Bottom-Up Approach to Explore Alpha-Amylase Assisted Membrane Remodelling

Soluble alpha-amylases play an important role in the catabolism of polysaccharides. In this work, we show that the enzyme can interact with the lipid membrane and further alter its mechanical properties. Vesicle fluctuation spectroscopy is used for quantitative measurement of the membrane bending rigidity of phosphatidylcholines lipid vesicles from the shape fluctuation based on the whole contour of Giant Unilamellar Vesicles (GUVs). The bending rigidity of the lipid vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in water increases significantly with the presence of 0.14 micromolar alpha-amylase in the exterior solution. However, as the concentration increases above 1 micromolar, the bending rigidity decreases but remains higher than estimated without the protein. Contact between the alpha-amylase in the outer solution and the outer leaflet leads to spontaneous membrane curvature and the corresponding morphological changes of the GUVs. The presence of outbuds directly demonstrates that AA has a preferable interaction with the membrane, giving a positive spontaneous curvature of $C_0 \leq 0.05 \ \mu \rm{m}^{-1}$ at $18 \ \mu$M $(\approx$ 1 mg/ml) of AA concentration. Above 1 mg/ml of AA concentration the shape of GUVs collapse completely suggesting a highly convoluted state.

[1]  R. Lipowsky,et al.  Generation of Bilayer Asymmetry and Membrane Curvature by the Sugar‐Cleaving Enzyme Invertase , 2022, ChemSystemsChem.

[2]  K. C. Mondal,et al.  Microbial Amylase: Old but still at the forefront of all major industrial enzymes , 2022, Biocatalysis and Agricultural Biotechnology.

[3]  T. Bhatia Micromechanics of Biomembranes , 2022, The Journal of Membrane Biology.

[4]  R. Lipowsky,et al.  Simple sugars shape giant vesicles into multispheres with many membrane necks. , 2020, Soft matter.

[5]  W. Pezeshkian,et al.  Fluctuations and conformational stability of a membrane patch with curvature inducing inclusions. , 2019, Soft matter.

[6]  J. Ipsen,et al.  Vesicle fluctuation analysis , 2019 .

[7]  R. Rajendran,et al.  Development of thermostable amylase enzyme from Bacillus cereus for potential antibiofilm activity. , 2018, Bioorganic chemistry.

[8]  M. Syamsunarno,et al.  The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View , 2017, Enzyme research.

[9]  L. Visai,et al.  Silver nanoparticles synthesized and coated with pectin: An ideal compromise for anti-bacterial and anti-biofilm action combined with wound-healing properties. , 2017, Journal of colloid and interface science.

[10]  Qiaoge Zhang,et al.  Microbial α-amylase: A biomolecular overview , 2017 .

[11]  R. S. Conlan,et al.  Clinical applications of amylase: Novel perspectives. , 2016, Surgery.

[12]  C. Hannig,et al.  Salivary amylase - The enzyme of unspecialized euryphagous animals. , 2015, Archives of oral biology.

[13]  A Vissink,et al.  The functions of human saliva: A review sponsored by the World Workshop on Oral Medicine VI. , 2015, Archives of oral biology.

[14]  L. Duelund,et al.  Buffers affect the bending rigidity of model lipid membranes. , 2014, Langmuir : the ACS journal of surfaces and colloids.

[15]  O. G. Mouritsen,et al.  Intrinsic reaction-cycle time scale of Na+,K+-ATPase manifests itself in the lipid–protein interactions of nonequilibrium membranes , 2012, Proceedings of the National Academy of Sciences.

[16]  L. Bagatolli,et al.  Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers. , 2010, Biochimica et biophysica acta.

[17]  Peter Westh,et al.  Role of electrostatic repulsion on colloidal stability of Bacillus halmapalus alpha-amylase. , 2009, Biochimica et biophysica acta.

[18]  J. Brask,et al.  Softening of POPC membranes by magainin. , 2008, Biophysical chemistry.

[19]  D. Marsh,et al.  Elastic curvature constants of lipid monolayers and bilayers. , 2006, Chemistry and physics of lipids.

[20]  M. D. Mitov,et al.  The influence of sucrose on the elasticity of SOPC lipid membrane studied by the analysis of thermally induced shape fluctuations , 2006 .

[21]  A. Rowat,et al.  Universal behavior of membranes with sterols. , 2006, Biophysical journal.

[22]  A. Rowat,et al.  Experimental evidence of the electrostatic contribution to membrane bending rigidity , 2004 .

[23]  A. Rowat,et al.  Vesicle fluctuation analysis of the effects of sterols on membrane bending rigidity , 2004, European Biophysics Journal.

[24]  B. Svensson,et al.  The structure of barley alpha-amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs. , 2003, Structure.

[25]  Gerhard Gompper,et al.  Advanced flicker spectroscopy of fluid membranes. , 2003, Physical review letters.

[26]  Torben Vedel Borchert,et al.  Industrial enzyme applications. , 2002, Current opinion in biotechnology.

[27]  J. Rothman,et al.  Budding vesicles in living cells. , 1996, Scientific American.

[28]  Netz,et al.  Inhomogeneous fluid membranes: Segregation, ordering, and effective rigidity. , 1995, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[29]  G. Niggemann,et al.  The Bending Rigidity of Phosphatidylcholine Bilayers: Dependences on Experimental Method, Sample Cell Sealing and Temperature , 1995 .

[30]  T. Lubensky,et al.  Measure Factors, Tension, and Correlations of Fluid Membranes , 1994, cond-mat/9401020.

[31]  Erich Sackmann,et al.  Bending elastic moduli of lipid bilayers : modulation by solutes , 1990 .

[32]  I. Bivas,et al.  Bending elasticity and thermal fluctuations of lipid membranes. Theoretical and experimental requirements , 1989 .

[33]  H. Lekkerkerker CONTRIBUTION OF THE ELECTRIC DOUBLE LAYER TO THE CURVATURE ELASTICITY OF CHARGED AMPHIPHILIC MONOLAYERS , 1989 .

[34]  W. Helfrich,et al.  Effect of surface charge on the curvature elasticity of membranes , 1988 .

[35]  W. Helfrich,et al.  Bilayer bending rigidity of some synthetic lecithins , 1985 .

[36]  E. Sackmann,et al.  Bilayer bending elasticity measured by Fourier analysis of thermally excited surface undulations of flaccid vesicles , 1985 .

[37]  Watt W. Webb,et al.  Thermal fluctuations of large quasi-spherical bimolecular phospholipid vesicles , 1984 .

[38]  Y. Matsuura,et al.  Structure and possible catalytic residues of Taka-amylase A. , 1982, Journal of biochemistry.

[39]  H. Zalkin,et al.  Membrane-bound and soluble extracellular alpha-amylase from Bacillus subtilis. , 1979, The Journal of biological chemistry.

[40]  W. Helfrich,et al.  Measurement of the curvature-elastic modulus of egg lecithin bilayers. , 1976, Biochimica et biophysica acta.

[41]  F. Brochard,et al.  Frequency spectrum of the flicker phenomenon in erythrocytes , 1975 .

[42]  E. Evans,et al.  Bending resistance and chemically induced moments in membrane bilayers. , 1974, Biophysical journal.

[43]  W. Helfrich Elastic Properties of Lipid Bilayers: Theory and Possible Experiments , 1973, Zeitschrift fur Naturforschung. Teil C: Biochemie, Biophysik, Biologie, Virologie.

[44]  L. Cowan,et al.  Biofilms in wounds and wound dressing , 2016 .

[45]  E. Veerman,et al.  Biochemical composition of human saliva in relation to other mucosal fluids. , 1995, Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists.

[46]  B. Ninham,et al.  Undulations of charged membranes , 1990 .

[47]  J. Rothman Assembly of cell membranes , 1979, Scientific American.