Simple Methods for the Preparation of Colloidal Chitin, Cell Free Supernatant and Estimation of Laminarinase

Background

Separation of insoluble solids from liquids may be achieved by either centrifugation or filtration. The use of centrifugation for the preparation of colloidal chitin (CC) and cell free supernatant (CFS) has limitations as a large volume of liquids are involved (Hsu and Lockwood, 1975; Mitsutomi et al., 1995). Preparation of CFS from lab-scale and pilot-scale fermented broth is particularly challenging (Sarkar et al., 2010). Thus, a large capacity centrifuge machine (preferably refrigerated) with suitable rotor must be used (e.g., Sorvall BIOS 16). These facilities may not be available to researchers, especially in developing countries. Under these circumstances, filtration can be a viable alternative to centrifugation.

Most of the filter elements described in the literature (viz. filter paper, paper coffee filter, Miracloth, cheese cloth, etc.) suffer from one or more of the drawbacks listed below (Sandhya et al., 2004; Javaid and Ali, 2011; Murthy and Bleakley, 2012; Nidheesh and Suresh, 2015; Park et al., 2015): (1) Expensive (Miracloth); (2) Fragile (filter paper and paper coffee filter); (3) Non-reusable (filter paper and paper coffee filter); (4) Poor filtration rate (cheese cloth, filter paper, and paper coffee filter) and (5) Not readily available (paper coffee filter and Miracloth).

In this work, a 280 g per square meter (GSM) 65/35 polyester-cotton blend LCCM was used as a filter element. LCCM was found to be perfectly suitable for the preparation of CC. CFS was prepared from Streptomyces rimosus AFM-1 using a similar set of materials and is most suitable for organisms exhibiting pellet growth (viz. fungi and actinomycetes) (Koteshwara et al., 2021).

Laminarin (β(1→3)-glucan interspersed with β(1→6)-branches) is the long-term carbon storage polymer of glucose found in brown algae (Beattie et al., 1961; Graiff et al., 2016). Laminarinases ((1→3)-β-glucan hydrolase) are a type of hemicellulase that act upon and hydrolyze laminarin. They can be classified as either endo-type (EC 3.2.1.39) or exo-type (EC 3.2.1.58) based on the substrate hydrolysis pattern (Wu et al., 2018). The laminarinase enzyme is produced by a wide variety of organisms and plays an important role in natural ecosystems for the recycling of carbon (Graiff et al., 2016). Bacteria usually only transport substrates with a molecular mass of <600Da (Weiss et al., 1991; Grinter and Lithgow, 2020). Consequently, many marine bacteria produce extracellular enzymes like laminarinases to hydrolyze high molecular mass substrates (e.g., laminarin: 2,000-7,000 Da) (Yang et al., 2020). β-Glucan hydrolases are used in the food industry, medicine, and biotechnology (Qin et al., 2017).

Commercial laminarin used in the laboratory is usually sourced from either Laminaria digitata (Sigma-Aldrich L9634) or Eisenia bicyclis (TCI L0088). Interestingly, laminarin has similarities to the β-glucans found in the fungal cell wall. Thus, it is used to evaluate the anti-fungal β-glucan hydrolases produced by various organisms, especially the biocontrol agents (Scott and Schekman, 1980; Watanabe et al., 1989; De la Cruz et al., 1995; Aktuganov et al., 2008). Under these circumstances, detection and estimation of laminarinase enzyme assumes importance.

AS precipitation continues to be the most preferred method for the separation of proteins from CFS due to the following reasons (Wingfield, 2001; Duong-Ly and Gabelli, 2014): (1) It is inexpensive, simple, germicidal, highly soluble in water, and exerts a stabilizing effect on the precipitated proteins; (2) For the precipitation of native and non-recombinant proteins; (3) It can inhibit proteolytic activity; and (4) Precipitation of recombinant protein without an affinity tag or an unexposed affinity tag.

AS precipitation is a non-specific method. Thus, optimization of the AS concentration must be carried out to reduce the amount of contaminating proteins. AS interferes with most enzyme assays, thus the precipitates must be dialyzed before evaluation. A simple and rapid assay that provides results without the need for dialysis would ease the AS optimization task; this idea was the motivation for this research work (Koteshwara et al., 2021).

Plate assays for enzymes are inherently much simpler than liquid assays. This is due to their simple design, ability to evaluate multiple samples in a single plate, and the fact that they provide direct qualitative/ semi-quantitative results (Teather and Wood, 1982; Samad et al., 1989; Gulati et al., 1997; Meddeb-Mouelhi et al., 2014; Sawant et al., 2015; Patil and Chaudhari, 2017). We have developed a plate assay for laminarinase using a laminarin-infused agarose gel. Laminarin is a translucent substrate necessitating the use of a dye for the detection of zones of hydrolysis. An easily available OFB Tinopal CBS-X (Disodium 4,4'-bis(2-sulfostyryl) biphenyl) was tested as a stain and found suitable. The plate assay provided accurate, reproducible results even in the presence of a high concentration of AS (Koteshwara et al., 2021). Congo red is an extensively used dye for the detection of polysaccharide hydrolysis but is not recommended for use with laminarin gel plate assay due to poor contrast (Wood et al., 1988). Many wide-ranging and complicated methods can be found in the literature for the preparation of CC, CFS, and laminarinase plate assay. Simple alternatives to complex protocols are described here.