Optimization of the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 by response surface methodology

The aim of this work was to study the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 using optimized nutritional and cultural conditions in a complex yeast medium under aerobic batch fermentation. By applying the conventional "one-variable-at-a-time" approach and the response surface methodology, the effect of four fermentation parameters (type of carbon source, initial culture pH, temperature, and incubation time) on the growth and α-amylase production was evaluated. The production of α-amylase during 60 h of fermentation increased 13-fold under optimized conditions (1% starch, pH 6.0, 30ºC) in comparison to the conventional optimization method. The initial pH value of 6.13 and temperature of 30.3ºC were optimal conditions by the response surface methodology, leading to further improvement (up to 13-fold) in the production of extracellular α-amylase. These results constituted first evidence that K. marxianus could be potentially used as an effective source of extracellular α-amylase.

Purification, kinetic characterization and properties of a novel thermo-tolerant extracellular protease from Kluyveromyces marxianus IFO 0288 with potential biotechnological interest.

A novel extracellular hydrolase of ∼45kDa molecular mass was purified from Kluyveromyces marxianus IFO 0288 cultures and characterized as serine protease. The K(m)-value of protease (designated protease-KM-IFO-0288-A), which was found active in media containing elevated [NaCl] but lacking EDTAK(2), decreased with increasing [Ca(2+)]. The protease maintained considerable activity at the range of 10-60°C and pH 6.00-10.25, with optimum k(cat)/K(m)-value at 35.5°C and pH 7.75. It was strongly affected by specific irreversible inhibitors of serine proteases while was unaffected by inhibitors of cysteine proteases. Significant rate constants, activation energies, and proton inventories were estimated from the profiles of Michaelis-Menten parameters, versus pH, temperature and deuterium atom fraction, in the hydrolysis of Suc-AAPF-pNA showing that protease-KM-IFO-0288-A performs catalysis via a charge-relay system. The properties of protease-KM-IFO-0288-A suggest that K. marxianus represents a valuable source of extracellular protease of biotechnological interest which, given its GRAS status, could find several important applications.

Aspects on the catalysis of lipase from porcine pancreas (type VI-s) in aqueous media: development of ion-pairs

This article reports a first contribution for the elucidation of catalytic mechanism of Lipase from porcine pancreas, type VI-s (PPL), in hydrolyzing an ester substrate in aqueous media. The conclusions were based on the pH-profiles of Michaelis-Menten parameters k cat/Km, k cat and Km, as well as on the absolute temperature profile of k cat/Km, obtained during the hydrolysis of p-nitrophenyl laurate by PPL. It was found that (a) PPL performs catalysis by means of ion pairs formed either as Ser152-Ο-/His263-Im+H and/or Carbonyl-Ο-/His263-Im+H, (b) the parameter k cat/Km equals to k1 and thus ES is formed and destroyed in the course of a series of consecutive reactions governed by the dynamic constant K S = k2/k1, and (c) the hydrolysis of substrate is assisted by a hydrogen bond developed between deprotonated Asp176 and the positively charged imidazole of His263 across a pKa-value 3.85, necessary for efficient catalysis.

Necessary auxiliary background for efficient use of an existing computer program of non-parametric fitting of nonlinear equations

This work constituted a significant contribution for more efficient use of a valuable computer program of non-parametric fitting of nonlinear multiparametric equations to experimental data. However, prerequisite in this context was the transformation of nonlinear multiparametric equations into linear hyperplane forms before their incorporation within the computer program; this latter was decisive and a matter of proper programming practice. Herein, a series of widely used equations useful in different fields of chemical processes, in biochemistry and/or in biotechnology, along with their suitable transformations as well as the appropriate programming support are being reported.

Este trabalho constitui uma contribuição significativa para um uso mais eficiente de um programa de computador valioso do encaixe não-paramétrico de equações multiparamétricas não lineares aos dados experimentais. Entretanto, o pré-requisito neste contexto é a transformação de equações multiparamétricas não lineares em formulários lineares do hyperplano antes de sua incorporação dentro do programa de computador; este último é decisive e uma matéria da prática de programação apropriada. Nisto, nós relatamos uma série das equações extensamente usadas úteis em campos diferentes de processos químicos, em bioquímia e/ou em biotechnologia, junto com suas transformações apropriadas além a sustentação de programação apropriada.

Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest.

Two thermophilic extracellular proteases, designated Lmm-protease-Lh ( approximately 29 kDa) and Hmm-protease-Lh ( approximately 62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK(2). Both novel proteases maintained full activity at 60 degrees C after 1-h incubation at 10 degrees C as well as at 80 degrees C, showing optimum k(cat)/K(m) values at pH 7.00 and 60 degrees C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis-Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis-Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k(cat)/K(m)) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis-Menten parameters.

Proton inventories constitute reliable tools in investigating enzymatic mechanisms: application on a novel thermo-stable extracellular protease from a halo-alkalophilic Bacillus sp.

A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.

The catalytic mode of cysteine proteinases of papain (C1) family.

The Proton Inventory (PI) method has been applied in the hydrolysis of synthetic substrates by papain, chymopapain and stem bromelain, comparing also their corresponding pH-(k(cat)/K(m)) profiles, and it was found: (a) k(cat)/K(m)=k(1), and thus K(S)=k(2)/k(1) is a dynamic equilibrium constant, (b) bowed-downward PI for k(cat)/K(m) exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for K(S), k(2) and k(3). A novel finding of this work is that the association of substrates onto all three studied cysteine proteinases proceeds via a stepwise pathway, in contrast to purely concerted pathways found previously for both acylation and deacylation. A hydrogen bond, which seems more likely to be developed across a pK(a)-value close to 4.00, connecting [see text] (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.