Compositions, systems, and methods for extraction of metals from phyllosilicates

Abstract

Provided herein are methods, systems, and compositions for degrading phyllosilicates. The methods, systems, and compositions provided herein involve the use of enzymes having silicase activity that have been engineered to have increased ability to degrade phyllosilicates as compared to a wild-type enzyme. The methods, systems, and compositions provided herein may be used to release metal from the phyllosilicates. The methods, systems, and compositions provided herein may further involve collecting, extracting, and / or purifying the metal released from the phyllosilicates.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR EXTRACTION OF METALS FROM PHYLLOSILICATESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 583,196, filed September 15, 2023, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Metals such as lithium, aluminum, iron, nickel, cobalt, strontium, and rare earth elements have vast industrial applications and are in high demand across various industries. For example, lithium is widely used for energy storage, rechargeable batteries, electronic motors, electric vehicles, air mobility, clean energy, energy storage from solar panels, and other applications. Lithium has pharmaceutical applications, such as in lithium-based bipolar disorder treatments. Currently available sources and technologies for obtaining metals for use in industrial applications and products are limited, inefficient, costly, energy -intensive, and harmful to the environment.SUMMARY

[0003] There is a significant unmet need for compositions, methods, and systems that facilitate access to existing sources of metals, such as to extract and / or collect the metals from their sources, separate them, process them, and make them available for use in various industrial applications and / or products, in a manner that is industrially scalable, efficient, and inexpensive. This is at least in part to meet the demand for such metals in the industrial applications and products in need thereof. Many of the currently available methods and techniques for doing so are limited with respect to efficiency, scalability, and high cost. In many cases, such existing technologies may require performing processes and reactions at high temperatures and pressures that are energy -intensive, costly, and harmful to the environment. The compositions, methods, and systems of the present disclosure address the aforementioned needs and shortcomings, in some aspects, by providing compositions, methods, and systems for extracting, separating, and / or collecting metals from phyllosilicates in an efficient, inexpensive, and scalable manner. In some cases, the disclosure further provides methods and systems for processing the extracted metals and / or using them in a product (e.g., rechargeable batteries). The extracted metal may be processed and turned into an industrial grade metal, battery grade metal, pharmaceutical grade metal, or other useful forms of metals.

[0004] In an aspect, provided herein is a method of degrading a phyllosilicate, the method comprising: contacting the phyllosilicate with a variant enzyme having increased ability to degrade the phyllosilicate relative to a wild-type enzyme under reaction conditions such that the variant enzyme degrades the phyllosilicate. In some embodiments, the variant enzyme comprises at least one amino acid modification relative to the wild -type enzyme that confers the increased ability to degrade the phyllosilicate. In some embodiments, the variant enzyme selectively degrades the phyllosilicate. In some embodiments, the phyllosilicate is contained within an ore or a rock. In some embodiments, the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof. In some embodiments, the phyllosilicate is selected from the group consisting of: lepidolite, muscovite, montmorillonite, and any combination thereof. In some embodiments, the method solubilizes and releases a metal contained within the phyllosilicate. In some embodiments, the method further comprises collecting the metal. In some embodiments, the method further comprises extracting the metal. In some embodiments, the method further comprises purifying the metal. In some embodiments, the method further comprises enriching for the metal. In some embodiments, the metal is lithium. In some embodiments, the metal is aluminum. In some embodiments, the metal is strontium. In some embodiments, the metal is iron. In some embodiments, the metal is released into a solution. In some embodiments, the variant enzyme comprises a sequence identity of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more with a carbonic anhydrase. In some embodiments, the carbonic anhydrase is a gamma carbonic anhydrase or alpha carbonic anhydrase. In some embodiments, the variant enzyme is a variant of a wild-type enzyme selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhy drase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransferase-like protein (Isoleucine patchsuperfamily), Fischer ella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus 'B>V- !(Thermosynechococcuselongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, Desulfojundulus thermobenzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combination thereof. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 - 18 or SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises any one of SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403-434. In some embodiments, the variant enzyme comprises any one of SEQ ID NOS: 403-434. In some embodiments, the variant enzyme has a pKd of at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or higher. In some embodiments, the variant enzyme has a Kcat value of at least about 2 mol per second (mol / s), at least about 10 mol / s, at least about 50 mol / s, at least about 100 mol / s, at least about 200 mol / s, at least about 300 mol / s, at least about 400 mol / s, at least about 500 mol / s, at least about 600 mol / s, at least about 700 mol / s, at least about 800 mol / s, at least about 900 mol / s, at least about 1000 mol / s, or higher. In some embodiments, the reaction conditions comprise a temperature from about 23 to about 85 degrees Celsius (C). In some embodiments, the reaction conditions comprise a temperature from about 45 to about 50 degrees Celsius (C). In some embodiments, the reaction conditions comprise a temperature of about 50 degrees Celsius (C). In some embodiments, the reaction conditions comprise a pH from about 4 to about 11. In some embodiments, the reaction conditions comprise a pH of about 5. In some embodiments, the reaction conditions comprise a pH of about 10. In some embodiments, the reaction conditions comprise contacting the variant enzyme with a co-factor. In some embodiments, the co-factor is zinc, copper, cobalt, nickel, magnesium, manganese, iron, or any combination thereof. In some embodiments, the cofactor is iron. In some embodiments, the variant enzyme depolymerizes the phyllosilicate. In some embodiments, the variant enzyme cleaves one or more Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4). In some embodiments, the method is performed in situ. In some embodiments, the variant enzyme is recombinantly produced in a host cell or in a cell-free production system. In some embodiments, the host cell is a bacterial cell or yeast cell. In some embodiments, the bacterial cell is Escherichia coli or the yeast cell is Pichia pastoris or Saccharomyces cerevisiae. In some embodiments, the reaction conditions comprise a rock to liquid ratio from about 1 -40% (w / v). In some embodiments, the reaction conditions comprise a rock to liquid ratio of about 30% (w / v). In some embodiments, the reaction conditions comprise a buffer. In some embodiments, the buffer is TRIS, PBS, citrate, monosodium glutamate, or a combination thereof.

[0005] In an aspect, provided herein is a method of degrading an phyllosilicate, the method comprising: contacting the phyllosilicate with a variant enzyme of any one of SEQ ID NOS: 403-434 under reaction conditions such that the variant enzyme degrades the phyllosilicate, wherein the reaction conditions comprise a temperature from about 23 -85 degrees Celsius (C), a pH from about 4-11, a co-f actor, and a rock to liquid ratio from about 1 -40% (w / v). In some embodiments, the reaction conditions proceed for about 1-48 hours. In some embodiments, the reaction conditions proceed for about 48 hours. In some embodiments, the reaction conditions comprise a temperature of about 50 degrees Celsius (C). In some embodiments, the reaction condition comprises a pH of about 10. In some embodiments, the co-f actor is zinc, iron, copper, cobalt, or any combination thereof. In some embodiments, the co-factor is iron. In some embodiments, the rock to liquid ratio is about 30% (w / v).

[0006] In an aspect, provided herein is a variant enzyme having increased ability to degrade phyllosilicate relative to a wild-type enzyme. In some embodiments, the variant enzyme comprises at least one amino acid modification relative to the wild -type enzyme that confers the increased ability to degrade phyllosilicate. In some embodiments, the variant enzyme selectively degrades phyllosilicate. In some embodiments, the variant enzyme does not have increased ability to degrade an amorphous silicate or a inosilicate relative to the wild -type enzyme. In some embodiments, the phyllosilicate is contained in a rock or an ore. In some embodiments, the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof. In some embodiments, the phyllosilicate is selected from the group comprising: lepidolite, muscovite, montmorillonite, and any combination thereof. In some embodiments, the variant enzyme has increased ability to solubilize and release a metal contained within the phyllosilicate as compared to the wild-type enzyme. In some embodiments, the variant enzyme is a variant of a wild-type enzyme selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gammacarbonic anhydrase, Me thanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransf erase-like protein (Isoleucine patch superfamily), Fischerella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus B>V- l(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, De sulfofundulus thermobenzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combination thereof. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 - 18 or SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises any one of SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403-434. In some embodiments, the variant enzyme comprises any one of SEQ ID NOS: 403-434. In some embodiments, the variant enzyme has a sequence identity of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or higher with a carbonic anhydrase. In some embodiments, the carbonic anhydrase is a gamma carbonic anhydrase or alpha carbonic anhydrase. In some embodiments, the variant enzyme has increased ability to depolymerize phyllosilicate as compared to the wild -type enzyme. In some embodiments, the variant enzyme has increased ability to cleave Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4) as compared to the wild-type enzyme. In some embodiments, the variant enzyme is recombinantly produced in a host cell or in a cell- free production system. In some embodiments, the host cell is a bacterial cell or yeast cell. In some embodiments, the bacterial cell is Escherichia coli or the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

[0007] In an aspect, provided herein is a polynucleotide comprising a nucleic acid sequence encoding the variant enzyme disclosed herein.

[0008] In an aspect, provided herein is a vector comprising the polynucleotide disclosed herein.

[0009] In an aspect, provided herein is a reaction mixture comprising an phyllosilicate and a variant enzyme having increased ability to degrade the phyllosilicate relative to a wild-type enzyme. In some embodiments, the variant enzyme comprises at least one amino acid modification relative to the wild-type enzyme that confers the increased ability to degrade phyllosilicate. In some embodiments, the variant enzyme selectively degrades phyllosilicate. In some embodiments, the phyllosilicate is contained within an ore or a rock. In some embodiments, the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof. In some embodiments, the phyllosilicate is selected from the group comprising: lepidolite, muscovite, montmorillonite, and any combination thereof. In some embodiments, the reaction mixture further comprises at least one metal solubilized and released from the phyllosilicate. In some embodiments, the metal comprises lithium. In some embodiments, the metal comprises iron. In some embodiments, the metal comprises strontium. In some embodiments, the metal comprises aluminum. In some embodiments, the reaction mixture has a pH from about 4 to about 11. In some embodiments, the reaction mixture has a pH of about 5. In some embodiments, the reaction mixture has a pH of about 10. In some embodiments, the reaction mixture has a temperature from about 23 to about 85 degrees Celsius (C). In some embodiments, the reaction mixture has a temperature from about 45 to about 50 degrees Celsius (C). In some embodiments, the reaction mixture has a temperature of about 50 degrees Celsius (C). In some embodiments, the reaction mixture further comprises a co-factor of the variant enzyme. In some embodiments, the co-factor is iron. In some embodiments, the co-factor is copper. In some embodiments, the reaction mixture further comprises a buffered saline solution. In some embodiments, the buffered saline solution is phosphate-buffered saline. In some embodiments, the reaction mixture further comprises an activator co-factor of the variant enzyme. In some embodiments, the activator co-factor is glycine. In some embodiments, the variant enzyme is a variant of a wild-type enzyme selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhy drase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceaebacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransferase-like protein (Isoleucine patch superfamily), Fischer ella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus 'B>V- !(Thermosynechococcuselongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, Desulfojundulus thermobenzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combination thereof. In some embodiments, the variant enzyme comprises an amino acid sequence having at least 50%, atleast 55%, atleast 60%, at least 65%, at least 70%, atleast 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1-18 or SEQ ID NOS: 19-434. In some embodiments, the variant enzyme is a carbonic anhydrase. In some embodiments, the carbonic anhydrase is a gamma carbonic anhydrase. In some embodiments, the variant enzyme has increased ability to depolymerize phyllosilicate as compared to the wild-type enzyme. In some embodiments, the variant enzyme has increased ability to cleave Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4) as compared to the wild- type enzyme. In some embodiments, the non-naturally occurring enzyme is recombinantly produced in a host cell or in a cell-free production system. In some embodiments, the host cell is a bacterial cell or yeast cell. In some embodiments, the bacterial cell is Escherichia coli or the yQ& i cAW 1’ichia pasloris or Saccharomyces cerevisiae. In some embodiments, the reaction conditions comprise a rock to liquid ratio from about 1 -40% (w / v). In some embodiments, the reaction conditions comprise a rock to liquid ratio of about 30% (w / v). In some embodiments, the reaction conditions comprise a buffer. In some embodiments, the buffer is TRIS, PBS, citrate, monosodium glutamate, or a combination thereof. In some embodiments, the reaction conditions proceed for about 1 -48 hours. In some embodiments, the reaction conditions proceed for about 48 hours.INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0012] FIG. 1 shows an example workflow according to the methods of the present disclosure.DETAILED DESCRIPTION

[0013] Metals such as lithium, aluminum, iron, nickel, cobalt, strontium and rare earth elements have vast applications across various industries and in different products. The demand for such metals continues to increase, and there is an unmet need for efficient technologies to facilitate access to metal sources, and to extract and collect the metals for use in products and industries in need thereof and to meet demand. As an example, lithium is highly in demand for rechargeable batteries which can be used in a variety of products such as electronics, electric motors and electric vehicles, clean energy industry, solar panels, and beyond. As these industries advance and become more prominent in global markets, so does the demand for lithium. Lithium can be found in a number of sources, including in brine, for example, in brine deposits generated as a result of accumulations of saline groundwater enriched in dissolved lithium. However, brine sources of lithium are limited in abundance and can only be found in limited geographical locations, mostly located in South America.

[0014] Another prominent and abundant source of metals are phyllosilicates, that are commonly available across the world in a diverse range of geographical areas, constituting a major primary source of metals. The currently available technologies for extracting metals from solid mineral materials / sources, rocks, and ores such as phyllosilicates are limited, inefficient, costly, and environmentally harmful. An example of such process is acid leaching or acid roasting which involves contacting a mineral with a strong acid (e.g., sulfuric acid) to extract a metal, such as lithium, from the mineral. Acid leaching / roasting usually requires reaction conditions involving highly acidic pH, high temperatures (e.g., 200 degrees Celsius (C) and above) and significant energy consumption (e.g., over 6000 megajoules (MJ) per ton of Li2O extracted). This process is expensive, energy -inefficient, and harmful to the environment. Therefore, there is an unmet need for improved compositions, methods, and systems to address these shortcomings.

[0015] Provided herein are compositions, methods, and systems that can efficiently extract metals from minerals (e.g., natural minerals (e.g., rock, ore, clay)) such as phyllosilicates in an efficient, industrially scalable, inexpensive, and environmentally friendly fashion. For example, in some cases, the methods may avoid reaction conditions requiring substances (e.g., highly acidic solvents), high temperatures and pressures, and the like, which may cause harm to the environment and / or increase the cost, energy demands, and / or environmental footprint of the process. In some cases, this is accomplished by performing an enzymatic reaction on a phyllosilicate, to extract and separate a metal (e.g., metal ion / atom) therefrom. The enzymatic reaction may have improved features such as higher reaction rate, specificity toward degrading a mineral material, acting on a certain substrate in the phyllosilicates with high / improved substrate specificity, such that performing the reaction can be industrially scaled and implemented for releasing and collecting the metal. For example, in some cases, the reaction may not require temperatures that are significantly higher than room temperature, pressures significantly higher than atmospheric pressure, highly acidic or highly basic pH conditions, and other conditions that are environmentally harmful and costly. Instead, in many cases, the reactions of the present disclosure may be efficiently performed in nearambienttemperature, near-atmospheric pressure, and / or near-neutral pH conditions, reducing their cost, energy demand, and environmental footprint. The details of such reaction conditions are further elaborated on herein.

[0016] In some cases, the enzymatic reaction may comprise using one or more enzymes and / or co-factors. The enzymatic reaction may comprise an enzymatic degradation / digestion reaction performed with the aid of one or more enzymes and / or co-factors. The enzymes may catalyze the reaction and facilitate the extraction of the metal from the phyllosilicate encasing it. For example, enzymes can be used to degrade, dissolve, and / or depolymerize silicates, and liberate metals (e.g., metal ions) therefrom at near-ambient temperatures without the need for an energy -intensive, high temperature acid separation process. This process significantly decreases the environmental impact of refining lithium and other metals deposited in phyllosilicates. Provided herein are also enzymes and co-factors with enhanced features and capabilities for performing such reactions. Such enzymes may be semi -synthetic and / or engineered enzymes with features, capabilities, sequences, methods of generation, and methods of use that are presented and further elaborated on in the present disclosure.

[0017] The term “sequence identity” as used herein generally refers to an exact nucleotide-to- nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identityinclude determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percentage (%) of “sequence identity” . The % of sequence identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent sequence identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol., 215 :403 -410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25 :3389-3402 (1997). The program may be used to determine percent sequence identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask -off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Ranges of desired degrees of sequence identity are approximately 50% to 100% and integer values therebetween. In general, this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, atleast 80%, atleast 85%, atleast 90%, atleast 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with any sequence provided herein.

[0018] The terms “variant enzyme”, “enzyme variant”, “modified enzyme”, “synthetic enzyme”, “truncated enzyme”, and “engineered enzyme” are used interchangeably throughout to generally refer to non-naturally occurring polypeptides. The non-naturally occurring polypeptides have been designed and sequences included herein.

[0019] The term “about” or “approximately” generally means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0020] In an aspect, provided herein is a method of degrading a phyllosilicate, the method comprising: contacting the phyllosilicate with a variant enzyme having increased ability to degrade the phyllosilicate relative to a wild-type enzyme under reaction conditions such that the variant enzyme degrades the phyllosilicate. FIG. 1 shows an example workflow according to the embodiments of the present disclosure.

[0021] In some cases, the variant enzyme comprises silicase activity such that the enzyme has the capability to digest or degrade a phyllosilicate. In some embodiments, the variant enzyme comprises at least one amino acid modification relative to the wild -type enzyme that confers an increased ability to degrade the phyllosilicate relative to the wild-type enzyme. In some embodiments, the variant enzyme selectively degrades the phyllosilicate. In some embodiments, the variant enzyme does not have increased ability to degrade an amorphous silicate or an Inosilicate relative to the wild-type enzyme.

[0022] In some cases, the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof. In some cases, the phyllosilicate is selected from the group consisting of: lepidolite, muscovite, montmorillonite, and any combination thereof. In some cases, the method solubilizes and releases a metal contained within the phyllosilicate. In some cases, the method further comprises collecting the metal. In some cases, the method further comprises extracting the metal. In some cases, the method further comprises purifying the metal. In some cases, the method further comprises enriching for the metal. In some cases, the metal comprises or is a metal ion. In some cases, the metal is lithium. In some cases, the metal is aluminum. In some cases, the metal is iron. In some cases, the metal is strontium. In some cases, the metal is released into a solution.

[0023] In some cases, the variant enzyme disclosed herein is an engineered enzyme with improved characteristics compared to a wild-type enzyme, such that the modified enzyme exhibits improved ability (e.g., increased reaction rate, increased efficiency, increased ability to degrade phyllosilicates, increased specificity for phyllosilicates) to release metals from phyllosilicate. In some cases, the enzyme is a modified, mutated, and / or engineered enzyme. In some cases, the enzyme used in a reaction / process of the present disclosure is a synthetic or semi-synthetic engineered enzyme. In some cases, the enzyme comprises or is a variant of a carbonic anhydrase, such as a gamma carbonic anhydrase, or an alpha carbonic anhydrase.In some cases, an enzyme of the present disclosure may share some similarities (e.g., sequence identity, enzymatic activity) with a carbonic anhydrase. Any combination of wildtype and engineered / modified enzymes may be used.

[0024] In some cases, the enzyme variant is an engineered enzyme. In some cases, the enzyme variant is an enzyme having at least one amino acid variation as compared to a wild- type enzyme. In some cases, the enzyme variant may comprise an amino acid sequence having at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99. 5%, at least about 99.5%, or greater, sequence identity to an amino acid sequence of a wild-type silicase enzyme. In some cases, the enzyme having silicase activity comprises an amino acid sequence having at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or at most about 10% sequence identity to an amino acid sequence of a wild -type silicase enzyme. In some cases, the enzyme having silicase activity comprises an amino acid sequence having about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35, about 30%, about 25%, about 20%, about 15%, or about 10% sequence identity to an amino acid sequence of a wild-type silicase enzyme.

[0025] In some cases, the enzyme variant is derived from an organism selected from the group consisting of: Methanosarcina thermophila, Bacillus licheniformis CG-B52, Pelobacter carhinolicus, Syntrophus acidilrophicus, Methanosarcina harkeri, Methanosarcina mazei. Bacillus halodurans, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii), Methanosarcina acetivorans, Kofleriaceae bacterium SLC26A / SulP, Thermodesulfitimonas autotrophica, Fischerella thermalis / Mastigocladus laminosus, Thermosynechococcus vestitus B>V- !(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila , Thermo syntropha lipolytica, isoleucine patch superfamily, De sulfo fundulus thermobenzoicus , Archaeoglobus veneficus, Suberites domuncula, and any combination thereof.

[0026] In some cases, the enzyme variant has a sequence identity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more with a Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG- B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransferase-like protein (Isoleucine patch superfamily), Fischer ella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly proteincarboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, De sulfo fundulus thermobenzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase.

[0027] In some cases, the enzyme variant has a sequence identity of at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10% or less with Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransf erase-like protein (Isoleucine patch superfamily), Fischerella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus B>V- !(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, De sulfofundulus thermobenzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase.

[0028] In some embodiments, the variant enzyme comprises a purification tag. The purification tag can comprise commonly used tags known in the art to aide in the purificationof the variant enzyme from a host cell. The purification tag can comprise, for example, a GST tag, a His tag, a NEXT tag, a FLAG tag, or any other tags known in the art. In some cases, the purification tag is conjugated to the N-terminus of the variant enzyme. In some cases, the purification tag is conjugated to the C-terminus of the variant enzyme. In some cases, the purification tag is cleaved from the variant enzyme after purification. In some cases, the purification tag is not cleaved from the variant enzyme after purification. In some cases, the purification tag aides in enzyme stability. In some cases, the purification tag does not affect enzymatic efficacy. In some cases, the purification tag does not affect variant enzyme polymerization.

[0029] In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80% %, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 84%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 -18 provided in Table 1.Table 1- Library of Carbonic Anhydrase Enzymes

[0030] In some embodiments, an enzyme of the present disclosure is an engineered enzyme. In some cases, the engineered enzyme may have the amino acid sequence of any one of SEQ ID NOS: 19-434 provided in Table 2. In some cases, the enzyme variant comprises an amino acid sequence having of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 84%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 19-434 provided in Table 2. In some embodiments, the enzyme variant has the sequence of any one of SEQ ID NOS: 19-434. In some cases, the enzyme variant comprises an amino acidsequence having of at least about 50%, at least about 60%, at least about 70%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 84%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403-434 provided in Table 2. In some embodiments, the enzyme variant has the sequence of any one of SEQ ID NOS: 403-434.Table 2- Library of modified or engineered enzymes

[0031] The enzyme variant may be specific or substantially specific to a phyllosilicate such that the enzyme acts specifically on the phyllosilicate and facilitates extraction of the metal from the phyllosilicate. In some cases, the enzyme variant may degrade, digest, and / or disintegrate the phyllosilicate. As a result, metals, such as in the form of metal ions, metal atoms, and / or metal precipitates, may be released from the phyllosilicate (e.g., into a solution). In some cases, the method may comprise collecting the metal or the solution containing the metal. In some cases, the method may comprise separating the metal from the solution. In some cases, the metal may be water soluble. Alternatively or in addition, the metal may precipitate in the solution. The solution may be an aqueous solution comprising water and / or a buffer described anywhere herein.

[0032] In some embodiments, the enzyme having silicase activity has a pKd of at least about 5, at least about 6, at least about 7, atleast about 8, at least about 9, at least about 10, at least about 11, or higher. In some embodiments, the enzyme having silicase activity has a Kcat value of at least about 2 mol per second (mol / s), at least about 10 mol / s, at least about 50 mol / s, at least about 100 mol / s, at least about 200 mol / s, at least about 300 mol / s, at least about 400 mol / s, at least about 500 mol / s, at least about 600 mol / s, at least about 700 mol / s, at least about 800, at least about 900, at least about 1000, or higher.

[0033] In some cases, the specificity of the enzyme variant to the substrate may be higher than a wild-type version of the enzyme. For example, a wild-type enzyme may be engineered to improve its specificity for a substrate. In some cases, the enzyme provided herein may be engineered by directed evolution. In some cases, machine learning and artificial intelligence may be used for performing such enzyme engineering. In some cases, directed evolution methods may be used to design the enzymes of the present disclosure. The engineered enzyme may be synthesized or semi-synthesized. In some embodiments, the enzyme (e.g., the engineered enzyme) has the amino acid sequence of any one of SEQ ID NOS: 19-434, or an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater, sequence identity to the amino acid sequence of any one of SEQID NOS: 19-434. In some embodiments, the enzyme (e.g., the engineered enzyme) has the amino acid sequence of any one of SEQ ID NOS: 403-434, or an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of any one of SEQ ID NOS: 403-434.

[0034] In some embodiments, the variant enzyme comprises a purification tag. The purification tag can comprise commonly used tags known in the art to aide in the purification of the variant enzyme from a host cell. The purification tag can comprise, for example, a GST tag, a His tag, a NEXT tag, a FLAG tag, or any other tags known in the art. In some cases, the purification tag is conjugated to the N-terminus of the variant enzyme. In some cases, the purification tag is conjugated to the C-terminus of the variant enzyme. In some cases, the purification tag is cleaved from the variant enzyme after purification. In some cases, the purification tag is not cleaved from the variant enzyme after purification. In some cases, the purification tag aides in enzyme stability. In some cases, the purification tag does not affect enzymatic efficacy. In some cases, the purification tag does not affect variant enzyme polymerization.

[0035] In some cases, the enzyme variant may have a catalytic activity that is superior to the wild-type version of the same enzyme. For example, the enzymes provided herein may have a higher catalytic rate. This may decrease the time required to extract a given amount of metal from phyllosilicate, compared to when using a wild-type enzyme. In some cases, the enzymes provided herein may have reduced energy requirements, as compared to a wild-type enzyme. In some cases, catalytic activity / rate / efficiency may be characterized by using one or more metrics.

[0036] In some embodiments, the enzyme variant has a catalytic efficiency of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the method has a maximum metal extraction rate of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the enzyme variant has a catalytic efficiency of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times orhigher. In some embodiments, the method has a maximum metal extraction rate of at least about l . l, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher.

[0037] In some embodiments, the enzyme having silicase activity has a pKd of at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or higher. In some embodiments, the enzyme having silicase activity has a Kcat value of at least about 2 mol per second (mol / s), at least about 10 mol / s, at least about 50 mol / s, at least about 100 mol / s, at least about 200 mol / s, at least about 300 mol / s, at least about 400 mol / s, at least about 500 mol / s, at least about 600 mol / s, at least about 700 mol / s, at least about 800 mol / s, at least about 900 mol / s, at least about 1000 mol / s, or higher.

[0038] The methods of the present disclosure, such as the enzymatic reactions provided herein, may be performed under a set of reaction conditions. In some cases, the reaction conditions may comprise a reaction temperature (e.g., a temperature under which the enzymatic reaction, or a portion thereof, is performed). In some cases, the reaction temperature is from about 20 to about 90 degrees Celsius (C). In some cases, the reaction temperature is from about 23 to about 90 degrees Celsius (C). In some cases, the reaction temperature is from about 23 to about 85 degrees Celsius (C). In some cases, the reaction temperature is from about 30 to about 90 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 30 to about 80 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 30 to about 70 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 30 to about 60 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 30 to about 50 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 45 to about 55 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 45 to about 50 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 20 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 23 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 25 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 30 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 35 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 40 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 45 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 50 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 55 degrees Celsius (C). Insome cases, the reaction conditions comprises a temperature about 60 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 70 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 75 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 80 degrees Celsius (C). In some cases, the reaction conditions comprises a temperature about 85 degrees Celsius (C). In some cases, the methods of the present disclosure may be performed in near-ambient temperatures and / or pressures. In some cases, the temperature ranges of the methods of the present disclosure may be significantly lower than temperatures required in acid roasting, (e.g., 200 degrees Celsius (C) and above). This may reduce the energy requirements of the process performed using the methods of the present disclosure.

[0039] In some cases, the enzymatic reaction may be performed at a pH from about 4 to about 11 . In some cases, the enzymatic reaction may be performed at a pH from about 4 to about 10. In some cases, the enzymatic reaction may be performed at a pH from about 4 to about 9. In some cases, the enzymatic reaction may be performed at a pH from about 4 to about 8. In some cases, the enzymatic reaction may be performed at a pH from about 4 to about 7. In some cases, the enzymatic reaction may be performed at a pH from about 4 to about 6. In some cases, the enzymatic reaction may be performed at a pH from about 5 to about 11 . In some cases, the enzymatic reaction may be performed at a pH from about 5 to about 10. In some cases, the enzymatic reaction may be performed at a pH from about 5 to about 9. In some cases, the enzymatic reaction may be performed at a pH from about 5 to about 8. In some cases, the enzymatic reaction may be performed at a pH from about 5 to about 7. In some cases, the enzymatic reaction may be performed at a pH from about 5 to about 6. In some cases, the enzymatic reaction may be performed at a pH from about 6 to about 11 . In some cases, the enzymatic reaction may be performed at a pH from about 6 to about 10. In some cases, the enzymatic reaction may be performed at a pH from about 7 to about 11 . In some cases, the enzymatic reaction may be performed at a pH from about 7 to about 10. In some cases, the enzymatic reaction may be performed at a pH from about 8 to about 11 . In some cases, the enzymatic reaction may be performed at a pH from about 8 to about 10. In some cases, the enzymatic reaction may be performed at a pH from about 9 to about 11 . In some cases, the enzymatic reaction may be performed at a pH from about 9 to about 10. In some cases, the enzymatic reaction may be performed at a pH of 4. In some cases, the enzymatic reaction maybe performed at a pH of 5. In some cases, the enzymatic reaction may be performed at a pH of 6. In some cases, the enzymatic reaction may be performed at a pH of 7. In some cases, the enzymatic reaction may be performed at a pH of 8.In some cases, the enzymatic reaction may be performed at a pH of 9. In some cases, the enzymatic reaction may be performed at a pH of 10. In some cases, the enzymatic reaction may be performed at a pH of 11. In some cases, the methods of the present disclosure may be performed in pH ranges that are substantially neutral, or in other words, not highly / strongly acidic or highly / strongly basic. In some cases, the method / reaction of the present disclosure may not comprise using a strong acid, such as is used in acid leaching. Instead, the enzymes presented throughout the disclosure may perform enzymatic degradation of the phyllosilicate in neutral pH conditions.

[0040] In some cases, the methods provided herein comprise crushing or grinding the rocks or ores to achieve a particulate size. In some cases, the ground ores comprise a size of about 50 pm to 1 mm. In some cases, the ground ores comprise a size from about 50 pm to 750 pm. In some cases, the ground ores comprise a size from about 50 pm to 500 pm. In some cases, the ground ores comprise a size from about 50 pm to 250 pm. In some cases, the ground ores comprise a size from about 50 pm to 150 pm. In some cases, the ground ores comprise a size from about 50 pm to 100 pm. In some cases, the ground ores comprise a size of about 50 pm. In some cases, the ground ores comprise a size of about 100 pm. In some cases, the ground ores comprise a size of about 150 pm. In some cases, the ground ores comprise a size of about 250 pm. In some cases, the ground ores comprise a size of about 500 pm. In some cases, the ground ores comprise a size of about 750 pm. In some cases, the ground ores comprise a size of about 1 mm.

[0041] In some cases, the methods provided herein comprise creating a slurry of crushed rock and liquid. In some cases, the rock to liquid ratio is from about 1 -40% (w / v). In some cases, the rock to liquid ratio is from about 1 -35% (w / v). In some cases, the rock to liquid ratio is from about 1-30% (w / v). In some cases, the rockto liquid ratio is from about 1 -25% (w / v). In some cases, the rock to liquid ratio is from about 1 -20% (w / v). In some cases, the rock to liquid ratio is from about 1 -15% (w / v). In some cases, the rock to liquid ratio is from about 1 - 10% (w / v). In some cases, the rock to liquid ratio is from about 1 -5% (w / v). In some cases, the rock to liquid ratio is from about 10-40% (w / v). In some cases, the rock to liquid ratio is from about 10-35% (w / v). In some cases, the rock to liquid ratio is from about 10 -30% (w / v). In some cases, the rock to liquid ratio is from about 10-25% (w / v). In some cases, the rock to liquid ratio is from about 15-35% (w / v). In some cases, the rock to liquid ratio is from about 15-30% (w / v). In some cases, the rock to liquid ratio is from about 20-35% (w / v). In some cases, the rock to liquid ratio is from about 20-30% (w / v). In some cases, the rock to liquid ratio is from about 25-35% (w / v). In some cases, the rock to liquid ratio is from about 25-30% (w / v). In some cases, the rock to liquid ratio is about 1% (w / v). In some cases, the rock to liquid ratio is about 5% (w / v). In some cases, the rock to liquid ratio is about 10% (w / v). In some cases, the rock to liquid ratio is about 15% (w / v). In some cases, the rockto liquid ratio is about 20% (w / v). In some cases, the rock to liquid ratio is about 25% (w / v). In some cases, the rock to liquid ratio is about 30% (w / v). In some cases, the rock to liquid ratio is about 35% (w / v). In some cases, the rock to liquid ratio is about 40% (w / v).

[0042] In some cases, the methods provided herein comprise an enzymatic reaction that proceeds for a set period of time. In some cases, the method comprises the enzymatic reaction proceeding for about 1-72 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 1 -60 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 1 -48 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 1-36 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 1 -24 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 1-12 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 1-6 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 12-72 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 12-60 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 12-48 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 12-36 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 12-24 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 24-72 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 24-60 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 24-48 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 24-36 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 36-72 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 36- 60 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 36-48 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 48-72 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 48-60 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 1 hour. In some cases, the method comprises the enzymatic reaction proceeding for about 6 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 12 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 24 hours. In some cases, the method comprises the enzymatic reactionproceeding for about 36 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 48 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 60 hours. In some cases, the method comprises the enzymatic reaction proceeding for about 72 hours.

[0043] In some cases, the methods provided herein comprise contacting the enzyme variant with a co-f actor. The co-f actor may help further increase the reaction rate when used in combination with the enzyme having silicase activity. In some cases, the co-factor is selected from the group consisting of : iron, zinc, copper, nickel, cobalt, and any combination thereof. Such co-factor may be used with any enzyme disclosed anywhere in the present disclosure. In some cases, the co-factor may be a non-natural co-factor, such as a co-factor that is typically not used by an enzyme as a co-factor in nature.

[0044] In some cases, the enzyme variant depolymerizes phyllosilicates. In some cases, the enzyme variant cleaves one or more Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4). In some cases, the metal is lithium, aluminum, iron, nickel, cobalt, uranium, strontium, and / or a rare earth element. In some cases, the metal is lithium. In some cases, the metal is aluminum. In some cases, the metal is strontium. In some cases, the metal is iron. As a result of enzymatic degradation and / or disintegration of the phyllosilicate, the metal may be released and extracted from the phyllosilicate, in some cases, in a solution, in some cases in the form of a metal ion or a metal atom. The solution may be an aqueous solution.

[0045] In some cases, the method comprises extracting the metal from the solution. In some cases, the method comprises purifying the metal from the solution, thereby generating a purified metal, a metal ion, a metal atom, a solid metal complex, a metal precipitate, or any combination thereof. In some cases, the purified metal has a purity of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or higher purity. In some cases, the purified metal is lithium, aluminum, iron, nickel, cobalt, strontium, a rare earth element, and / or uranium. In some cases, the purified metal is industry -grade, battery -grade, and / or pharmaceutical grade. In some cases, the purified metal is industry -grade lithium, battery -grade lithium, and / or pharmaceutical -grade lithium.

[0046] In some embodiments, the enzyme variant has a catalytic efficiency of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the method has a maximum metalextraction rate of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the enzyme variant has a catalytic efficiency of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher. In some embodiments, the method has a maximum metal extraction rate of at least about l . l, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher.

[0047] In some aspects, the enzyme variant is recombinantly produced in a host cell or in a cell-free production system. In some cases, the host cell is a bacterial cell or yeast cell. In some cases, the bacterial cell is Escherichia coli. In some cases, the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

[0048] In some embodiments, the methods of the present disclosure comprise performing a reaction involving an enzyme and phyllosilicates, as described throughout the disclosure. The reaction may be performed inside any suitable container. In some cases, the reaction may be performed on a rock or ore. In some cases, the phyllosilicate may be placed inside a container and added to a solution comprising the enzyme, water, a buffer, and / or potential other reagents for performing the reaction. In some cases, the reaction may be performed in one or more of a container, a dish, a beaker, a device, a tank, a reactor, and / or any combination thereof. The containers (e.g., one or more reactors and / or tanks) may be connected to one another to perform one or more reactions according to the embodiments of the present disclosure. The containers may also be reaction units and / or process units each of which may serve a function as part of the method and / or in combination with the method. For example, one or more tanks, reactors, and / or processing units, may be connected to each other with any configuration, such as in series, in parallel, or any combination thereof to perform the method steps such as contact the enzyme with the phyllosilicate, extracting the metal, separating the metal from the solution, purifying the metal, processing the metal, and converting the metal into an industry -grade, battery -grade, or pharmaceutical-grade metal. In some cases, the method further comprises grinding the phyllosilicate prior to performing the reaction (e.g., the enzymatic degradation). In some cases, the method further comprises using a filtration / chelating system, a precipitation system, a recycle system, or any combination thereof.

[0049] In an aspect, provided herein is an enzyme variant which may be a non-naturally occurring enzyme having silicase activity, the enzyme comprising at least one amino acidvariation relative to a wild-type enzyme, and having increased ability to release metals from phyllosilicate as compared to the wild-type enzyme. In some cases, the enzyme variant is used in a reaction / process of the present disclosure for metal extraction, as described anywhere in the present disclosure. In some cases, a wild -type carbonic anhydrase, gamma carbonic anhydrase, or alpha carbonic anhydrase may be used as a starting point for the enzyme engineering and performing the modifications to design and synthesize the non- naturally occurring enzyme having silicase activity. The non -naturally occurring enzyme having silicase activity may in some cases be a modified, engineered, and semi-synthetic enzyme (e.g., the enzyme having silicase activity for performing the methods and reactions of the present disclosure). As such, the resulting modified enzyme may have some similarities, for example with respect to characteristics and sequence, to a wild -type carbonic anhydrase, such as, for example, a gamma carbonic anhydrase or alpha carbonic anhydrase, and some differences, for example with respect to characteristics and sequence, to a wild -type carbonic anhydrase (e.g., gamma carbonic anhydrase or alpha carbonic anhydrase).

[0050] In some cases the wild-type enzyme is selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhy drase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain -containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransferase-like protein (Isoleucine patch superfamily), Fischerella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus B>V- !(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, Desulfofundulus thermobenzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combination thereof.

[0051] In some cases, the non-naturally occurring enzyme is derived from an organism selected from the group consisting of: Methanosarcina thermophila, Bacillus licheniformis CG-B52, Pelobacter carbinolicus, Syntrophus aciditrophicus, Methanosarcina barkeri, Methanosarcina mazei, Bacillus halodurans, Alkalihalobacillus clausii (strain KSM-K16)(Bacillus clausii), Methanosarcina acetivorans, Kofleriaceae bacterium, Thermodesulfitimonas autotrophica, Fischer ella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus BP- \!(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila , Thermo syntropha lipolytica, De sulfofundulus thermob enzoicus, Archaeoglobus veneficus, Suberites domuncula, and any combination thereof.

[0052] In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1-18. In some embodiments, the variant enzyme may be any one of the SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 -18. In some embodiments, the variant enzyme may be any one of the SEQ ID NOS: 403-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403-434.

[0053] In some cases, the methods of the present disclosure are performed on a mineral material such as phyllosilicate. The method may be accordingto the embodiments described anywhere herein. According to the embodiments described anywhere in the present disclosure, in some cases, an enzyme having silicase activity acts on a phyllosilicate. Theenzyme and the method of the present disclosure may degrade, digest, or disintegrate the phyllosilicate and extract a metal therefrom. The phyllosilicate maybe an abundant source of valuable metals such as lithium, aluminum, iron, nickel, cobalt, copper, strontium, rare earth elements, uranium, and other metals with vast industrial use and applications. The methods and enzymes of the present disclosure facilitate access to such sources. In some embodiments, the phyllosilicate is selected from the group consisting of : lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof. In some embodiments, the phyllosilicate is selected from the group consisting of: lepidolite, muscovite, montmorillonite, and any combination thereof.

[0054] In some cases, the non-naturally occurring enzyme has an increased ability to depolymerize the phyllosilicate as compared to the wild-type enzyme, increased selectivity or specificity toward a mineral structure in the phyllosilicate, or both. In some cases, the non- naturally occurring enzyme has increased ability to cleave one or more Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4) as compared to the wild-type enzyme. In some cases, the metal comprises lithium, aluminum, iron, nickel, cobalt, strontium, or a rare earth element. In some cases, the metal comprises lithium. In some cases, the metal comprises aluminum. In some cases, the metal comprises strontium. In some cases, the non- naturally occurring enzyme is recombinantly produced in a host cell or in a cell -free production system. In some cases, the host cell is a bacterial cell or yeast cell. In some cases, the bacterial cell is Escherichia coli. In some cases, the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

[0055] In an aspect, provided herein is a reaction mixture comprising a phyllosilicate and a non-naturally occurring enzyme having silicase activity or an enzyme variant described anywhere in the present disclosure, wherein the enzyme variant comprises at least one amino acid variation relative to a wild-type enzyme, and has increased ability to release metal from the phyllosilicate as compared to the wild-type enzyme. The phyllosilicate comprises Si-0 bonds. In some cases, the phyllosilicate. In some cases, the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, and any combination thereof. The phyllosilicate may be a source of a metal.

[0056] The metal may be extracted from the phyllosilicate (in some cases a rock or ore) by using the methods and enzymes described anywhere in the present disclosure. The enzyme variant may be any enzyme disclosed anywhere in the present disclosure. In some cases, the reaction mixture has a pH from about 4 to about 11. In some cases, the reaction mixture has a pH from about 4 to about 10. In some cases, the reaction mixture has a pH from about 4 toabout 9. In some cases, the reaction mixture has a pH from about 4 to about 8. In some cases, the reaction mixture has a pH from about 4 to about 7. In some cases, the reaction mixture has a pH from about 4 to about 6. In some cases, the reaction mixture has a pH from about 5 to about 11 . In some cases, the reaction mixture has a pH from about 5 to about 10. In some cases, the reaction mixture has a pH from about 5 to about 9. In some cases, the reaction mixture has a pH from about 5 to about 8. In some cases, the reaction mixture has a pH from about 5 to about 7. In some cases, the reaction mixture has a pH from about 5 to about 6. In some cases, the reaction mixture has a pH from about 6 to about 11. In some cases, the reaction mixture has a pH from about 6 to about 10. In some cases, the reaction mixture has a pH from about 7 to about 11. In some cases, the reaction mixture has a pH from about 7 to about 10. In some cases, the reaction mixture has a pH from about 8 to about 11. In some cases, the reaction mixture has a pH from about 8 to about 10. In some cases, the reaction mixture has a pH from about 9 to about 11 . In some cases, the reaction mixture has a pH from about 9 to about 10. In some cases, the reaction mixture has a pH of 4. In some cases, the reaction mixture has a pH of 5. In some cases, the reaction mixture has a pH of 6. In some cases, the reaction mixture has a pH of 7. In some cases, the reaction mixture has a pH of 8. In some cases, the reaction mixture has a pH of 9. In some cases, the reaction mixture has a pH of 10. In some cases, the reaction mixture has a pH of 11. In some cases, the reaction mixture has a temperature from about 20 to about 90 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 23 to about 90 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 23 to about 85 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 30 to about 90, from about 30 to about 80, from about 30 to about 70, from about 30 to about 60, or from about 30 to about 50 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 45 to about 55 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 45 to about 50 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 20 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 23 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 25 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 30 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 35 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 40 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 45 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 50 degrees Celsius (C). In some cases, the reaction mixture has atemperature about 55 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 60 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 70 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 75 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 80 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 85 degrees Celsius (C). In some cases, the reaction mixture further comprises a co-factor of the non-naturally occurring enzyme. In some cases, the co-factor is selected from the group consisting of iron, zinc, copper, nickel, and cobalt. In some cases, the co-factor is copper. In some cases, the co-factor is iron. In some cases, the reaction mixture further comprises a buffered saline solution. In some cases, the buffered solution comprises saline, glycine, iron ions, or any combination thereof. In some cases the buffered solution comprises TRIS, PBS, citrate, monosodium glutamate, or any combination thereof. In some cases, the reaction mixture further comprises an activator co-factor of the non-naturally occurring enzyme. In some cases, the activator co-factor is glycine. The reaction mixture may be used to perform any method described anywhere herein using any enzyme, any co-factor, and / or any reaction condition disclosed anywhere herein.

[0057] In some cases, the reaction mixture provided herein comprise crushing or grinding the rocks or ores to achieve a particulate size. In some cases, the ground ores comprise a size of about 50 pm to 1 mm. In some cases, the ground ores comprise a size from about 50 pm to 750 pm. In some cases, the ground ores comprise a size from about 50 pm to 500 pm. In some cases, the ground ores comprise a size from about 50 pm to 250 pm. In some cases, the ground ores comprise a size from about 50 pm to 150 pm. In some cases, the ground ores comprise a size from about 50 pm to 100 pm. In some cases, the ground ores comprise a size of about 50 pm. In some cases, the ground ores comprise a size of about 100 pm. In some cases, the ground ores comprise a size of about 150 pm. In some cases, the ground ores comprise a size of about 250 pm. In some cases, the ground ores comprise a size of about 500 pm. In some cases, the ground ores comprise a size of about 750 pm. In some cases, the ground ores comprise a size of about 1 mm.

[0058] In some cases, the reaction mixture provided herein comprise creating a slurry of crushed rock and liquid. In some cases, the rockto liquid ratio is from about 1 -40% (w / v). In some cases, the rock to liquid ratio is from about 1 -35% (w / v). In some cases, the rock to liquid ratio is from about 1 -30% (w / v). In some cases, the rock to liquid ratio is from about 1 - 25% (w / v). In some cases, the rock to liquid ratio is from about 1 -20% (w / v). In some cases, the rock to liquid ratio is from about 1 -15% (w / v). In some cases, the rock to liquid ratio isfrom about 1-10% (w / v). In some cases, the rockto liquid ratio is from about 1 -5% (w / v). In some cases, the rock to liquid ratio is from about 10-40% (w / v). In some cases, the rock to liquid ratio is from about 10-35% (w / v). In some cases, the rock to liquid ratio is from about 10-30% (w / v). In some cases, the rock to liquid ratio is from about 10-25% (w / v). In some cases, the rock to liquid ratio is from about 15-35% (w / v). In some cases, the rock to liquid ratio is from about 15-30% (w / v). In some cases, the rock to liquid ratio is from about 20- 35% (w / v). In some cases, the rock to liquid ratio is from about 20 -30% (w / v). In some cases, the rock to liquid ratio is from about 25 -35% (w / v). In some cases, the rock to liquid ratio is from about 25-30% (w / v). In some cases, the rock to liquid ratio is about 1% (w / v). In some cases, the rock to liquid ratio is about 5% (w / v). In some cases, the rock to liquid ratio is about 10% (w / v). In some cases, the rock to liquid ratio is about 15% (w / v). In some cases, the rock to liquid ratio is about 20% (w / v). In some cases, the rock to liquid ratio is about 25% (w / v). In some cases, the rock to liquid ratio is about 30% (w / v). In some cases, the rock to liquid ratio is about 35% (w / v). In some cases, the rockto liquid ratio is about 40% (w / v).

[0059] In some cases, the reaction mixture provided herein comprise an enzymatic reaction that proceeds for a set period of time. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -36 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1-24 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1-12 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -6 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12 -48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-36 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-24 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24-60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24-48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24-36hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36-60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36-48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 48-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 48-60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 hour. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 6 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 72 hours.

[0060] The reaction mixture comprises the enzyme variant or non-naturally occurring enzyme described anywhere herein. As described throughout the present disclosure, in some cases, the enzyme variant (e.g., non-naturally occurring enzyme) may be an engineered and / or semi-synthetic enzyme having a modification compared to a wild-type enzyme. In some cases, the wild-type enzyme is selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransf erase-like protein (Isoleucine patch superfamily), Fischerella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus B>V- !(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, De sulfofundulus thermobenzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combinationthereof. In some cases, the wild-type enzyme is a carbonic anhydrase. In some cases, the carbonic anhydrase is a gamma carbonic anhydrase or alpha carbonic anhydrase.

[0061] In some cases, the non -naturally occurring enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80% at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 -18 or SEQ ID NOS: 19-434. In some cases, the wild-type enzyme is a carbonic anhydrase. In some cases, the carbonic anhydrase is a gamma carbonic anhydrase or alpha carbonic anhydrase.

[0062] In some cases, the enzyme variant has increased ability to depolymerize phyllosilicate as compared to the wild-type enzyme. In some cases, the non-naturally occurring enzyme has increased ability to cleave one or more Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4) as compared to the wild-type enzyme. In some cases, the metal comprises lithium. In some cases, the metal comprises aluminum. In some cases, the metal comprises strontium. In some cases, the metal may comprise lithium, aluminum, nickel, iron, cobalt, copper, strontium, uranium, a rare earth element, another metal, or any combination thereof. In some cases, the non-naturally occurring enzyme is recombinantly produced in a host cell or in a cell-free production system. In some cases, the host cell is a bacterial cell or yeast cell. In some cases, the bacterial cell is Escherichia coli. In some cases, the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

[0063] In an aspect, provided herein is a polynucleotide comprising a nucleotide sequence encoding the enzyme variant of any one of the preceding embodiments. In an aspect, provided herein is a vector comprising the polynucleotide comprising the nucleotide sequence encoding the enzyme variant. The enzyme variant may be according to any embodiment mentioned anywhere in the present disclosure. The polynucleotide and vector may be designed and engineered. The polynucleotide and the vector may get synthesized. The polynucleotide and / orvector may be used to generate and / or produce the non-naturally occurring enzyme of the present disclosure.

[0064] In an aspect, provided herein is a method of increasing silicase activity of an enzyme, the method comprising contacting or combining the enzyme with a non -natural co-f actor. In some cases, the enzyme and the co-factor may be added to a solution. In some cases, the cofactor may be brought in proximity of the enzyme. In some cases, the enzyme and the cofactor may be part of the same system, the same reaction mixture, or the same kit for- Il l -performing the methods of the present disclosure. In some cases, the non-natural co-factor increases silicase activity of the enzyme as compared to the enzyme in the presence of a natural co-factor. In some cases, the non-natural co-factor may be copper. In some cases, natural co-factor is zinc. In some cases, natural co-factor is iron. In some cases, the method is performed in the absence of the natural co-factor. In some cases, the non-natural co-factor does not act as a co-factor for the enzyme having silicase activity in nature. In some cases, the method further comprises contacting the enzyme and the non-natural co-factor with the phyllosilicate under reaction conditions such that a metal contained within the phyllosilicate is solubilized and released from the phyllosilicate (e.g., phyllosilicate rock / ore).

[0065] As an example, a method of the present disclosure comprises using an enzyme having silicase activity (wild-type or engineered / modified / semi-synthetic) to degrade a phyllosilicate so as to extract the metal from the mineral material. Zinc may act as a natural co-factor for the enzyme comprising silicase activity in nature. For example, a natural silicate rock may get degraded by a wild-type silicase enzyme such as a gamma carbonic anhydrase in nature, zinc may act as a co -factor for the wild-type enzyme, catalyze the digestion / degradation reaction, and speed it up and / or increase its efficiency. Alternatively, in some cases, the method of the present disclosure may comprise bringing an enzyme having silicase activity to a phyllosilicate such as a phyllosilicate, and further provide a co-factor other than zinc (the natural co-factor) to increase the catalytic effects of the enzyme having silicase activity on the mineral material. The co-factor other than zinc may be a non-natural co-factor. The non- natural co-factor is in some cases, copper, iron, nickel, cobalt, or glycine. In some cases, the non-natural co-factor may work better than the natural co-factor. For example, copper, iron, nickel, cobalt, or glycine may work better than zinc in increasing the catalytic efficiency of the enzyme and increasing the reaction rate. The enzyme used in the method of the present disclosure may comprise using a wild-type enzyme or a modified enzyme described anywhere in the present disclosure.

[0066] In some cases, the amount of metal extracted, solubilized / precipitated in the extraction solution, and / or released from the phyllosilicate is greater than an amount of metal extracted from the phyllosilicate when the enzyme is contacted with the natural co-factor (e.g., in unit time when other conditions are equal). In some cases, the amount of metal extracted from the phyllosilicate is greater than an amount of metal extracted from the phyllosilicate when the enzyme is contacted with the natural co-factor. In some cases, the enzyme having silicase activity is a carbonic anhydrase. In some cases, the carbonic anhydrase is a gamma carbonic anhydrase or an alpha carbonic anhydrase. In some cases, theenzyme is a wild-type enzyme. In some cases, the enzyme is a modified or engineered enzyme.

[0067] In some cases, the enzyme variant is derived from an organism selected from the group consisting of: Methanosarcina thermophila, Bacillus licheniformis CG-B52, Pelobacter carhinolicus, Syntrophus acidilrophicus, Methanosarcina harkeri, Methanosarcina mazei. Bacillus halodurans, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii), Methanosarcina acetivorans, Kofleriaceae bacterium, Thermodesulfitimonas autotrophica, Fischer ella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus BP- \!(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila , Thermo syntropha lipolytica, De sulfofundulus thermob enzoicus, Archaeoglobus veneficus, Suberites domuncula, and any combination thereof.

[0068] In some cases, the enzyme variant is an engineered enzyme. In some cases, the enzyme variant is an enzyme having at least one amino acid variation as compared to a wild- type enzyme. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, tale at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5%, atleast about 99.9%, or more sequence identity to an amino acid sequence ofany one of SEQ ID NOS: 1 -18. In some embodiments, the variant enzyme may be any one of the SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence ofany one of SEQ ID NOS: 19-434. In some embodiments, the variant enzyme may be any one of the SEQ ID NOS: 403-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403-434.

[0069] In some embodiments, the enzyme having silicase activity has a catalytic efficiency of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the method has a maximum metal extraction rate of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the enzyme having silicase activity has a catalytic efficiency of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75,5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher. In some embodiments, the method has a maximum metal extraction rate of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher. In some embodiments, the enzyme having silicase activity has a pKd of at least about 5, at least about6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or higher. In some embodiments, the enzyme having silicase activity has a Kcat value of at least about 2 mol per second (mol / s), at least about 10 mol / s, atleast about 50 mol / s, at least about 100 mol / s, at least about 200 mol / s, at least about 300 mol / s, at least about 400 mol / s, at least about 500 mol / s, at least about 600 mol / s, at least about 700 mol / s, at least about 800 mol / s, at least about 900 mol / s, at least about 1000 mol / s, or higher.

[0070] In some cases, the reaction conditions comprise a temperature from about 20 to about 90 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 23 to about 90 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 23 to about 85 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 30 to about 90, from about 30 to about 80, from about 30 to about 70, from about 30 to about 60, or from about 30 to about 50 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature from about 45 to about 55 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about45 to about 50 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 20 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 23 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 25 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 30 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 35 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 40 degrees Celsius (C). In some cases, the reaction conditions comprise atemperature about 45 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 50 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 55 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 60 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 70 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 75 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 80 degrees Celsius (C). In some cases, the reaction conditions comprise a temperature about 85 degrees Celsius (C). In some cases, the reaction conditions comprise a pH from about 4 to about 11 . In some cases, the reaction conditions comprise a pH from about 4 to about 10. In some cases, the reaction conditions comprise a pH from about 4 to about 9. In some cases, the reaction conditions comprise a pH from about 4 to about 8. In some cases, the reaction conditions comprise a pH from about 4 to about 7. In some cases, the reaction conditions comprise a pH from about 4 to about 6. In some cases, the reaction conditions comprise a pH from about 5 to about 11 . In some cases, the reaction conditions comprise a pH from about 5 to about 10. In some cases, the reaction conditions comprise a pH from about 5 to about 9. In some cases, the reaction conditions comprise a pH from about 5 to about 8. In some cases, the reaction conditions comprise a pH from about 5 to about 7. In some cases, the reaction conditions comprise a pH from about 5 to about 6. In some cases, the reaction conditions comprise a pH from about 6 to about 11 . In some cases, the reaction conditions comprise a pH from about 6 to about 10. In some cases, the reaction conditions comprise a pH from about 7 to about 11 . In some cases, the reaction conditions comprise a pH from about 7 to about 10. In some cases, the reaction conditions comprise a pH from about 8 to about 11. In some cases, the reaction conditions comprise a pH from about 8 to about 10. In some cases, the reaction conditions comprise a pH from about 9 to about 11 . In some cases, the reaction conditions comprise a pH from about 9 to about 10. In some cases, the reaction conditions comprise a pH of 4. In some cases, the reaction conditions comprise a pH of 5. In some cases, the reaction conditions comprise a pH of 6. In some cases, the reaction conditions comprise a pH of 7. In some cases, the reaction conditions comprise a pH of 8. In some cases, the reaction conditions comprise a pH of 9. In some cases, the reaction conditions comprise a pH of 10. In some cases, the reaction conditions comprise a pH of 11 . In some cases, the reaction conditions comprise contacting the enzyme having silicase activity with a co-factor. In some cases, the co-factor is selected from the group consisting of: iron, zinc, copper, nickel, and cobalt.

[0071] In some cases, the reaction conditions provided herein comprise crushing or grinding the rocks or ores to achieve a particulate size. In some cases, the ground ores comprise a size of about 50 pm to 1 mm. In some cases, the ground ores comprise a size from about 50 pm to 750 pm. In some cases, the ground ores comprise a size from about 50 pm to 500 pm. In some cases, the ground ores comprise a size from about 50 pm to 250 pm. In some cases, the ground ores comprise a size from about 50 pm to 150 pm. In some cases, the ground ores comprise a size from about 50 pm to 100 pm. In some cases, the ground ores comprise a size of about 50 pm. In some cases, the ground ores comprise a size of about 100 pm. In some cases, the ground ores comprise a size of about 150 pm. In some cases, the ground ores comprise a size of about 250 pm. In some cases, the ground ores comprise a size of about 500 pm. In some cases, the ground ores comprise a size of about 750 pm. In some cases, the ground ores comprise a size of about 1 mm.

[0072] In some cases, the reaction conditions provided herein comprise creating a slurry of crushed rock and liquid. In some cases, the rockto liquid ratio is from about 1 -40% (w / v). In some cases, the rock to liquid ratio is from about 1 -35% (w / v). In some cases, the rock to liquid ratio is from about 1 -30% (w / v). In some cases, the rock to liquid ratio is from about 1 - 25% (w / v). In some cases, the rock to liquid ratio is from about 1 -20% (w / v). In some cases, the rock to liquid ratio is from about 1 -15% (w / v). In some cases, the rock to liquid ratio is from about 1-10% (w / v). In some cases, the rockto liquid ratio is from about 1 -5% (w / v). In some cases, the rock to liquid ratio is from about 10-40% (w / v). In some cases, the rock to liquid ratio is from about 10-35% (w / v). In some cases, the rock to liquid ratio is from about 10-30% (w / v). In some cases, the rock to liquid ratio is from about 10-25% (w / v). In some cases, the rock to liquid ratio is from about 15-35% (w / v). In some cases, the rock to liquid ratio is from about 15-30% (w / v). In some cases, the rock to liquid ratio is from about 20- 35% (w / v). In some cases, the rock to liquid ratio is from about 20-30% (w / v). In some cases, the rock to liquid ratio is from about 25 -35% (w / v). In some cases, the rock to liquid ratio is from about 25-30% (w / v). In some cases, the rock to liquid ratio is about 1% (w / v). In some cases, the rock to liquid ratio is about 5% (w / v). In some cases, the rock to liquid ratio is about 10% (w / v). In some cases, the rock to liquid ratio is about 15% (w / v). In some cases, the rock to liquid ratio is about 20% (w / v). In some cases, the rock to liquid ratio is about 25% (w / v). In some cases, the rock to liquid ratio is about 30% (w / v). In some cases, the rock to liquid ratio is about 35% (w / v). In some cases, the rockto liquid ratio is about 40% (w / v).

[0073] In some cases, the reaction conditions provided herein comprise an enzymatic reaction that proceeds for a set period of time. In some cases, the reaction conditionscomprise the enzymatic reaction proceeding for about 1 -72 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 1 -60 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 1 -48 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 1 - 36 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 1 -24 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 1 -12 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 1 -6 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 12-72 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 12-60 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 12- 48 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 12-36 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 12-24 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 24-72 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 24-60 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 24 -48 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 24 - 36 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 36-72 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 36-60 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceedingfor about 36-48 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 48-72 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 48-60 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 1 hour. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 6 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceedingfor about 12 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceedingfor about 24 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 36 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 48 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 60 hours. In some cases, the reaction conditions comprise the enzymatic reaction proceeding for about 72 hours.

[0074] In some cases, the enzyme having silicase activity depolymerizes phyllosilicate. In some cases, the enzyme having variant cleaves one or more Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4). In some cases, the metal (e.g., metal ion) extracted from the phyllosilicate is lithium, aluminum, iron, nickel, cobalt, uranium, strontium, a rare earth element, or any combination thereof. In some cases, the metal is lithium. In some cases, the metal ion is aluminum. In some cases, the metal ion is strontium.

[0075] In some cases, the metal is released into a solution. In some cases, the metal is extracted and released in form of metal ion. In some cases, the metal is extracted and released in form of a metal atom. In some cases, the metal is solubilized in a solution comprising water and / or buffer. In some cases the buffer comprises TRIS, PBS, citrate, monosodium glutamate, or any combination thereof. In some cases, the metal precipitates in the solution. In some cases, the metal is released and / or extracted in form of a metal complex. In some cases, the method further comprises extracting, and / or separating the metal from the solution. Any proper separation technique may be used. In some cases, an electromagnetic force may be used to separate metal ions from the solution. In some cases, a solid -liquid separation technique may be used to separate a metal precipitate from the solution. Any combination of separation and processing methods may be used. The method may comprise collecting the metal from the phyllosilicate (e.g., source rock / ore) and from the system or solution used to perform the extraction according to the embodiments of the present disclosure and provide the metal for use in its intended application. In some cases, the metal may be processed as an industry -grade metal, battery -grade metal, or pharmaceutical-grade metal. An example of this may comprise industry -grade metal, battery -grade metal, or pharmaceutical-grade lithium.

[0076] In some cases, the method further comprises comprising purifying the metal from the solution, thereby generating a purified metal, a metal ion, a metal atom, a solid metal complex, a metal precipitate, or any combination thereof. In some cases, the purified metal ion a purity of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999% or greater.

[0077] In some cases, the method is performed in situ or ex-situ. In some embodiments, the enzyme variant has a catalytic efficiency of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the method has a maximum metal extraction rate of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the enzyme variant has a catalytic efficiency of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, lOtimes or higher. In some embodiments, the method has a maximum metal extraction rate of at least about 1. 1, 1 .2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher.

[0078] In some cases, the enzyme variant is recombinantly produced in a host cell or in a cell-free production system. In some cases, the host cell is a bacterial cell or a yeast cell. In some cases, the bacterial cell is Escherichia coli or the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

[0079] In an aspect, provided herein is a reaction mixture comprising an enzyme variant described anywhere herein, and a non-natural co-factor. In some cases, the non -natural cofactor is bound to enzyme variant. In some cases, the non-natural co-factor increases a function of the enzyme variant as compared to a reaction mixture comprising the enzyme variant and a natural co-factor. In some cases, the non-natural co-factor is copper, iron, nickel, cobalt, or glycine. In some cases, the natural co-factor is zinc. In some cases, the natural co-factor is iron. In some cases, the reaction mixture does not contain the natural cofactor. In some cases, the non-natural co-factor does not act as a co-factor for the enzyme variant in nature. In some cases, the reaction mixture further comprises the phyllosilicate. In some cases, the reaction mixture has reaction conditions such that a metal contained within the phyllosilicate is extracted, released, or solubilized, or precipitated into a solution from the phyllosilicate. For example, a solution may be provided in proximity of a phyllosilicate comprising a metal. An enzyme variant of the present disclosure and a non-natural co-factor according to the embodiments disclosed anywhere herein may be present in the solution. The enzyme may facilitate breaking Si-0 bonds in the phyllosilicate, thereby digesting and / or degrading the phyllosilicate and releasing the metal encased in the phyllosilicate in form of metal ion, metal atom, metal solubilized in solution, metal precipitated in solution, or any combination thereof. The non-natural co-factor combined with or bound to the enzyme may further enhance the catalytic efficiency of the enzyme in releasing the metal from the phyllosilicate in any of the mentioned forms.

[0080] In some cases, the enzyme variant has increased ability to release metal in any form mentioned anywhere herein, from phyllosilicates in the presence of the non-natural co-factor as compared to the enzyme having silicase activity in the presence of the natural co-factor.

[0081] In some cases, the enzyme variant has a sequence identity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or more identity with a carbonic anhydrase. In some cases, the enzyme variant has a sequence identity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or more identity with an alpha carbonic anhydrase or a gamma carbonic anhydrase. In some cases, the enzyme variant is derived from an organism selected from the group consisting of: Methanosarcina thermophila, Bacillus licheniformis CG-B52, Pelobacter carhinolicus, Syntrophus aciditrophicus, Methanosarcina harkeri, Methanosarcina mazei. Bacillus halodurans, Alkalihalobacillus clausii (strain KSM- K16) (Bacillus clausii), Methanosarcina acetivorans , Kofleriaceae bacterium SLC26A / SulP, Thermodesulfitimonas autotrophica, Fischerella thermalis / Mastigocladus laminosus, Thermosynechococcus vestitus B>V- !(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila , Thermo syntropha lipolytica, isoleucine patch superfamily, De sulfo fundulus thermobenzoicus, Archaeoglobus veneficus, Suberites domuncula, and any combination thereof.

[0082] In some embodiments, the variant enzyme comprises an amino acid sequence at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 -18. In some embodiments, the variant enzyme may be any one of the SEQ ID NOS: 19-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 19-434. In some embodiments, the variant enzyme may be any one of the SEQ ID NOS: 403-434. In some embodiments, the variant enzyme comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403-434.

[0083] In some embodiments, the enzyme having silicase activity has a catalytic efficiency of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the method has a maximum metal extraction rate of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% , at least about 95%, at least about 98% at least about 99%, at least about 99.5% or higher. In some embodiments, the enzyme variant has a catalytic efficiency of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher. In some embodiments, the method has a maximum metal extraction rate of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 8, 9, 10 times or higher.

[0084] In some embodiments, the enzyme having silicase activity has a pKd of at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or higher. In some embodiments, the enzyme having silicase activity has a Kcat value of at least about 2 mol per second (mol / s), at least about 10 mol / s, at least about 50 mol / s, at least about 100 mol / s, at least about 200 mol / s, at least about 300 mol / s, at least about 400 mol / s, at least about 500 mol / s, at least about 600 mol / s, at least about 700 mol / s, at least about 800 mol / s, at least about 900 mol / s, at least about 1000 mol / s, or higher.

[0085] In some cases, the reaction mixture has a pH from about 4 to about 11 . In some cases, the reaction mixture has a pH from about 4 to about 10. In some cases, the reaction mixture has a pH from about 4 to about 9. In some cases, the reaction mixture has a pH from about 4 to about 8. In some cases, the reaction mixture has a pH from about 4 to about 7. In some cases, the reaction mixture has a pH from about 4 to about 6. In some cases, the reaction mixture has a pH from about 5 to about 11 . In some cases, the reaction mixture has a pH from about 5 to about 10. In some cases, the reaction mixture has a pH from about 5 to about 9. In some cases, the reaction mixture has a pH from about 5 to about 8. In some cases, the reaction mixture has a pH from about 5 to about 7. In some cases, the reaction mixture has a pH from about 5 to about 6. In some cases, the reaction mixture has a pH from about 6 to about 11 . In some cases, the reaction mixture has a pH from about 6 to about 10. In some cases, the reaction mixture has a pH from about 7 to about 11 . In some cases, the reaction mixture has a pH from about 7 to about 10. In some cases, the reaction mixture has a pH from about 8 to about 11 . In some cases, the reaction mixture has a pH from about 8 to about 10. In some cases, the reaction mixture has a pH from about 9 to about 11. In some cases, thereaction mixture has a pH from about 9 to about 10. In some cases, the reaction mixture has a pH of 4. In some cases, the reaction mixture has a pH of 5. In some cases, the reaction mixture has a pH of 6. In some cases, the reaction mixture has a pH of 7. In some cases, the reaction mixture has a pH of 8. In some cases, the reaction mixture has a pH of 9. In some cases, the reaction mixture has a pH of 10. In some cases, the reaction mixture has a pH of 11 . In some cases, the reaction mixture has a temperature from about 20 to about 90 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 23 to about 90 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 23 to about 85 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 30 to about 90, from about 30 to about 80, from about 30 to about 70, from about 30 to about 60, or from about 30 to about 50 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 45 to about 55 degrees Celsius (C). In some cases, the reaction mixture has a temperature from about 45 to about 50 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 20 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 23 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 25 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 30 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 35 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 40 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 45 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 50 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 55 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 60 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 70 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 75 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 80 degrees Celsius (C). In some cases, the reaction mixture has a temperature about 85 degrees Celsius (C). In some cases, the reaction mixture further comprises a buffered saline solution. In some cases, the buffered solution comprises saline, glycine, iron ions, or any combination thereof. In some cases the buffered solution comprises TRIS, PBS, citrate, monosodium glutamate, or any combination thereof. In some cases, the reaction mixture further comprises an activator co-factor of the non-naturally occurring enzyme. In some cases, the activator co-factor comprises glycine, iron ion, or both. In some cases, the metal is lithium, aluminum, iron, nickel, cobalt, strontium or a rare earth element. In some cases, the metal is lithium. In some cases, the metal is aluminum. In some cases, themetal is strontium. In some cases, the enzyme having silicase activity is recombinantly produced in a host cell or in a cell-free production system. In some cases, the host cell is a bacterial cell or yeast cell. In some cases, the bacterial cell is Escherichia coh, or the yeast cell is Pichia pastoris or Saccharomyces cerevisiae .

[0086] In some cases, the reaction mixture provided herein comprise crushing or grinding the rocks or ores to achieve a particulate size. In some cases, the ground ores comprise a size of about 50 pm to 1 mm. In some cases, the ground ores comprise a size from about 50 pm to 750 pm. In some cases, the ground ores comprise a size from about 50 pm to 500 pm. In some cases, the ground ores comprise a size from about 50 pm to 250 pm. In some cases, the ground ores comprise a size from about 50 pm to 150 pm. In some cases, the ground ores comprise a size from about 50 pm to 100 pm. In some cases, the ground ores comprise a size of about 50 pm. In some cases, the ground ores comprise a size of about 100 pm. In som e cases, the ground ores comprise a size of about 150 pm. In some cases, the ground ores comprise a size of about 250 pm. In some cases, the ground ores comprise a size of about 500 pm. In some cases, the ground ores comprise a size of about 750 pm. In some cases, the ground ores comprise a size of about 1 mm.

[0087] In some cases, the reaction mixture provided herein comprise creating a slurry of crushed rock and liquid. In some cases, the rockto liquid ratio is from about 1 -40% (w / v). In some cases, the rock to liquid ratio is from about 1 -35% (w / v). In some cases, the rock to liquid ratio is from about 1 -30% (w / v). In some cases, the rock to liquid ratio is from about 1 - 25% (w / v). In some cases, the rock to liquid ratio is from about 1 -20% (w / v). In some cases, the rock to liquid ratio is from about 1 -15% (w / v). In some cases, the rock to liquid ratio is from about 1-10% (w / v). In some cases, the rockto liquid ratio is from about 1 -5% (w / v). In some cases, the rock to liquid ratio is from about 10-40% (w / v). In some cases, the rock to liquid ratio is from about 10-35% (w / v). In some cases, the rock to liquid ratio is from about 10-30% (w / v). In some cases, the rock to liquid ratio is from about 10-25% (w / v). In some cases, the rock to liquid ratio is from about 15-35% (w / v). In some cases, the rock to liquid ratio is from about 15-30% (w / v). In some cases, the rock to liquid ratio is from about 20- 35% (w / v). In some cases, the rock to liquid ratio is from about 20 -30% (w / v). In some cases, the rock to liquid ratio is from about 25-35% (w / v). In some cases, the rock to liquid ratio is from about 25-30% (w / v). In some cases, the rock to liquid ratio is about 1% (w / v). In some cases, the rock to liquid ratio is about 5% (w / v). In some cases, the rock to liquid ratio is about 10% (w / v). In some cases, the rock to liquid ratio is about 15% (w / v). In some cases, the rock to liquid ratio is about 20% (w / v). In some cases, the rock to liquid ratio is about25% (w / v). In some cases, the rock to liquid ratio is about 30% (w / v). In some cases, the rock to liquid ratio is about 35% (w / v). In some cases, the rockto liquid ratio is about 40% (w / v).

[0088] In some cases, the reaction mixture provided herein comprise an enzymatic reaction that proceeds for a set period of time. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -36 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1-24 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -12 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 -6 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12 -48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-36 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12-24 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24-60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24-48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24 -36 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36-60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36-48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 48-72 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 48 -60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 1 hour. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 6 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 12 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 24 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 36 hours. In some cases, the reaction mixture comprises theenzymatic reaction proceeding for about 48 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 60 hours. In some cases, the reaction mixture comprises the enzymatic reaction proceeding for about 72 hours.

[0089] In some cases, provided herein are engineered enzymes. In some cases, the method of enzyme engineering comprises performing directed molecular evolution on an enzyme sequence and generating an evolved enzyme sequence, wherein the evolved enzyme sequence has a higher specificity to phyllosilicate, a higher catalytic rate for acting on the phyllosilicate, or both, compared to the first enzyme sequence.

[0090] The evolved enzyme, the wild-type enzyme, or both may cleave Si-0 bonds in the substrate and extract a metal from the phyllosilicate, in some cases a silicate rock. In some examples, directed molecular evolution is performed to generate the engineered enzyme sequences. In some cases, one or more Machine Learning (ML) or Artificial Intelligence (Al) Algorithms may be used to generate the engineered enzyme sequences. In some cases, performing directed molecular evolution comprises deoxyribonucleic acid (DNA) shuffling. In some cases, the ML or Al algorithm comprises one or more of structural sequence generation, sequence ranking, and sequence fine-tuning. In some cases, the ML or Al algorithm comprises a transformer model system. In some cases, the ML or Al algorithm comprises using natural language processing (NLP). In some cases, the evolved enzyme sequence is the sequence of the synthetic enzyme used in the methods of any of the embodiments of the present disclosure.EXAMPLESExample 1. Silica Degradation With Synthetic Enzymes

[0091] The enzyme gamma carbonic anhydrase from A7. thermophila degrades silicate mineral material which allows for the extraction of metals such as lithium and aluminum, for example. Synthetic enzymes were designed from Gamma Carbonic Anhydrase to improve the efficiency of the degradation reaction of silicate mineral material, specifically phyllosilicates, such as lepidolite, montmorillonite, and muscovite.

[0092] The 17 amino acid residue signal sequence or the 17 amino acid residue signal sequence and part of the disordered region (about residues 18 to 64, starting from the N- terminus) of wildtype Gamma Carbonic Anhydrase from M. thermophila were truncated (SEQ ID NO: 403 and SEQ ID NO: 424 respectively). From there, further mutations were generated to optimize for enzymatic efficacy, as shown in SEQ ID NOs: 404-423 and 425- 434. Table 3 shows the percentage identity, as calculated by comparing sequence informationusing the advanced BLAST computer program, of the synthetic enzymes compared to wildtype gamma carbonic anhydrase from M. thermophila.Table 3 - Percentage Identity of Synthetic Enzymes to Wildtype Gamma CarbonicAnhydrase

[0093] Synthetic enzymes of SEQ ID NOs: 403-434 were generated comprising a GST tag and expressed in E. coli BL21 using a pGEX-6Pl GST codon -optimized vector. E. coli cultures were grown at 37 °C with shaking and induced. After induction, the cells were harvested by centrifugation and lysed. The lysate was supplemented with ferrous gluconate to provide a source of Fe. The lysate was heated to 55 °C to aide in protein folding and stability. Enzymes were generated comprising an N-terminal GST tag. Other common purification tags, such as NEXT or 6-HIS, or no tags may be utilized in purification and experimental use of the synthetic enzymes. The synthetic enzymes were purified using a GST affinity column. The GST tag was found to not affect enzymic activity under reaction conditions.

[0094] The degradation reactions were tested for the various synthetic enzymes lepidolite, montmorillonite, and muscovite. Degradation reactions were conducted in plastic containers to avoid silicate dissolution from glass. A range of reaction conditions were tested. The reaction was operational in a buffer comprising TRIS, PBS, 0.1 M citrate, or 0.9% monosodium glutamate in a pH range of 4-11, the rock to liquid rock ratio was tested between 1-40% (w / v), the minerals were crushed with a small grinder to ground ore sizes between 50 pm to 1 mm, the reaction was shaken with a range of 1 -220 RPM, at a temperature between 23 -85 °C, the synthetic enzymes of SEQ ID NOs: 403 -434 with GST, NEXT, 6-His or no N-terminal tag were tested, Zn, Fe, Cu, or Co metal cofactors were included, and the reaction ran for a time range of 1 -48 hours. The range of reaction conditions tested resulted in degradation of silicate material. Data not shown.

[0095] Optimal reaction conditions were developed and used to compare the synthetic enzymes of SEQ ID NOs: 403-434. The synthetic enzymes were tested with lepidolite, montmorillonite, and muscovite. Optimized reaction conditions were found to be the following: the minerals were crushed with a small grinder and sifted to achieve a particulate size between 50 and 150 pm. The reaction proceeded in a 0.1 M TRIS buffer at pH 10 in arock to liquid rock ration of 30% (w / v) at a temperature of 51 °C and shaking at 220 RPM for 48 hours. Reactions were completed in triplicate. After the reaction proceeded for 48 hours, the suspensions were centrifuged at 14,000 RPM for 15 minutes. The supernatant was filtered using a 45 pm, 13 mm diameter syringe filter.

[0096] The elemental composition of the supernatant was determined using a Laser Induced Breakdown Spectroscopy (LIBS) lithium brine analyzer and X-ray fluorescence analyzer. The results of the extracted metals were measured as PPM in solution as shown in Table 4. Degradation results were normalized to compared to SEQ ID NO: 403 or SEQ ID NO: 424, which corresponds to two truncated wildtype Gamma Carbonic Anhydrases from M. thermophila.

[0097] The hydrolytic activity of the synthetic enzymes on the crystalline structure was also assessed using a silica degradation assay. The assay measured the amount of free silica (Si(OH)4, orthosilicic acid) using a molybdenum blue assay. The reaction proceeded as described above. 100 pL of the supernatant was deposited into a 96-well plate. 10 pL 1 :1 sulfuric acid solution was added to the sample and mixed with the sample. 20 pL of 5% ammonium molybdate solution was added to the acidified sample and allowed to rest. Next, 20 pL of 0.5% ascorbic acid reducing reagent was added to the mixture and allowed the color to develop for 10 minutes. The molybdenum blue assay reaction mixture was transferred to a microcuvette and the absorbance was measured at 810 nm. The amount of free orthosilicic acid was calculated against a calibrated standard curve. The results of the degradation of synthetic enzymes of SEQ ID NOs: 404-423 were normalized to activity of SEQ ID NO: 403 and the results of the degradation of synthetic enzymes of SEQ ID NOs: 425 -434 were normalized to activity of SEQ ID NO: 424 as shown in Table 5. The results show superior extraction of Fe, Li and Al as well as enzyme degradation activity in the mutated synthetic enzymes of SEQ ID NOs: 404-423 and 425-434 as compared to the designed truncated wildtype synthetic enzyme of SEQ ID NO: 403 and SEQ ID NO: 424, respectively.Table 4 - Metal Extraction Reaction ResultsTable 5 - Degradation Activity of Synthetic EnzymesN.D. = no dataExample 2. Silica Degradation With Host Cells Expressing Synthetic Enzymes

[0098] The enzyme Gamma Carbonic Anhydrase from M. thermophila degrades silicate mineral material which allows for the extraction of metals such as lithium and aluminum, for example. Synthetic enzymes are designed to improve the efficiency of the degradation reaction of silicate mineral material, specifically phyllosilicates, such as lepidolite, montmorillonite, and muscovite.

[0099] Synthetic enzymes are generated comprising a tag and expressed in a bacteria host cell, such as E. co , using a host cell appropriate vector. Host cell cultures are grown and induced. After induction, the cells are harvested.

[0100] The degradation reactions are tested using the host cells expressing the synthetic enzymes with phyllosilicate materials, such as lepidolite, montmorillonite, and muscovite. Degradation reactions are conducted in plastic containers to avoid silicate dissolution from glass. A range of reaction conditions are tested. The reaction is tested in a buffer comprising TRIS, PBS, 0.1 M citrate, or 0.9% monosodium glutamate in a pH range of 4-11, the rock to liquid rock ratio is tested between 1 -40% (w / v), the minerals are crushed with a small grinder to ground ore sizes between 50 pm to 1 mm, the reaction is shaken with a range of 1 -220 RPM, at a temperature between 23-85 °C, Zn, Fe, Cu, or Co metal cofactors are included, and the reaction is run for a time range of 1 -48 hours.

[0101] The elemental composition of the supernatant is determined using a Laser Induced Breakdown Spectroscopy (LIBS) lithium brine analyzer and X-ray fluorescence analyzer. The results of the extracted metals are measured as PPM in solution.

[0102] The hydrolytic activity of the synthetic enzymes on the crystalline structure is also assessed using a silica degradation assay. The assay measures the amount of free silica (Si(OH)4, orthosilicic acid) using a molybdenum blue assay. Supernatant is deposited into a 96-well plate. 1 :1 sulfuric acid solution is added to the sample and mixed with the sample. 5% ammonium molybdate solutionis added to the acidified sample and allowed to rest. 0.5% ascorbic acid reducing reagent is added to the mixture and allowed the color to develop for 10 minutes. The molybdenum blue assay reaction mixture is transferred to a microcuvette and the absorbance is measured at 810 nm. The amount of free orthosilicic acid is calculated against a calibrated standard curve.

[0103] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:1 . A method of degrading a phyllosilicate, the method comprising: contacting the phyllosilicate with a variant enzyme having increased ability to degrade the phyllosilicate relative to a wild-type enzyme under reaction conditions such that the variant enzyme degrades the phyllosilicate.

2. The method of claim 1, wherein the variant enzyme comprises at least one amino acid modification relative to the wild-type enzyme that confers the increased ability to degrade the phyllosilicate.

3. The method of claim 1 or 2, wherein the variant enzyme selectively degrades the phyllosilicate.

4. The method of any one of claims 1 -3, wherein the phyllosilicate is contained within an ore or a rock.

5. The method of any one of claims 1 -4, wherein the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof.

6. The method of any one of claims 1 -4, wherein the phyllosilicate is selected from the group consisting of: lepidolite, muscovite, montmorillonite, and any combination thereof.

7. The method of any one of claims 1 -6, wherein the method solubilizes and releases a metal contained within the phyllosilicate.

8. The method of claim 7, further comprising collecting the metal.

9. The method of claim 7 or 8, further comprising extracting the metal.

10. The method of any one of claims 7-9, further comprising purifying the metal.11 . The method of any one of claims 7-10, further comprising enriching for the metal.

12. The method of any one of claims 7-11, wherein the metal is lithium.

13. The method of any one of claims 7-11, wherein the metal is aluminum.

14. The method of any one of claims 7-11, wherein the metal is strontium.

15. The method of any one of claims 7-11, wherein the metal is iron.

16. The method of any one of claims 1 -15, wherein the metal is released into a solution.

17. The method of any one of claims 1 -16, wherein the variant enzyme comprises a sequence identity of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or more with a carbonic anhydrase.

18. The method of claim 17, wherein the carbonic anhydrase is a gamma carbonic anhydrase or alpha carbonic anhydrase.

19. The method of any one of claims 1-18, wherein the variant enzyme is a variant of a wild-type enzyme selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransf erase-like protein (Isoleucine patch superfamily), Fischerella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus B>?- l(Thermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, De sulfofundulus thermob enzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combination thereof.

20. The method of any one of claims 1-19, wherein the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 -18 or SEQ ID NOS: 19-434.21 . The method of any one of claims 1-20, wherein the variant enzyme comprises any one of SEQ ID NOS: 19-434.

22. The method of any one of claims 1-21, wherein the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403 -434.

23. The method of any one of claims 1-22, wherein the variant enzyme comprises any one of SEQ ID NOS: 403-434.

24. The method of any one of claims 1-23, wherein the variant enzyme has a pKd of at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least ab out 10, at least about 11, or higher.

25. The method of any one of claims 1-24, wherein the variant enzyme has a Kcat value of at least about 2 mol per second (mol / s), at least about 10 mol / s, at least about 50 mol / s, at least about 100 mol / s, at least about 200 mol / s, at least about 300 mol / s, at least about 400 mol / s, at least about 500 mol / s, at least about 600 mol / s, at least about 700 mol / s, at least about 800 mol / s, at least about 900 mol / s, at least about 1000 mol / s, or higher.

26. The method of any one of claims 1 -25, wherein the reaction conditions comprise a temperature from about 23 to about 85 degrees Celsius (C).

27. The method of any one of claims 1 -26, wherein the reaction conditions comprise a temperature from about 45 to about 50 degrees Celsius (C).

28. The method of any one of claims 1 -27, wherein the reaction conditions comprise a temperature of about 50 degrees Celsius (C).

29. The method of any one of claims 1-28, wherein the reaction conditions comprise a pH from about 4 to about 11 .

30. The method of any one of claims 1-29, wherein the reaction conditions comprise a pH of about 5.31 . The method of any one of claims 1-30, wherein the reaction conditions comprise a pH of about 10.

32. The method of any one of claims 1 -31, wherein the reaction conditions comprise contacting the variant enzyme with a co -factor.

33. The method of claim 32, wherein the co-factor is zinc, copper, cobalt, nickel, magnesium, manganese, iron, or any combination thereof.

34. The method of claim 32, wherein the co-factor is iron.

35. The method of any one of claims 1 -34, wherein the variant enzyme depolymerizes the phyllosilicate.

36. The method of any one of claims 1 -35, wherein the variant enzyme cleaves one or more Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4).

37. The method of any one of claims 1 -36, wherein the method is performed in situ.

38. The method of any one of claims 1 -37, wherein the variant enzyme is recombinantly produced in a host cell or in a cell-free production system.

39. The method of claim 38, wherein the host cell is a bacterial cell or yeast cell.

40. The method of claim 39, wherein the bacterial cell is Escherichia coli or the yeast cell is Pichia pastoris or Saccharomyces cerevisiae .41 . The method of any one of claims 1 -40, wherein the reaction conditions comprise a rock to liquid ratio from about 1 -40% (w / v).

42. The method of any one of claims 1 -41, wherein the reaction conditions comprise a rock to liquid ratio of about 30% (w / v).

43. The method of any one of claims 1 -42, wherein the reaction conditions comprise a buffer.

44. The method of claim 43, wherein the buffer is TRIS, PBS, citrate, monosodium glutamate, or a combination thereof.

45. A method of degrading an phyllosilicate, the method comprising: contacting the phyllosilicate with a variant enzyme of any one of SEQ ID NOS: 403-434 under reaction conditions such that the variant enzyme degrades the phyllosilicate, wherein the reaction conditions comprise a temperature from about 23 -85 degrees Celsius (C), a pH from about 4-11, a co-factor, and a rock to liquid ratio from about 1-40% (w / v).

46. The method of any one of claims 1 -45, wherein the reaction conditions proceed for about 1-48 hours.

47. The method of any one of claims 1 -46, wherein the reaction conditions proceed for about 48 hours.

48. The method of any one of claims 1 -47, wherein the reaction conditions comprise a temperature of about 50 degrees Celsius (C).

49. The method of any one of claims 1 -48, wherein the reaction condition comprises a pH of about 10.

50. The method of any one of claims 1 -49, wherein the co-factor is zinc, iron, copper, cobalt, or any combination thereof.51 . The method of any one of claims 1 -50, wherein the co-factor is iron.

52. The method of any one of claims 1 -51, wherein the rock to liquid ratio is about 30% (w / v).

53. A variant enzyme having increased ability to degrade phyllosilicate relative to a wildtype enzyme.

54. The variant enzyme of claim 53, wherein the variant enzyme comprises at least one amino acid modification relative to the wild -type enzyme that confers the increased ability to degrade phyllosilicate.

55. The variant enzyme of claim 53 or 54, wherein the variant enzyme selectively degrades phyllosilicate.

56. The variant enzyme of claim 55, wherein the variant enzyme does not have increased ability to degrade an amorphous silicate or a inosilicate relative to the wild-type enzyme.

57. The variant enzyme of any one of claims 53-56, wherein the phyllosilicate is contained in a rock or an ore.

58. The variant enzyme of any one of claim 53-57, wherein the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof.

59. The variant enzyme of any one of claims 53-58, wherein the phyllosilicate is selected from the group comprising: lepidolite, muscovite, montmorillonite, and any combination thereof.

60. The variant enzyme of any one of claims 53-59, wherein the variant enzyme has increased ability to solubilize and release a metal contained within the phyllosilicate as compared to the wild-type enzyme.

61. The variant enzyme of any one of claims 53-60, wherein the variant enzyme is a variant of a wild-type enzyme selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransf erase-like protein (Isoleucine patch superfamily), Fischer ella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus BP- \!(Ihermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, Desulfofundulus thermob enzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combination thereof.

62. The variant enzyme of any one of claims 53-61, wherein the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 -18 or SEQ ID NOS: 19-434.

63. The variant enzyme of any one of claims 53-62, wherein the variant enzyme comprises any one of SEQ ID NOS: 19-434.

64. The variant enzyme of any one of claims 53-63, wherein the variant enzyme comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100% sequence identity to an amino acid sequence of any one of SEQ ID NOS: 403 -434.

65. The variant enzyme of any one of claims 53-64, wherein the variant enzyme comprises any one of SEQ ID NOS: 403-434.

66. The variant enzyme of any one of claims 53-65, wherein the variant enzyme has a sequence identity of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or higher with a carbonic anhydrase.

67. The variant enzyme of claim 53-66, wherein the carbonic anhydrase is a gamma carbonic anhydrase or alpha carbonic anhydrase.

68. The variant enzyme of any one of claims 53-67, wherein the variant enzyme has increased ability to depolymerize phyllosilicate as compared to the wild-type enzyme.

69. The variant enzyme of any one of claims 53-68, wherein the variant enzyme has increased ability to cleave Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4) as compared to the wild-type enzyme.

70. The variant enzyme of any one of claims 53-69, wherein the variant enzyme is recombinantly produced in a host cell or in a cell-free production system.

71. The variant enzyme of any one of claims 53-70, wherein the host cell is a bacterial cell or yeast cell.

72. The variant enzyme of claim 71, wherein the bacterial cell is Escherichia coli or the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

73. A polynucleotide comprising a nucleic acid sequence encoding the variant enzyme of any one of claims 53-72.

74. A vector comprising the polynucleotide of claim 73.

75. A reaction mixture comprising an phyllosilicate and a variant enzyme having increased ability to degrade the phyllosilicate relative to a wild-type enzyme.

76. The reaction mixture of claim 75, wherein the variant enzyme comprises at least one amino acid modification relative to the wild -type enzyme that confers the increased ability to degrade phyllosilicate.

77. The reaction mixture of any one of claims 75-76, wherein the variant enzyme selectively degrades phyllosilicate.

78. The reaction mixture of any one of claims 75-77, wherein the phyllosilicate is contained within an ore or a rock.

79. The reaction mixture of any one of claims 75-78, wherein the phyllosilicate is selected from the group consisting of: lepidolite, hectorite, kaolinite, vermiculite, muscovite, montmorillonite, and any combination thereof.

80. The reaction mixture of any one of claims 75-79, wherein the phyllosilicate is selected from the group comprising: lepidolite, muscovite, montmorillonite, and any combination thereof.

81. The reaction mixture of any one of claims 75-80, further comprising at least one metal solubilized and released from the phyllosilicate.

82. The reaction mixture of claim 81, wherein the metal comprises lithium.

83. The reaction mixture of claim 81, wherein the metal comprises iron.

84. The reaction mixture of claim 81, wherein the metal comprises strontium.

85. The reaction mixture of claim 81, wherein the metal comprises aluminum.

86. The reaction mixture of any one of claims 75-85, wherein the reaction mixture has a pH from about 4 to about 11.

87. The reaction mixture of any one of claims 75-86, wherein the reaction mixture has a pH of about 5.

88. The reaction mixture of any one of claims 75-87, wherein the reaction mixture has a pH of about 10.

89. The reaction mixture of any one of claims 75-88, wherein the reaction mixture has a temperature from about 23 to about 85 degrees Celsius (C).

90. The reaction mixture of any one of claims 75-89, wherein the reaction mixture has a temperature from about 45 to about 50 degrees Celsius (C).

91. The reaction mixture of any one of claims 75-90, wherein the reaction mixture has a temperature of about 50 degrees Celsius (C).

92. The reaction mixture of any one of claims 75-91, wherein the reaction mixture further comprises a co-factor of the variant enzyme.

93. The reaction mixture of claim 92, wherein the co-factor is iron.

94. The reaction mixture of claim 92, wherein the co-factor is copper.

95. The reaction mixture of any one of claims 75-94, further comprising a buffered saline solution.

96. The reaction mixture of claim 95, wherein the buffered saline solution is phosphate- buffered saline.

97. The reaction mixture of any one of claims 75-96, further comprising an activator cofactor of the variant enzyme.

98. The reaction mixture of claim 97, wherein the activator co-factor is glycine.

99. The reaction mixture of any one of claims 75-98, wherein the variant enzyme is a variant of a wild-type enzyme selected from the group consisting of: Methanosarcina thermophila gamma carbonic anhydrase, Bacillus licheniformis CG-B52 gamma carbonic anhydrase, Pelobacter carbinolicus gamma carbonic anhydrase, Syntrophus aciditrophicus gamma carbonic anhydrase, Methanosarcina barkeri gamma carbonic anhydrase, Methanosarcina mazei carbonic anhydrase, Bacillus halodurans alpha carbonic anhydrase, Alkalihalobacillus clausii (strain KSM-K16) (Bacillus clausii) alpha carbonic anhydrase, Methanosarcina acetivorans carbonate dehydratase, Kofleriaceae bacterium SLC26A / SulP transporter domain-containing protein, Thermodesulfitimonas autotrophica carbonic anhydrase / acetyltransf erase-like protein (Isoleucine patch superfamily), Fischerella thermalis / Mastigocladus laminosus JSC-11 carboxysome assembly protein CcmM, Thermosynechococcus vestitus BP- \!(Ihermosynechococcus elongatus BP-1) carboxysome assembly protein CcmM, Methanothrix thermoacetophila carbonate dehydratase, Thermo syntropha lipolytica carbonic anhydrase or acetyltransferase, isoleucine patch superfamily, Desulfofundulus thermob enzoicus transferase, Archaeoglobus veneficus carbonate dehydratase, Suberites domuncula carbonic anhydrase, and any combination thereof.

100. The reaction mixture of any one of claims 75-99, wherein the variant enzyme comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOS: 1 -18 or SEQ ID NOS: 19-434.

101. The reaction mixture of any one of claims 75-100, wherein the variant enzyme is a carbonic anhydrase.

102. The reaction mixture of claim 101, wherein the carbonic anhydrase is a gamma carbonic anhydrase.

103. The reaction mixture of any one of claims 75-102, wherein the variant enzyme has increased ability to depolymerize phyllosilicate as compared to the wild-type enzyme.

104. The reaction mixture of any one of claims 75-103, wherein the variant enzyme has increased ability to cleave Si-0 bonds in the phyllosilicate to generate silicic acid (Si(OH)4) as compared to the wild-type enzyme.

105. The reaction mixture of any one of claims 75-104, wherein the non-naturally occurring enzyme is recombinantly produced in a host cell or in a cell-free production system.

106. The reaction mixture of claim 105, wherein the host cell is a bacterial cell or yeast cell.

107. The reaction mixture of claim 106, wherein the bacterial cell is Escherichia coli or the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

108. The reaction mixture of any one of claims 75-107, wherein the reaction conditions comprise a rock to liquid ratio from about 1 -40% (w / v).

109. The reaction mixture of any one of claims 75-108, wherein the reaction conditions comprise a rock to liquid ratio of about 30% (w / v).

110. The reaction mixture of any one of claims 75-109, wherein the reaction conditions comprise a buffer.

111. The reaction mixture of claim 110, wherein the buffer is TRIS, PBS, citrate, monosodium glutamate, or a combination thereof.

112. The reaction mixture of any one of claims 75-111, wherein the reaction conditions proceed for about 1-48 hours.

113. The reaction mixture of any one of claims 75-112, wherein the reaction conditions proceed for about 48 hours.

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