impediment, another recombinant E. coli carrying plasmid pTAHEF is cloned with gltA encoding citrate synthase, which produced 1.98 g/l of Vanillin (Lee et al., 2009). For improving the potential of microbial sources for higher production of vanillin, many metabolic optimizations and genetic manipulations have been carried out so far using various organic compounds as substrates, though more attempts are needed to further enhance yield of biovanillin production to meet higher demand of vanillin.

2.5 Bioactive Properties of Vanillin

      2.5.1 Antimicrobial Activity

      Resistance of pathogenic bacteria toward majority of antimicrobial drugs has been prominently developed, which warrants the need for new antivirulence drugs for their effective control. Research and development of innovative antimicrobial agents to target various pathogenic bacterial virulence factors is going on since back two decades, but few drugs showing significance in their efficacy. Phytochemicals and microbial transformed metabolites or bioactive metabolites show promising antimicrobial activity by targeting various virulence factors. As we know quorum sensing is a bacterial signaling pathway for communicating to their external environments and in turn quorum sensing facilitate the pathogenic bacteria to enhance their pathogenesis by regulating the expression patterns of various virulence factors gene and associated factors (Deep et al., 2011). Targeting various pathways related to quorum sensing has become a novel and innovative approach for designing antibacterial quorum‐sensing agents. Antiquorum‐sensing drugs have the ability to affect bacterial pathogenicity because drug affects various factors and pathways involved in quorum sensing pathway in different ways. Vanilla and vanilla‐containing foods might promote antimicrobial activity by suppressing quorum sensing and their intermediate pathways particularly colonization and biofilms formation (Jakobsen et al., 2012). For example, Chromobacterium violaceum is a Gram‐negative bacterium that produces violacein pigment, a molecule that helps this bacterium to communicate with external environment, whose expression is controlled by quorum sensing. Recently, it has been evidenced that vanilla‐containing food materials promote inhibition of violacein production and thus help in breaking quorum sensing and in turn also arrest the communication among bacteria and environments around it (Asfour, 2018).

      2.5.2 Antioxidant Activity

Reactive oxygen and nitrogen species (ROS and RNS) are generated by normal metabolic pathway in normal body, and the production of ROS and RNS increased day by day, if body is exposed more and persistently to the unwanted higher intake of dietary xenobiotics and as well as unhealthy food. The ROS and RNS create oxidative stress by altering normal cellular functions of our body resulting in various diseases such as diabetes, hypertension, cancer, and many other complications. Naturally produced antioxidants such as various bioactive phenolic compounds naturally found in dietary sources can be effectively toiled to lower down these oxidative stresses. Certain genes encoding the enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx) help to neutralize oxidative stress, and findings of naturally derived compounds to enhance expression of these genes are now days prominently explored by researchers (Kurutas, 2016). The antioxidant effect of vanillin has been reported by Deters (2008), where vanillin had moderate free radical scavenging activity (IC₅₀ of 2945 ± 247 μM) and also showed mild antioxidant activity in mouse liver microsomes (Yan‐Chun and Rong‐Liang, 1991). Likewise, antioxidant activity of vanillin is investigated using multiple antioxidant assays, which has shown potent antioxidant activity in the ABTS^∙+ scavenging assay, ORAC assay, and in OxHLI assay, while no activity has been noticed in the DPPH radical scavenging assays (Tai et al., 2011).

      2.5.3 Anticancer Activity

      2.5.3.1 Apoptosis Pathway

      Mitochondrial‐dependent apoptosis is an important pathway for the induction of apoptosis, and any disturbance in this pathway could inhibit apoptosis. The apoptosis pathway is regulated by the B‐cell lymphoma‐2 (Bcl‐2) family proteins and this Bcl‐2 regulates (elevation or downregulation) the mitochondrial membrane permeability for the release of cytochrome‐c and other apoptotic proteins. Furthermore, caspase activation and DNA fragmentation are the main features of induced apoptosis. Xie et al. (2020) have shown that vanilla‐containing food‐induced apoptosis in HT‐29 colon cancer cells by inducing oxidative damage and also modulated the expression of apoptotic markers. Gupta et al. (2010) have reported that vanilla and vanilla‐containing food downregulates Bcl‐2, which causes upregulation of Bax and thereby increases the permeability of the mitochondria to apoptosis‐inducing factor (AIF), which is then released to cytosol from the mitochondria. AIF in the cytosol‐activated caspases‐3, 7, and 9, which causes DNA fragmentation and finally induced cell death by apoptosis.

      2.5.3.2 Tumor Necrosis Factor‐induced Apoptosis

      Tumor necrosis factor (TNF) also triggers apoptosis, which is a promising anticancer target by which cancer cells are killed selectively with negligible effect on normal cells. In the HeLa cells, pretreatment of vanillin‐enhanced tumor necrosis factor‐related apoptosis‐induced cell death by induced phosphorylation of p65 and transcriptional activity of NF‐κB (Lirdprapamongkol et al., 2010).

      2.5.3.3 Cell Cycle Arrest

      Cells are regulated at different checkpoints by the interactions of various cyclins and cyclin‐dependent kinases (CDKs). Before the progression to the next phase of the cell cycle, cell growth is regulated at each checkpoints by cyclin and CDKs. In cell cycle regulation, cyclin, CDKs, and p53 play a key role. Recently, vanilla has shown its potential to arrest cell cycle in colorectal cancer cells at the G1/G0 stage at lower concentration (200 μg/ml), СКАЧАТЬ