**2. Enzyme biosynthesis**

Biocatalysis can be defined as the utilization of living materials or molecules to speed up the rate of chemical reactions. The usage of recombinant enzymes, groups of enzymes, naturally occurring enzymes, cell extracts and whole cells, modified or engineered enzymes inclusive. These biological materials have consequential edge over conventional chemical catalysis. Ideally, biochemical reactions occur in aqueous solution, at moderate temperatures and atmospheric pressure which can result in both environmental and economic values as compared with the existing processes at upraised temperatures and pressures, and in organic medium [5]. High cost of enzymes and potential environmental damages resulting from high temperatures and pressures requires substantial energy inputs. Comparatively little known aspect of biocatalysis is the role of protein potency compare to the overall average structure of a protein, mobility/potency is much difficult to estimate experimentally. Like other molecules, proteins are in sustained motion, with stretching, and rotating bonds bending. These motions can bring about to much greater, for example conformational rearrangements following substrate binding or the movement of two domains relative to one another. It is now clear that these motions play important in catalysis and the regulation of enzyme activity [6]. Moreover, recognizing those motions which come up with catalysis will be experimentally challenging. Likewise, modeling them precisely enough to predict their effects in novel arrangement will be tasking.

As important hydrolytic enzymes, amylase and protease represent the two largest groups of industrial enzymes and account for approximately 85% of total enzyme sales all over the globe. At present, more than 3000 different enzymes have been characterized and many of them found their way into biotechnological and industrial applications [7]. One technicality in enzyme technology especially those of starch biosynthetic enzymes is their manipulations to meet the enormous demand of teeming population, safeguard our environment from non-degradable biopolymer and of course satisfy the food industries' need [8]. Some commercially available biocatalysts do not resist industrial processing conditions due to severity of such conditions. Therefore, certain desirable characteristics during isolation and screening of novel enzymes viz.; alkaline stability, halophilicity, and thermostability are foremost to meet the industrial demand. Great deal of attention has been drawn on extremophiles, which are the valuable source of

#### *Prospects of Biocatalyst Purification Enroute Fermentation Processes DOI: http://dx.doi.org/10.5772/intechopen.97382*

novel biocatalysts [9]. Extracellular enzymes from these halophiles with polymerdegrading ability at low water activity are of significant in many task processes where concentrated salt solutions hinder enzymatic production. The potency of enzymes to maintain its activeness via organic solvents has attracted considerable interest over the past twenty years.

In contrast to in water, numerous advantages of using enzymes in organic solvents or aqueous solutions containing organic solvents have been observed. Generally, enzymes are easily denatured and their activities disappear in the presence of organic solvents. Therefore, enzymes that remain stable in the presence of organic solvents might be useful for biotechnological applications in which such solvents are used [10]. Because salt reduces water activity, a feature in common with organic solvent systems, halophilic enzymes are thought to be valuable tools as biocatalysts in other low-water-activity environments, such as in aqueous/organic and non-aqueous media.

#### **2.1 Purification protocols and applications**

There are considerable ways of maintaining enzyme efficiency. Purification technique is a very powerful device has been arduously used to make some economically attainable and high performance enzymes with improved stability [11]. Enzyme purification is imperative for a full apprehension of the description and established process of enzymes. This is usually a multistep process involving biomass separation, concentration, primary isolation, and purification [12]. The contractual methods for the removal of enzymatic debrises or impurities and colloidal particles from fermentation broth include ammonium sulphate precipitation which encompassing dialysis for almost 16 hours before product could be recovered and also results into protein denaturation due to conformational changes [13].

Large volume of industrial enzymes are usually not purified. Their recovery is often accomplished by an ultrafiltration step. During enzyme production, desired products are synthesis after several concentration and separation techniques known as downstream processing (DSP). Two factors (time and cost) are the major challenges confronting these conventional techniques and their sustainability and efficiency depend on precise choice of purification methods [14, 15]. Here are some examples of strategies undertaken to improve the performance of enzymes with applications in food industry. Wong et al. [16] investigated strategies employed in starch liquefaction with targeted improvement of thermostability using α-amylase, protein engineering through site-directed mutagenesis and mutant displayed increased half-life between 15 min and 70 min at 100*°*C evolved. Glucoamylase with specific role as starch saccharifier and targeted improvement of substrate specificity, thermostability and pH optimum was characterized with protein engineering through site-directed mutagenesis alone [17]. Xylose (glucose) isomerase displayed isomerization/epimerization of hexoses, pentoses and tetroses as significance role of pH-activity profile with targeted improvement which resulted in protein engineering through directed evolution and the yield number on D-glucose in wild type was sustained between pH 6.0 and 7.5 and improvably at pH 7.3 as compared with mutant strains enhanced by 30–40%.

The application of polyvinyl alcohol or carbowax for protein and enzyme concentration is being restricted by poor water holding capacity. Moreover, gel filtration technique is also considered arduous and costly to the developing nations [18]. Carboxy-methyl cellulose, tannic acid, edible gum and some organic solvents as precipitants also poses the problem of product recovery [13]. In fishing industry, the use of fast, simple and low cost techniques such as using organic solvents vis ethanol and acetone, successive stages of centrifugation and filtration; and

saline solution (ammonium sulfate) were adopted for the separation and partial purification of protein biomolecules obtained from fish by-products beneficiation remains [19–21], aiming to improve the degree of biomolecule purity [21]. The use of ammonium sulphate precipitates during enzyme purification needs protracted separation technique between 12 and 16 h for recovery of product that frequently bring about protein denaturation as gel chromatography is high priced and moderate for developing economies [22]. Application of chromatographic techniques such as gel filtration and ion exchange give rise to purer enzyme fractions, with significant increase in specific activity. These are often used to estimate the molecular mass of the enzyme by comparing protein mixtures of known molecular mass (reference standards) with the unknown.

Pectinase enzyme was precipitated by dissolving it in a 0.1 M, pH 4.2 sodium acetate buffer after mixing with 3 volumes of ice-cold acetone and allowed to stay for 15 min [23]. Chimbekujwo et al. [24] reported the application of SDS-PAGE analysis of purified fungal protease of major protein band with molecular weight of 68 KDa, 13.3 fold and 28% yield. This partially purified enzyme was stable between 30 and 40°C temperature and pH 4–6 which enhanced the activity by Tween-20 and Calcium ions. Moreover, during the production, characterization and anti-cancer application of extracellular L-glutaminase from the marine bacterial isolate, the enzyme was purified through QFF technique by engaging ethanol precipitation and ion-exchange chromatography and resulted into 2-fold purification with molecular weight 54.8 kDa, specific activity 89.78 U/mg, maximum enzyme activity at 40°C and pH 8.2 and ultimately retained 90% activity for an hour [25].
