JOUFWAL OP BI~~~IBN~B AND BIOBNOINBERRW Vol. 90, No. 6, 681-683. 2000

Effect of Additives on Transesterification Activity of Rhizopus chinensis Lipase

MASAHIRO YASUDA, * TAR0 KIGUCHI, HIROKI KASAHARA, HIROYASU OGINO, AND HARUO ISHIKAWA

Department of Chemical Engineering, Graduate School of Engineering, Osaka Prefecture University, I-l Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received 26 July 2OOWAccepted 26 September 2000

The transesterificatlon activity of powder lipase prepared from the porilkd llpase of Rhizopur chinens& cells by freeze-drying was quite low compared with that of acetone-dried cells. Additives which could enhance the transesteriflcation activity of the freeze-dried powder lipase were screened. The freeze-dried Iipases prepared with certain fatty add methylesters or certain types of sarfactants exhibited high transesterlfication activity. It was shown that not only the solnbility of the freeze-dried lipase in n-hexane but also the organic solvent-stability was enhanced when methyl stearate was added to the Iipase solution at the freeze-drying step.

mey words: Rhizopus chinensis, Iipase, transesterification, additive]

Rhizopus chinensis, a filamentous fungus, produces a lipase with a 1,3-positional specificity. When the cells were cultivated in a medium containing olive oil, high hydrolytic lipase activity was observed in both the acetonsdried cells and culture supernatant. The acetone- dried cells also exhibited transesterification activity on olive oil and methyl stearate, whereas a lipase prepared from the culture supematant by freeze-drying did not (1). Yasuda et al. (2) purified a lipase from R. chine&s cells and studied its catalytic properties. They found that the purified lipase exhibited high hydrolytic activity. Powder lipase prepared from the purified lipase by freezedrying exhibited hydrolytic activity as well as trans- esterification activity on olive oil and methyl laurate in n-hexane, although the latter was low. Clarifying why the transesteritication activity of the freeze-dried lipase was low compared with that of the acetone-dried cells is very important for obtaining acetone-dried cells and freeze-dried lipase which exhibit high transesterification activity.

Recently, several lipases that were modified with lipids (3, 4), surfactants (5, 6) and synthetic polymers (7) have been developed. These modified lipases exhibit high activ- ity in organic solvents compared with unmodified pow- der lipases. This is because the modified enzymes are highly soluble in organic solvents while the unmod&d ones are not. Furthermore, they have good stability in organic solvents (5, 7). Therefore, the difference in trans- esterification activity between the freeze-dried powder samples prepared from the purified lipase of R. chinensti cells and the acetone-dried R. chinens& cell samples may be attributed to the differences in the stability of these two biocatalysts and the accessibility of the substrates to them.

The objective of the present study is to verify the above speculation by screening additives that can en- hance the transesterification activity of the freeze-dried lipase.

A purified lipase was prepared from R. chine&s IF0 4768 cells according to the method described in a previ- ous paper (2). A fine-grade lipase of R. delemar was pur-

* Corresponding author.

chased from Seikagaku Kougyo Co. (Tokyo) and used for comparison without purification.

The protein concentration was determined according to the method of Bradford (8) using bovine serum albu- min as a standard. The hydrolytic activity of the lipase was assayed by the method using 2,3dimercaptopropan- l-01 tributyrate and 5,5-dithiobis(Znitrobenzoic acid) (9). One unit (U) of hydrolytic activity was defined as the amount of enzyme that liberated 1 pmol of SH groups per min at 30C.

In general, the transesterification activity is assayed in an organic solvent using dry lipase samples. Therefore, the freeze-dried lipase was prepared as follows: an addi- tive was added to 1Oml of 1OmM potassium phosphate buffer (pH 5.5) containing 3.5 mg of lipase. The mixture was well mixed with a magnetic stirrer and frozen in a freezer. It was then lyophilized in a freeze-drying appara- tus (FD-SN, Tokyo Rikakikai Co., Tokyo). As listed in Table 1, the following chemicals were examined as the additives: fatty acid methyl esters, anionic surfactants, cationic surfactants, nonionic surfactants, amino com- pounds, water-soluble polymers, alcohols, chloride salts, soluble starch, gelatin, and skim milk.

The transesterification activity was determined by mea- suring the extent of transesterification of olive oil and fatty acid methyl esters. Methyl laurate and methyl palmitate were used as fatty acid methyl esters for activ- ity measurements of the R. chinensis lipase and the R. delemar lipase, respectively. The reaction mixture con- tained 2 g of olive oil, 4 g of fatty acid methyl esters and 5 g of n-hexane. The concentrations of methyl laurate and methyl palmitate in the reaction mixture were 1.2 M and 0.95 M, respectively. The freeze-dried powder lipase was added to the reaction mixture which was then stirred at 500 rpm at 37C for 5 h. The methyl esters produced were analyzed using a Yanagimoto G80 gas chromato- graph (Yanagimoto Kogyo, Kyoto) equipped with a hydrogen flame ionization detector. A stainless steel col- umn (3 mm ID x 2m) packed with Unisole 3000 (GL Science, Tokyo) was used. The temperatures of the injec- tor and the column were 270C and 23OC, respectively. One unit (U) of transesterification activity was defined as the amount of enzyme that produced 1 pmol of methyl

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682 YASUDA ET AL. J. Bmsm. BIOHNO.,

TABLE 1. Effect of additives on transesteri8cation activity of TABLE 2. Effect of additives on transesterification activity of K. chinens& lipase R. delemar lipase

Additive

No additive Methyl stearate Methyl pahnitate Methyl myristate Methyl laurate

Transesteriiication activity

(ku/g-lipase)

0.047 0.424 0.144 0.393 0.410

Methyl decanoate 0.142 Methyl decanoate 0.186 Methyl octanoate 0.262 Methyl octanoate 0.192 Methyl hexanoate 0.181 Methyl butyrate N.D. Methyl acetate N.D. Sodium cholate 0.464 Sodium deoxycholate 0.513 Sodium taurocholate N.D. Sodium dodecyl sulfate N.D. Diisooctyl sodium sulfosuccinate (Aerosol OT) N.D. Hexadecyltrimethyl ammonium bromide N.D. Tricaprylyhnethyl ammonium chloride (Aliquat 336) N.D. Triton X-45 0.405 Triton X-100 0.333 Triton X-165 0.269 Triton X-405 N.D. Triton N-57 0.330 Tween 20 Tween 80 NN*:. I,l-Dimethyl urea N:D: 1,3-Dimethyl urea N.D. Urea N.D. Guanidine thiocyanate N.D. Guanidine chloride N.D. Guanidme carbonate N.D. Dimethyl acetoamide N.D. n-Butyl amide N.D. Acetoamide N.D. Benxamide N.D. Succinamide N.D. Polyvinyl alcohol 500 0.262 Polyvinyl alcohol 1500 0.033 Polyethylene imine 70 N.D. Polyvinyl pyrrohdone K-30 N.D. Polyvinyl pyrrolidone K-90 N.D. Polyethylene glycol200 0.031 Polyethylene plvcol 1000 0.040 Polyethylene g&o1 20000 N.D. Soluble starch N.D. Gelatin N.D. Skim milk 0.334 n-Hexanol N.D. n-Decyl alcohol N.D. n-Hexadecauol N.D. Zinc chloride N.D. Magnesium chloride N.D. Cobalt chloride N.D. Lithium chloride N.D. Aluminum chloride N.D. N.D., Not detected.

esters per min at 37C. To determine what types of additives are effective for

enhancing the transesterification activity of the freeze- dried lipase, various additives were added to the lipase solution before freeze-drying. In the preliminary experi- ments, the effect of the amount of the additives in the reaction mixture on the transesterification activity of the unmodified lipase was studied, and the amount of the ad- ditives that did not affect the transesterification activity was noted. The amount of additives added to 10ml of

Additive

No additive Methyl stearate Methyl palmitate Methyl myristate Methyl laurate

Transesterification activity

(kU/g-lipase)

0.026 0.671 0.105 0.361 0.281

Methyl hexanoate Methyl butyrate Sodium cholate Sodium deoxycholate Tween 20 Aerosol OT

0.438 0.086

:.zz ND. N.D.

Aliquat 336 N.D. Triton X-45 0.098 Triton X-100 0.114 Triton X-165 0.104 Triton N-57 0.099 Skim milk 0.066

N.D., Not detected.

phosphate buffer (pH 5.5) containing 3.5 mg of lipase was 1 mmol each except for the surfactants, the water- soluble polymers, soluble starch, gelatin, and skim milk. The amount of the surfactants, soluble starch, gelatin, and skim milk added to 1Oml of phosphate buffer was 0.1 g each and that of the water-soluble polymers was 0.2g each. The nominal molecular masses of polyvinyl alcohol 500 and 1500 were about 22,000 and 66,000, respectively. Those of polyvinyl pyrrolidone K-30 and K-90 were about 30,000 and 90,000, respectively, that of polyethylenimine 70 was about 70,000, and those of poly- ethylene glycol200, 1000, and 20,000 were 8800, 44,000, and 880,000, respectively.

The effect of the additives on the transesterification ac- tivity of the R. chine&s lipase is shown in Table 1. The transesterification activity of the freeze-dried lipase pre- pared without an additive was only about 0.047 kU/g- lipase. As can be seen in the table, fatty acid methyl es- ters with long alkyl chains, sodium salt chelates, certain types of Triton X, Triton N-57, polyvinyl alcohol 500, and skim milk were very effective for enhancing the trans- esterification activity of the freeze-dried lipase. These transesterification activities were about 10 times higher than that of the freeze-dried lipase prepared without an additive.

The effects of some of the additives which enhanced the transesterification activity of the R. chinensis lipase on the transestetication activity of the R. defemar li- pase are shown in Table 2. The table shows that these additives were also effective in enhancing the transesteri- fication activity of the R. delemar lipase. However, the effectiveness of the additives for enhancing the transes- teri6cation activity was slightly different between the two lipases .

Finally, the enhancement of the transesterification ac- tivity of the freeze-dried lipase prepared in the presence of an additive was studied. The freeze-dried lipases pre- pared with fatty acid methyl esters, sodium salt chelates, or a nonionic surfactant were soluble in n-hexane, while that prepared without an additive was not. However, the transesterification activities of the freeze-dried lipases pre-

VOL. 90, 2oGu NOTES 683

pared with these additives differed from one another. Therefore, these results suggest that in addition to the solubility in n-hexane of the freeze-dried lipases pre- pared with these additives, the inactivation of the lipase due to freeze-drying and the organic solvent-stability of the freeze-dried lipase affect transesteritkation activity. When methyl stearate was used as an additive, its effect on the inactivation of the lipase by freeze-drying and the organic solvent-stability of the freeze-dried lipase were in- vestigated. Three milliliter mixtures of 10mM potas- sium phosphate buffer (pH 5.5) and 3 mg of lipase with and without 1OOmM methyl stearate were freeze-dried. After freeze-drying, 3 ml of distilled water was added to the powder samples. The hydrolytic activities of the pow- der lipase of R. chine&s cells prepared by freeze-drying with and without methyl stearate were 63% and 55% of those before freeze-drying, respectively. This result shows that the addition of methyl stearate at the freeze- drying step had little effect on the hydrolytic activity, and that the decrease in the activity due to freeze-drying was not significant.

To study the organic solvent-stability of the powder li- pase of R. chinensis cells prepared by freeze-drying with and without methyl stearate, the lipase was incubated in n-hexane for 5 h. The freeze-dried powder lipase pre- pared with methyl stearate was soluble in n-hexane, but that prepared without methyl stearate was not. After the incubation, n-hexane was removed under vacuum, and the resulting powder samples were suspended in 3 ml of distilled water. The remaining hydrolytic activity of the freeze-dried lipase prepared without methyl stearata decreased by about 75% after 5 h incubation in n- hexane. However, the remaining hydrolytic activity of the freeze-dried lipase prepared with methyl stearate was almost unchanged during 5 h incubation in n-hexane. Therefore, the stability as well as the solubility of the freeze-dried powder lipase in n-hexane was improved by methyl stearate.

In this study, the screening of additives with which the transesterification activity of the freeze-dried lipase was enhanced was performed. The freeze-dried lipase pre- pared with certain fatty acid methyl esters or certain

types of surfactants exhibited higher transesterification activities than that prepared without an additive. Although the level of effectiveness of the additives in en- hancing the transesterification activity of R. chinensis li- pase differed slightly, these additives also enhanced the transesterification activity of the R. delemar lipase. The addition of methyl stearate to the lipase solution at the freeze-drying step increased not only the solubility of the resulting freeze-dried lipase in n-hexane but also its or- ganic solvent stability. The present results indicate that the organic solvent-stability of the lipase plays an im- portant role in transesterification reactions in n-hexane.

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