ENZYME CATALYSTS FOR A BIOTECHNOLOGY-BASED CHEMICAL INDUSTRY

Professor Frances H. Arnold California Institute of Technology

DOE Contract DE-FG02-93CH10578 Quarterly Progress Report, April 1-June 28, 1996

July 22, 1996

DISCLAIMER

This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or respnsi- bility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights, Refer- ence herein td any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recom- mendation, or favozing by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.

DISCLAIMER

Portions of this document may be illegible in electronic image products. Images are produced from the best avaiiable originaI document.

The goal of this research is to engineer enzymes to be efficient and economically attractive catalysts for the chemical industry. We are attempting to demonstrate generally- applicable approaches to enzyme improvement as well as develop specific catalysts for potential industrial application.

Progress during quarter April 1 -June 28, 1996

1. Random mutagenesis of pNB esterase: imtxoved activity and stability. A major goal of this DOE contract is to extend our basic enzyme engineering work

to developing specific catalysts of industrial importance. Thus part of our effort includes a collaboration with Eli Lilly & Co. to engineer an esterase for use in antibiotics production. This quarter has been devoted to further efforts to evolve a thermostable esterase.

The thermostability of the wild type pNB esterase was measured by incubating the enzyme at various temperatures for 1 hour and then chilling promptly in a ice bath. Residual esterase activity was assayed at 23 "C using para-nitrophenyl acetate (pNPA). The results show that the enzyme is half-deactivated around 50 "C. The loss of enzyme activity was irreversible under the conditions employed in this experiment. The kinetics of thermal denaturation at 55 "C are shown in Figure 1. In this experiment we also compared the inactivation rate of the purified protein with the protein inside the E. coli cells. Fig. 1 shows that inactivation rates inside and outside the cell are indistinguishable. This is an important result since the goal is to eventually find enzyme variants that are more thermostable outside the cell in purified form, while the screening reaction takes place intracellularly. The irreversible thermoinactivation of the pNB esterase obeys first-order kinetics, with a half-life of inactivation of 1.44 minutes. The screening assay described in the previous report was developed on the basis of this information.

We have screened 690 pNB esterase mutants created by shuffling the genes of four fourth-generation variants evolved for increased pNE3 esterase activity in 15% DMF. The result of screening these mutants for thermostability is shown in Figure 2, where the clones have been ordered in decreasing order of their Ra&. A significant fraction of the shuffled fourth-generation pNB esterases are more thermostable than the wild-type enzyme. These have been isolated. During the next quarter we will attempt to recombine these mutations in a single, improved enzyme.

1.2 c

0

0

mo 0

\ .\ "\ \

2 4 6

Time (min) 8 10 12

Figure 1. Kinetics of thermal inactivation of purified pNB esterase (filled circles) and pNl3 esterase in the E. coEi. cell (open circles) at 55 O C (0.1 M Tris-HC1, pH 7.5,O. 1 M NaCl). The lines are the least-squares fit for a first-order decay reaction.

0.6 0.5 x

Q) a E n 0.4 u a $ 0.3

9 - e 0.1 '1 .d d 0.2

3; 0

-0.1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .._.__.. ..__.. ...I .OQ ...__...._... .200 ._...___

Number of Clones

Figure 2. Thermal stability profile of 690 mutant variant of pNB esterase generated by DNA shuffling of four evolved pNl3 esterases. The data are displayed in decreasing order of RaAa. The Ra/Ia value for wildtype pNB esterase is -0.1.

2. Subtilisin mutants exhibiting; improved ligase activity in organic solvents.

Work is continuing on the development of subtilisin E variants which exhibit improved peptide ligase activity in organic solvents, using directed evolution methods. In addition, we have begun screening random mutant libraries for subtilisins with improved thermostabilities. It is envisioned that thermostability and peptide ligase activity are independent properties (Le. involve independent sets of amino acids) and can therefore be evolved in parallel and subsequently combined into a single variant.

Three positive variants from 5000 random mutants were isolated, characterized and sequenced. The DNA substitutions from these three variants are shown below. All three variants have been purified by ion-exchange chromatography and characterized for their ability to catalyze the polymerization of L- methionine methyl ester. Kinetic analysis shows that mutant P9D12 has a hydrolysis rate constant one-half that of wt subtilisin E, while its aminolysis rate is the same. Thus the partition coefficient for this enzyme is increased approximately 2-fold over that of wt.

Improved thermostability: A screening method for thermostabilization has been developed, and 2000 variants from the random mutant library have been screened. Three positive variants have been isolated and further characterized.

Improved peptide Eigase activity:

To screen for thermostable variants, the transformed colonies are picked up and grown in SG medium on 96-well plates overnight. The plates are then centrifuged and two copy plates for each master plate are made by transferring 5ul of supernatant from each well. One of the two copies is used for the initial hydrolytic activity assay. The other is used for the residual hydrolytic activity assay in which the whole plate is incubated at 60C for 30 min before activity assay. The length of incubation time is chosen to avoid the possible incomplete heat transfer while keep one-third to half of initial activity for the wild type. The hydrolytic activity is assayed by monitoring the hydrolysis of peptide substrate sAAPF-pNa at room temperature using plate reader. The normalized residual activity (ratio of residual activity and initial activity) is used as a criterion for the rate of thermoinactivation. All the colonies are wild type and the normalized residual activity is about 35% with error less than 5% . This screening method is quite sensitive and reliable.

To validate this screening method, about two hundred variants were screened. The variants with normalized residual activity higher than wild type is about 4%, the variants with similar activity as wild type is about lo%, the variants with lower activity than wild type is about 30% and the rest 50% are inactive. Their culture supernants are used for the thermoinactivation assay (Figure below).

P9D12

P20E8

P34B 1 1

Base substitution

CAG + CGG (A->G) GCA - GCG (A->G)

GGC + AGC (G->A) TCA - CCA (T->C)

TAT-AAT (T->A)

Amino acid substitution

Gln59 -Arg silent

Gly166-Thr Ser188 + Pro

silent silent silent

Val 143 - Asp Gly166- Cys

Tyr217- Asn

Table 1: The DNA and amino acid substitutions in the three variants P9D12, P20E8 and P34B 1 1 identified from the first generation.

.

U a> N

(b .- - E 0 z L

1.2

1

0.8

0.6

0.4

0.2

0

y = 0.9866 * e/\(-0.045548~) R2= 0.99841 - - - y = 0.7954 * &'(-0.077976~) R2= 0.99207

y = 1 .0191 * e/\(-0.059242~) R2= 0.99582 - - - - -

--I- 2-AI 2 - C -2-81

1 -E8

-1 0 0 10 20 30 40

Time (min) 50 60

Significant invited lectures:

Invited speaker for "Design of Protein Structure and Function" Conference, Royal Swedish Academy of Sciences, Stockholm, Sweden, May 22-25, 1996.

Invited speaker for 50th Anniversary Celebration of the Office of Naval Research, at the National Academy of Sciences, May, 1996.

(Je8 Moore):

Invited speaker, Forced and Natural Molecular Evolution Workshop, Biotechnology and Biological Sciences Research Council, U. Warwick, UK, May 8-9, 1996.

Upcoming invited lectures (F. Arnold):

70

Invited speaker, Gordon Conference on Biocatalysis, July, 1996.

Invited plenary speaker, 15th Biotechnology Symposium, Tokyo, November 15, 11996.

C A L I F O R N I A I N S T I T U T E O F T E C H N O L O G Y CHEMICAL ENGINEERING 210-41

PASADENA, CA 91 125

Frances H. Arnold Professor

July 23, 1996

Phone 818 395-4162 FAX 818 568-8743

frances@macpost.caltech.edu

Mr. Matthew A. Barron U.S. Department of Energy Golden Field Office 16 17 Cole Boulevard Golden, CO SO401

Dear Mr. Barron:

Enclosed is my quarterly Technical Progress Report for DOE contract DE-FG02-93-CH10578 for the period of April l-June 28, 1996.

If you have questions, please contact me.

Sincerely,

A- L A Frances H. Arnold %-*

FHA:dj Enclosure