Arthrobacter lipase hydrolysis

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. 

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

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. 

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).

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)]. 

---