Displaying publications 1 - 20 of 52 in total

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  1. Mohamed RA, Salleh AB, Leow ATC, Yahaya NM, Abdul Rahman MB
    Mol Biotechnol, 2017 Jul;59(7):284-293.
    PMID: 28580552 DOI: 10.1007/s12033-017-0012-0
    An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53.8%. Docking of T1 lipase with P(3HB) gave forth high binding energy of 5.4 kcal/mol, with the distance of 4.05 Å between serine hydroxyl (OH) group of TI lipase to the carbonyl carbon of the substrate, similar to the native PhaZ7 Pl . This suggests the possible ability of T1 lipase to bind P(3HB) in its active site. The ability of T1 lipase in degrading amorphous P(3HB) was investigated on 0.2% (w/v) P(3HB) plate. Halo zone was observed around the colony containing the enzyme which confirms that T1 lipase is indeed able to degrade amorphous P(3HB). Results obtained in this study highlight the fact that T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation activity but amorphous P(3HB) degradation activity as well.
  2. Sahebi M, Hanafi MM, Azizi P, Hakim A, Ashkani S, Abiri R
    Mol Biotechnol, 2015 Oct;57(10):880-903.
    PMID: 26271955 DOI: 10.1007/s12033-015-9884-z
    Suppression subtractive hybridization (SSH) is an effective method to identify different genes with different expression levels involved in a variety of biological processes. This method has often been used to study molecular mechanisms of plants in complex relationships with different pathogens and a variety of biotic stresses. Compared to other techniques used in gene expression profiling, SSH needs relatively smaller amounts of the initial materials, with lower costs, and fewer false positives present within the results. Extraction of total RNA from plant species rich in phenolic compounds, carbohydrates, and polysaccharides that easily bind to nucleic acids through cellular mechanisms is difficult and needs to be considered. Remarkable advancement has been achieved in the next-generation sequencing (NGS) field. As a result of progress within fields related to molecular chemistry and biology as well as specialized engineering, parallelization in the sequencing reaction has exceptionally enhanced the overall read number of generated sequences per run. Currently available sequencing platforms support an earlier unparalleled view directly into complex mixes associated with RNA in addition to DNA samples. NGS technology has demonstrated the ability to sequence DNA with remarkable swiftness, therefore allowing previously unthinkable scientific accomplishments along with novel biological purposes. However, the massive amounts of data generated by NGS impose a substantial challenge with regard to data safe-keeping and analysis. This review examines some simple but vital points involved in preparing the initial material for SSH and introduces this method as well as its associated applications to detect different novel genes from different plant species. This review evaluates general concepts, basic applications, plus the probable results of NGS technology in genomics, with unique mention of feasible potential tools as well as bioinformatics.
  3. Woon JS, King PJH, Mackeen MM, Mahadi NM, Wan Seman WMK, Broughton WJ, et al.
    Mol Biotechnol, 2017 Jul;59(7):271-283.
    PMID: 28573450 DOI: 10.1007/s12033-017-0015-x
    Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris. Expressed GH1254 had an apparent molecular mass of 42 kDa, but purification was hampered by its low expression levels in shaken flasks. To obtain more of the enzyme, GH1254 was produced in a bioreactor that resulted in a fourfold increase in crude enzyme levels. The purified enzyme was active towards soluble synthetic substrates such as 4-methylumbelliferyl-β-D-cellobioside, 4-nitrophenyl-β-D-cellobioside and 4-nitrophenyl-β-D-lactoside but was non-hydrolytic towards Avicel or carboxymethyl cellulose. GH1254 catalyzed optimally at 35 °C and maintained 70% of its activity at 25 °C. This enzyme is thus potentially useful in food industries employing low-temperature conditions.
  4. Vakhshiteh F, Allaudin ZN, Lila MA, Abbasiliasi S, Ajdari Z
    Mol Biotechnol, 2015 Jan;57(1):75-83.
    PMID: 25218408 DOI: 10.1007/s12033-014-9803-8
    Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.
  5. Patil RV, Hadawale KN, Ramli ANM, Wadkar SS, Bhuyar P
    Mol Biotechnol, 2023 Jun;65(6):833-848.
    PMID: 36544065 DOI: 10.1007/s12033-022-00633-7
    In plant development, flowering is the most widely studied process. Floral forms show large diversity in different species due to simple variations in basic architecture. To determine the floral gene expression during the past decade, MADS-box genes have identified as key regulators in both reproductive and vegetative plant development. Traditional genetics and functional genomics tools are now available to elucidate the expression and function of this complex gene family on a much larger scale. Moreover, comparative analysis of the MADS-box genes in diverse flowering and non-flowering plants, boosted by various molecular technologies such as ChIP and next-generation DNA sequencing, contributes to our understanding of how this important gene family has expanded during the evolution of land plants. Likewise, the big data analysis revealed combined activity of transcriptional regulators and floral organ identity factors regulate the flower developmental programs. Thus, with the help of cutting-edge technologies like RNA-Sequencing, sex determination is now better understood in few non-model plants Therefore, the recent advances in next-generation sequencing (NGS) should enable researchers to identify the full range of floral gene functions, which will significantly help to understand plant development and evolution. This review summarizes the floral homeotic genes in model and non-model species to understand the flower development genes and dioecy evolution.
  6. Gammatantrawet N, Nguyễn CT, Susawaengsup C, Ramli ANM, Tongkoom K, Chatsungnoen T, et al.
    Mol Biotechnol, 2024 Mar 20.
    PMID: 38507018 DOI: 10.1007/s12033-024-01122-9
    The world of pharmaceutical research has been increasingly turning its gaze toward the treasure trove of natural products in search of novel drugs and therapeutic agents. Amidst the vast array of medicinal plants that dot our planet, the Asclepiadaceae family unexplored species have piqued the interest of researchers. Both medicinal plants are indigenous to specific regions and have been integral to traditional medicine systems for centuries. This systematic review aims to provide a comprehensive summary of the current knowledge regarding the phytochemical profile of these plants and their potential implications in the pharmaceutical industry. These plants are rich in phytochemical constituents such as alkaloids, flavonoids, terpenoids, phenolic compounds, glycosides, and saponins. These constituents have been found to exhibit a range of pharmacological activities. They have antimicrobial properties, providing a defense against various microorganisms. They also show anti-inflammatory properties, helping to reduce inflammation in the body. In addition, these plants have antioxidant properties, which help protect cells from damage by harmful free radicals. They have shown anticancer activity, offering potential for cancer treatment. Their neuroprotective properties could be beneficial in treating neurological disorders. The analgesic properties of these plants could be harnessed for pain relief. Furthermore, they have antidiabetic properties, offering potential for diabetes management. The hope is that this review will stimulate further research into these fascinating plants and contribute to discovering new drugs from natural herbs.
  7. Ng YS, Chan DJC
    Mol Biotechnol, 2023 Aug 31.
    PMID: 37651079 DOI: 10.1007/s12033-023-00853-5
    Membrane distillation (MD) has lower operating temperature and potential to recycle waste heat for desalination which catches much attention of the researchers in the recent years. However, the biofouling is still a challenging hurdle to be overcome for such applications. The microbial growth rate, secretion and biofilm formation are sensitive to heat. Membrane distillation is a thermally driven separation, so the increase of temperature in the seawater feed could influence the extent of biofouling on the unit parts. In this review, we present the effect of temperature on algal growth, the range of temperature the microbes, marine algae and planktons able to survive and the changes to those planktons once exceed the critical temperature. Thermal effect on the biofilm, its composition and properties are discussed as well, with association of the biofilm secreting microbes, but the study related to membrane distillation unit seems to be lacking and MD biofouling factors are not fully understood. Characterization of the algae, biofilm and EPS that govern biofouling are discussed. This information not only will help in designing future studies to fill up the knowledge gaps in biofouling of membrane distillation, but also to some extent, assist in pointing out possible fouling factors and predicting the degree of biofouling in the membrane distillation unit.
  8. Nizan IEF, Kamaruddin K, Ong PW, Ramli Z, Singh R, Rose RJ, et al.
    Mol Biotechnol, 2022 Feb 02.
    PMID: 35107753 DOI: 10.1007/s12033-022-00450-y
    EgENOD93 was first identified in a cDNA microarray study of oil palm tissue culture where it was highly expressed in leaf explants with embryogenic potential. Functional characterization via an RNA interference study of its orthologue in Medicago truncatula demonstrated a significant role of this gene in somatic embryo formation. In this study, EgENOD93 was overexpressed in the important model plant Arabidopsis thaliana to investigate the embryogenic potential of EgENOD93 transgenic Arabidopsis explants compared to explants from control plants (pMDC140 and WT). Experiments using leaf explants revealed higher numbers of regenerated shoots at day 27 in all the homozygous transgenic Arabidopsis cultures (Tg01, Tg02 and Tg03) compared to controls. The expression level of EgENOD93 in Arabidopsis cultures was quantified using reverse transcription quantitative real-time PCR (RT-qPCR). The results supported the overexpression of this gene in transgenic Arabidopsis cultures, with 6 and 10 times higher expression of EgENOD93 in callus at Day 9 and Day 20, respectively. Overall, the results support the role of EgENOD93 in the enhancement of shoot regeneration in transgenic Arabidopsis. This together with the previous results observed in oil palm and Medicago truncatula suggests that ENOD93 plays a key role in the induction of somatic embryogenesis. A similarity to early nodulation-like ontogeny is possible.
  9. Chee MJ, Lycett GW, Khoo TJ, Chin CF
    Mol Biotechnol, 2017 Jan;59(1):1-8.
    PMID: 27826796 DOI: 10.1007/s12033-016-9986-2
    Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.
  10. Ng CL, Lim TS, Choong YS
    Mol Biotechnol, 2023 Sep 24.
    PMID: 37742298 DOI: 10.1007/s12033-023-00885-x
    Since the advent of hybridoma technology in the year 1975, it took a decade to witness the first approved monoclonal antibody Orthoclone OKT39 (muromonab-CD3) in the year 1986. Since then, continuous strides have been made to engineer antibodies for specific desired effects. The engineering efforts were not confined to only the variable domains of the antibody but also included the fragment crystallizable (Fc) region that influences the immune response and serum half-life. Engineering of the Fc fragment would have a profound effect on the therapeutic dose, antibody-dependent cell-mediated cytotoxicity as well as antibody-dependent cellular phagocytosis. The integration of computational techniques into antibody engineering designs has allowed for the generation of testable hypotheses and guided the rational antibody design framework prior to further experimental evaluations. In this article, we discuss the recent works in the Fc-fused molecule design that involves computational techniques. We also summarize the usefulness of in silico techniques to aid Fc-fused molecule design and analysis for the therapeutics application.
  11. Tan KL, Chia WC, How CW, Tor YS, Show PL, Looi QHD, et al.
    Mol Biotechnol, 2021 Sep;63(9):780-791.
    PMID: 34061307 DOI: 10.1007/s12033-021-00339-2
    The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs' size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.
  12. Duff-Farrier CRA, Mbanzibwa DR, Nanyiti S, Bunawan H, Pablo-Rodriguez JL, Tomlinson KR, et al.
    Mol Biotechnol, 2019 Feb;61(2):93-101.
    PMID: 30484144 DOI: 10.1007/s12033-018-0139-7
    Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.
  13. Mohd Rasid NH, Abdul Halid N, Song AA, Sabri S, Saari N, Hasan H
    Mol Biotechnol, 2023 Jun;65(6):861-870.
    PMID: 36273370 DOI: 10.1007/s12033-022-00584-z
    There is an increasing demand for natural food preservatives due to consumers' concern on the negative effects of chemical preservatives in food products. Nisin (bacteriocin) is an effective food biopreservative that has been approved globally. However, its low yield proves to be a limiting factor and must be addressed to meet the increasingly high demand from the food industry. The present work thus investigated the effects of individual and combined fermentation factors on Lactococcus lactis ATCC 11454 growth and nisin activity using the one-factor-at-a-time (OFAT) method. The level of each factor that gave the highest nisin production was then selected and combined to further improve its activity. The best combined conditions for highest cell growth and nisin activity were 30 °C, pH 6.0, and mild agitation with the addition of 1.0% w/v glucose, 1.0% w/v skim milk, and 0.5% v/v Tween 20. This increased nisin production by 22.7% as compared to control (basic condition). The present work provided critical information on the relationship between fermentation conditions, growth, and nisin activity of L. lactis ATCC 11454 that could be explored to understand the potential and limitation of the strain. This fermentation strategy can also serve as a benchmark to further enhance the production of bacteriocin or other biopreservative compounds.
  14. Chelliah S, Velappan RD, Lim KT, Swee CWK, Nor Rashid N, Rothan HA, et al.
    Mol Biotechnol, 2020 May;62(5):289-296.
    PMID: 32185600 DOI: 10.1007/s12033-020-00244-0
    Pasteurella multocida is the main cause of haemorrhagic septicaemia (HS) outbreak in livestock, such as cattle and buffaloes. Conventional vaccines such as alum-precipitated or oil-adjuvant broth bacterins were injected subcutaneously to provide protection against HS. However, the immunity developed is only for short term and needed to be administered frequently. In our previous study, a short gene fragment from Pasteurella multocida serotype B was obtained via shotgun cloning technique and later was cloned into bacterial expression system. pQE32-ABA392 was found to possess immunogenic activity towards HS when tested in vivo in rat model. In this study, the targeted gene fragment of ABA392 was sub-cloned into a DNA expression vector pVAX1 and named as pVAX1-ABA392. The new recombinant vaccine was stable and expressed on mammalian cell lines. Serum sample collected from a group of vaccinated rats for ELISA test shows that the antibody in immunized rats was present at high titer and can be tested as a vaccine candidate with challenge in further studies. This successful recombinant vaccine is immunogenic and potentially could be used as vaccine in future against HS.
  15. Abd-Aziz N, Tan BC, Rejab NA, Othman RY, Khalid N
    Mol Biotechnol, 2020 Apr;62(4):240-251.
    PMID: 32108286 DOI: 10.1007/s12033-020-00242-2
    In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
  16. Wilawan B, Chan SS, Ling TC, Show PL, Ng EP, Jonglertjunya W, et al.
    Mol Biotechnol, 2024 Mar;66(3):402-423.
    PMID: 37270443 DOI: 10.1007/s12033-023-00768-1
    The demand for astaxanthin has been increasing for many health applications ranging from pharmaceuticals, food, cosmetics, and aquaculture due to its bioactive properties. Haematococcus pluvialis is widely recognized as the microalgae species with the highest natural accumulation of astaxanthin, which has made it a valuable source for industrial production. Astaxanthin produced by other sources such as chemical synthesis or fermentation are often produced in the cis configuration, which has been shown to have lower bioactivity. Additionally, some sources of astaxanthin, such as shrimp, may denature or degrade when exposed to high temperatures, which can result in a loss of bioactivity. Producing natural astaxanthin through the cultivation of H. pluvialis is presently a demanding and time-consuming task, which incurs high expenses and restricts the cost-effective industrial production of this valuable substance. The production of astaxanthin occurs through two distinct pathways, namely the cytosolic mevalonate pathway and the chloroplast methylerythritol phosphate (MEP) pathway. The latest advancements in enhancing product quality and extracting techniques at a reasonable cost are emphasized in this review. The comparative of specific extraction processes of H. pluvialis biological astaxanthin production that may be applied to large-scale industries were assessed. The article covers a contemporary approach to optimizing microalgae culture for increased astaxanthin content, as well as obtaining preliminary data on the sustainability of astaxanthin production and astaxanthin marketing information.
  17. Mohamed ME, Pahirulzaman KA, Lazarus CM
    Mol Biotechnol, 2016 Mar;58(3):172-8.
    PMID: 26718544 DOI: 10.1007/s12033-015-9911-0
    Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone). Pyrethrolone is generated from 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid, which originates from α-linolenic acid via the jasmonic acid biosynthetic cascade. The first four genes in this cascade, encoding lipoxygenase 2, allene-oxide synthase, allene-oxide cyclase 2 and 12-oxophytodienoic acid reductase 3, were amplified from an Arabidopsis thaliana cDNA library, cloned in a purpose-built fungal multigene expression vector and expressed in Aspergillus oryzae. HPLC-MS analysis of the transgenic fungus homogenate gave good evidence for the presence of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid.
  18. Alias FL, Nezhad NG, Normi YM, Ali MSM, Budiman C, Leow TC
    Mol Biotechnol, 2023 Nov;65(11):1737-1749.
    PMID: 36971996 DOI: 10.1007/s12033-023-00725-y
    Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.
  19. Ramli N, Abd-Aziz S, Alitheen NB, Hassan MA, Maeda T
    Mol Biotechnol, 2013 Jul;54(3):961-8.
    PMID: 23338983 DOI: 10.1007/s12033-013-9647-7
    Regulation of RNA transcription in controlling the expression of genes at promoter and terminator regions is crucial as the interaction of RNA polymerase occurred at both sites. Gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. NR5 UPM isolated in the previous study was used for further construction of pTZCGT-SS, pTZCGT-BS and pTZCGT-BT expression systems for enhancement of CGTase production. The putative promoter regions, -35 and -10 sequences were found in the upstream of the mature gene start codon. Whereas, long inverted repeats sequences which can form a stable stem and loop structure was found downstream of the open reading frame (ORF) of Bacillus sp. NR5 UPM CGTase. The construction of E. coli strain harbouring pTZCGT-BS showed increment of 3.2-fold in CGTase activity compared to the wild type producer. However, insertion of terminator downstream of CGTase gene in E. coli strain harbouring pTZCGT-BT only resulted in 4.42 % increment of CGTase production compared to E. coli strain containing pTZCGT-BS, perhaps due to low intrinsic termination efficiency. Thus, it is suggested that the insertion of the putative promoter regions upstream of the coding sequence for the construction of CGTase expression system will further enhance in the recombinant enzyme production.
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