Arthrobacter lipase

A lipase from Arthrobacter species yielded pure (R)-HMPC at 50% hydrolysis with the smallest amount of the enzyme. Lipases from Pseudomonas fluorescens, Chromobacterium viscosum and Alcaligenes species were of less interest to us than the Arthrobacter lipase among others, judging from the optical purity of the product, degree... 

Enantioselective Hydrolysis with Arthrobacter Lipase. Reaction performance with the Arthrobacter lipase was studied in detail. The pH profile curve of the zero-order reaction exhibited a pH-optimum around 7.0, and spontaneous hydrolysis was not significant at pH... 

Kinetic Study of Hydrolysis. It is of great interest to know the nature of the high enantioselectivity of the Arthrobacter lipase. The initial velocity measurements were conducted for the purpose of knowing which is the main factor of the enantioselectivity, the apparent Michaelis constant K m or the catalytic constant k cat. 

A study of the influence of stirring cycles on the specific activity of the Arthrobacter lipase revealed that the activity increased with the stirring cycles and leveled off at 800 rpm. This might imply that the maximal interfacial area was obtained at more than 800 rpm of the agitation. This was supported by the effect of detergents on the activity at 1, 000 rpm. Addition of various detergents to the reaction mixture decreased the activity. This is attributed to the decrease of the interfacial area, a part of which... 

Figure 1. pH Profile for Arthrobacter Lipase-catalyzed Hydrolysis of Acetate of Racemic HMPC. 

Figure 2. Reaction Performance of Acetate of Racemic HMPC with Arthrobacter Lipase.

According to the kinetic treatment of Lavayre et al (10) on two insoluble enantiomeric isomers, the relative initial velocities, V/VR were plotted against various ratios in the (S)-enantiomer. All of the initial velocity measurements were made at 50°C instead of 40°C to avoid crystallization of the acetate of pure (R)-HMPC, and at pH 6.0 to prevent spontaneous hydrolysis of the substrate at 50°C. The result gives a concave type of curve as illustrated in Figure 3. This implies that K mS < K mR and that the (S)-enantiomer is a strong competitive inhibitor. Thus, it is concluded that a very high optical purity of (R)-HMPC liberated with Arthrobacter lipase is entirely the result of the great catalytic constant of the (R)-HMPC ester. 

Table III. Apparent Kinetic Constants of Hydrolysis of Acetate of HMPC with Arthrobacter Lipase...

Figure 3. Initial Velocity of Hydrolysis of Mixtures of (R)- and (S)-HMPC Acetate with Arthrobacter Lipase.

Enantioselective Hydrolysis with Arthrobacter Lipase. The results of the enantioselective hydrolysis of the acetate of racemic CPBA are summarized in Table V for several commercial lipases that liberate very optically pure CPBA. The experimental conditions were chosen to give approximately 50% hydrolysis for each enzyme. It is noticed that all of the lipases in Table V hydrolyzed the ester of (S)-CPBA preferentially to give the insecticidally active (S)-isomer. This is apparently different from the case of HMPC. The highest activity and optical purity were again given by the Arthrobacter lipase. Spontaneous termination of the reaction at 50% hydrolysis was observed with this enzyme as was the case of HMPC. 

For the production of various pyrethroid insecticides the (S)-4-hydroxy-3-methyl-2-(2-propynyl)-2-cyclopenten-l-one was required. Arthrobacter lipase hydrolysed only the P-enantiomer of the racemic acetate and, after extraction, the mixture of the alcohol and acetate were submitted to methanesulfonyl chloride and triethylamine. The thus-obtained acetate/mesylate mixture was hydrolysed/ inverted yielding 82% of (S)-4-hydroxy-3-methyl-2-(2-propynyl)-2-cyclopenten-l-one with an ee of 90% (Scheme 6.17 B). The procedure was also proved to work with other secondary alcohols and instead of the mesylate a Mitsunobu inversion could be applied [35]. 

Efficient biochemical processes were developed for the preparation of the two optically active pyrethroid insecticides by a combination of enzyme-catalyzed reactions and chemical transformations. These are based on the findings that a lipase from Arthrobacter species hydrolyzes the acetates of the two important secondary alcohols of synthetic pyrethroids with high enantioselectivity and reaction rate. The two alcohols are 4-hydroxy-3-methy1-2-(2 -propynyl)-2-cyclopentenone (HMPC) and a-cyano-3-phenoxybenzyl alcohol (CPBA). The enzyme gave optically pure (R)-HMPC or (S)-CPBA and the unhydrolyzed esters of their respective antipodes. 

Table 11.1-16. Lipase-catalyzed enantiomer-differentiating hydrolysis of esters of racemic cyclic secondary and tertiary alcohols in aqueous solution (PFL Pseudomonasfluorescens lipase, PSL Pseudomonas sp. lipase, CCL Candida cylindracea lipase, ABL Arthrobacter sp. lipase, PCL Pseudomonas cepacia lipase, CRL Candida rugosa lipase, CE cholesterol esterase).

The resolution of the commercially important esters of (5)-a-cyano-3-pheno-xybenzyl alcohol was only moderately efficient using lipases from Candida rugosa. Pseudomonas, and Alcaligenes sp. (Scheme 2.70). The best selectivities were obtained with lipases from Chromobacterium and Arthrobacter sp. [485], respectively. 

Yang, G., J. Wu, G. Xu, and L. Yang. 2009. Improvement of Catalytic Properties of Lipase from Arthrobacter Sp. By Encapsulation in Hydrophobic Sol-Gel Materials. Bioresource Technology 100 (19) 4311-4316. 

In a further example, a biocatalytic route for the production of optically pure 3-substituted cyclohexylamine derivatives from prochiral bicychc P-diketones was established by employing three biocatalytic reaction steps (Scheme 4.16) [53]. The sequence combined the stereoselective hydrolysis of a C-C bond catalyzed by a P-diketone hydrolase [54] (6-oxocamphor hydrolase (OCH) from Rhodococcus sp. [55]), followed by an Upase-catalyzed esterification [Candida antarctica lipase B (CAL-B), Novozyme 435], and a subsequent asymmetric amination by either an (S)-or (1 )-selective m-TA [V.fluvialis [27] or a variant of the Arthrobacter sp. TA [16a] (ArRmutll)]. 

Chaubey,A., Parshad, R, Ihneja, S. C.,and Qazi, G. N. [2008).Arthrobacter sp. lipase immobilization on magnetic sol-gel composite supports for enantioselectivity improvement. Process Biochem., 44,154-160. 

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