ß-Galaktosidase (ß-Gal) dikenali sebagai laktase adalah salah satu enzim kerap digunakan dalam pemprosessan makanan. ß-Gal adalah pemangkin hidrolisis laktosa kepada glukosa dan galaktosa. Perencatan galaktosa dalam tindakbalas bermangkin ß-Gal adalah masalah yang dihadapi dalam industri tenusu. Kajian mendalam tentang perencatan ß-Gal penting bagi mengoptimumkan sistem enzim secara menyeluruh. Matlamat kajian ini ialah, memahami kesan suhu terhadap mekanisma perencatan. Seperti diketahui, aktiviti enzim boleh berubah pada suhu yang berbeza. Oleh itu, perencat boleh bersifat berbeza pada suhu berlainan. Kajian pertama adalah, memahami kesan kepekatan substrat terhadap kadar tindak balas ß-Gal. Kadar tindak balas ß-Gal meningkat jika kepekatan awal substrat ditingkatkan dari 3 mM kepada 20 mM. Pada kepekatan substrat yang rendah, kadar tindak balas adalah berkadar kepada kepekatan substrat. Manakala, pada kepekatan tinggi, kadar tindak balas akan menjadi sama kerana enzim menjadi tepu. Kesan suhu terhadap aktiviti ß-Gal dikaji dan didapati, kadar tindak balas meningkat pada suhu 30 ⁰C kepada 40 ⁰C disebabkan kinetik tindak balas meningkat merangsang akiviti enzim. Akhirnya, mekanisma perencatan pada 30 ⁰C, 40 ⁰C dan 50 ⁰C adalah perencat yang berdaya saing. Ini boleh diatasi dengan menggunakan kepekatan substrat yang tinggi. Nilai pemalar bagi Vmax, Km and Ki ditentukan. Vmax tidak berubah jika kepekatan perencat ditingkatkan manakala, pengurangan Km dilihat apabila suhu ditingkatkan disebabkan pemisahan enzim-subtrat yang tinggi dan seterusnya meningkatkan kadar tindak balas. Ki menurun apabila suhu dinaikkan kerana galaktosa memberi kesan tinggi dalam menurunkan kadar tindak balas. Kesimpulannya, mekanisma perencatan tindak balas ß-Gal tidak berubah pada suhu 30 ⁰C hingga 50 ⁰C namun, perencat memberi kesan tinggi pada suhu yang tinggi.
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ß-Galactosidase (ß-Gal) known as lactase is one of the most important enzymes used in food processing. For instance, ß-Gal catalyses the hydrolysis of lactose to glucose and galactose. In dairy industries, inhibition by galactose is a common problem for ß-Gal catalysed reaction. In depth study on inhibition behaviour is required for optimising the enzymatic system in a whole. This work aims to study effect of temperature on inhibition mechanism. As we know, activity of the enzyme can be regulated by reaction temperature. Hence, it is possible that the way inhibitor act on the enzyme is changed at different temperature. Study was done to understand effects of initial substrate concentration on reaction rate of ß-Gal. Reaction rate increases as the initial substrate concentration is increased from 3 mM to 20 mM. At low substrate concentration, reaction rate is proportional to substrate concentration. While, at high concentration of substrate, reaction rate will become constant due to saturation of enzyme. Next, effect of temperature on ß-Gal activity was studied. Reaction rate increases when temperature is increased from 30 ⁰C to 40 ⁰C due to increase in kinetics of reaction resulting in high enzyme activity. Finally, inhibition mechanism at 30 ⁰C to 50 ⁰C was found to be the same which is competitive inhibition. This inhibition can be reduced by increasing initial substrate concentration. Constants Vmax, Km and Ki were determined. Vmax remained same even though inhibitor concentration was increased while reduction of Km is obtained when temperature is increased due to higher enzyme-substrate complex leading to higher reaction rate. Ki decreases when temperature is increased because galactose gives higher effect in lowering reaction rate. In short, the mechanism of inhibition for ß-Gal catalysed reaction does not change with temperature between 30 ⁰C to 50 ⁰C, however, the inhibitor is more potent at high temperatures.
The effect of temperature on the inhibition mechanism of ß-galactosidase catalysed reactions / Jasvinderjit Singh Harbhajan Singh