Purification, physico-chemico-kinetic characterization and thermal inactivation thermodynamics of milk clotting enzyme from Bacillus subtilis MTCC 10422

Narwal, Rajesh Kumari; Bhushan, Bharat; Pal, Ajay; Panwar, Anil; Malhotra, Sarla P.

doi:10.1016/j.lwt.2015.08.065

Cited by 30 publications

(21 citation statements)

References 32 publications

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“…In addition, the microbial rennet seemed to be more effective in degrading κ‐casein than α‐ and β‐casein components since the κ‐casein band faded away at an early stage with a low rennet concentration. Similar proteolytic activity on casein components by the rennet preparation from B. subtilis MTCC 10,422 was also reported (Kumari Narwal, Bharat, Ajay, Anil, & Sarla, ). The SDS‐PAGE results also indicated a broad specific activity of the microbial rennet from the rice wine on milk caseins, as reported for other microbial proteases, such as an extracellular metalloprotease from the edible mushroom Termitomyces clypeatus (Majumder, Banik, & Khowala, ) and the milk‐clotting enzyme from Paenibacillus spp .…”

Section: Resultssupporting

confidence: 82%

Change of proteolysis and sensory profile during ripening of Cheddar‐style cheese as influenced by a microbial rennet from rice wine

Zhao

Zheng

Zhang

et al. 2019

Food Science & Nutrition

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To test the potential of a novel microbial rennet isolated from traditional fermented rice wine for cheese making, Cheddar‐style cheese made with this enzyme was studied for changes in composition, proteolysis, and sensory profile during 90 days of ripening in comparison with a control cheese made with a commercial rennet. The initial proteolysis assay of the microbial rennet on milk proteins indicated a notable increase in the hydrolysis of casein components (α‐, β‐, and κ‐caseins) but no effect on whey proteins upon increasing the concentration of the enzyme. Correspondingly, compared to cheese made with commercial rennet, the use of the microbial rennet in Cheddar‐style cheese resulted in significantly higher primary and secondary proteolysis in the later stages of ripening (60–90 days ripening) and thus a softer texture and the formation of more volatile compounds and free amino acids (FAAs) despite its lower moisture content (41.7%, w/w). Though the cheese made with the microbial rennet was found to contain bitter‐taste FAAs (1,000 mg/100 g), the combined effect of other‐taste FAAs, including sweet (231 mg/100 g), umami (225 mg/100 g), and tasteless (361 mg/100 g) FAAs, in the cheese attenuated the bitter taste of the cheese. This analysis was in accordance with the sensory evaluation, which showed no significantly different sensory scoring between the cheeses made with the microbial and commercial rennets. The present study demonstrated a novel approach to evaluate the bitter taste of ripened cheese. The results of this study suggest the potential of the microbial rennet from rice wine to serve as a new source of milk‐clotting agents in cheese making.

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Section: Resultssupporting

confidence: 82%

Change of proteolysis and sensory profile during ripening of Cheddar‐style cheese as influenced by a microbial rennet from rice wine

Zhao

Zheng

Zhang

et al. 2019

Food Science & Nutrition

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“…However, for practical application, crude enzyme extracts have to be purified to improve their apparent activity and catalytic efficiency [15]. The reported enzyme purification processes such as multi-step solvent partitioning [16], the aqueous two-phase system [17], ammonium sulfate precipitation [18] and sequential chromatographic process [19,20] are tedious and time-consuming. In addition, these steps also incur extra downstream processing costs.…”

Section: Introductionmentioning

confidence: 99%

Direct immobilization of laccase on titania nanoparticles from crude enzyme extracts of P. ostreatus culture for micro-pollutant degradation

Nguyen

Hou

et al. 2017

Separation and Purification Technology

136

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“…However, its activity decreased by more than 50% at pH 7.0 and was insignificant at pH 8.0. Therefore, the stability of the enzyme in MCA in the present study corresponds with a milk pH (5.0 to 6.5) at the time of enzyme addition that is appropriate for cheese production (Kumari et al 2016). The instability of the enzyme at alkaline pH"s suggested an irreversible inactivation of enzyme (Kumari et al 2016) or alkaline pH"s may cause a protein denaturation in the dialyzed enzyme (Fazouane-Naimi et al 2010).…”

Section: Discussionmentioning

confidence: 67%

“…The dialyzed enzyme from A. oryzae DRDFS13 was shown best milk-clotting activity between pH values 4.5-6.5 with highest activity at pH 5.0. This implies that the enzyme is active at acidic pH and appropriate for cheese production (Kumari et al 2016). Similarly, the optimal milkclotting activity for aspartyl proteinase from Mucor mucedo DSM 809 , milkclotting enzyme from Rhizomucor miehei (Moghaddaml et al, 2008), acid protease from A.…”

Section: Discussionmentioning

confidence: 99%

“…The effect of varying monovalent and divalent metal ions on the milk-clotting activity in the forms of chloride and sulfate salts (Na, K, Ca 2+, Mg 2+ Mn 2+ , Fe 2+ , Zn 2+ , Ni 2+ , Cu 2+, and Co 2+) were added to the substrate at a final concentration of 10 mM and the milk-clotting assay was conducted after 30 min (Table 2). For comparison, the control (without the addition of CaCl 2 or any metal ion) was taken as 100% and the effect of various metal ions was expressed as residual or relative milk-clotting activity (Kumari et al, 2016). were incorporated in the reaction mixture (Table 3).…”

Section: Effect Of Metal Ions On the Milk-clotting Activitymentioning

confidence: 99%

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Purification and Characterization of Aspartic Protease Produced fromAspergillus oryzaeDRDFS13 under Solid-State Fermentation

Mamo

Orellana

Yelemane

et al. 2020

Preprint

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Aspartic proteases (E.C.3.4.23.) are endopeptidases with molecular masses ranging between 30-45 kDa. They depend on aspartic acid residues for their catalytic activity and show maximal activity at low pH. Thus the main objective of the present study was to purify and characterize aspartic protease from locally identified fungi by solid-state fermentation. The aspartic protease in the current study was obtained from A. oryzae DRDFS13 under SSF. The crude enzyme extract was purified by size-exclusion (SEC) and ion-exchange (IEC) chromatography. The protein contents of crude enzyme and IEC fractions were determined by BCA methods while the presence of N-glycosylation was checked using Endo-H. Inhibition studies were conducted using protease inhibitors. The milk-clotting activity (MCA), protease activity (PA); molecular weight and enzyme kinetics were determined using standard methods. Optimum temperature and stability, optimum pH and stability, and the effect of cations on MCA were assessed using standard methods. The maximum MCA (477.11 U/mL) was recorded from IEC fraction A8. The highest specific activity (183.50 U/mg), purification fold (6.20) and yield (9.2%) were also obtained from the same fraction (IEC A8). The molecular weight of 40 kDa was assigned for the purified enzyme (IEC A8). However, its molecular weight was decreased to 30 KDa upon deglycosylation assay which infers that the protein is glycosylated. Incubation of the pure enzyme (IEC A8) with pepstatin A caused a 94 % inhibition on MCA. The dialyzed enzyme showed a Km and Vmax values of 17.50 mM and 1369 U, respectively. The enzyme showed maximum MCA at 60 oC and pH 5.0 with stability at pH 4.5-6.5 and temperature 35-45 oC. Most cat-ions stimulate the activity of the enzyme; moreover, the highest MCA was detected at 50 mM of MnSO4. Furthermore, the results obtained in the present study confirmed that the aspartic protease enzyme produced from A. oryzae DRDFS13 and purified in ion-exchange chromatography could be used as a substitute source of rennet enzyme for cheese production.

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Purification, physico-chemico-kinetic characterization and thermal inactivation thermodynamics of milk clotting enzyme from Bacillus subtilis MTCC 10422

Cited by 30 publications

References 32 publications

Change of proteolysis and sensory profile during ripening of Cheddar‐style cheese as influenced by a microbial rennet from rice wine

Change of proteolysis and sensory profile during ripening of Cheddar‐style cheese as influenced by a microbial rennet from rice wine

Direct immobilization of laccase on titania nanoparticles from crude enzyme extracts of P. ostreatus culture for micro-pollutant degradation

Purification and Characterization of Aspartic Protease Produced fromAspergillus oryzaeDRDFS13 under Solid-State Fermentation

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