BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Preparation and comparative characterization
of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic
biodiesel production
Shinji Hama&Sriappareddy Tamalampudi&
Yuya Suzuki&Ayumi Yoshida&Hideki Fukuda&
Akihiko Kondo
Received:12July2008/Revised:23August2008/Accepted:26August2008
#Springer-Verlag2008
Abstract In this paper,we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production.Aspergillus oryzae,transformed with a heterologous lipase-encoding gene from Fusarium heter-osporum,produced fully processed and active forms of recombinant F.heterosporum lipase(FHL).Cell immobili-zation within porous biomass support particles enabled the convenient usage of FHL-producing A.oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis.The addition of5%water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides,resulting in the final methyl ester content of94%even in the tenth batch cycle.A comparative study showed that FHL-producing A.oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae,the previously developed whole-cell biocatalyst.Although both FHL and R.oryzae lipase exhibit1,3-regiospecificity towards triglyceride,R.oryzae accumulated a much higher amount of sn?2isomers of partial glycerides,whereas FHL-producing A.oryzae maintained a low level of the sn?2 isomers.This is probably because FHL efficiently facilitates the acyl migration from the sn?2to the sn?1(3)position in partial glycerides.These findings indicate that the newly developed FHL-producing A.oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production. Keywords Whole-cell biocatalyst.
Biomass support particles.Filamentous fungi.
Fusarium heterosporum lipase.Methanolysis Introduction
The enzymatic production of fatty acid methyl esters(i.e., biodiesel fuel)has been investigated extensively using extracellular or intracellular lipases,both of which can catalyze the transesterification of triglycerides in either aqueous(Kaieda et al.2001;Kamini and Iefuji2001)or nonaqueous systems(Iso et al.2001;Shimada et al.1999). Although the application of lipases in biodiesel production is considered as an effective means of avoiding conventional drawbacks(e.g.,contamination of products with alkaline),the cost of lipase preparation can be an obstacle to the widespread use of enzymatic processing.
Utilizing lipase-producing microorganisms immobilized within porous biomass support particles(BSPs;Atkinson et al.1979)is effective in improving cost efficiency because no complex purification procedures are required (Fukuda et al.2001).Several researchers(Ban et al.2001; Hama et al.2004;Li et al.2007)have attempted to use
Appl Microbiol Biotechnol
DOI10.1007/s00253-008-1689-6
S.Hama
:S.Tamalampudi:A.Yoshida
Bio-energy Corporation,Research and Development Laboratory, 2-9-7Minaminanamatsu,
Amagasaki660-0053,Japan
S.Tamalampudi
:A.Yoshida:H.Fukuda
Organization of Advanced Science and Technology,
Kobe University,
1-1Rokkodai,Nada,
Kobe657-8501,Japan
Y.Suzuki
:A.Kondo(*)
Department of Chemical Science and Engineering,
Faculty of Engineering,Kobe University,
1-1Rokkodai,Nada,
Kobe657-8501,Japan
e-mail:akondo@kobe-u.ac.jp
BSP-immobilized Rhizopus oryzae for biodiesel production and reported that immobilized R.oryzae can efficiently catalyze the methanolysis of plant oils with the addition of a certain amount of water.For methanolysis in a water-containing system,the following reaction mechanism has been proposed:the oil is first hydrolyzed to free fatty acids, and the fatty acids produced are esterified with methanol. Although R.oryzae lipase(ROL)exhibits1,3-regiospecificity towards triglyceride,the methyl ester(ME)content in the reaction mixture exceeds80%owing to the acyl migration in partial glycerides(Kaieda et al.1999).Moreover,studies using R.oryzae cells also include stabilizing the lipase activity with glutaraldehyde(Ban et al.2002)and a packed-bed reactor system(Hama et al.2007).Such extensive research studies have made the immobilized R.oryzae a promising candidate for enzymatic biodiesel production.
However,the detailed analysis of R.oryzae-catalyzed methanolysis led to a new finding that the accumulation of partial glycerides,which have acyl chains in their sn?2 position,can be one of the rate-limiting steps(Oda et al. 2005).This trend was found to be more pronounced in repeated-batch methanolysis.Therefore,it has become necessary to develop a new whole-cell biocatalyst possessing catalytic properties different from those of ROL.
A useful approach to meeting the aforementioned requirement is to use a recombinant microorganism carrying a heterologous lipase-encoding gene.Among the several microorganisms utilized so far as a host,Aspergillus species are well-recognized for their ability to produce a large amount of proteins(Christensen et al.1988). Recently,the molecular biology of Aspergillus species has been extensively studied(Abe et al.2006),and improved promoters have been developed for the high-level production of heterologous proteins(Minetoki et al.1998).Therefore, BSP-immobilized Aspergillus species overexpressing and accumulating recombinant lipases are expected to be an effective whole-cell biocatalyst for industrial biodiesel production.
In previous studies,several recombinant lipase genes were expressed in Aspergillus oryzae under the control of the improved promoter,P-No8142(Minetoki et al.1998; Ozeki et al.1996).Fusarium heterosporum lipase(FHL), isolated by Shimada et al.(1993),is a solvent-tolerant1,3-regiospecific lipase.In a study using lipases immobilized on anion exchange resins,Nagao et al.(2001)reported that FHL is effective for acidolysis in a water-containing system and found that the half-life of FHL is significantly longer than those of Rhizopus and Rhizomucor lipases.Therefore, in this study,we focused on the preparation of an FHL-producing recombinant A.oryzae and its application to methanolysis in a water-containing system.
Here,we report that the immobilized A.oryzae carrying FHL exhibits high conversion of triglycerides to MEs.Additionally,the characteristics of reaction products
obtained using recombinant A.oryzae were investigated
and compared with those of reaction products obtained
using immobilized R.oryzae,the previously developed
whole-cell biocatalyst.Thus,in this study,we provide the
first example of utilizing recombinant fungal whole cells
for biodiesel fuel production.
Materials and methods
Strains,media,and culture conditions
The Escherichia coli strain used for genetic manipulation
was Novablue(Novagen,Madison,WI,USA)and was
grown in Luria–Bertani(LB)medium(1%tryptone,0.5%
yeast extract,0.5%sodium chloride,w/v)containing
0.1mg/ml ampicillin.The host strain A.oryzae niaD300
was a niaD mutant derived from the wild-type strain
RIB40(Unkles et al.1989)and routinely maintained on
Czapek-Dox(CD)medium[2%glucose,0.2%NaNO2,
0.1%KH2PO4,0.05%MgSO4·7H2O,0.2%KCl(w/v),
0.8M NaCl,0.001%(v/v)trace element solution(2%
CuSO4·5H2O,1%FeSO4·7H2O,0.1%ZnSO4·7H2O,0.1%
MnSO4·7H2O,0.1%AlCl3,w/v),adjusted to pH 5.5]
containing 1.5%(w/v)agar.Fungal transformants were
selected on1.5%(w/v)agar-containing CD-NO3medium,
in which NaNO3was used instead of NaNO2,and their
spores were suspended in0.01%(w/v)Tween80.The
solution containing spores was aseptically inoculated to a
Sakaguchi flask(500ml)containing100ml of DP medium
(2%glucose,2%polypeptone,1%KH2PO4,0.2%NaNO3,
0.05%MgSO4·7H2O,adjusted to pH6.8).The flasks were
incubated at30°C for96h on a reciprocal shaker(150
oscillations/min;amplitude,50mm).After cultivation,the
culture broth was collected by filtration,whereas the
cultivated mycelia were washed thoroughly with distilled
water and lyophilized for24h before use in subsequent
experiments.
R.oryzae IFO4697was routinely maintained on an agar
slant made from4%potato dextrose agar and2%agar.Per
liter of distilled water,the basal medium contained:olive
oil3g;polypeptone70g;NaNO31.0g;KH2PO41.0g;
MgSO4?7H2O0.5g;adjusted to pH5.6.BSP-immobilized R.oryzae cells were prepared as described previously(Ban
et al.2001).
Construction of expression plasmids
The gene encoding F.heterosporum lipase(FHL;GenBank
accession number S77816)was amplified from pYGF2
(Nagao et al.1996)using two primers:FHL-fw-Hin dIII(5′-
TACAAGCTTATGATGCTCGTCCTATCTC-3′)and FHL-
Appl Microbiol Biotechnol
rv-Spe I (5′-GAACTAGTCTAAATCATCTGCTTAAC-3′).Polymerase chain reaction was carried out using KOD Plus polymerase (Toyobo,Osaka,Japan).The amplified fragment was digested with Hin dIII and Spe I,and inserted into pNAN8142(Minetoki et al.1998;Ozeki et al.1996).The resulting plasmid was designated pNAN8142FHL and represented schematically in Fig.1.The successful construc-tion of the desired plasmid was confirmed by nucleotide sequencing (ABI Prism 3100-Avant Genetic Analyzer,Applied Biosystems,Tokyo,Japan).Transformation procedure
The transformations of E.coli and A.oryzae were carried out according to the methods described by Hanahan (1983)and Gomi et al.(1987),respectively.A.oryzae protoplasts were prepared from mycelia grown at 30°C for 48h using Yatalase (Takara Bio,Shiga,Japan).The constructed plasmids were digested with Bam HI prior to transformation.Linearization of the plasmids facilitated the single-copy integration at the niaD locus in the genome.Preparation of BSP-immobilized cells
Sakaguchi flasks (500ml)containing 100ml of DP medium,fungal spores,and 150BSPs were incubated at
30°C for 96h on a reciprocal shaker (150oscillations/min;amplitude,50mm).The BSPs used for cell immobilization were 6-mm cubes of reticulated polyurethane foam (Bridge-stone,Osaka,Japan)with a particle voidage of more than 97%and a pore size of 50pores per linear inch (ppi).The A.oryzae cells became well immobilized within the BSPs as a natural consequence of their growth during shake-flask cultivation.The resulting BSP-immobilized cells were separated from the culture broth by filtration,washed with distilled water,and lyophilized for 24h before use in subsequent experi-ments.Immobilized cell weight within a BSP was measured as described previously (Ban et al.2001).Detection of recombinant lipases
Analytical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE)was performed using a 12.5%gel.The proteins were then stained with Coomassie brilliant blue R-250.For N-terminal amino acid sequencing analysis,the separated proteins were electro-blotted onto a polyvinylidene difluoride membrane (Milli-pore,Boston,MA,USA)for 1h at 2.0mA/cm 2and room temperature.The N-terminal sequence of proteins blotted onto the membrane was determined by automated Edman degradation using a protein sequencer (model 492;Applied Biosystems,Tokyo,Japan).Measurement of lipase activity
The lipase hydrolytic activity in culture broth and cells was measured with Lipase Kit S (Dainippon Pharmaceutical,Osaka,Japan)according to the protocol specified by the supplier,and the resulting values were expressed in interna-tional units (IU).One unit of lipase activity was defined as the amount of enzyme catalyzing the formation of 1μmol of 2,3-dimercaptopropan-1-ol from 2,3-dimercaptopropan-1-ol tributyl ester per minute.Methanolysis reaction
Methanolysis reaction was carried out in a screw-capped bottle.The reaction mixture contained 9.65g of soybean oil (Wako Pure Chemical Industries,Osaka,Japan),0.35g of methanol (one molar equivalent to 9.65g of soybean oil),0.1–1.5ml of distilled water,and 50BSPs retaining dry immobilized cells.To fully convert the oil to its corresponding methyl esters,0.35g of methanol was added stepwise at 24,48,and 72h.The screw-capped bottles were incubated at 30°C on a reciprocal shaker (150oscillations/min;amplitude,50mm).For the repeated use of the enzyme,the BSP-immobilized cells were dried at room temperature after washing with tap water,and methanolysis was carried out ten times with a fresh reaction mixture.
I
97
1002Mature
Fig.1Expression plasmid pNAN8142FHL.The FHL precursor consists of a signal sequence (pre;16amino acids),a prosequence (pro;16amino acids),and a mature region (mature;301amino acids).The plasmid construct was expressed under the control of P-No8142,which is the improved promoter region of No.8AN genes (Ozeki et al.1996).The niaD gene was used as a selectable marker
Appl Microbiol Biotechnol
Gas chromatography analysis
Samples were obtained from the reaction mixture at specified intervals and centrifuged at12,000rpm for 5min.The upper oil layer was analyzed using a GC-2010 gas chromatograph(Shimadzu,Kyoto,Japan)connected to a ZB-5HT capillary column(0.25mm×15m;Phenomenex, USA).The temperatures of the injector and detector were set at320°C and370°C,respectively.The column temperature was initially maintained at130°C for 2min,increased to350°C at10°C/min,then to370°C at 7°C/min,and finally maintained at this temperature for 10min.Chromatographic peaks were identified by com-parison of their retention times with those of a standard solution.Tricaprylin served as the internal standard for the quantification of the methyl ester(ME),free fatty acid (FFA),monoglyceride(MG),diglyceride(DG),and triglyc-eride(TG)in the reaction mixture.The contents were calculated as the ratio of each material present in the reaction mixture without water and glycerol.The detailed procedure for the determination of ME content was described in a previous paper(Ban et al.2001).
The regioisomers of partial glycerides such as MG and DG were analyzed by the method of Satou et al.(2004). Samples(upper oil layer)and tricaprylin were dissolved in 1ml of hexane,into which200μl of N,O-bis(trimethyl-silyl) acetamide was added.The resulting solution was kept at room temperature for1h and analyzed using a gas chromatograph under the conditions described above.The silylation of the samples enabled the separation of the partial glycerides into the each isomer,1-MG,2-MG,1,2-DG,and 1,3-DG.
Results
Expression and hydrolytic activity of recombinant FHL Cell immobilization within the polyurethane foam BSPs induces pellet formation of A.oryzae near the surface of the support particle.Since such a morphological change in the wild-type filamentous fungi markedly affects enzyme secretion(Hama et al.2006),we investigated the lipase activities of the recombinant A.oryzae cultivated in suspension and immobilized cell cultures.As shown in Table1,cells carrying pNAN8142FHL in suspension culture showed9890IU/L of extracellular lipase activity, which is56times that of the parent strain, A.oryzae niaD300.In immobilized cell culture,the extracellular lipase activity of the transformant showed only504IU/L, whereas the specific intracellular lipase activity reached 54IU/g-dry cell weight,which is18times that of the parent strain.As illustrated in Fig.2,SDS-PAGE analysis of the supernatant in suspension culture showed a clear band at 29kDa with the N-terminal amino acid sequence of Ala-Val-Thr-Val-Thr-Thr-Gln-Asp-Leu-Ser,corresponding to the N-terminus of the mature FHL(Shimada et al.1993). In contrast,immobilized cells showed a much lower amount of the29kDa protein in the culture supernatant. The inhibition of lipase secretion is consistent with the previous study using Candida antarctica lipase(Tamalampudi et al.2007).These results indicate that A.oryzae carrying pNAN8142FHL produced fully processed and active forms of recombinant FHL and suggest that the immobilized recombinant A.oryzae inhibits lipase secretion,thereby retaining a significant amount of FHL.Subsequent experi-ments were therefore carried out using immobilized cells. Effect of water content on methanolysis
To achieve a high conversion during methanolysis,the effect of water content on methanolysis was investigated using FHL-producing A.oryzae(Fig.3).In the cases of1% and2%water,the ME content was low at less than65% even after a96-h reaction.Lipase inactivation should be responsible for the low ME content because the final ME content in the second and third methanolysis cycles in the presence of2%water reached only41.2%and8.44%, respectively(data not shown).The best result of methanolysis was obtained in the presence of5%water,with the ME
Table1Extra-and intracellular lipase activities of A.oryzae cultivated in suspension and immobilized cell cultures
Cell Extracellular lipase activity(103×IU/L)Intracellular lipase activity(10×IU/g-dry cell)
Suspension a Immobilization b Suspension Immobilization Transformant c9.89±0.410.504±0.057 2.48±0.71 5.40±0.50 niaD300d0.176±0.0600.147±0.0320.152±0.0660.297±0.066
a Cells cultivated in suspension culture for96h
b Cells cultivated in immobilized culture for96h
c A.oryzae carrying pNAN8142FHL
d A.oryza
e niaD mutant
Appl Microbiol Biotechnol
contents of88.6%and94.3%at72and96h,respectively. Although the final ME content reached90.3%in the presence of15%water,the progress of methanolysis was much slower than that in the presence of5%water.These results indicate that there exists an optimal water content,which results in both higher ME content and higher reaction rate.Subsequent experiments were therefore carried out using the reaction medium with5%water.
Repeated-batch methanolysis
Figure4shows the time course of ME content during ten repeated-batch cycles of methanolysis using FHL-producing A.oryzae or R.oryzae.Although FHL-producing A.oryzae showed a relatively lower initial reaction rate,as represented by the ME content(20.7%)after5h,the final ME content in the first cycle reached a high value of over94%.On the other hand,immobilized R.oryzae,which is the previously developed whole-cell biocatalyst,showed a higher initial reaction rate(ME content of26.6%at5h)compared with FHL-producing A.oryzae.Nevertheless,the final ME content resulted in relatively low level,with86.7%at the end of the first cycle.A significant difference in lipase stability was also observed.In the case of the FHL-producing A.oryzae,the final ME content reached a high
Marker kDa
97.4
66.2
45.0
31.0
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Fig.2SDS-PAGE of FHL in culture https://www.doczj.com/doc/ec4039361.html,ne1Suspension culture,lane2immobilized cell culture. A.oryzae carrying pNAN8142FHL was cultivated in DP medium for4days.Fifteen microliters of culture supernatant was subjected to SDS-PAGE.The gel was stained with Coomassie blue
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Fig.3Effect of water content on methanolysis.Methanolysis was carried out using a water-containing reaction medium(1–15wt.% based on oils and methanol)as a starting material.One molar equivalent of methanol to oil was added stepwise at0,24,48,and 72h.The vertical axis shows ME content in the reaction mixture after each reaction time(24–96h)
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Fig.4Time course of ME content during ten repeated-batch cycles of methanolysis.A.oryzae carrying pNAN8142FHL(closed circles)and native R.oryzae(open circles)without chemical treatment were used as catalysts.Stepwise addition of methanol was conducted
Appl Microbiol Biotechnol
value of94%even in the tenth cycle,whereas R.oryzae showed a decrease in the production rate with increasing numbers of batch cycles,to yield an ME content of only 5.79%at the end of the tenth cycle.As for R.oryzae,such a decrease in the lipase activity was easily presumed because our previous study showed the necessity of glutaraldehyde treatment for stabilizing the cells(Ban et al.2002).The result thus indicates that the FHL-producing A.oryzae exhibits high lipase stability even without chemical treatment. Comparison of reaction characteristics between FHL
and ROL
The results in Fig.4prompt the question of why FHL-producing A.oryzae,despite its lower initial reaction rate, shows a higher ME conversion in the later period of reaction.Thus,the reactant components were analyzed and compared between FHL-producing A.oryzae and R.oryzae.Figure5shows the time course of the remaining TG,DG,MG,and FFA contents in the first batch cycle of methanolysis.In the case of TG(Fig.5a),R.oryzae showed the lower content at5h,and the values rapidly decreased after the stepwise addition of methanol(see closed arrows in Fig.5a).The result suggests that,on comparison with FHL,ROL has a higher ability to produce ME from TG. This can be presumed from the fact that R.oryzae shows the higher reaction rate,which is defined as the ME production rate during5-h methanolysis from initiation (Fig.4).
As can be noted in Fig.5b,c,the FHL-producing A. oryzae showed less amounts of DG and MG in all the reaction stages,whereas R.oryzae accumulated the high levels of DG and MG,with contents of5.48%and2.74%, respectively,even after96h.As indicated by the open arrows in Fig.5c,an increase in MG content was observed, which suggests that the conversion of MG to ME is one of
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Fig.5Comparison of reactant components between FHL-producing
A.oryzae and R.oryzae.The vertical axes show triglyceride(a),
diglyceride(b),monoglyceride(c),and free fatty acid(d)contents.
Symbols represent FHL-producing A.oryzae(closed circles,triangles,
squares,and diamonds)and R.oryzae(open circles,triangles,
squares,and diamonds),respectively.The closed arrows indicate the
addition of methanol into the reaction mixture.Note that R.oryzae
shows an increase in MG content(open arrows)
Appl Microbiol Biotechnol
the rate-limiting steps during the R.oryzae -catalyzed methanolysis.When the cells were used for repeated-batch methanolysis,the difference in the accumulation level of MG became even more significant,to the extent of 0.316%(FHL-producing A.oryzae )and 20.2%(R.oryzae )at the end of the fifth batch cycle (data not shown).The FFA contents decreased rapidly when methanol was added,
indicating that MEs were produced by condensation reaction using FFA and methanol as well as by methanolysis using glycerides and methanol (Fig.5d).Also note that the FFA contents after 24–53h of methanolysis by the FHL-producing A.oryzae were relatively higher than those by R.oryzae .
Because a key to explaining the difference in the ME conversion was assumed to be the accumulation level of partial glycerides such as MG and DG,their regioisomers were analyzed.As shown in Fig.6,the FHL-producing A.oryzae produced a higher amount of 1-MG than 2-MG during methanolysis,whereas in the case of R.oryzae ,a significant amount of 2-MG remained in the reaction mixture.Moreover,in contrast to the FHL-producing A.oryzae ,R.oryzae accumulated a higher amount of 1,2-DG than 1,3-DG.Table 2shows the ratios of regioisomers (2-MG/1-MG and 1,2-DG/1,3-DG)in the reaction mixture during methanolysis by the FHL-producing A.oryzae or R.oryzae .The FHL-producing A.oryzae showed lower ratios than R.oryzae ,and this trend was more distinctly observed in the later period of the reaction.These results suggest that the FHL-producing A.oryzae can keep the sn ?2isomers of partial glycerides at a low level even in the later period of the reaction,thereby leading to a substantial improvement in ME conversion.
Discussion
In this study,we successfully prepared the immobilized A.oryzae cells expressing a heterologous lipase gene from F .heterosporum .To develop recombinant cells with a satisfactory level of lipase activity,the recombinant lipase-encoding genes should be highly expressed in the host organism.As a preliminary experiment,we evaluated the extracellular lipase production of A.oryzae carrying the single-copy integrated genes of FHL (F .heterosporum ),tglA (A.oryzae ;Kaieda et al.2004),ROL (R.oryzae ;Hama
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a
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Fig.6Representative chromatograms showing regioisomers of partial glycerides.Samples obtained from 72-h methanolysis using the FHL-producing A.oryzae (a )and R.oryzae (b )were analyzed by gas chromatography.Peak 12-MG,peak 21(3)-MG,peak 3tricaprylin as the internal standard,peak 41,2-DG,peak 51,3-DG
Table 2Ratio of regioisomers of partial glycerides Ratio (wt/wt)
Cell
Reaction time (h)24
4872962-MG/1-MG a A.oryzae c 0.2590.2560.2670.324R.oryzae d 0.6730.798 1.90 1.771,2-DG/1,3-DG b
A.oryzae c 1.52 1.39 1.73 2.32R.
oryzae d
3.11
3.71
7.31
6.01
a
Ratio of regioisomer of monoglyceride,which represents an accumulation level of the sn ?2isomer.b
Ratio of regioisomer of diglyceride,which represents an accumulation level of the sn ?2isomer.c
Immobilized A.oryzae carrying pNAN8142FHL.d
Immobilized R.oryzae cells.
Appl Microbiol Biotechnol
et al.2008),and CALB(C.antarctica;Tamalampudi et al. 2007),respectively.The production level of FHL(approx-imately200mg/l-culture broth)was similar to that of the homologous lipase,tglA,suggesting that A.oryzae is a suitable host organism for producing the recombinant FHL. Additionally,when the cells were utilized as a whole-cell biocatalyst immobilized within polyurethane foam BSPs, the FHL-producing cells showed the highest ME conversion among the four lipases described above(data not shown). These findings,together with data in Table1and Figs.2,3, and4,indicate that the immobilized A.oryzae overexpressing the recombinant FHL may be a promising whole-cell biocatalyst for enzymatic biodiesel production.
Adding a certain amount of water into the reaction mixture is effective in both preventing the lipase inactiva-tion by methanol(Kaieda et al.2001)and facilitating the acyl migration in partial glycerides(Oda et al.2005).Since FHL exhibits1,3-regiospecificity towards TG(Shimada et al.1993),the high ME content in the reaction mixture seems to be attributable to the acyl migration from the sn?2 to the sn?1(3)position in MG and DG.Our results show that adding5%water is sufficient to achieve the high ME content(Fig.3)and high lipase stability(Fig.4).
A comparative study of FHL-producing A.oryzae and R. oryzae in methanolysis interestingly showed that the FHL-producing A.oryzae,despite its lower initial reaction rate, attained the higher final ME content than R.oryzae(Fig.4). Further quantitative analysis of regioisomers clearly showed a significant accumulation of the sn?2isomers of partial glycerides in R.oryzae-catalyzed methanolysis, which is consistent with the thin-layer chromatography data of Oda et al.(2005).In a study of methanolysis using R.oryzae,Oda et al.(2005)reported that the acyl migration of partial glycerides,promoted by the presence of water,is also subject to the facilitatory effect of lipase and suggested that increasing numbers of reaction cycles decreased the lipase activity,resulting in the marked accumulation of the sn?2isomers.Therefore,the result that the FHL-producing A.oryzae maintained a low level of the sn?2isomers (Table2)may imply a high ability of FHL to promote the acyl migration from the sn?2to the sn?1(3)position in partial glycerides.Although we cannot unequivocally exclude the possibility that FHL directly catalyzed the sn?2position,our results clearly reflect the differences in the catalytic properties between FHL and ROL.
It is clear that the FHL-producing A.oryzae presents higher enzyme reusability than R.oryzae(Fig.4).FHL is reported to be highly stable in organic solvents such as dimethylformamide,hexane,and benzene(Shimada et al. 1993).The solvent tolerance of FHL therefore may contribute to the higher reusability of FHL-producing A. oryzae.Another possible explanation is the high ability of FHL to promote the acyl migration in partial glycerides.Because the FHL-producing A.oryzae efficiently converts partial glycerides to MEs,accumulation of methanol and MG can be maintained at low levels.This may prevent lipase inactivation by methanol and/or inhibitory effect of MG,resulting in the high reusability of the FHL-producing A.oryzae.
In this study,we provide the first example of developing recombinant fungal whole cells to improve the enzymatic biodiesel production.For scaled-up operation,the recom-binant A.oryzae can be treated similarly as R.oryzae, where an airlift bioreactor(Oda et al.2005)and a packed-bed reactor(Hama et al.2007)are used in the cultivation and methanolysis reaction,respectively.We thus consider that a combination of fungal whole cells with different catalytic properties is beneficial in the actual production of biodiesel.
Further improvement would be possible by designing a novel recombination system including the enhancement of promoter activity(Koda et al.2004),multicopy integration of genes,and coexpression of different types of lipases. These investigations are currently under consideration to encourage the application of whole-cell biocatalyst to industrial biodiesel production.
Acknowledgements This work was partly supported by the Japanese Ministry of the Environment for Technical Development of Measures to Prevent Global Warming(2007).Generous gifts of the fungal expression vector pNAN8142and Aspergillus oryzae niaD300from Ozeki(Hyogo,Japan)are gratefully acknowledged.We also thank Dr. Yuji Shimada(Osaka Municipal Technical Research Institute,Osaka, Japan)for providing pYGF2.
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