A method for processing enzymes

Abstract

The invention relates to a method for processing enzymes comprising preparing an enzyme composition, exposing the enzyme composition to multiple transitions from a high pressure to a low pressure to provide a processed enzyme composition. The invention extends to a method for processing enzymes using an apparatus, a processed enzyme composition and methods of using the processed enzyme composition.

Description

[0001] A METHOD FOR PROCESSING ENZYMES

[0002] FIELD

[0003]

[0001] The present invention relates to a method for processing enzymes. More specifically, the invention relates to a method for processing enzymes using a mixing apparatus.

[0004] BACKGROUND

[0005]

[0002] Enzymes are proteins that act upon substrate molecules and decrease the activation energy necessary for a chemical reaction to occur by stabilizing the transition state. There are factors such as temperature and pH that can affect an enzymes activity. There are known processes to increase the activity of an enzyme, such as the immobilization of enzymes.

[0006]

[0003] The immobilization of enzymes (i.e. fixing the enzyme location) may increase the enzymatic activity due to enzymes only coming into contact with the substrates.

[0007]

[0004] However, these known methods of increasing enzymatic activity are expensive and / or require a significantly long time to carry out. For example, a known process has improved enzymatic activity through creation of pepsin-colloidal gold conjugates, however this process has not been widely adopted due to both the cost and the complexity of the process.

[0008]

[0005] There is a need for a process for increasing enzymatic activity that can be performed at industrial scale, that is also quick, cheap, and efficient.

[0009]

[0006] It is therefore an object of aspects of the present invention to address one or more of the abovementioned or other problems.

[0010] SUMMARY

[0011]

[0007] According to a first aspect of the present invention there is provided a method for processing enzymes comprising preparing an enzyme composition, exposing the enzyme composition to multiple transitions from a high pressure to a low pressure to provide a processed enzyme composition.

[0012]

[0008] According to a second aspect of the present invention there is provided a method for processing enzymes using an apparatus, the method comprising contacting an enzyme composition with the apparatus, wherein the apparatus comprises at least partially opposing surfaces which are operable to at least partially define a fluid flow channel, wherein the separation distance between the opposing surfaces varies in the direction of bulk fluid flow, so that the fluid flow channel comprises multiple narrowed and expanded portions.

[0013]

[0009] According to a third aspect of the present invention there is provided a processed enzyme composition formed from the method according to the first or second aspect.

[0014]

[0010] According to a fourth aspect of the present invention there is provided a method for recycling paper using a processed enzyme composition according to the third aspect.

[0011] According to a fifth aspect of the present invention there is provided a method for producing high-fructose corn syrup using a processed enzyme composition according to the third aspect.

[0015]

[0012] The processed enzyme composition may comprise enzymes with improved activity. Advantageously, the method for processing enzymes provides processed enzymes with improved enzymatic activity without denaturing or damaging the enzymes. The enzyme composition may be exposed to high temperatures and pressures during the process without denaturing or damaging the enzymes.

[0016]

[0013] By “processing enzymes”, it is intended to mean that the enzymes themselves are processed, thereby providing enzymes which have, for example, improved activity as the product.

[0017]

[0014] The terms “at least partially opposing surfaces” and “opposing surfaces” are used interchangeably unless stated otherwise, to mean at least partially opposing surfaces.

[0018]

[0015] As used herein, the term “multiple” refers to at least two, such as least 3, such as at least 4, such as at least 5.

[0019]

[0016] The fluid flow channel may comprise multiple narrowed and expanded portions relative to the longitudinal axis. As used herein, the “longitudinal axis” refers to the axis along the length of the opposing surfaces in the direction of bulk fluid flow of the enzyme composition.

[0020]

[0017] The apparatus may comprise two at least partially opposing surfaces.

[0021]

[0018] The at least partially opposing surfaces may be static. The opposing surfaces may be operable to cause flow of fluid on relative motion of the surfaces. The opposing surfaces may be rotated relative to each other. The apparatus may comprise at least two partially opposing surfaces wherein one of the opposing surfaces may be static while the other opposing surface is operable to be rotated.

[0022]

[0019] The apparatus may be operable to generate a pressure differential before and / or after each narrowed portion of the fluid flow channel. The enzyme composition may be exposed to transitions of high pressure at each expanded portion of the fluid flow channel.

[0023]

[0020] The enzyme composition may be exposed to increases and decreases in pressure and / or increases and decreases in velocity (flow rate). The transitions from a high pressure to a low pressure may be due to the enzyme composition moving from a region of higher pressure to a region of lower pressure. The region of lower pressure may be a region of lower volume and the region of higher pressure may be a region of higher volume. Suitably, the enzyme composition may pass through multiple regions of higher volume and lower volume.

[0024]

[0021] The multiple transitions from high to low pressure may comprise different pressure changes. For instance, the high pressure at one transition may be different to the high pressure at a second transition and the low pressure at one transition may be different to the low pressure at a second transition. The pressure change may be different at each of the transitions from high to low pressure. The pressure change may be the same at one or more of the transitions from high to low pressure.

[0025]

[0022] The enzyme composition may be exposed to transitions of low pressure to high pressure. The transitions of low pressure to high pressure may precede or succeed the transitions from high pressure to low pressure.

[0026]

[0023] The narrowed portion of the fluid flow channel may provide a region of lower pressure. The expanded portion of the fluid flow channel may provide a region of higher pressure. The narrowed portion of the fluid flow channel may provide a region of lower volume. The expanded portion of the fluid flow channel may provide a region of higher volume.

[0027]

[0024] The high pressure may be a pressure of from 2 to 15 bar. The high pressure may be a pressure of from 5 to 14 bar, such as from 7 to 13 bar, such as from 10 to 12 bar. The high pressure may be at least 2 bar, such as at least 4 bar, such as at least 5 bar, such as at least 6 bar, such as at least 7 bar, such as at least 8 bar, such as at least 9 bar, such as at least 10 bar. The high pressure may be up to 15 bar, such as up to 14 bar, such as up to 13 bar, such as up to 12 bar. The high pressure at each transition may be different.

[0028]

[0025] The low pressure may be a pressure of from 0.5 to 3 bar, such as from 1 to 2 bar, such as, from 1 to 1 .5 bar. The low pressure may be at least 0.5 bar, such as at least 1 bar. The low pressure may be up to 3 bar, such as up to 2 bar such as up to 1 .5 bar. The low pressure at each transition may be different.

[0029]

[0026] The pressure change between the high and low pressure may be at least 2 bar, preferably, at least 3 bar, more preferably at least 4 bar, more preferably at least 5 bar, more preferably at least 6 bar, more preferably at least 7 bar, more preferably at least 8 bar, more preferably at least 9 bar, most preferably at least 10 bar. The pressure change at each transition may be different.

[0030]

[0027] The enzyme composition may be exposed to multiple transitions from a high velocity to a low velocity. The enzyme composition may be exposed to multiple transitions from a low velocity to a high velocity. The velocity and pressure may be directly related such that the enzyme composition is exposed to a high pressure when it is exposed to a low velocity and a low pressure when it is exposed to a high velocity.

[0031]

[0028] The high velocity may be a velocity of up to 110 kg / h, such as up to 100 kg / h. The high velocity may be a velocity of at least 60 kg / h, such as at least 70 kg / h, such as at least 80 kg / h. The low velocity may be a velocity of at least 25 kg / h, such as at least 30 kg / h, such as at least 40 kg / h. The low velocity may be a velocity of up to 50 kg / h, such as up to 40 kg / h, such as up to 30 kg / h.

[0032]

[0029] The transition from a high pressure to a low pressure may be a direct result of a change in velocity (i.e. the flow rate) of the enzyme composition. An increase in the speed of the enzyme composition through the apparatus may occur due to multiple narrowed and expanded portions of the fluid flow channel.

[0033]

[0030] The enzyme composition may be exposed to a pressure of at least 2 bar, preferably, at least 4 bar, more preferably, at least 5 bar, more preferably, at least 8 bar, more preferably, at least 9 bar, most preferably, at least 10 bar.

[0034]

[0031] The enzyme composition may be exposed to a pressure of from 2 to 15 bar, such as from 5 to 12 bar, such as from 10 to 12 bar during an expanded portion of the fluid flow channel.

[0035]

[0032] The enzyme composition may be exposed to a pressure of from 0.5 to 3 bar, preferably from 1 to 2 bar, preferably from 1 to 1 .5 bar during a narrowed portion of the fluid flow channel.

[0036]

[0033] The enzyme composition may flow through the fluid flow channel. The fluid flow channel may be non-uniform. The flow path of the enzyme composition may be non-linear. The bulk flow may flow through the fluid flow channel causing flow of the enzyme composition in both the radial and axial direction. As used herein, the term “bulk fluid flow” or “bulk flow” refers to the overall direction of flow of the enzyme composition from entering the apparatus to leaving the apparatus. The bulk fluid flow may be along the longitudinal axis.

[0037]

[0034] The velocity (flow rate) of the enzyme composition may be increased upon relative motion of the opposing surfaces. The velocity (flow rate) of the enzyme composition may increase upon rotation of at least one of the opposing surfaces.

[0038]

[0035] The apparatus may further comprise a pump. The velocity (flow rate) of the enzyme composition may be controlled by the pump. The velocity (flow rate) of the enzyme composition entering the apparatus may be up to 110 kg / h, such as up to 100 kg / h. The velocity (flow rate) of the enzyme composition entering the apparatus may be at least 25 kg / h, such as at least 30 kg / h, such as at least 40 kg / h, such as at least 50 kg / h, such as at least 60 kg / h, such as at least 70 kg / h, such as at least 80 kg / h.

[0039]

[0036] The velocity at which the enzyme composition is passed through the apparatus may cause the enzymes to be exposed to a pressure change.

[0040]

[0037] The narrowed portions of the fluid flow channel are where the shear rate within the apparatus tends to be the highest. It is possible to confine the most intense shear to relatively few portions.

[0041]

[0038] The narrowed and expanded portions of fluid flow channel may alternate. Suitably, an expanded portion of fluid flow channel directly follows a narrowed portion of fluid flow channel. Suitably, a narrowed portion of the fluid flow channel directly follows an expanded portion of the fluid flow channel.

[0039] The fluid flow channel may comprise at least two narrowed and expanded portions. The fluid flow channel may comprise at least three narrowed and expanded portions, such as at least four portions, such as at least five portions.

[0042]

[0040] There may be from 4 to 15 expanded portions of the fluid flow channel (distributive mixing) and a comparable number of the narrowed portions of the fluid flow channel (dispersive mixing). Suitably, there may be 5 to 12 narrowed and expanded portions of the fluid flow channel. The fluid flow channel may comprise from 4 to 11 narrowed portions, such as from 5 to 10, such as from 6 to 9 narrowed portions. Suitably, there may be 5 to 12 expanded portions of the fluid flow channel. The fluid flow channel may comprise from 4 to 11 expanded portions, such as from 5 to 10, such as from 6 to 9 expanded portions.

[0043]

[0041] The fluid flow channel may comprise up to 12 narrowed portions. The fluid flow channel may comprise up to 11 narrowed portions, such as up to 10 portions, such as up to 9 portions. The fluid flow channel may comprise up to 12 expanded portions. The fluid flow channel may comprise up to 11 expanded portions, such as up to 10 portions, such as up to 9 portions.

[0044]

[0042] At least one of the opposing surfaces may comprise a series of cavities which define the narrowed and expanded portions of the fluid flow channel. Suitably, when the apparatus comprises two opposing surfaces, both of the opposing surfaces comprise cavities. The cavities may be substantially hemispherical and / or substantially cuboid The cavities may be substantially hemispherical on one of the opposing surfaces and substantially cuboid on the other opposing surface.

[0045]

[0043] Typical embodiments of the invention take the form of a stator / rotor drum / sleeve mixer. However, the apparatus can be embodied in a "flat" form where the drum and the sleeve are replaced with a pair of disks mounted for relative rotation and the cavities are provided in the opposing surfaces of the disks.

[0046]

[0044] The opposing surfaces may be cylindrical. In such a configuration, the apparatus will generally comprise a cylindrical drum and co-axial sleeve. The opposing surfaces will be defined by the outer surface of the drum and the inner surface of the sleeve. However, there are alternative configurations in which the opposing surfaces are circular. Between these two extremes of configurations are those in which the opposing surfaces are conical or frusto-conical. Non-cylindrical embodiments allow for further variation in the shear in different parts of the flow through the apparatus.

[0047]

[0045] The apparatus may comprise a “stepped” drum comprising multiple cylindrical regions of differing diameters. The sleeve is similarly stepped, so as to maintain the separation between the outer surface of the drum and the inner surface of the sleeve and to define an annular space between them of varying radius. In one such configuration, a region of axial flow is either followed or preceded by a region of radial flow.

[0046] The opposing surfaces may be operable to be adjusted relative to each other in the direction of bulk fluid flow (i.e. along the longitudinal axis). The opposing surfaces may be operable to be adjusted so that the position of the cavities on each of the opposing surfaces may be altered. This can be done by ensuring that the cavities on opposing surfaces are generally aligned, slightly offset or completely offset in the axial direction (i.e. along the longitudinal axis). Axial flow of the enzyme composition from cavity to cavity therefore requires the enzyme composition to pass through narrow spaces and good dispersive mixing is obtained.

[0048]

[0047] Each of the opposing surfaces may comprise from 4 to 12 cavities in the direction of bulk fluid flow. Each of the opposing surfaces may comprise from 5 to 11 cavities, such as from 6 to 10 cavities, such as from 7 to 9 cavities in the direction of bulk fluid flow.

[0049]

[0048] Suitably, each of the opposing surfaces may comprise at least 4 cavities, such as at least 5 cavities, such as at least 6 cavities, such as at least 7 cavities in the direction of bulk fluid flow.

[0050]

[0049] Each of the opposing surfaces may comprise up to 12 cavities, such as up to 11 cavities, such as up to 10 cavities, such as up to 9 cavities in the direction of bulk fluid flow.

[0051]

[0050] Each cavity along the opposing surface in the direction of bulk fluid flow may be part of a layer of cavities extending perpendicular to the row of cavities extending in the direction of bulk fluid flow. The one or both opposing surfaces may have layers of cavities extending from each cavity along the opposing surface in the direction of bulk fluid flow. The one or both opposing surfaces may comprise from 5 to 24 layers of cavities. Suitably, the one or both opposing surfaces may comprise from 7 to 18 layers of cavities, such as 10 to 15 layers of cavities, such as 12 layers of cavities.

[0052]

[0051] Suitably, in the drum and co-axial sleeve configuration, the sleeve may comprise at least 1 more cavity than the drum along the sleeve in the direction of bulk fluid flow.

[0053]

[0052] The cavities may have a depth of at least 1 mm, such as at least 1.1 mm, such as at least 1 .2 mm, such as at least 1 .3 mm, such as at least 1 .4 mm.

[0054]

[0053] The cavities may have a depth of up to 2 mm, such as up to 1 .9 mm, such as up to 1 .8 mm, such as up to 1 .7 mm, such as up to 1 .6 mm.

[0055]

[0054] The distance between the opposing surfaces is from 40 to 2,500 pm, such as from 50 to 2,000 pm, such as 50 to 1 ,500 pm, such as from 50 to 1 ,000 pm, such as from 50 to 500 pm. Suitably, the distance between the narrowed portions of the fluid flow channel (i.e. the closest opposing surfaces) may be from 50 to 200 pm, such as from 50 to 100 pm. Suitably, the distance between the expanded portions of the fluid flow channel (i.e. the widest opposing surfaces) may be from 1 ,000 to 2,500 pm, such as 1 ,400 to 2,000 pm.

[0056]

[0055] The distance between each cavity on each of the opposing surfaces may be at least 0.5 mm, such as at least 0.75 mm, such as at least 1 mm. The distance between each cavity on each of the opposing surfaces may be up to 1 .5 mm, such as at least 1 .25 mm, such as at least 1 mm.

[0056] Each cavity may have length of at least 2 mm, such as at least 3 mm, such as at least 4 mm, such as 5 mm. Each cavity may have length of up to 8 mm, such as up to 7 mm, such as up to 6 mm, such as 5 mm.

[0057]

[0057] Suitably, the distance between the opposing surfaces is up to 2,500 pm, such as up to 2,000 pm, such as up to 1 ,500 pm, such as up to 1 ,000 pm, such as up to 500 pm, such as up to 100 pm.

[0058]

[0058] The opposing surfaces may be provided with means to heat or cool it. The apparatus may further comprise a heating and / or cooling means. The heating and / or cooling means may be operable to increase or decrease the temperature of the opposing surfaces.

[0059]

[0059] Where cavities are provided in the opposing surfaces these may have a different geometry in different parts of the mixer to as to further vary the shear conditions. The operating parameters of the apparatus according to the present invention will vary according to the enzymes being processed.

[0060]

[0060] At least one of the opposing surfaces may be operable to rotate at a continuous or varied speed, suitably, at a continuous speed. At least one of the opposing surfaces may be operable to rotate at a speed of from 1 to 20,000 rpm, such as from 2,500 to 15,000 rpm, such as 4,000 to 12,000 rpm, such as from 5,000 to 10,000 rpm. Suitably, at least one of the opposing surfaces may be operable to rotate at a speed of 4,000 rpm, 5,000 rpm, 6,000 rpm, 7,000 rpm, 8,000 rpm, 9,000 rpm, or 10,000 rpm.

[0061]

[0061] At least one of the opposing surfaces may be operable to rotate at a speed of up to 20,000 rpm, such as up to 15,000 rpm, such as up to 12,500 rpm, or even up to 10,000 rpm.

[0062]

[0062] At least one of the opposing surfaces may be operable to rotate at a speed of at least 5 rpm, such as at least 100 rpm, such as at least 1 ,000 rpm, such as at least 2,000 rpm, such as at least 3,000 rpm, or even 4,000 rpm.

[0063]

[0063] The enzyme composition may comprise a carrier. The carrier may be water. The enzyme composition may be prepared by adding the selected enzyme to water. The enzyme composition may be an aqueous enzyme composition.

[0064]

[0064] The enzyme composition may comprise at least 50 wt% of enzymes based on the total weight of solutes in the composition. Suitably, the enzyme composition comprises at least 60 wt% of enzymes based on the total weight of solutes in the composition, such as at least 70 wt%, such as at least 80 wt%, such as at least 90 wt%, such as at least 95 wt%, or even 99 wt% of enzymes, based on the total weight of solutes in the composition.

[0065]

[0065] The enzyme composition may comprise enzymes selected from hydrolases, oxidoreductases, transferases, lyases and / or isomerases. Preferably, the enzyme composition may comprise an enzyme selected from oxidoreductases, hydrolases and / or isomerases.

[0066] The enzyme composition may comprise an enzyme selected from laccase, xylanase and / or a-amylase. Suitable commercially available enzymes include those sold under the trade name Novozym 51003, Shearzyme® 480 L, and Aquazym® 480.

[0066]

[0067] The enzyme composition may be subjected to a pressure of at least 2 bar, such as at least 3 bar, such as at least 4 bar, such as at least 5 bar, such as at least 6 bar, such as at least 7 bar, such as at least 8 bar, such as at least 9 bar, such as at least 10 bar during the method according to the first or second aspect of the invention. The enzyme composition may be subjected to a pressure change of from 5 bar to 1 bar, such as from 8 bar to 1 bar, such as from 10 bar to 1 bar, such as from 11 bar to 1 bar, such as from 12 bar to 1 bar.

[0067]

[0068] The enzyme composition may be subjected to a pressure of up to 15 bar, such as up to 14 bar, such as up to 13 bar, such as up to 12 bar.

[0068]

[0069] The temperature of the enzyme composition during the process may be from 10 to 55 °C, such as from 11 to 50 °C, such as from 12 to 45 °C, such as from 13 to 40 °C, such as from 14 to 35°C, such as from 15 to 30°C, such as from 17 to 25°C.

[0069] BRIEF DESCRIPTION OF DRAWINGS

[0070]

[0070] Figure 1 shows an axial section through a relieved rotating cylindrical drum and static coaxial sleeve apparatus according to the invention;

[0071]

[0071] Figure 2 shows the apparatus of Figure 1 in a different configuration;

[0072]

[0072] Figure 3 shows the apparatus of Figure 1 in a different configuration to Figure 1 and 2;

[0073]

[0073] Figure 4 shows a detailed view of region “A” in Figure 1 ;

[0074]

[0074] Figure 5 shows a preferred embodiment of Figure 1 , wherein one opposed surface comprises hemispherical cavities and the other comprises cuboid cavities;

[0075]

[0075] Figure 6 shows the enzymatic activity of a laccase enzyme which has been processed according to the method of the present invention.

[0076]

[0076] Figure 7 shows the enzymatic activity of a laccase enzyme which has been processed at different rotation speeds according to the method of the present invention.

[0077]

[0077] Figure 8 shows the enzymatic activity of a xylanase enzyme which has been processed according to the method of the present invention.

[0078]

[0078] Figure 9 shows the enzymatic activity of a xylanase enzyme which has been processed according to the method of the present invention.

[0079]

[0079] Figure 10 shows the enzymatic activity of a a-amylase enzyme which has been processed according to the method of the present invention.

[0080]

[0080] Figure 11 shows the enzymatic activity of a glucose isomerase enzyme which has been processed according to the method of the present invention. DESCRIPTION OF EMBODIMENTS

[0081]

[0081] Figure 1 shows a portion of a mixer 100 comprising an inner drum 102 and an outer sleeve 104. Cavities 106 are provided in the drum 102 and the sleeve 104 so that as the drum 102 rotates about its axis (shown dashed), the drum 102 and the sleeve 104 co-operate to form a controlled deformation dynamic mixer (CDDM). Ports (not shown) are provided for input and output of the process flow. Means for rotating the drum 102 relative to the sleeve 104 and end seals are not shown. Flow of an enzyme composition (not shown) within the mixer in from one side to the other. A pump (not shown) may be used to control the flow of the enzyme composition. The flow of the enzyme composition is shown with arrows.

[0082]

[0082] Cavities 106 are hemispherical and disposed on the opposing surfaces of the drum and 102 and sleeve 104 so that the outer face of the drum 102 and inner face of the sleeve 104 form opposing surfaces. A fluid flow channel 108 is formed therebetween and the enzyme composition flows through the channel in use, along the longitudinal axis (shown as a dashed line).

[0083]

[0083] Figure 2 shows a portion of a mixer 100 comprising an inner drum 102 and an outer sleeve 104. The mixer 100 of Figure 2 is the same as that of Figure 1 except in a different configuration. The position of the drum 102 has been altered along the longitudinal axis relative to the position of the sleeve 104. In the configuration of Figure 2, the cavities 106 on the drum 102 and sleeve 104 are misaligned so that as the drum 102 rotates about its axis (shown dashed), the drum 102 and the sleeve 104 co-operate to form a cavity transfer mixer (CTM). The cavities on the opposing surfaces of the drum 102 and sleeve 104 are misaligned so that a cavity on the drum opposes a region with no cavities on the sleeve 104. The flow path of the enzyme composition (shown with arrows) is altered compared to the flow path in the configuration of Figure 1 . A fluid flow channel 108 is formed between the opposing surfaces and the enzyme composition flows through the channel in use, along the longitudinal axis (shown as a dashed line).

[0084]

[0084] Figure 3 shows a portion of a mixer 100 comprising an inner drum 102 and an outer sleeve 104. The mixer 100 of Figure 3 is the same as that of Figure 1 except in a different configuration. The position of the drum 102 has been altered along the longitudinal axis relative to the position of the sleeve 104. In the configuration of Figure 3, the cavities 106 on the drum 102 and sleeve 104 are partially misaligned so that as the drum 102 rotates about its axis (shown dashed), the drum 102 and the sleeve 104 co-operate to form a controlled deformation dynamic mixer (CDDM). The flow path of the enzyme composition (shown with arrows) is altered compared to the flow path in the configuration of Figure 1 and Figure 2. A fluid flow channel 108 is formed between the opposing surfaces and the enzyme composition flows through the channel in use, along the longitudinal axis (shown as a dashed line).

[0085]

[0085] Figure 4 provides a more detailed view of the region "A" in Figure 1 . It can be seen that in region "X” the surface of the drum 102 is relieved and the radial spacings of the opposing surfaces of the drum 102 and the sleeve 104 are relatively large as compared with the corresponding radial spacings in region "Y”. In region X the cavities 106 promote CTM-like distributive mixing while in region Y the narrow spacing in the flow path induces extensional flow and CDDM-like dispersive mixing. In this particular embodiment of the mixer the radial spacings in regions X are constant, and the radial spacings in regions Y are also constant. The distance between the opposing surfaces on the drum 102 and sleeve 104 in region X is larger than the distance between the opposing surfaces in region Y.

[0086]

[0086] The regions “X” and “Y” in Figures 2 and 3 (not shown) differ in size compared to regions “X” and “Y” in Figure 4.

[0087]

[0087] Figure 5 shows a further embodiment of a portion of a mixer 100 comprising an inner drum 102 and an outer sleeve 104. Cavities 106a located on the opposing surface of the drum are substantially hemispherical and the cavities 106b located on the opposing surface of the sleeve are substantially cuboid. A fluid flow channel 108 is formed therebetween and the enzyme composition flows through the channel in use, along the longitudinal axis (shown as a dashed line).

[0088]

[0088] As used herein, unless otherwise expressly specified, all numbers such as those expressing values, ranges, amounts or percentages may be read as if prefaced by the word "about", even if the term does not expressly appear. Also, the recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within that range (e.g. 1 to 5 can include 1 , 2, 3, 4 when referring to, for example, a number of elements, and can also include 1.5, 2, 2.75 and 3.80, when referring to, for example, measurements). The recitation of end points also includes the end point values themselves (e.g. from 1 .O to 5.0 includes both 1.0 and 5.0). Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[0089]

[0089] Singular encompasses plural and vice versa. For example, although reference is made herein to "a" cavity, “an” opposing surface, “an” enzyme, and the like, one or more of each of these and any other components can be used.

[0090]

[0090] The terms "comprising", "comprises" and "comprised of’ as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Additionally, although the present invention has been described in terms of “comprising”, the coating compositions detailed herein may also be described as “consisting essentially of’ or “consisting of’.

[0091]

[0091] As used herein, the term "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and / or C, the list can comprise A alone; B alone; C alone; A and B in combination; A and C in combination, B and C in combination; or A, B, and C in combination.

[0092]

[0092] All of the features contained herein may be combined with any of the above aspects in any combination.

[0093] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the following examples.

[0093] EXAMPLES

[0094]

[0094] The enzyme compositions were diluted to the respective concentration to allow for enzymatic activity calculation, and processed using the apparatus of the present invention at varying flow rates and impeller speeds (from O to 15,000 rpm) to identify the optimum combination to increase enzymatic activity.

[0095] Laccase analysis

[0096]

[0095] Laccase Analysis Determination of the enzymatic activity of Laccase was performed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) which changes colour as the enzyme reacts with the substrate. ABTS is a nonphenolic dye and is oxidized by laccase to its more stable and preferred state of the cation radical. Analysis was performed using UV-Vis spectroscopy which provides quantitative measurements to determine activity accurately. The enzyme composition comprised 1 part laccase to 5 parts water.

[0097]

[0096] The processed samples are Laccase processed using the method of the present invention, with the control samples being enzyme taken from the same batch, prior to it entering the apparatus.

[0098]

[0097] Processed sample SA04 was processed using the method according to the present invention, with a rotation speed of 15,000 rpm. Processed sample SA06 was processed using the method according to the present invention, with a rotation speed of 12,500 rpm.

[0099]

[0098] Figure 6 shows that samples that have been processed with the method according to the present invention with a rotation speed of 12,500 and 15,000 rpm have a slight increase in absorbance compared to unprocessed samples. The absorbance value corresponds to the amount of substrate processed and therefore the processed samples show an increase in enzymatic activity.

[0100]

[0099] In Figure 7, processed sample SA02 was processed using the method according to the present invention, with a rotation speed of 5,000 rpm. Processed sample SA03 was processed using the method according to the present invention, with a rotation speed of 10,000 rpm. Processed sample SA07 was processed using the method according to the present invention, with a rotation speed of 7,500 rpm.

[0101]

[0100] Figure 7 shows that the samples which were processed with the method according to the present invention with a rotation speed of from 5,000 rpm to 10,000 rpm have a substantially greater absorbance than the control samples and the samples processed at different rotation speeds. The processed samples in Figure 6 show an increase in enzymatic activity of ~250% compared to the unprocessed (control) samples. Xylanase Analysis

[0102]

[0101] The method for Xylanase Activity determination was carried out according to Enzymatic Assay of XYLANASE Activity in DRISELASE (EC 3.2.1.8). The method involves creating a standard glucose concentration curve and comparing xylan treated with xylanase to the curve, to determine how much glucose has been produced by the enzyme, through quantitative analysis using UV-Vis. The enzyme composition comprised 1 part xylanase to 1000 parts water.

[0103]

[0102] Figure 8 shows a standard curve produced using standard glucose composition, along with two curves produced from treating xylan with xylanase, one which has been treated (processed) according to the present invention and one which is untreated (unprocessed), followed by analysis with UV-Vis and addition of p-Hydroxybenzoic Acid Hydrazide Solution (PAHBAH) as an indicator.

[0104]

[0103] The data in Figure 8 shows that the processed (treated) enzyme has an increased enzymatic activity of~100% based on the quantity of glucose produced at each of the time points, with the time points on the graph taken at 15, 30, 45, 60 minutes and final time point on the graph being taken 75 minutes after the reaction commenced. The absorbance value corresponds to the amount of substrate processed.

[0105]

[0104] In Figure 9, treatment A relates to a processed enzyme sample which was processed according to the method of the present invention, wherein the two opposing surfaces where static (stationary). Treatment B relates to a processed enzyme sample which was processed according to the method of the present invention, wherein the one of the opposing surfaces where static (stationary) and the other opposing surface was rotating. Figure 9 shows that both of the samples processed by the method of the present invention show an improvement in enzyme activity compared to an unprocessed sample and further show that in an embodiment where at least one of the opposing surfaces is rotating relative to the other, the increase in enzymatic activity lasts longer. a-Amylase Analysis

[0106]

[0105] A quantitative starch-iodine method for measuring a-amylase and glucoamylase activities, published by Xiao et al., was used to determine the enzymatic activity of the a-amylase (Xiao et al., 2006). A visible colour change occurs when using this method, as the enzyme breaks down the substrate, with a change from black to orange. The enzyme composition comprised 1 part a- amylase to 1 ,000,000 parts water.

[0107]

[0106] The “Benchmark” sample was the enzyme composition taken from the same batch as the “Treatment” samples, prior to it being processed with the method of the present invention.

[0108]

[0107] For Treatment A the enzyme composition was processed according to the method of the present invention with a rotation speed of 0 rpm (the apparatus was static). For Treatment B, the enzyme composition was processed according to the method of the present invention with a rotation speed of 5000 rpm. For Treatment C, the enzyme composition was processed according to the method of the present invention with a rotation speed of 10,000 rpm.

[0109]

[0108] Figure 10 shows that processing a-amylase with the method of the present invention improves the enzymatic activity. For example, “Treatment C” shows a colour change after only 15 minutes, compared to the benchmark colour changing after 45 minutes, showing an increase in enzymatic activity of ~200%.

[0110] Glucose Isomerase Analysis

[0111]

[0109] Glucose Isomerase Analysis Determination of the enzymatic activity of Glucose Isomerase was performed using Thermo Scientific 201 UV-Visible spectrophotometer. Analysis was performed using UV-Vis spectroscopy which provides quantitative measurements to determine activity accurately. The enzyme composition comprised of glucose isomerase distributed within water at a concentration of a concentration of 0.0363 g.mL-1.

[0112]

[0110] The processed samples are Glucose Isomerase processed using the method of the present invention, with the control samples being enzyme taken from the same batch, prior to it entering the apparatus.

[0113]

[0111] Figure 11 shows that samples that have been processed with the method according to the present invention with a rotation speed of 5,000 and 10,000 rpm have an increase in absorbance compared to the unprocessed sample. The absorbance value corresponds to the amount of substrate processed and therefore the processed samples show an increase in enzymatic activity. The sample processed at 5,000 rpm also increases the yield of product obtained within the same timeframe.

Claims

CLAIMS1. A method for processing enzymes comprising preparing an enzyme composition, exposing the enzyme composition to multiple transitions from a high pressure to a low pressure to provide a processed enzyme composition.

2. A method for processing enzymes using an apparatus, the method comprising contacting an enzyme composition with the apparatus, wherein the apparatus comprises at least partially opposing surfaces which are operable to at least partially define a fluid flow channel, wherein the separation distance between the opposing surfaces varies in the direction of bulk fluid flow, so that the fluid flow channel comprises multiple narrowed and expanded portions.

3. A method according to claim 2, wherein the fluid flow channel comprises multiple narrowed and expanded portions relative to the longitudinal axis.

4. A method according to claims 2 or 3, wherein the at least partially opposing surfaces are operable to cause flow of fluid on relative motion of the surfaces.

5. A method according to any of claims 2 to 4, wherein the apparatus comprises two at least partially opposing surfaces.

6. A method according to any of claims 2 to 5, wherein the apparatus is operable to generate a pressure differential before and / or after each narrowed portion of the fluid flow channel, such as a transition from high to low pressure.

7. A method according to any of claims 2 to 6, wherein the fluid flow channel is non- uniform.

8. A method according to any preceding claims, wherein the flow path of the enzyme composition is non-linear9. A method according to any preceding claims, wherein the enzyme composition is exposed to a pressure of at least 2 bar, preferably, at least 4 bar, more preferably, at least 5 bar, more preferably, at least 8 bar, more preferably, at least 9 bar, most preferably, at least 10 bar.

10. A method according to any preceding claims, wherein the high pressure is a pressure of from 2 to 15 bar, such as from 5 to 14 bar, such as from 7 to 13 bar, such as from 10 to 12 bar and / or the low pressure is a pressure of from 0.5 to 3 bar, such as 1 to 2 bar, such as 1 to 1 .5 bar.

11. A method according to any preceding claims, wherein the enzyme composition is exposed to multiple transitions from a high velocity to a low velocity.

12. A method according to any of claims 2 to 10, wherein the fluid flow channel comprises from 4 to 15 expanded portions, such as 4 to 12 expanded portions, such as from 5 to 11 expanded portions.

13. A method according to any of claims 2 to 11 , wherein the fluid flow channel comprises from 4 to 15 narrowed portions, such as 4 to 12 narrowed portions, such as from 5 to 11 narrowed portions.

14. A method according to any of claims 2 to 12, wherein distance between the narrowed portions of the fluid flow channel is from 50 to 200 pm, such as from 50 to 100 pm, and / or wherein distance between the expanded portions of the fluid flow channel is from 1 ,000 to 2,500 pm, such as 1 ,400 to 2,000 pm.

15. A method according to any of claims 2 to 14, wherein at least one of the opposing surfaces comprises a series of cavities which define the narrowed and expanded portions of the fluid flow channel.

16. A method according to any of claims 15, wherein the opposing surfaces comprise from 4 to 12 cavities in the direction of bulk fluid flow.

17. A method according to any of claims 15 or 16, wherein each of the opposing surfaces comprises at least 4 cavities, such as at least 5 cavities, such as at least 6 cavities, such as at least 7 cavities in the direction of bulk fluid flow.

18. A method according to any of claims 2 to 17, wherein at least one of the opposing surfaces is operable to rotate at a speed of from 1 to 20,000 rpm, such as from 2,500 to 15,000 rpm, such as from 4,000 to 12,000 rpm, such as from 5,000 to 10,000 rpm.

19. A method according to any of claims 2 to 18, wherein at least one of the opposing surfaces is static.

20. A method according to any preceding claims, wherein the enzyme composition comprises enzymes selected from hydrolases, oxidoreductases, transferases, lyases and / or isomerases.21 . A method according to any of claims 2 to 20, wherein the apparatus further comprises a pump.

22. A method according to any of claims 2 to 20, wherein the apparatus is a drum / sleeve mixer.

23. A processed enzyme composition formed from the method according to any preceding claim.

24. A method for recycling paper using a processed enzyme composition according to claim 23.

25. A method for producing high-fructose corn syrup using a processed enzyme composition according to claim 23.

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