Kobe University Repository : Thesis

学位論文題目Tit le

Development of a process model for enzymatic biodiesel product ionfrom a broad range of feedstocks using Aspergillus oryzae whole-cellbiocatalysts(麹菌全細胞触媒を用いた多様な原料からの酵素法バイオディーゼル燃料生産を目指したプロセスモデルの開発)

氏名Author 足立, 大輔

専攻分野Degree 博士（工学）

学位授与の日付Date of Degree 2013-03-25

資源タイプResource Type Thesis or Dissertat ion / 学位論文

報告番号Report Number 甲5764

権利Rights

JaLCDOI

URL http://www.lib.kobe-u.ac.jp/handle_kernel/D1005764※当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。

PDF issue: 2021-06-17

博士論文 Development of a process model for enzymatic biodiesel production from a broad range of feedstocks using Aspergillus oryzae whole-cell biocatalysts 麹菌全細胞触媒を用いた多様な原料からの酵素法バイオディーゼル燃料生産を

目指したプロセスモデルの開発

2013 年 1 月

神戸大学大学院工学研究科

足立 大輔

PREFACE This is a thesis submitted by the author to Kobe University for the degree of

Doctor of Engineering. The studies performed here were conducted between 2007 and

2013 under the direction of Professor Akihiko Kondo in the Laboratory of Biochemical

Engineering, Department of Chemical Science and Engineering, Graduate school of

Engineering, Kobe University.

First of all, the author would like to express his sincerest gratitude to his research

adviser, Professor Akihiko Kondo, for continuous guidance and invaluable suggestions

during the course of his studies. The author would also like to express his hearty

gratitude to Professor Hideki Fukuda, Associate Professor Chiaki Ogino, Ph. D. Shinji

Hama (Bioenergy Corp.), Assistant Professor Kazunori Nakashima (Tohoku university)

for invaluable suggestion, discussion and kind support during the conduct of this

research. The author is also deeply grateful to Professor Naoto Ohmura, Professor

Minoru Mizuhata, Assistant Professor Ryosuke Yamada, Assistant Professor Fumiyoshi

Okazaki (Sumitomo Chemical Co., Ltd.) for their informative advice and hearty

encouragement through the work.

The author further pays his acknowledgement to Professor Tsuneo Yamane

(Chubu university), Professor Hideki Yamaji, Associate Professor Tomohisa Hasunuma,

Associate Professor Kiyotaka Hara, Associate Professor Tsutomu Tanaka, Associate

Professor Jun Ishii, Assistant Professor Naoko Okai, Mr. Takao Numata (Chugai

Pharmaceutical Co., Ltd.), Ph. D. Ayumi Yoshida (Nippon Suisan Kaisha, Ltd), Ph. D.

Sriappareddy Tamalampudi, Ph. D. Nobuo Fukuda (National Institute of Advanced

Industrial Science and Technology) for the helpful discussion and advice.

The generous gift of the fungal expression vector pUSC from Professor Katsuya

Gomi (Tohoku university) and pSENSU, pSENSU-FHL, pSENSU-CALB and

pNAN8142 from Ozeki Co. Ltd. (Hyogo, Japan) are also sincerely acknowledged.

The author further pays his acknowledgement to Mr. Nobuyuki Kuratani (Kansai

Chemical Engineering Co. Ltd.) and Mr. Naoki Tamadani (Bioenergy Corp.) for kind

technical assistance of bioethanol distillation, and unbounded appreciation to Ms.

Miyuki Ueyama, Ms. Naomi Ohtani and Ms. Ayako Okamoto for many kind supports

and to Ph. D. Yuki Matano, Ms. Risa Koda (TonenGeneral Sekiyu K.K.), Mr. Tomohiro

Takaya (Meiji Seika Kaisha, Ltd.), Mr. FookHee Koh (Fuji Oil Co., Ltd.), Mr. Tetsuya

Kosaka, Mr. Takuya Matsumoto, Mr. Hironori Tezuka (Mitsubishi Plastics, Inc.), Mr.

Junichi Aoki (Nippon Shokubai Co., Ltd.), Mr. Toshihide Yoshie, Ms. Nanami

Nakashima, and all the members of Professor Kondo’s laboratory for their technical

assistance and encouragement.

The present work was partially supported by Regional Innovation Creation R&D

Programs, the Ministry of Economy, Trade and Industry (METI) and Special

Coordination Funds for Promoting Science and Technology, Creation of Innovation

Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction

Kobe), MEXT, Japan.

Last but not least, the author expresses deep appreciation to his parents, Kenichi

and Kazuyo Adachi for the constant assistance and financial support, and to his family

Yuka Adachi for her encouragement.

Daisuke Adachi Biochemical Engineering Laboratory Department of Chemical Science and Engineering Graduate School of Engineering Kobe University

CONTENTS

Introduction

Synopsis

Chapter 1

Development of an Aspergillus oryzae whole-cell biocatalyst coexpressing

triglyceride and partial glyceride lipases for biodiesel production

Chapter 2

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant

lipase into an immobilized Aspergillus oryzae cells: Characterization and

application to enzymatic biodiesel production

Chapter 3

Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae

whole-cell biocatalyst highly expressing Candida antarctica lipase B

Chapter 4

An integrative process model of enzymatic biodiesel production through ethanol

fermentation of brown rice followed by lipase-catalyzed ethanolysis in a

water-containing system

General conclusion

Publication lists

1

17

21

40

61

80

100

103

1

INTRODUCTION

Biodiesel fuel

Recent consideration of the possible exhaustion of fossil fuels and environmental

concerns such as global warming and acid rain, have made biofuel—a renewable and

environmentally friendly energy—an attractive alternative. Among the many examples

of biofuel, biodiesel, fatty acid alkyl esters produced by alcoholysis of plant oils or

animal fats (Fig. 1), has been studied extensively because of its many attractive features:

(i) it has high octane numbers and flash points, which makes it a promising candidate to

replace conventional diesel fuel; (ii) its combustion does not increase current

atmospheric levels of CO2 since it is derived from plants, not petroleum; (iii) it is

biodegradable; (iv) it provides a substantial reduction in SOx emissions and a

considerable reduction in CO and suspended particulate matter; and, (v) it has lower

well-to-wheel emissions of methane (Ma and Hanna, 1999). As shown in Fig. 2, the

demand for biodiesel has gradually increased worldwide, and the increase is predicted

to continue. A life-cycle assessment of biodiesel has been conducted with regard to its

reduction ratio for emissions of greenhouse gases (Fig. 3). The results indicate that

biodiesel shows promise for a reduction in the emissions of greenhouse gases, which is

similar to other biofuels.

Fig. 1 Reaction scheme of alcoholysis.

CH2OCOR3

CH2OCOR1CHOCOR2 + 3R4OH

Triglycerides

R1COOR4

R2COOR4

R3COOR4

CH2OH

CHOHCH2OH

（BDF）Glycerol

Catalyst

Alcohol Alkyl esters

++

+

2

Fig. 2 The transition of production amounts of biodiesel from the main producers1).

Reduction ratio of the emission of greenhouse gas (%)

0 10 20 30 40 50 60 70 80 90

WasteWastewood

Wheat strawSugar cane

CornWheat

Sugar beetWaste oil or animal fat

Palm oil (recovery of methane gas)Palm oil

Soybean oilSunflower oilRapeseed oil

Biodiesel

Bioethanol

Biogas

Fig. 3 Reduction ratio for the emissions of greenhouse gases of biofuels from various

forms of feedstocks2).

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

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2003

2004

2005

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2007

2008

2009

2010

2011

BrazilUnited StatesTurkeyEUCanadaAustralia

Pro

duct

ion

amou

nt o

f bio

dies

el (M

L)

3

Production processes for biodiesel fuel

A number of processes have been developed for biodiesel production: catalytic

processes use homogeneous catalysts such as alkali and acid, heterogeneous catalysts,

or enzymes; and, non-catalytic processes use supercritical alcohol or other solvents.

Although an alkali-catalysis process can convert triglycerides to their corresponding

alkyl esters in a short amount of time, there are several drawbacks that include a high

amount of energy consumption, the difficulty of glycerol recovery, the need for the

removal of the alkaline catalyst from the product, and the treatment of highly alkaline

wastewater. In addition, the presence of water or fatty acids (FA) in feedstock can lead

to soap formation, which reduces the yield and makes product separation difficult (Ma

and Hana 1999; Lotero et al., 2005). Although acid catalysts are less sensitive to the

presence of FA in feedstocks, the process also has several disadvantages such as the

requirement of a high alcohol-to-oil molar ratio, the need for high processing

temperatures, and the high cost of equipment (Bankovic-Ilic et al., 2012; Wang et al.,

2006). Heterogeneous catalysts such as metal oxides, carbonates, zeolites, and

heteropolyacids have also been investigated for biodiesel production. These catalysts

have several advantages including easy separation from products and tolerance to water

and FA in feedstocks. However, compared with homogeneous catalysts, heterogeneous

catalysts generally require a relatively higher temperature and pressure in order to

catalyze alcoholysis (Sagiroglu et al., 2011). Moreover, long-term use of a catalyst is

sometimes limited because of deactivation by the clogging of pores due to the

adsorption of triglycerides, water or glycerol. The treatment of supercritical alcohol

(Saka and Kusdiana, 2001) or other solvents (Chen et al., 2010) is sufficient to convert

triglycerides and FA to their corresponding methyl esters in a short amount of time

using no catalyst; however, the energy consumption is very high because the process

requires the highest settings for temperature and pressure among all the processes

presented here.

4

The related enzymatic process using lipase, which catalyzes a variety of reactions

such as hydrolysis, transesterification and ester synthesis at the oil-water interface

(Sarda and Desnuelle, 1958; Zaks and Klibanov, 1984) offers several advantages over

the aforementioned processes: low energy consumption, easy product purification, and

no generation of waste water. Thus far, numerous studies have reported on enzymatic

biodiesel production using commercial immobilized lipases such as Novozym435 (Du

et al., 2004; Samukawa et al., 2000; Shimada et al., 1999; Talukder et al., 2009),

Lipozyme TL IM (Sim et al., 2010; Hernandez-Martin et al., 2008) and Lipozyme RM

IM.

In order to identify the optimal conditions for lipase-catalyzed transesterification,

the reaction mechanisms have been investigated. The complete conversion of

triglycerides to biodiesel involves three reversible steps that produce DG and MG as

intermediates (Li et al., 2010; Türkan and Kalay 2006). Ping Pong Bi Bi mechanisms

were proposed to describe the kinetics of transesterification and hydrolysis (Al-Zuhair

et al., 2007; Cheirsilp et al., 2008). Fig. 4 shows the hydrolysis and esterification steps

with the free enzyme (E) reacting with triglyceride (T) to form the first complex (E・T)

and then T was hydrolyzed to diglyceride (D) and FA (F). Subsequently, D was released

from the second complex (E・F・D) to form the third complex (E・F). This complex

might react with alcohol (Al) through alcoholysis to form an alkyl ester or with water

(W) through hydrolysis to form free F. The mechanisms for the hydrolysis of D and

monoglyceride (M) are similar to that described above. The kinetic models indicate that

in the presence of alcohol, the glycerides (T, D and M) are more easily converted to

alkyl ester through alcoholysis rather than to FA through hydrolysis (Al-Zuhair et al.,

2007; Cheirsilp et al., 2008).

Although such intensive studies have been performed, the cost of lipases that is

associated with the preparation processes, which includes purification from culture

broth and immobilization on a carrier, seems to be one of the main obstacles to

industrial application of enzymatic biodiesel production.

5

Fig. 4 Schematic diagram of Ping Pong Bi Bi mechanisms for stepwise

transesterification.

Whole-cell biocatalyst

In order to make the enzymatic process more convenient, enzymes were

immobilized on different types of supports after recovery from the cell or culture broth

and additional purification steps. However, enzymes recovered through such operations

are generally unstable and expensive (Macrae, 1989), and consequently there has been

considerable attention in the direct use of immobilized cells as whole-cell biocatalysts

(Nikolova et al., 1993). Immobilized cells have several advantages: (i) operations for

enzyme extraction and purification are unnecessary; (ii) products can easily be

recovered from immobilized cells; (iii) operational stability is generally high; and, (iv)

application to multiple enzyme reactions is possible. Such attractive features and

potential have allowed immobilized cell systems to be utilized in a wide variety of

processes. Several studies have previously reported on the direct use of

lipase-producing cells including use with fungi (Ban et al., 2001; Hama et al., 2008,

2009), yeast (Tanino et al., 2007), and Escherichia coli (Li et al., 2012; Narita et al.,

2006). Fig. 5 compares lipase production processes using conventional immobilized

T D Al (W) Es (F)

D M Al (W) Es (F)

M G Al (W) Es (F)

E

E

E

E

E

E

E・T

E・D

E・M

E・F

E・F

E・F

E・D・F

E・M・F

E・G・F

6

lipase and whole-cell biocatalyst, suggesting that the process is obviously simplified by

introducing a whole-cell biocatalyst. Since the main hurdle to commercializing the

enzymatic process is the cost of lipase production, the use of a whole-cell biocatalyst

seems to be significantly advantageous. Therefore, our thesis was focused on whole-cell

biocatalysis using immobilized lipase-producing cells.

Among the several microorganisms, Aspergillus species are promising hosts: (i)

they are robust microorganisms, which can grow at wide ranges of temperatures (10-50

oC), pH values (2.0–11.0), and osmolarities in nearly pure water up to 34% salt

(Kis-Papo et al., 2003); (ii) they can easily be immobilized on porous biomass support

particles (BSPs) during submerged culture; (iii) they can produce a large amount of

proteins; and, (iv) intensive research studies have made many advances, including those

in molecular biology and the development of improved promoters for the high-level

expression of heterologous genes (Minetoki et al., 1998; Koda et al., 2004). These

advantages prompted us to develop Aspergillus oryzae whole-cell biocatalysts carrying

heterologous lipase-encoding genes.

Fig. 5: Comparison of lipase production processes using (a) conventional immobilized

lipase and (b) whole-cell biocatalyst

Enzymatic biodiesel production using A. oryzae whole-cell biocatalysts from a

broad range of feedstocks

Cultivation Separation Purification Immobilization Reaction

(a) Conventional immobilized lipase

(b) Whole-cell biocatalyst

Cultivation Recovery Reaction

7

In a previous study, a biodiesel conversion of more than 90% was achieved using

an A. oryzae whole-cell biocatalyst expressing a triglyceride lipase from Fusarium

heterosporum (FHL) (Hama et al., 2008). However, biodiesel production from a broad

range of feedstocks is desired from the viewpoint of cost effectiveness because

community-based biodiesel production using oil obtained from a local crop could

efficiently reduce the transportation cost of feedstocks, and lead to a lower cost of

biodiesel. Since the composition of FA varies depending on the kind of plant oil,

characteristics such as fluidity and reactivity also vary. Therefore, the application of all

feedstocks to same reaction system is difficult. In the present study, we focused on

various forms of feedstocks including soybean oil, palm oil or FA, and attempted to

produce biodiesel efficiently from each feedstock (Chapter 1-3). In traditional biodiesel

production, alcohol that is used as an acyl accepter has been derived from petroleum.

Therefore, to produce biodiesel from only biomass-derived feedstock, a fermentative

bioethanol solution from brown rice was used instead of petroleum derived methanol

(Chapter 4). There were various drawbacks for efficient biodiesel production using such

various feedstocks and alcohols. To solve the problems, we attempted to develop a

process model by using an appropriate whole-cell biocatalyst for each process. A

summary of each chapter in the present study is shown in Table 1.

When using soybean oil, a biodiesel conversion of more than 90% was achieved

using an A. oryzae whole-cell biocatalyst expressing FHL (r-FHL). However, an

accumulation of partial glycerides that occurred as intermediates during methanolysis

was a problem. In the present study, we focused on a partial glyceride lipase that

specifically catalyzes the conversion of intermediates into corresponding alkyl esters

and developed an A. oryzae whole-cell biocatalyst coexpressing FHL and mdlB to

decrease the residual glycerides. (as described in Chapter 1).

Since palm oil is an abundant and inexpensive oil, it has received attention as an

attractive biodiesel feedstock. However, palm oil becomes solid at room temperature

(25-30 oC), so that higher temperatures are required to dissolve it and to effectively

8

proceed to transesterification. Since the conventional whole-cell biocatalyst, r-FHL,

showed an inadequate thermostability and was obviously deactivated at moderately high

temperatures (40-50 oC), the higher thermostability of a lipase was desirable for palm oil

methanolysis. Therefore, we focused on a lipase from Geobacillus thermocatenulatus

(BTL2) that has shown high stability toward moderately high temperatures (40 oC), and

we introduced the A. oryzae whole-cell biocatalyst. The resultant catalyst was

characterized and applied to palm oil methanolysis at moderately high temperature

(described in Chapter 2).

The cost of feedstocks currently constitutes a large percentage of the total

production cost of biodiesel. Therefore, an investigation into low-cost feedstock has

recently been of considerable interest in related research fields. Low-cost feedstocks

such as waste oil, crude oil, and acid oil contain more FA from the oxidation of plant oil

compared with feedstocks from purified oils. When using feedstocks containing FA,

alkali catalysis methods and enzymatic methods using commercial immobilized lipase

Novozym 435 cannot produce biodiesel effectively. In the case of alkali catalysis, FA

were converted into soap via a saponification reaction, whereas with Novozym 435, FA

generated water that inhibited methanolysis through esterification. Therefore, an

efficient biodiesel production is desired from feedstocks containing FA. To produce

biodiesel enzymatically from such feedstocks, we investigated a 2-step process

involving the hydrolysis of acylglycerols using Candida rugosa lipase, followed by

methyl esterification of the resultant hydrolysates using an A. oryzae whole-cell

biocatalyst expressing Candida antarctica lipase B with an excellent esterification

activity (described in Chapter 3).

In traditional biodiesel production, feedstock from plant oil is derived from

biomass. However, methanol that is used as an acyl accepter is derived from petroleum.

Therefore, we attempted to produce biodiesel from only biomass-derived feedstock by

using a fermentative bioethanol solution after distillation instead of methanol. In the

present study, we used a recombinant A. oryzae expressing FHL, which catalyzes

9

ethanolysis efficiently and tolerates a high water concentration in reaction media (Hama

et al., 2008; Koda et al., 2010) (described in Chapter 4).

10

Feedstock Alcohol Problem Solution Whole-cell biocatalyst

Chapter 1 Soybean oil MethanolAccumulation of much amount of residual glycerides

Utilization of a lipase specified toward partial

glycerides (mdlB)

Chapter 2 Palm oil MethanolDifficult to an efficient reaction in moderately-high temperature (40-50℃)

Utilization of a thermostable lipase

(BTL)

Chapter 3 Fatty acid (FA) MethanolDifficult to an efficient biodiesel production from a feedstock containing FA

Utilization of a lipase with high esterification activity

(CALB)

Chapter 4 Rapeseed oil Bioethanol Utilization of petroleum-derived alcohol

Utilization of bioethanol that is derived from brown

rice

Recombinant A. oryzae expressing BTL (r-BTL)

Recombinant A. oryzae expressing CALB (r-CALB)

Recombinant A. oryzae expressing FHL (r-FHL)

Recombinant A. oryzae coexpressing

FHL and mdlB

Table 1 List of research contents in the present study

11

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14

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Web references 1)http://stats.oecd.org/viewhtml.aspx?QueryId=36356&vh=0000&vf=0&l&il=blank&la

ng=en

Database - OECD-FAO Agricultural Outlook

2) http://www.meti.go.jp/press/20100305002/20100305002-2.pdf

Ministry of Economy, Trade and Industry (METI)

17

SYNOPSIS

Chapter 1 Development of an Aspergillus oryzae whole-cell biocatalyst coexpressing

triglyceride and partial glyceride lipases for biodiesel production

To improve biodiesel production using lipase-expressing fungal cell as a

biocatalyst, an Aspergillus oryzae whole-cell biocatalyst which coexpress Fusarium

heterosporum lipase (FHL) and mono- and di-acylglycerol lipase B (mdlB) in the same

cell has been developed. Screening a number of transformants revealed that a

combination of an integrated lipase and a selectable marker is important for the

development of cells showing a high methyl ester content in methanolysis. The best

transformant was obtained when FHL was integrated into the host strain using sC gene

as a selection marker while mdlB was integrated using niaD selection marker. The

reaction system using lipase-coexpressing whole-cells was found to be superior to

others such as lipase-mixing and two-step reactions, resulting in the highest reaction

rate and the highest ME content (98%) of the first batch. Moreover, an ME content of

more than 90% was maintained during 10 repeated batch cycles. The developed fungal

cells are, therefore, promising biocatalysts for biodiesel production.

18

Chapter 2 A robust whole-cell biocatalyst that introduces a thermo- and

solvent-tolerant lipase into an immobilized Aspergillus oryzae: Characterization

and application to enzymatic biodiesel production

To develop a robust whole-cell biocatalyst that works well at moderately high

temperatures (40-50 oC) with organic solvents, a thermostable lipase from Geobacillus

thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell

biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was

highest at 50 oC and was maintained even after an incubation of 24-h at 60 oC. In

addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate,

ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also

worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at

elevated temperatures from 40 to 50 oC. Moreover, r-BTL catalyzed methanolysis at a

high methanol concentration without a significant loss of lipase activity. In particular,

when 2 molar equivalents of methanol were added 2 times, a methyl ester content of

more than 90% was achieved; the yield was higher than those of conventional

whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On

the basis of an efficient biodiesel production at elevated temperature and high methanol

concentration, the developed whole-cell biocatalyst would be a promising biocatalyst in

a broad range of applications including biodiesel production.

19

Chapter 3

Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae

whole-cell biocatalyst highly expressing Candida antarctica lipase B

For enzymatic biodiesel production from plant oil hydrolysates, an Aspergillus

oryzae whole-cell biocatalyst that expresses Candida antarctica lipase B (r-CALB) with

high esterification activity was developed. Each of soybean and palm oils was

hydrolyzed using Candida rugosa lipase, and the resultant hydrolysates were subjected

to esterification where immobilized r-CALB was used as a catalyst. In esterification,

r-CALB afforded a methyl ester content of more than 90% after 6 h with the addition of

1.5 molar equivalents of methanol. Favorably, stepwise additions of methanol and a

little water were unnecessary for maintaining the lipase stability of r-CALB during

esterification. During long-term esterification in a rotator, r-CALB can be recycled for

20 cycles without a significant loss of lipase activity, resulting in a methyl ester content

of more than 90% even after the 20th batch. Therefore, the presented reaction system

using r-CALB shows promise for biodiesel production from plant oil hydrolysates.

20

Chapter 4

An integrative process model of enzymatic biodiesel production through ethanol

fermentation of brown rice followed by lipase-catalyzed ethanolysis in a

water-containing system

We attempted to integrate lipase-catalyzed ethanolysis into fermentative

bioethanol production. To produce bioethanol, ethanol fermentation from brown rice

was conducted using a tetraploid Saccharomyces cerevisiae expressing α-amylase and

glucoamylase. The resultant ethanol was distilled and separated into three fractions with

different concentrations of water and fusel alcohols. In ethanolysis using the first

fraction with 89.3% ethanol, a recombinant Aspergillus oryzae whole-cell biocatalyst

expressing Fusarium heterosporum lipase (r-FHL) afforded the highest ethyl ester

content of 94.0% after 96 h. Owing to a high concentration of water in the bioethanol

solutions, r-FHL, which works best in the presence of water when processing

ethanolysis, was found to be more suitable for the integrative process than a commercial

immobilized Candida antarctica lipase. In addition, r-FHL was used for repeated-batch

ethanolysis, resulting in an ethyl ester content of more than 80% even after the fifth

batch. Fusel alcohols such as 1-butanol and isobutyl alcohol are thought to decrease the

lipase activity of r-FHL. Using this process, a high ethyl ester content was obtained by

simply mixing bioethanol, plant oil, and lipase with an appropriate adjustment of water

concentration. The developed process model, therefore, would contribute to biodiesel

production from only biomass-derived feedstocks.

21

Chapter 1 Development of an Aspergillus oryzae whole-cell biocatalyst coexpressing

triglyceride and partial glyceride lipases for biodiesel production

Introduction

Biodiesel fuel (BDF) is expected to serve as an alternative to fossil fuel. Methyl

esters (ME) of long-chain fatty acids, which are produced by methanolysis of vegetable

oils, can be directly used as BDF (Clark et al., 1984; Fukuda et al., 2001). BDF has

environmental advantages, such as low amounts of suspended particulate matter and

emissions containing sulfur oxide. There are several conventional methods for BDF

production using alkaline catalysts, supercritical fluid, and lipase. The

alkaline-catalyzed method widely employed has so many disadvantages that purification

of the product and the by-product, glycerol, is complex and laborious. Since the

supercritical method, using supercritical alcohol (Tan et al., 2010) and other solvents

(Chen et al., 2010), needs a much higher temperature and pressure than the other two

methods, its energy consumption is very high. In contrast, the lipase-catalyzed method

provides several advantages, such as low energy consumption and easy product

purification. BDF production using immobilized lipase has, therefore, been extensively

studied (Li et al., 2006; Nelson et al., 1996; Sakai et al., 2010; Shimada et al., 1999;

Talukder et al., 2009). However, the cost of the lipase associated with its preparation

processes, including purification from culture broth and immobilization on a carrier,

seems to be one of the main obstacles to industrial application.

To overcome these drawbacks, we focused on a whole-cell biocatalyst for

biodiesel production, which enables the direct use of lipase-producing microorganisms

(Ban et al., 2001, 2002; Hama et al., 2007; Oda et al., 2005). Among several

microorganisms, Aspergillus species are promising hosts because (i) they can be

spontaneously immobilized within porous biomass support particles (BSPs) during

cultivation, (ii) they can produce a large amount of proteins, and (iii) intensive research

studies have made many advances, including those in molecular biology and the

22

development of improved promoters for the high-level expression of heterologous genes

(Minetoki et al., 1998; Koda et al., 2004). These advantages led us to develop

Aspergillus oryzae whole-cell biocatalysts carrying heterologous lipase-encoding genes.

In our previous work (Hama et al., 2008), a biodiesel conversion of more than

90% was achieved using an A. oryzae whole-cell biocatalyst expressing Fusarium

heterosporum lipase (FHL), which is a solvent-tolerant triglyceride (TG) lipase that

possesses 1,3- regiospecificity (Shimada et al., 1993; Nagao et al., 2001). However, the

methyl ester and residual glyceride content did not meet the standard value for BDF in

Europe (EN14214). On the other hand, several lipases that show specificity for partial

glycerides have been reported (Sakiyama et al., 2001; Watanabe et al., 2004). Mono-

and di-acylglycerol lipase B (mdlB) from A. oryzae (Tuchiya et al., 1996) can catalyze

the hydrolysis of mono- (MG) and di-glycerides (DG), regardless of the position of the

fatty acid (Toida et al., 1995). Because the complete conversion of TG to biodiesel

involves three reversible steps that produce DG and MG as intermediates (Li et al.,

2010; Türkan and Kalay 2006), enhancement of methanolysis activities toward these

intermediates may be effective in improving biodiesel production. Accumulation of the

intermidiates was greately decreased when mdlB-producing A. oryzae was applied to

reaction mixtures obtained from methanolysis using TG lipase-producing Rhizopus

oryzae (Hama et al., 2009).

In the present study, a novel A. oryzae whole-cell biocatalyst coexpressing TG

lipase (FHL) and partial glyceride lipase (mdlB) was developed in order to improve

biodiesel production through whole-cell biocatalysis. The host strain, A. oryzae NS4,

has two auxotrophic selectable markers, sC (Yamada et al., 1997) and niaD (Unkles et

al., 1989), which show different integration characteristics. The plasmid possessing the

sC gene is integrated into an A. oryzae chromosome with more than three copies in

random multiple forms (Yamada et al., 1997). On the other hand, the plasmid bearing

the niaD gene shows a high homologous integration rate, namely a single copy (Yamada

et al., 1997). Because the different characteristics of the selectable markers would affect

23

the performance of the transformants, the combinations of lipase-encoding genes and

selectable markers was investigated. In addition, several reaction systems, including

lipase-mixing, two-step, and lipase-coexpression systems was compared with regard to

their performance in biodiesel production, and then used the best transformant for

long-term batch methanolysis.

Materials and methods

Strains and chemicals

A. oryzae NS4, an sC and niaD mutant derived from the wild-type RIB40 strain

(Yamada et al, 1997), which has been deposited at the National Research Institute of

Brewing (Hiroshima, Japan), was used as a host strain for transformation. Reticulated

polyurethane foam BSPs (Bridgestone Co Ltd., Osaka Japan) measuring 6 mm × 6 mm

× 3 mm cuboids were used to immobilize A. oryzae. The Escherichia coli strain used for

gene manipulation was Nova Blue {endA1 hsdR17 (rK12- mK12+) supE44 thi-1 recA1

gyrA96 relA1 lac [F' proAB+ lacIqZΔM15::Tn10 (Tetr)]} (Novagen, Madison, WI, USA).

Soybean oil, rapeseed oil, olive oil or glycerol dioleate (Wako Pure Chemical Industries,

Osaka, Japan) was used as the substrate for methanolysis.

Construction of lipase expression plasmids

Two parent plasmids, pNAN8142 and pUSC8142, into which lipase genes for

FHL and mdlB, were inserted to obtain four kinds of plasmids, pNAN8142FHL,

pNAN8142mdlB, pUSC8142FHL, and pUSC8142mdlB (Fig. 1). The first two,

pNAN8142FHL and pNAN8142mdlB, were prepared as described previously (Hama et

al., 2008, 2009). To construct pUSC8142FHL and pUSC8142mdlB, fragments including

a promoter, P-No8142 (Minetoki et al., 1998; Ozeki et al., 1996), a terminator, T-addA,

and the gene encoding FHL or mdlB (Tuchiya et al., 1996; Shimada et al., 1993) were

amplified by polymerase chain reaction using KOD plus polymerase, digested with

BamHI, and inserted into pUSC (Yamada et al., 1997).

24

Fig.1 Lipase expression plasmids used in the present study. Either FHL (a and c) or

mdlB (b and d) genes were inserted into fungal expression vectors carrying two

selectable markers: niaD (pNAN8142) and sC (pUSC8142). All lipase genes were

expressed under the control of P-No8142 (Minetoki et al., 1998).

Transformation of A. oryzae

Fungal strains prepared in this study were summarized in Table 1. A. oryzae

protoplasts were prepared using Yatalase (Takara Bio Inc, Shiga, Japan) from mycelia

grown in dextrin-peptone (DP) medium at 30 °C for 48 h. The DP medium contained

2% glucose, 2% polypeptone, 0.5% KH2PO4, 0.1% NaNO3, and 0.05% MgSO4·7H2O.

The constructed plasmids were transformed into the A. oryzae chromosome according

to the method of Gomi et al. (Gomi et al., 1987), with minor modifications. The sC and

niaD genes were used as selectable markers. First, pUSC8142FHL was transformed into

A. oryzae NS4 to obtain the first transformant, sC-FHL (Table 1). After selecting the

Pre Pro49 971 1002

HindIII SpeIpNAN81429.50 kbp

niaD

T-addA

P-No8142

Ampr

pUSC81428.39 kbp

sC

Ampr

P-No8142

T-addA

BamHI

BamHI

Fusarium heterosporum lipase (FHL)

Mature

Mono-and diacylglycerol lipase (mdlB)

(a)

ExonExonExon86 137 306 3581 1024

SalI SphI

(b)

(c)

(d)

FHL

ExonExonExon86 137 306 3581 1024

SalI SphI

Pre Pro49 971 1002

HindIII SpeI

mdlB

25

sC-FHL strain with the highest activity, the cells were again transformed with

pNAN8142mdlB, resulting in sC-FHL-niaD-mdlB (Table 1). Another

lipase-coexpressing cell changing the combination of selectable markers and lipase

genes was also prepared. A. oryzae NS4 was transformed with pUSC8142mdlB and

then with pNAN8142FHL, resulting in sC-mdlB-niaD-FHL. All transformants were

maintained on Czapek-Dox (CD) plate medium composed of 2% glucose, 0.1%

KH2PO4, 0.2% KCl, 0.8M NaCl, 0.05% MgSO4.7H2O, 1.5% agar and 0.2% NaNO2

(first transformants) or NaNO3 (second transformants).

Table 1 Abbreviations of fungus strains and plasmids used in this study.

Strain Plasmids carried by fungus strain Reference

sC-FHL pUSC8142FHL This study

sC-mdlB pUSC8142mdlB This study

sC-FHL-niaD-mdlB pUSC8142FHL and pNAN8142mdlB This study

sC-mdlB-niaD-FHL pUSC8142mdlB and pNAN8142FHL This study

niaD-FHL pNAN8142FHL Hama et al., 2008

niaD-mdlB pNAN8142mdlB Hama et al., 2009

Immobilization of the whole-cell biocatalysts

Each A. oryzae transformant was grown at 30 oC for 5-6 days on a CD agar plate,

and spores were harvested with 5 ml of 0.01 wt% Tween 80. The spore solution was

aseptically inoculated into a 500 ml Sakaguchi flask containing 300 BSPs in 100 ml of

DP medium and cultivated at 30 oC on a reciprocal shaker at 150 oscillations per min.

After cultivation for 96 h, the fungal cells immobilized on the BSPs were collected by

filtration, washed with distilled water, and lyophilized for 48 h. The immobilized lipase-

26

expressing cells on BSPs were directly used as biocatalysts for the following reaction.

The amount of immobilized cells on the BSPs was calculated by measuring the weight

of cell before and after cell removal by the treatment with 10 vol% sodium hypochlorite

solution (Oda et al., 2005). The hydrolytic activity of each whole-cell biocatalyst was

determined using hydrolysis of p-nitrophenyl butyrate (pNPB) as a chromogenic

substrate.

Methanolysis of oil

Methanolysis was carried out in a screw-capped bottle. The reaction mixture

contained 9.65 g of soybean oil (unless otherwise noted), 0.35 g of methanol (one molar

equivalent to oil), 0.5 g of distilled water and 100 particles of BSP-immobilized cells.

To evaluate the activity of sC-mdlB expressesing only mdlB, glycerol dioleate was used

as a substrate instead of soybean oil, because mdlB shows substrate specificity for

mono- and di-acylglycerol (Hama et al., 2009). Methanol (0.35 g) was added stepwise

to the reaction mixture after 24, 48 and 72 h to avoid lipase inactivation by an excess

amount of methanol. The reaction was conducted at 30 oC for 96 h on a reciprocal

shaker at 150 oscillations per min.

Comparison of the methanolysis efficiency among the three reaction systems (i.e.

lipase-mixing, two-step, and lipase-coexpression system) was investigated.

Methanolysis was performed fixing the number of BSPs to acquire homogeneity in the

reaction mixture. Therefore, catalytic efficiencies of the reaction systems were

evaluated by adjusting the total number of BSPs subjected to the reaction in this study.

The lipase-mixing system is a one-step reaction system using two kinds of lipases at the

same time. In this system, methanolysis was conducted by the mixture of the two

whole-cell biocatalysts changing the ratio of FHL to mdlB. For two-step reaction,

methanolysis was carried out using two kinds of lipases separately. First, 96-h

methanolysis was conducted using 80 particles of BSPs immobilizing FHL-producing

cells (niaD-FHL, Table 1). Subsequently, the resulting reaction mixture was removed by

27

filtration and then subjected to the second reaction, where 20 particles of BSPs

immobilizing mdlB-producing cells (niaD-mdlB, Table 1) were used for 24-h

methanolysis. In a lipase-coexpression system, the reaction was conducted with 100

particles of BSPs which immobilized lipase-coexpression cells (sC-FHL-niaD-mdlB).

Gas chromatography analysis

Samples were obtained from the reaction mixture at the specified times and

centrifuged at 12,000 rpm for 5 min. The upper oil layer was analyzed using a GC-2014

gas chromatography system (Shimadzu, Kyoto, Japan). The ME content and other

reaction components (MG, DG, TG contents) were analyzed using a DB-5 capillary

column (0.25 mm × 15 m; J&W Scientific, USA) and a ZB-5HT capillary column (0.25

mm × 15 m; Phenomenex, USA), respectively.

The temperature conditions of the injector and detector using DB-5 were set at

245 °C and 320 °C, respectively. The column temperature was set at 150 °C for 0.5 min,

increased to 300 °C at 10 °C/min, and finally maintained at this temperature for 10 min.

When ZB-5HT was used, the temperature conditions of the injector and detector were

set at 320 °C and 380 °C, respectively. The column temperature was set at 130 °C for 2

min, increased to 350 °C at 10 °C/min, then to 370 °C at 7 °C/min, and finally

maintained at 370 °C for 10 min.

The contents of each material present in the reaction mixture were calculated

based on the standard curves, using each standard.

Recycling of immobilized lipase-coexpressing cells

Repeated batch methanolysis was performed using the sC-FHL-niaD-mdlB strain.

After one cycle of methanolysis, immobilized cells were recovered from the reaction

mixture, washed with distilled water, and dried in air at room temperature. The

recovered cells were added to a fresh reaction mixture for the next cycle. This cycle was

repeated for 10 batches.

28

Results and discussion

Screening of lipase-expressing cells with high activity

The A. oryzae strain with the sC gene glows when MgSO4 is added to the plate

medium as the sole sulfur source; however, the strain without this gene does not glow

under the same conditions. Likewise, the strain with the niaD gene can glow with the

addition of NaNO3 to the plate medium as the sole nitrogen source, however, the strain

without this gene cannot do so. Lipase-expressing strains were selected, utilizing such

autotrophic characterizations of these selectable markers.

To obtain lipase-expressing cells with high activity, screening of sC-FHL,

sC-FHL-niaD-mdlB, sC-mdlB, and sC-mdlB-niaD-FHL was carried out (Fig. 2). The

general strategy was that the strain producing the highest ME content was screened

using sC as a selectable marker (Fig. 2(a) and (c)), and the resulting strain was subjected

to a second screening using niaD as a selectable marker (Fig. 2(b) and (d)). Because the

characteristics of the selectable markers would affect the screening results, the order of

introduction of the lipase-encoding genes also was investigated (FHL and mdlB).

Colony No. 5 in Fig. 2(b), which showed the highest ME content, was selected

and used in the following experiment as sC-FHL-niaD-mdlB. Although sC-mdlB

(colony No.1 in Fig. 2 (c)) showed a low ME content (10%) during soybean oil

methanolysis (data not shown), probably because of its substrate specificity toward

mono- and di-acylglycerol (Toida et al., 1995; Hama et al., 2009), the expression of

FHL (sC-mdlB-niaD-FHL) significantly increased the ME content during the 96-h

methanolysis of soybean oil (Fig. 2(d)). Colony No.3 in Fig. 2(d), which showed the

highest ME content, was selected and used in the following experiment as

sC-mdlB-niaD-FHL. Two kinds of lipase-coexpressing cells, sC-FHL-niaD-mdlB and

sC-mdlB-niaD-FHL were thus obtained.

To determine which fungus strain was more suitable for biodiesel production,

soybean oil methanolysis was performed and the components of the reaction mixture

were analyzed. ME is a target product in biodiesel production, while MG and DG are

29

intermediates and TG is unreacted oil; therefore, these glyceride contents should be

suppressed at low levels. As shown in Table 2, both sC-FHL-niaD-mdlB and

sC-mdlB-niaD-FHL attained MG and DG contents that met the biodiesel standard. In

particular sC-FHL-niaD-mdlB produced a high ME content, whereas

sC-mdlB-niaD-FHL did not meet ME product specifications. These results suggest that

a combination of an integrated lipase and a selectable marker is important in order to

develop cells showing a high ME content in methanolysis. sC-FHL-niaD-mdlB as an

optimized strain coexpressing FHL and mdlB for biodiesel production was thus

selected.

We then investigated the catalytic properties of whole-cell biocatalysts such as the

amount of cells on BSPs and hydrolytic activity of the cell (Table 3). The amounts of

the cells on BSPs were similar in the three biocatalysts while hydrolytic activity of the

cell was different, i.e. sC-FHL-niaD-mdlB exhibited the highest hydrolytic activity.

Table 2 Comparison of reaction components after methanolysis catalyzed by two

recombinant A. oryzae cells.

Content (wt%)

ME MG DG TG

Standarda >96.5

30

Table 3 Catalytic performance of lipase-expressing A. oryzae cells.

Comparison of reaction systems using lipase-expressing cells

There are several possible reaction systems using lipase-expressing cells. To

confirm the performance of lipase-coexpression systems catalyzed by the newly

developed strain, this system was compared with several other reaction systems,

including lipase-mixing and two-step systems. In the lipase-mixing system, FHL- and

mdlB-producing cells were used simultaneously for methanolysis. The mixing ratio was

varied (FHL: mdlB=100:0, 90:10, 80:20, 70:30) to optimize the lipase-mixing system.

As shown in Table 3, an increase in the mdlB ratio decreased the MG and DG contents,

however, increased the TG content. Adjusting the ratio of the two lipases appeared to be

important for an effective reaction. Based on these results, a mixing ratio of 80:20

(FHL: mdlB) was selected for the following experiment. In the two-step reaction system,

the sequential methanolysis was performed using niaD-FHL or niaD-mdlB

independently. The same ratio of 80:20 (FHL: mdlB) was also employed in this system,

namely, 80 particles of FHL-expressing cells were used in the first reaction and 20

particles of mdlB-expressing cells were used in the second one. Fig. 3(a) indicates the

time course of methanolysis in each reaction system by stepwise addition of methanol at

0, 24, 48, 72 h. Compared with the two other systems, lipase-coexpression system

showed a little higher reaction rate. In addition, the final ME content in the

lipase-coexpression system was higher than the two other systems with low contents of

Immobilized cell

weight on BSPs

(mg/BSP)

Hydrolytic activity

of the cell

(U/g-dry cell)

Hydrolytic activity

of lipases per BSP

(U/BSP)

niaD-FHL 3.03 12.9 0.0782

niaD-mdlB 2.49 5.95 0.0296

sC-FHL-niaD-mdlB 2.51 18.5 0.0928

31

residues (Fig. 3(b)). The ME content of BDF produced in the lipase-coexpression

system met the biodiesel standards (>96.5%, EN14214), which was not achieved in our

previous reports (Hama et al., 2007, 2008, 2009). Higher yield in coexpression system

could possibly be related to the deactivation of lipase by methanol left in the reaction

mixture. The reaction rate in the coexpression system was high enough to consume

methanol in the reaction, while those in lipase-mixing and two-step systems were lower

and excess methanol would be accumulated, resulting in the deactivation of lipase to

cause low methyl ester yield. The difference in the reaction rate could be attributed to

the difference in total lipase activity in each system. In coexpression system, total lipase

activity in initial of reaction was 9.28 U, while two-step and lipase-mixing systems were

6.26 and 6.85 U, respectively. The result showed that the catalytic efficiency of the

lipase-coexpression system was superior to the two other systems.

The two-step sequential reaction requires a long reaction time, because each

reaction step should be conducted separately. Moreover, in both the two-step and

lipase-mixing systems, two operations are necessary for the preparation of cells

expressing FHL and mdlB. On the other hand, the lipase-coexpression system allows

the utilization of both FHL and mdlB in one step. From the viewpoint of process

simplification, the latter system would be superior to the other two systems, which

require two operations for catalyst preparation. For these reasons, lipase-coexpression

system using sC-FHL- niaD-mdlB was determined the most effective among the three

systems examined. The subsequent experiments were, therefore, carried out using

sC-FHL-niaD-mdlB.

32

Fig. 3 Comparison of biodiesel productivity in three reaction systems. (a) Time course

of methanolysis in each system. (b) Methyl ester and residual glyceride contents after

methanolysis in each system. Lipase-mixing: reaction mixing niaD-FHL and

niaD-mdlB at a ratio of 80-to-20; Two-step: two-step reaction using niaD-FHL or

niaD-mdlB independently. 80 particles of FHL-expressing cells were used in the first

96-h reaction and 20 particles of mdlB-expressing cells were used in the second 24-h

reaction; Lipase-coexpression: reaction using sC-FHL-niaD-mdlB.

Two-stepLipase-mixing

Lipase-coexpression

Met

hyl e

ster

con

tent

(wt%

)

Methyl esterResidual

glycerides

Res

idua

l gly

cerid

es(w

t%)

0

5

10

15

20

88

90

92

94

96

98

100

0102030405060708090

100

0 30 60 90 120Reaction time (h)

Met

hyl e

ster

con

tent

(wt%

)

Lipase-mixingTwo-stepLipase-coexpression

(b)

(a)

33

Application of the whole-cell biocatalyst for industrial BDF production

In the present study, soybean oil was used as a feedstock for the production of

BDF. However, in order to apply our methodology to industrial biodiesel production, an

investigation of various oils with different fatty acid compositions was required.

Therefore, the versatility of the developed cells using several vegetable oils, including

soybean oil, rapeseed oil, and olive oil was tested. A high ME content of more than 95

wt% was achieved for each oil (data not shown), which suggests that the reaction

system using sC-FHL-niaD-mdlB can be applied to biodiesel production from various

oils with different fatty acid compositions.

In industrial applications, recycling of lipase is required to reduce the production

cost. Thus, repeated-batch methanolysis was studied using sC-FHL-niaD-mdlB. Fig. 4

shows the time course of ME content during ten batch reactions. The final ME content

in each batch decreased gradually, but a high ME content, above 90%, was maintained

even after the tenth batch cycle. Further analysis of the final reaction mixture in each

batch showed that the 1st-3rd batches produced ME content that met the biodiesel

standard (>96.5%), and the MG content was suppressed at a low level (

34

purification process. Although the present system was improved biodiesel production,

this method is still inferior to other methods (Sakai et al., 2010; Sim et al., 2010;

Talukder et al., 2009). Further improvement could be achieved by several approaches

including the enhancement of promoter activity (Koda et al., 2004), the introduction of

a 5’ untranslated region (5’UTR) to improve translation efficiency (Koda et al. 2006)

and an increase in gene copy number. Also, in lipase-coexpression system, optimization

of expression balance of lipases could improve the activity of the whole-cell biocatalyst.

From the viewpoint of bioprocess engineering, utilization of a packed-bed reactor

instead of a shaken bottle would provide a significant advantage, since immobilized

cells could be protected from physical damage under vigorous shaking as reported

previously (Hama et al., 2007). Investigations to make whole-cell biocatalysis more

feasible for industrial biodiesel production are currently in progress.

Fig. 4 Recycling of immobilized lipase-expressing cells for repeated batch methanolysis

using sC-FHL-niaD-mdlB.

0

20

40

60

80

100

0 200 400 600 800 1000

Met

hyl e

ster

con

tent

(wt%

)

Reaction time (h)

35

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1367–1369.

40

Chapter 2 A robust whole-cell biocatalyst that introduces a thermo- and

solvent-tolerant lipase into an immobilized Aspergillus oryzae cells:

Characterization and application to enzymatic biodiesel production

Introduction

Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) make up a versatile group of

enzymes that have the ability to hydrolyze triglycerides at the lipid-water interface.

Lipases show wide substrate specificity and they are often used in chemo-, enantio- and

stereo-selective reactions of biotechnological importance (Schmid and Verger 1998;

Jaeger and Eggert 2002). Although immobilization of enzymes may produce some

improvements in enzyme features, like stability, activity, selectivity or specificity (lyer

and Ananthanarayan 2008; Mateo et al., 2007), this process has a cost that in certain

cases may promote some difficulties to the implementation of the processes.

To overcome this drawback, we focused on a whole-cell biocatalyst, which

enables the direct use of lipase-producing microorganisms. Among several

microorganisms, Aspergillus oryzae is a promising host because it has high protein

productivity in the expression of heterologous genes using improved promoters

(Minetoki et al., 1998; Koda et al., 2004) and can easily be immobilized on porous

biomass support particles (BSPs; Fukuda et al., 2008). Thus far, A. oryzae strains that

have been genetically engineered to express several microbial lipases have been

developed for use as whole-cell biocatalysts in biodiesel production and

enantioselective transesterification (Adachi et al., 2012; Hama et al., 2008, 2009;

Tamalampudi et al., 2007). Since practical conditions that promote the reaction include

a high temperature and a high concentration of organic solvents, there is a necessity to

develop a robust whole-cell biocatalyst. Therefore, introducing robust lipases into A.

oryzae would further expand the application of this technology.

Lipases from thermophiles often show their extreme stability at elevated

temperatures and in organic solvents (Bornscheuer et al., 1994). Thus, they have

41

become objects of special interest for structural investigations and also for industrial

applications (Jaeger et al., 1994). The lipase from Geobacillus thermocatenulatus

(BTL2), isolated by Schmidt-Dannert et al. (1996), is an interesting lipase possessing

unique structural characteristics. BTL2 comprises 389-amino-acid residues with a

molecular mass of 43 kDa, which is relatively larger than those of other microbial

lipases (Carrasco-Lopez et al., 2009). In contrast to most lipases, BTL2 has two lids and

a zinc-binding domain that is typically observed in the same family (Carrasco-Lopez et

al., 2009). Moreover, BTL2 shows high stability toward moderately high temperature

(40 oC) and organic solvents (Schmidt-Dannert et al., 1996). Such excellent

characteristics of BTL2 have led to extensive studies such as high level expression in

Escherichia coli (Rua et al., 1998) and Pichia pastoris (Quyen et al., 2003),

immobilization on various kinds of supports (Fernandez-Lorente et al., 2008; Godoy et

al., 2011; Mendes et al., 2012; Palomo et al., 2004), and applications including the

kinetic resolution of chiral substrates (Palomo et al., 2003) and aliphatic ester synthesis

(Mendes et al., 2012).

In the present study, a recombinant A. oryzae whole-cell biocatalyst expressing

BTL2 (r-BTL) was developed. The characteristics of r-BTL were evaluated and

compared with those of a conventional A. oryzae whole-cell biocatalyst expressing

Fusarium heterosporum lipase (r-FHL), which provides high alkyl ester contents of

more than 90% during alcoholysis (Koda et al., 2010). In addition, r-BTL was used for

biodiesel production from palm oil, which is one of the most abundant, cheapest and

available vegetable oils and would be a more sustainable biodiesel-feedstock than

rapeseed oil, as shown in life cycle assessments (Yee et al. 2009; Papong et al. 2010).

However, because of the low fluidity of palm oil at room temperature, a moderately

high temperature is desirable for processing reaction mixtures in transesterification.

Given the high thermostability of BTL2, r-BTL was applied to the methanolysis of palm

oil at a moderately high temperature.

42

Materials and methods

Strains and chemicals

A. oryzae niaD300, which is a niaD mutant derived from the wild type strain

RIB40, was used as a recipient strain for transformation. The Escherichia coli strain

used for gene manipulation was Nova Blue {endA1 hsdR17 (rK12- mK12+) supE44 thi-1

recA1 gyrA96 relA1 lac [F' proAB+ lacIqZΔM15::Tn10 (Tetr)]} (Novagen, Madison, WI,

USA). Reticulated polyurethane foam BSPs (Bridgestone Co Ltd., Osaka, Japan) with 6

mm × 6 mm × 3 mm cuboids were used to immobilize A. oryzae. Palm oil was obtained

from Wako Pure Chemical Industries (Osaka, Japan).

Construction of lipase expression vectors

The gene encoding BTL2 (Genebank accession number X95309) was isolated

from G. thermocatenulatus NBRC 15316 by PCR using following two primers; BTL

fw-SpeI (5’-ggAAGCTTatgatgaaaggctgccgggtgatggttgtg-3’) and BTL rv-NdeI

(5’-ggCATATGtcattaaggccgcaaactcgccaactgctc -3’). The isolated fragment was digested

with restriction enzymes SpeI and NdeI and inserted into the multi-cloning site of

pNAN8142 (Minetoki et al., 1998; Ozeki et al., 1996), and the resultant plasmid named

pNAN8142BTL. The construction of pNAN8142FHL for expressing F. heterosporum

lipase (FHL) was described in our previous paper (Hama et al., 2008).

Transformation of A. oryzae

Transformation of A. oryzae was carried out according to a method previously

described by Gomi et al. (1987). A. oryzae protoplasts were prepared using Yatalase

(Takara, Shiga, Japan) from mycelia grown at 30 oC for 48 h in dextrin-peptone medium,

which consists of 2% dextrin, 1% polypeptone, 0.5% KH2PO4, and 0.05%

MgSO4·7H2O. The recombinant A. oryzae strains carrying pNAN8142BTL and

pNAN8142FHL were designated r-BTL and r-FHL, respectively.

43

Preparation of Aspergillus oryzae whole-cell biocatalysts

Each member of the recombinant A. oryzae strain was grown at 30 oC for 5-6

days on a CD agar plate (2% glucose, 0.1% KH2PO4, 0.2% KCl, 0.8M NaCl, 0.05%

MgSO4.7H2O, 1.5% agar and 0.2% NaNO3), and spores were harvested with 5 ml of

0.01% Tween 80. The spore solution was aseptically inoculated into a 500 ml Sakaguchi

flask containing 300 BSPs in 100 ml of DP medium (2% glucose, 2% polypeptone,

0.5% KH2PO4, 0.1% NaNO3, and 0.05% MgSO4·7H2O) and cultivated at 30 oC on a

reciprocal shaker at 150 oscillations per min. After cultivation for 96 h, the fungal cells

immobilized on the BSPs were collected by filtration, washed with distilled water, and

lyophilized for 48 h. The lipase-expressing cells thus obtained were used as whole-cell

biocatalysts.

Lipase activity assay

Lipase activity assays of r-BTL and r-FHL under various conditions were

performed using p-nitrophenyl butyrate (pNPB) as a chromogenic substrate. The

lipase-hydrolytic activities at various temperatures were determined by an assay at

temperatures that varied from 30 to 70°C. The effect of temperature on lipase stability

was determined by the lipase activity assay at 40°C using r-BTL and r-FHL after a 24-h

incubation with 0.1 M Tris-HCl buffer (pH 8.0) at temperatures that varied from 30 to

80 °C. The relative activity was calculated as the ratio of the lipase-hydrolytic activities

of r-BTL and r-FHL incubated under each temperature to those at 4°C.

The effect of the organic solvents on lipase stability was also investigated by the

lipase activity assay at 40°C using r-BTL and r-FHL after a 24 h-incubation with 0.1 M

Tris (pH 8) containing 30% (v/v) of each organic solvent (dimethyl carbonate (DMC),

ethanol, methanol, 2-propanol, and acetone) at 30°C. The relative activity was

calculated as the ratio of the lipase-hydrolytic activities of r-BTL and r-FHL incubated

with the buffer containing each organic solvent to those without each organic solvent.

44

Methanolysis of palm oil

Methanolysis of palm oil using r-BTL and r-FHL was conducted at temperatures

that ranged from 40 to 60°C in a thermo block rotator (NISSIN, Tokyo, Japan) at 35

rpm. The composition of the reaction mixtures were as follows: palm oil 9.63 g,

methanol 0.37 g, and distilled water 0.5 g (unless otherwise noted). The reaction

mixtures were incubated at the respective temperatures, and the reaction were initiated

by adding 100 pieces of either r-BTL or r-FHL. To avoid lipase deactivation by an

excess amount of methanol, the methanol (0.37 g) was added stepwise to the reaction

mixture at 0, 24, 48 and 72 h, corresponding to the total amount of four molar

equivalents of palm oil.

To investigate the effect of the methanol addition pattern on methanolysis, an

appropriate amount of methanol was added 1-4 times to adjust the total amount to 4

molar equivalents of palm oil. As described elsewhere, 0.4 g of Novozym 435

(Novozymes, Bagsvaerd, Denmark) was added to each reaction mixture as a reference

biocatalyst.

Gas chromatography analysis

Samples obtained from the reaction mixture were centrifuged at 12,000 rpm for 5

min. The upper oil layer was analyzed using a GC-2010 gas chromatograph (Shimadzu,

Kyoto, Japan) connected to a DB5 capillary column (0.25 mm × 15 m; J&W Scientific,

USA). The column temperature was set at 150 °C for 0.5 min, increased to 300 °C at

10 °C/min, and finally maintained at this temperature for 10 min. Tricaprylin served as

the internal standard for the quantification of the alkyl esters in the reaction mixture.

The detailed procedure for the determination of alkyl ester content was described in a

previous paper (Ban et al. 2001).

Results

Evaluation of the catalytic performance of r-BTL

45

The gene sequence of BTL2, cloned into pNAN8142 completely corresponded to

the GenBank reference sequence (X95309). Among several transformants carrying

pNAN8142BTL2, the transformant that provided the highest reaction rate in

methanolysis was selected and employed in the subsequent experiments.

To evaluate the catalytic performance of r-BTL, the lipase-hydrolytic activities at

high temperature and organic solvent tolerance of r-BTL were investigated and

compared with those of r-FHL. The optimum temperature of both immobilized cells was

determined by the lipase activity assay at each temperature from 30 to 70 °C (Fig. 1).

r-BTL showed a maximum activity of 0.034 U per BSP at 50 °C, while r-FHL showed a

maximum activity of 0.010 U per BSP at 40 °C (Fig. 1). From 30 to 50 °C, the lipase

activity of r-BTL increased from 0.016 to 0.034 U per BSP. Although the lipase activity

gradually decreased at temperatures above 60 °C, r-BTL maintained an activity of 0.032

U per BSP even at the highest temperature of 70 °C. The activity of r-BTL in the present

temperature range was significantly higher than that of r-FHL. The thermostability of

both immobilized cells was also investigated (Fig. 2). Although the hydrolytic activity

of r-BTL decreased steeply at temperatures above 70 °C, it was retained without

significant deactivation at temperatures below 60 °C. In contrast, the lipase stability of

r-FHL was greatly affected by incubation temperature, and the residual activity of

r-FHL significantly decreased with an increase in incubation temperature. From these

results, the thermostability of r-BTL was found to be higher than that of r-FHL.

To investigate the organic solvent tolerance of r-BTL and r-FHL, the

lipase-hydrolytic activities were investigated after a 24 h-incubation with buffer

containing 30% (v/v) of each organic solvent at 30 °C (Fig. 3). r-BTL retained higher

activity than r-FHL in all cases. Among the five kinds of organic solvents, the addition

of either DMC or acetone slightly decreased the lipase activity of r-BTL, whereas the

addition of other organic solvents (ethanol, methanol and 2-propanol) had no significant

influence on the lipase activity of r-BTL. In the case of r-FHL, more than 50% of the

lipase activity was decreased by the addition of any organic solvents. These results

46

show that r-BTL is highly tolerant to various kinds of organic solvents.

Fig. 1 Effect of temperature on lipase activity of r-BTL (diamond) and r-FHL (circle).

The lipase activity was analyzed as described in “Lipase activity assay” in the Materials

and Methods section

Fig. 2 Effect of temperature on lipase stability of r-BTL (diamond) and r-FHL (circle).

The relative activity was analyzed as described in “Lipase activity assay” in the

Materials and Methods section.

Temperature (℃)

Rel

ativ

e ac

tivity

(%)

r-BTL

r-FHL

0

20

40

60

80

100

120

20 30 40 50 60 70 80 90

Temperature (℃)

Hyd

roly

tic a

ctiv

ity (U

/BS

P)

r-BTL

r-FHL

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

20 30 40 50 60 70 80

47

Rel

ativ

e ac

tivity

(%)

r-BTL

r-FHL

0

20

40

60

80

100

120

140

160

Fig. 3 Effects of various organic solvents on the lipase stability of r-BTL (gray bar) and

r-FHL (white bar). The relative activity was analyzed as described in “Lipase activity

assay” in the Materials and Methods section.

Application of r-BTL to palm oil methanolysis at elevated temperature

Given its high thermostability, r-BTL was applied to the methanolysis of palm oil

at a moderately high temperature (Fig. 4). During methanolysis, r-BTL afforded a high

methyl ester (ME) content of nearly 100% after 96 h at 40-50 °C. Meanwhile, the

performance of r-FHL decreased significantly with increasing reaction temperature,

resulting in the final ME contents of 81, 44, and 5% at 40, 45, and 50 °C, respectively.

By comparison with r-FHL, these results show that r-BTL was more stable for palm oil

methanolysis at high temperature. The reaction rate using r-BTL in each temperature

was shown in Fig. 5. Higher reaction rate was obtained in higher temperature, however,

a little decrease in ME content at 24 h was observed.

48

Fig. 4 Effect of reaction temperature on the methanolysis of palm oil using (a) r-BTL

and (b) r-FHL at 40 (diamond), 45 (square), and 50 °C (triangle).

Fig. 5 Reaction rate of palm oil methanolysis using r-BTL at 40 (diamond), 45 (square),

and 50 °C (triangle).

0102030405060708090

100

0 25 50 75 1000

102030405060708090

100

0 25 50 75 100

40 ℃ 45 ℃ 50 ℃

(a) (b)

Reaction time (h)M

ethy

l est

er c

onte

nt (w

t%)

Reaction time (h)

Met

hyl e

ster

con

tent

(wt%

)

0

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16 18 20 22 24

Met

hyl e

ster

con

tent

(wt%

)

Reaction time (h)

49

Effect of the methanol addition pattern on methanolysis using r-BTL

In a previous study of soybean oil methanolysis, we found that the stepwise

addition of methanol is effective for maintaining the lipase activity of whole-cell

biocatalysts (Hama et al., 2008, 2009). However, for practical application, reducing the

addition number of methanol is effective in simplifying the process. Because r-BTL

shows a high tolerance toward methanol, we investigated the effect of the methanol

addition pattern on methanolysis (Fig. 6). In the present study, an appropriate amount of

methanol was added 1-4 times to adjust the total amount to 4 molar equivalents of palm

oil. r-BTL afforded a much higher ME content than r-FHL when decreasing the number

of methanol additions, namely, a greater amount of methanol could be added each time

(Fig. 6 (a)). In particular, when 2 molar equivalents of methanol were added 2 times, the

final ME content was almost the same as when 1 molar equivalent was added 4 times.

With r-FHL, however, the methanolysis proceeded efficiently only when 1 molar

equivalent methanol was added 4 times (Fig. 6 (b))—fewer additions of greater amounts

significantly lowered the ME content to less than 10%.

In addition, the reaction profile using r-BTL was compared with that using a

commercial lipase, Novozym 435. To confirm the effect of a small amount of water,

methanolysis was performed with or without the addition of 0.5 g of water (Fig. 7).

When 2 molar equivalents of methanol were added at 0 and 24 h, r-BTL worked best in

the presence of water, whereas the ME contents during methanolysis using Novozym

435 was less than 20% regardless of the addition of water. These results suggest that the

high methanol tolerance of r-BTL contributed to a decrease in the number of methanol

additions in palm oil methanolysis in the presence of water.

50

Fig. 6 Effect of methanol addition on the methanolysis of palm oil using (a) r-BTL and

(b) r-FHL at 40° C. Methanol was added stepwise 1-4 times to adjust the total amount of

methanol to 4 molar equivalents of palm oil: 1 molar equivalent methanol was added at

0, 24, 48 and 72 h (diamond); 2 molar equivalents of methanol were added a