Heterologous Expression and Characterization of Plant Lipase LIP2 from Elaeis guineensis Jacq. Oil Palm Mesocarp in Escherichia coli
Round 1
Reviewer 1 Report
In their manuscript Din et al. showed biochemical and structural features of Elaeis guineensis Jacq. oil palm mesocarp lipase, in which LIP2 gene was isolated, expressed, purified and characterized through Escherichia coli microbial recombinant system. The results are interesting in the way of increasing existing knowledge of plant lipases, although not completely new. Design of experiments is mostly well done, with some exceptions indicated in my comments and with some discussible results. Chapter ˝Materials and methods˝ is not sufficiently well written, references were not cited in accordance with instructions to authors, as well as literature list. The manuscript is written in quite poor English, resulting in some parts that are hardly comprehensible. My opinion is that in the present form the manuscript is not acceptable for publishing.
Here are my comments through chapters of the manuscript:
Chapter ˝Results˝:
In the line 132 there is a sentence: ˝Primary screening was carried out by streaking the transformant on tributyrin-LB agar (50 pg/mL ampicillin) due to its sensitivity over other detection methods.˝ Authors should state wich other methods, what sensitivity? It is not clear what the authors were want to say in this sentence. Methods listed further in the chapter are not satisfactorily described in the Materials and methods chapter as well.
Fig. 3. Which samples are 1, 2 and 3 in the a) part of the picture? What is extracellular lysate?? Which sample(s) are separated in the gel for the zymogram (b)? Why the same gel is not used for protein staining and His-tag staining?
In the line 177 the sentence: ˝A simple and rapid colorimetric assay to determine lipase activity by the amount of free fatty acids released.˝ is not finished? What were authors want to say with it?
Text in the lines 184-188 is extremely poor written and should be cleared and improved.
In the line 190 authors’ state: ˝The co-expression with pET51b+ vector in facilitating the release of LIP2 lipase….˝. What is co-expressed with vector??
In the line 194. sentence :˝Low level expression of soluble enzymes upon over induction with higher concentration of IPTG.˝ - has no any meaning? What were authors want to say with it?
In the line 200. authors stated that ˝Low temperatures (20-40 °C) increased the solubility of the expressed protein and hence secretory expression.˝ On which basis authors conclude that the increased solubility is reason for increased expression?
Sentences in lines 201 – 203: ˝While high temperature whereas more than 50 °C have low secretion of LIP2 lipase. While the effect of postinduction time of LIP2 lipase release was investigated by inducing the culture with 0.3 mM of IPTG at 37 °C up to 24 h as in Figure 4 d.˝ - have extremely bad syntax and should be clarified and rewritten.
Is it possible that the authors cultivated bacterial cells for 12 hours at 50 – 70 °C? As far as I know the highest temp. that E. coli could be growth at is 48.5 °C. Furthermore, later on they find that Tm of the enzyme is around 65 °C. So, even if they succeeded to grow cells at so high temperatures, how could they state that the secretion is lower instead that the enzyme is denatured (12 hours on high temperature!!). The authors should additionally explain and give some evidence for that result. The OD600 should be shown in the fig 4b and 4c as well. It is not clear which Series (1 or 2) is shown on which axes (OD600 or enzyme activity) in fig 4d.
Figure 5B shows fractions with unbound material – where are the proteins? In thy Lysate (4A) there is a bunch of proteins, and in unbound material there is no proteins whatsoever?
In line 292 – according to the results shown in fig 8. Rb+, Cu2+, Ca2+, Na+, and K+ caused decrease of activity TO 88.57%, 37.28%, 26.27%, 20.6%, and 8.01% - not BY. How did authors explain different effects of ions in 5 mM and 1 mM concentrations? What was control?
The sentence: ˝The enzyme was pre-incubated for 30 min in the presence of different organic solvents with different organic solvents with different level of resistance in different reaction systems.˝- is not clear. What were authors want to say with it?
Chapter ˝Discussion˝:
In the line 461. Table 2 is mentioned – I don't see any tables in the manuscript? (˝Nevertheless, LIP2 was purified successfully with higher yield and fold purification (Table 2) compared with other oil palm mesocarp lipase through the microbial recombinant system.˝)? Anyway, I don’t think that is correct to compare purification of recombinant protein overexpressed in E. coli with purification of native enzyme from plant material.
In the line 532 authors stated that ˝In some cases, if it is too much or too little percentage of metal ion added, there are possibilities that they will get access to the site more often than the substrate in terms of competitiveness or disrupt the folding of the enzymes near the pocket and active site˝. Mechanism of competitive inhibition is well known – too little percentage of anything cannot act as competitive inhibitor. Furthermore, to be competitive inhibitor the structure of the molecule has to be similar to the structure of original substrate at least partly – and that is not case here.
Chapter ˝Materials and methods˝:
Authors stated that synthesis of cDNA was prepared from tRNA sample? tRNA usually stands for transport DNA? I suppose the authors were meant to say total RNA? Further in the text is indicated that some tags were added to the gene – which tags and how were they added? Furthermore, it was stated that transformants are ˝selected on Tributyrin agar plates for resistance to the corresponding antibiotics˝ - which antibiotics? Why did they used Tributyrin agar plates for selection on antibiotics?
Methods for testing lipolytic activity are inadequately explained. Media composition, conditions and procedure should be described in details.
Authors stated that Zymogram analysis and His-tag staining is done according to reference 41., that is complete book (Pratt, C. W., & Cornely, K. (2004). Essential biochemistry. Hoboken, NJ: Wiley). Methods should be described in more details and exact pages in the book should be stated in the reference, not complete book.
In the chapter ˝Optimization of secretory expression˝ the method for isolation of intracellular material is described instead of optimization of ˝secretory expression˝ as stated in the chapter title? Furthermore, in this chapter is stated that the cells are cultivated in ˝blue cap bottles˝. Any kind of bottle could have blue cap. If authors for some reason need to specify in which exactly kind of bottles the cells were cultivated it should be more specific than ˝blue caps˝. On the other hand, conditions of cultivation are not given at all (temperature, rpm, time of cultivation, media composition etc.). What cannot be seen from this chapter at all is in which way is the extracellular enzyme activity collected and assayed? Is growth media concentrated in some way, or partly purified, or used in original dilution and composition? There is no data of the quantity of material used in assaying activities at all, for neither fraction. In connection with this, in the lane 149 in text, there is the sentence ˝Two main samples were prepared: extracellular crude lysate and intracellular crude lysate (soluble and insoluble)˝. I don’t understand what should be ˝extracellular lysate˝? Lysate is material obtained after cells lysis, so what is extracellular lysate? Furthermore it is stated that ˝ Crude lysate was subjected to treatments by performing lipase assay from Wickler & Stuckman, (1979) with various parameters on lipase secretion˝(line 635). This sentence is incomprehensible. If authors are to assay effect of various parameters on lipase secretion why and how should they treat crude cell lysate in some way (whichever it might be – and that is not stated or explained in here at all as well)? In conclusion – from this chapter it is absolutely not clear how and why ˝Optimization of secretory expression˝ is conducted.
˝Lipase assay˝ chapter is poor written – there is no type nor concentration of pNP substrates used, as well as definition of enzyme activity (rate in which fatty acids are released in 1 min does not mean anything).
˝ Biochemical characterization of LIP2˝ chapter is not finished – I don’t know how much of the text is missing. Furthermore, it is not stated in which way the measurement of activity after testing enzyme stability at different temperatures and pH values is done (in which way are pH and T adjusted to standard conditions of measurement)?
After all corrections, additions and clarifications are done the manuscript might be reconsidered for publication.
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript submitted by Mohd Hadzdee Mohd Din summarizes the main results regarding the heterologous expression and characterization of plant lipase (LIP2) from Elaeis guineensis Jacq. oil palm mesocarp using Escherichia coli as host for protein expression. The topics fits to the scope of the journal and it is of low soundness because is a conventional study of heterologous expression of a enzyme with potential uses in biotechnology.
The manuscript is in general organized and figures support the discussion. English grammar must be revised by a native English speaker. Comments has been embedded through the MS in order to help the authors to improve this version.
Comments for author File: Comments.pdf
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Dear Sir,
I have checked the revised version of the manuscript ˝Heterologous expression and characterization of plant lipase LIP2 from Elaeis guineensis Jacq. oil palm mesocarp in Escherichia coli˝. Manuscript is generally improved, but there are still some points that should be addressed before publication.
The points that should be improved are stated below:
line 332 (and later 553-555) Authors stated that ˝In contrast, it has better activity towards the esters of long-chain and medium-chain fatty acids as compared those of small chain fatty acids, except for C:16, as LIP2 did not show any activity at all.˝. In the same time, it can be seen (Fig 10) that enzyme has better activity towards C2 than to C8, C10 and C12, while towards C4 and C10 has practically the same activity. So why the authors concluded that it has better activity towards long-chain fatty acids?
line 654 -657 .............. I think that the method for His-tag staining is not proper, as well as the reference given (51 - review on proteinase zymograms)? That is described as practically the same method as the method for activity staining, the only difference is in the period of gel incubation on tributyrine plates? In the Fig 3b and 3c it can be seen that the results of these two methods are different (one is performed on tributyrine plates - activity staining Fig3b - and clear zones are visible on the plate, while on Fig 3c the PAG is shown in which distinct bands can be seen, including standard, that were visualised by some sort of colour).
There are also some minor corrections that should be done as indicated below:
line 19 ˝Gene analysis LIP2 lipase...˝ should be ˝Gene analysis of LIP2 lipase...˝
line 59 ˝The lipases from the mesocarp were proven to be serine hydrolase˝ should be ˝The lipases from the mesocarp were proven to 59 be serine hydrolases˝
line 637 ..... in the sentence ˝Protein samples were heated at 100 for 5 min to...˝ 0C is omitted (it should be ˝Protein samples were heated at 100 0C for 5 min to˝....)
My opinion is that after these corrections manuscript might be acceptable for publication.
Author Response
Please see the attachment, thank you.
Author Response File: Author Response.pdf
Reviewer 2 Report
Thank you very much for your time and effrot addressing all the comments made by this reviewer.
Author Response
Thank you for the previous comments on the manuscript.
